Leukocyte TNFR1 and TNFR2 Expression Contributes to the Peripheral Immune Response in Cases with Ischemic Stroke.
Hansen Rikke B,Laursen Cathrine C H,Nawaz Niala,Madsen Jonna S,Nielsen Helle H,Kruuse Christina,Møller Arne,Degn Matilda,Lambertsen Kate L
Cells
Tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2) have been found in brain parenchyma of stroke patients, and plasma levels are increased in the acute phase of stroke. We evaluated associations between TNFR1 and TNFR2 plasma levels and stroke severity, infarct size, and functional outcome. Furthermore, we examined cellular expression of TNFR1 and TNFR2 on leukocyte subpopulations to explore the origin of the increased receptor levels. Blood samples were taken from 33 acute ischemic stroke patients and 10 healthy controls. TNFR1 and TNFR2 plasma concentrations were measured and correlated against the Scandinavian Stroke Scale at admission, infarct volume, and the modified Rankin Scale score three months after stroke onset. Classical, intermediate, and non-classical monocytes as well as neutrophils were purified, and cellular expression of TNFR1 and TNFR2 was examined using flow cytometry. TNFR1 and TNFR2 plasma levels were both increased after ischemic stroke, but we found no correlation with patient outcome measurements. Compared to healthy controls, ischemic stroke patients had decreased non-classical monocyte and neutrophil populations expressing TNFR1 and increased neutrophils expressing TNFR2, and decreased non-classical populations co-expressing both TNFR1 and TNFR2. This study supports the hypothesis of an acute immunological response orchestrated by the peripheral immune system following an ischemic stroke. However, the origin of the increased TNFR1 and TNFR2 plasma levels could not be clearly linked to peripheral monocytes or neutrophils. Future studies are needed and will help clarify the potential role as treatment target.
10.3390/cells10040861
Central Nervous System-Infiltrating T Lymphocytes in Stroke Are Activated via Their TCR (T-Cell Receptor) but Lack CD25 Expression.
Schulze Juliane,Gellrich Juliane,Kirsch Michael,Dressel Alexander,Vogelgesang Antje
Stroke
Background and Purpose:T lymphocytes contribute to secondary brain damage after stroke. It has not been fully investigated whether this contribution is caused by antigen-specific or antigen-nonspecific activation of T lymphocytes. Lymphocytes from Nur77GFP transgenic mice express a fluorescent protein upon activation via the TCR (T-cell receptor), allowing the differentiation of activation mode in a natural repertoire of immune cells and antigens. Methods:Middle cerebral artery occlusion or sham surgery was performed, and T-lymphocyte activation was analyzed by flow cytometry in the brain, spleen, and blood 16 hours, 2 days, 3 days, 4 days, and 7 days after surgery. Results:Ipsilateral hemispheric T-lymphocyte invasion peaked on day 4 poststroke. Here, we observed PD-1 (programmed cell death protein 1) expression on almost all invading T lymphocytes, while CD25 expression was low. CD25+, CD69+, or PD-1+ T lymphocytes predominantly displayed antigen-specific activation; the opposite was observed for T lymphocytes isolated from the blood. A mixed activation that favored antigen-specific activation was observed in the spleen. PD-1 was upregulated within the brain, whereas CD25 was not. Antigen-specific T lymphocytes home to the brain, while antigen-nonspecifically activated cells remain within the blood. Conclusions:Our data clearly demonstrate antigen-specific activation of T lymphocytes infiltrating ischemic brain lesions in stroke. The high expression of inhibitory PD-1 and low expression of CD25 on activated T lymphocytes in the brain most likely reflect immunosuppressive mechanisms.
10.1161/STROKEAHA.120.032763
Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.
Frontiers in immunology
Background and Objectives:Inhibition of pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide. Methods:High-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles. Results:We found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory T follicular helper T cell and helper T cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism. Discussion:Overall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.
10.3389/fimmu.2021.730342
Interleukin-9 regulates macrophage activation in the progressive multiple sclerosis brain.
Donninelli Gloria,Saraf-Sinik Inbar,Mazziotti Valentina,Capone Alessia,Grasso Maria Grazia,Battistini Luca,Reynolds Richard,Magliozzi Roberta,Volpe Elisabetta
Journal of neuroinflammation
BACKGROUND:Multiple sclerosis (MS) is an immune-mediated, chronic inflammatory, and demyelinating disease of the central nervous system (CNS). Several cytokines are thought to be involved in the regulation of MS pathogenesis. We recently identified interleukin (IL)-9 as a cytokine reducing inflammation and protecting from neurodegeneration in relapsing-remitting MS patients. However, the expression of IL-9 in CNS, and the mechanisms underlying the effect of IL-9 on CNS infiltrating immune cells have never been investigated. METHODS:To address this question, we first analyzed the expression levels of IL-9 in post-mortem cerebrospinal fluid of MS patients and the in situ expression of IL-9 in post-mortem MS brain samples by immunohistochemistry. A complementary investigation focused on identifying which immune cells express IL-9 receptor (IL-9R) by flow cytometry, western blot, and immunohistochemistry. Finally, we explored the effect of IL-9 on IL-9-responsive cells, analyzing the induced signaling pathways and functional properties. RESULTS:We found that macrophages, microglia, and CD4 T lymphocytes were the cells expressing the highest levels of IL-9 in the MS brain. Of the immune cells circulating in the blood, monocytes/macrophages were the most responsive to IL-9. We validated the expression of IL-9R by macrophages/microglia in post-mortem brain sections of MS patients. IL-9 induced activation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 and reduced the expression of activation markers, such as CD45, CD14, CD68, and CD11b in inflammatory macrophages stimulated in vitro with lipopolysaccharide and interferon (IFN)-γ. Similarly, in situ the number of activated CD68 macrophages was significantly reduced in areas with high levels of IL-9. Moreover, in the same conditions, IL-9 increased the secretion of the anti-inflammatory cytokine, transforming growth factor (TGF)-β. CONCLUSIONS:These results reveal a new cytokine expressed in the CNS, with a role in the context of MS. We have demonstrated that IL-9 and its receptor are both expressed in CNS. Moreover, we found that IL-9 decreases the activation state and promotes the anti-inflammatory properties of human macrophages. This mechanism may contribute to the beneficial effects of IL-9 that are observed in MS, and may be therapeutically potentiated by modulating IL-9 expression in MS.
10.1186/s12974-020-01770-z
High-throughput investigation of molecular and cellular biomarkers in NMOSD.
Yandamuri Soumya S,Jiang Ruoyi,Sharma Aditi,Cotzomi Elizabeth,Zografou Chrysoula,Ma Anthony K,Alvey Jessica S,Cook Lawrence J,Smith Terry J,Yeaman Michael R,O'Connor Kevin C,
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To identify candidate biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD) using high-throughput technologies that broadly assay the concentrations of serum analytes and frequencies of immune cell subsets. METHODS:Sera, peripheral blood mononuclear cells (PBMCs), and matched clinical data from participants with NMOSD and healthy controls (HCs) were obtained from the Collaborative International Research in Clinical and Longitudinal Experience Study NMOSD biorepository. Flow cytometry panels were used to measure the frequencies of 39 T-cell, B-cell, regulatory T-cell, monocyte, natural killer (NK) cell, and dendritic cell subsets in unstimulated PBMCs. In parallel, multiplex proteomics assays were used to measure 46 serum cytokines and chemokines in 2 independent NMOSD and HC cohorts. Multivariable regression models were used to assess molecular and cellular profiles in NMOSD compared with HC. RESULTS:NMOSD samples had a lower frequency of CD16CD56 NK cells. Both serum cohorts and multivariable logistic regression revealed increased levels of B-cell activating factor associated with NMOSD. Interleukin 6, CCL22, and CCL3 were also elevated in 1 NMOSD cohort of the 2 analyzed. Multivariable linear regression of serum analyte levels revealed a correlation between CX3CL1 (fractalkine) levels and the number of days since most recent disease relapse. CONCLUSIONS:Integrative analyses of cytokines, chemokines, and immune cells in participants with NMOSD and HCs provide congruence with previously identified biomarkers of NMOSD and highlight CD16CD56 NK cells and CX3CL1 as potential novel biomarker candidates.
10.1212/NXI.0000000000000852
Hookworm Treatment for Relapsing Multiple Sclerosis: A Randomized Double-Blinded Placebo-Controlled Trial.
Tanasescu Radu,Tench Christopher R,Constantinescu Cris S,Telford Gary,Singh Sonika,Frakich Nanci,Onion David,Auer Dorothee P,Gran Bruno,Evangelou Nikos,Falah Yasser,Ranshaw Colin,Cantacessi Cinzia,Jenkins Timothy P,Pritchard David I
JAMA neurology
Importance:Studies suggest gut worms induce immune responses that can protect against multiple sclerosis (MS). To our knowledge, there are no controlled treatment trials with helminth in MS. Objective:To determine whether hookworm treatment has effects on magnetic resonance imaging (MRI) activity and T regulatory cells in relapsing MS. Design, Setting, and Participants:This 9-month double-blind, randomized, placebo-controlled trial was conducted between September 2012 and March 2016 in a modified intention-to-treat population (the data were analyzed June 2018) at the University of Nottingham, Queen's Medical Centre, a single tertiary referral center. Patients aged 18 to 61 years with relapsing MS without disease-modifying treatment were recruited from the MS clinic. Seventy-three patients were screened; of these, 71 were recruited (2 ineligible/declined). Interventions:Patients were randomized (1:1) to receive either 25 Necator americanus larvae transcutaneously or placebo. The MRI scans were performed monthly during months 3 to 9 and 3 months posttreatment. Main Outcomes and Measures:The primary end point was the cumulative number of new/enlarging T2/new enhancing T1 lesions at month 9. The secondary end point was the percentage of cluster of differentiation (CD) 4+CD25highCD127negT regulatory cells in peripheral blood. Results:Patients (mean [SD] age, 45 [9.5] years; 50 women [71%]) were randomized to receive hookworm (35 [49.3%]) or placebo (36 [50.7%]). Sixty-six patients (93.0%) completed the trial. The median cumulative numbers of new/enlarging/enhancing lesions were not significantly different between the groups by preplanned Mann-Whitney U tests, which lose power with tied data (high number of zeroactivity MRIs in the hookworm group, 18/35 [51.4%] vs 10/36 [27.8%] in the placebo group). The percentage of CD4+CD25highCD127negT cells increased at month 9 in the hookworm group (hookworm, 32 [4.4%]; placebo, 34 [3.9%]; P = .01). No patients withdrew because of adverse effects. There were no differences in adverse events between groups except more application-site skin discomfort in the hookworm group (82% vs 28%). There were 5 relapses (14.3%) in the hookworm group vs 11 (30.6%) receiving placebo. Conclusions and Relevance:Treatment with hookworm was safe and well tolerated. The primary outcome did not reach significance, likely because of a low level of disease activity. Hookworm infection increased T regulatory cells, suggesting an immunobiological effect of hookworm. It appears that a living organism can precipitate immunoregulatory changes that may affect MS disease activity. Trial Registration:ClinicalTrials.gov Identifier: NCT01470521.
10.1001/jamaneurol.2020.1118
The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity.
Croxford Andrew L,Lanzinger Margit,Hartmann Felix J,Schreiner Bettina,Mair Florian,Pelczar Pawel,Clausen Björn E,Jung Steffen,Greter Melanie,Becher Burkhard
Immunity
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.
10.1016/j.immuni.2015.08.010
Trained Immunity Characteristics Are Associated With Progressive Cerebral Small Vessel Disease.
Noz Marlies P,Ter Telgte Annemieke,Wiegertjes Kim,Joosten Leo A B,Netea Mihai G,de Leeuw Frank-Erik,Riksen Niels P
Stroke
Background and Purpose- Cerebral small vessel disease (cSVD) is the major vascular cause of cognitive decline and dementia. The pathogenesis of cSVD remains largely unknown, although several studies suggest a role for systemic inflammation. In certain pathophysiological situations, monocytes can reprogram toward a long-term proinflammatory phenotype, which has been termed trained immunity. We hypothesize that trained immunity contributes to the progression of cSVD. Methods- Individuals with mild-to-severe cSVD participated in the study. Severity of cSVD was determined by the white matter hyperintensities (WMH) volume (mL) on magnetic resonance imaging in 2006, 2015, and the progression between 2006 and 2015 (ΔWMH). Cytokine production was assessed after ex vivo stimulation of peripheral blood mononuclear cells and monocytes. Additionally, monocyte subsets were identified by flow cytometry. Results- Fifty-one subjects (70±6 years, 60% men, 5.1±6.4 mL ΔWMH) were included. Circulating hsIL (high-sensitivity interleukin)-6 correlated with cSVD ( P=0.005, r=0.40). Cytokine production capacity by monocytes was associated with cSVD progression. Basal IL-8 and IL-17 production ( P=0.08, r=0.25; P=0.03, r=0.30) and IL-6 production after Pam3Cys stimulation in monocytes was associated with cSVD (n=35: P=0.008, r=0.44). Conversely, interferon (IFN)-γ production in Candida albicans stimulated peripheral blood mononuclear cells was negatively correlated with cSVD ( P=0.009, r=-0.36). Flow cytometry revealed a correlation of the intermediate monocyte subset with cSVD ( P=0.01, r=0.36). Conclusions- Severity and progression of cSVD are not only correlated with systemic inflammation (hsIL-6) but also with trained immunity characteristics of circulating monocytes, in terms of an altered cytokine production capacity and a shift toward the proinflammatory intermediate monocyte subset.
10.1161/STROKEAHA.118.023192
NK Cell Patterns in Idiopathic Inflammatory Myopathies with Pulmonary Affection.
Pawlitzki Marc,Nelke Christopher,Rolfes Leoni,Hasseli Rebecca,Tomaras Stylianos,Feist Eugen,Schänzer Anne,Räuber Saskia,Regner Liesa,Preuße Corinna,Allenbach Yves,Benveniste Olivier,Wiendl Heinz,Stenzel Werner,Meuth Sven G,Ruck Tobias
Cells
BACKGROUND:Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. METHODS:To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with ( = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA ( = 12). RESULTS:We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56 NK cells, while CD56 NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D on NK cells from patients with PA compared with non-PA patients, especially on the CD56 subset. NKG2D and NKp46 cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. CONCLUSION:Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches.
10.3390/cells10102551
Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease.
Yamamoto Yohei,Matsui Naoko,Uzawa Akiyuki,Ozawa Yukiko,Kanai Tetsuya,Oda Fumiko,Kondo Hiroyuki,Ohigashi Izumi,Takizawa Hiromitsu,Kondo Kazuya,Sugano Mikio,Kitaichi Takashi,Hata Hiroki,Kaji Ryuji,Kuwabara Satoshi,Yamamura Takashi,Izumi Yuishin
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:To investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact. METHODS:Thymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19B220 thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated. RESULTS:The frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19B220 thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy. DISCUSSION:Our findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.
10.1212/NXI.0000000000001087
CDC6 is a prognostic biomarker and correlated with immune infiltrates in glioma.
Molecular cancer
BACKGROUND:Cell division cycle 6 (CDC6) has been proven to be associated with the initiation and progression of human multiple tumors. However, it's role in glioma, which is ranked as one of the common primary malignant tumor in the central nervous system and is associated with high morbidity and mortality, is unclear. METHODS:In this study, we explored CDC6 gene expression level in pan-cancer. Furthermore, we focused on the relationships between CDC6 expression, its prognostic value, potential biological functions, and immune infiltrates in glioma patients. We also performed vitro experiments to assess the effect of CDC6 expression on proliferative, apoptotic, migrant and invasive abilities of glioma cells. RESULTS:As a result, CDC6 expression was upregulated in multiple types of cancer, including glioma. Moreover, high expression of CDC6 was significantly associated with age, IDH status, 1p/19q codeletion status, WHO grade and histological type in glioma (all p < 0.05). Meanwhile, high CDC6 expression was associated with poor overall survival (OS) in glioma patients, especially in different clinical subgroups. Furthermore, a univariate Cox analysis showed that high CDC6 expression was correlated with poor OS in glioma patients. Functional enrichment analysis indicated that CDC6 was mainly involved in pathways related to DNA transcription and cytokine activity, and Gene Set Enrichment Analysis (GSEA) revealed that MAPK pathway, P53 pathway and NF-κB pathway in cancer were differentially enriched in glioma patients with high CDC6 expression. Single-sample gene set enrichment analysis (ssGSEA) showed CDC6 expression in glioma was positively correlated with Th2 cells, Macrophages and Eosinophils, and negative correlations with plasmacytoid dendritic cells, CD8 T cells and NK CD56bright cells, suggesting its role in regulating tumor immunity. Finally, CCK8 assay, flow cytometry and transwell assays showed that silencing CDC6 could significantly inhibit proliferation, migration, invasion, and promoted apoptosis of U87 cells and U251 cells (p < 0.05). CONCLUSION:In conclusion, high CDC6 expression may serve as a promising biomarker for prognosis and correlated with immune infiltrates, presenting to be a potential immune therapy target in glioma.
10.1186/s12943-022-01623-8
Biological activity of glatiramer acetate on Treg and anti-inflammatory monocytes persists for more than 10years in responder multiple sclerosis patients.
Spadaro Michela,Montarolo Francesca,Perga Simona,Martire Serena,Brescia Federica,Malucchi Simona,Bertolotto Antonio
Clinical immunology (Orlando, Fla.)
Glatiramer acetate (GA) is a widely used treatment for multiple sclerosis (MS), with incompletely defined mechanism of action. Short-term studies suggested its involvement in the modulation of anti-inflammatory cytokines and regulatory T cells (Treg), while long-term effect is still unknown. To investigate this aspect, we analyzed by flow-cytometry peripheral-blood Treg, natural killer (NK), CD4 and CD8 T-cells and anti-inflammatory CD14CD163 monocytes from 37 healthy donor and 90 RRMS patients divided in untreated, treated with GA for 12months and from 34 to 192months. While NK, CD4 and CD8 T-cells did not show any significant differences among groups over time, we demonstrated that GA increased the anti-inflammatory monocytes and restored the Treg level in both GA-treated groups. Both these effects are a characteristic of responder patients and are observed not just in short-term but even after as long as a decade of GA treatment.
10.1016/j.clim.2017.06.006
Alterations of host-gut microbiome interactions in multiple sclerosis.
EBioMedicine
BACKGROUND:Multiple sclerosis (MS) has a complex genetic, immune and metabolic pathophysiology. Recent studies implicated the gut microbiome in MS pathogenesis. However, interactions between the microbiome and host immune system, metabolism and diet have not been studied over time in this disorder. METHODS:We performed a six-month longitudinal multi-omics study of 49 participants (24 untreated relapse remitting MS patients and 25 age, sex, race matched healthy control individuals. Gut microbiome composition and function were characterized using 16S and metagenomic shotgun sequencing. Flow cytometry was used to characterize blood immune cell populations and cytokine profiles. Circulating metabolites were profiled by untargeted UPLC-MS. A four-day food diary was recorded to capture the habitual dietary pattern of study participants. FINDINGS:Together with changes in blood immune cells, metagenomic analysis identified a number of gut microbiota decreased in MS patients compared to healthy controls, and microbiota positively or negatively correlated with degree of disability in MS patients. MS patients demonstrated perturbations of their blood metabolome, such as linoleate metabolic pathway, fatty acid biosynthesis, chalcone, dihydrochalcone, 4-nitrocatechol and methionine. Global correlations between multi-omics demonstrated a disrupted immune-microbiome relationship and a positive blood metabolome-microbiome correlation in MS. Specific feature association analysis identified a potential correlation network linking meat servings with decreased gut microbe B. thetaiotaomicron, increased Th17 cell and greater abundance of meat-associated blood metabolites. The microbiome and metabolome profiles remained stable over six months in MS and control individuals. INTERPRETATION:Our study identified multi-system alterations in gut microbiota, immune and blood metabolome of MS patients at global and individual feature level. Multi-OMICS data integration deciphered a potential important biological network that links meat intakes with increased meat-associated blood metabolite, decreased polysaccharides digesting bacteria, and increased circulating proinflammatory marker. FUNDING:This work was supported by the Washington University in St. Louis Institute of Clinical and Translational Sciences, funded, in part, by Grant Number # UL1 TR000448 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (Zhou Y, Piccio, L, Lovett-Racke A and Tarr PI); R01 NS10263304 (Zhou Y, Piccio L); the Leon and Harriet Felman Fund for Human MS Research (Piccio L and Cross AH). Cantoni C. was supported by the National MS Society Career Transition Fellowship (TA-180531003) and by donations from Whitelaw Terry, Jr. / Valerie Terry Fund. Ghezzi L. was supported by the Italian Multiple Sclerosis Society research fellowship (FISM 2018/B/1) and the National Multiple Sclerosis Society Post-Doctoral Fellowship (FG-190734474). Anne Cross was supported by The Manny & Rosalyn Rosenthal-Dr. John L. Trotter MS Center Chair in Neuroimmunology of the Barnes-Jewish Hospital Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
10.1016/j.ebiom.2021.103798
Prospective phase II clinical trial of autologous haematopoietic stem cell transplant for treatment refractory multiple sclerosis.
Moore John J,Massey Jennifer C,Ford Carole D,Khoo Melissa L,Zaunders John J,Hendrawan Kevin,Barnett Yael,Barnett Michael H,Kyle Kain A,Zivadinov Robert,Ma Kris C,Milliken Sam T,Sutton Ian J,Ma David D F
Journal of neurology, neurosurgery, and psychiatry
BACKGROUND:Autologous haematopoietic stem cell transplantation (AHSCT) has been explored as a therapeutic intervention in multiple sclerosis (MS) over the last two decades; however, prospective clinical trials of the most common myeloablative conditioning regimen, BEAM, are limited. Furthermore, patient selection, optimal chemotherapeutic regimen and immunological changes associated with disease response require ongoing exploration. We present the outcomes, safety and immune reconstitution (IR) of patients with active, treatment refractory MS. METHODS:This study was a single-centre, phase II clinical trial of AHSCT for patients with active relapsing remitting (RRMS) and secondary progressive MS (SPMS). Patients underwent AHSCT using BEAM (carmustine, etoposide, cytarabine, melphalan)+antithymocyte globulin chemotherapeutic regimen. OUTCOMES:The primary outcome was event-free survival (EFS); defined as no clinical or radiological relapses and no disability progression. Multiparameter flow cytometry was performed for evaluation of post-transplant IR in both MS and lymphoma patients receiving the same chemotherapy regimen. RESULTS:Thirty-five patients (20 RRMS, 15 SPMS) completed AHSCT, with a median follow-up of 36 months (range 12-66). The median Expanded Disability Status Scores (EDSS) was 6 (2-7) and patients had failed a median of 4 (2-7) disease modifying therapies. 66% failed treatment with natalizumab. EFS at 3 years was 60%, (70% RRMS). Sustained improvement in EDSS was seen in 15 (44%) of patients. There was no treatment-related mortality. A sustained rise in CD39 T regulatory cells, immunosuppressive CD56 natural killer cells and ablation of proinflammatory mucosal-associated invariant T cells was seen for 12 months following AHSCT in patients with MS. These changes did not occur in patients with lymphoma receiving the same chemotherapy for AHSCT. CONCLUSIONS:The EFS in our MS cohort is significantly greater than other high-efficacy immunosuppressive therapies and similar to other AHSCT studies despite a more heavily pretreated cohort. TRIAL REGISTRATION NUMBER:ACTRN12613000339752.
10.1136/jnnp-2018-319446
Effect of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice.
Neurology
OBJECTIVE:To assess functional changes in lymphocyte repertoire and subsequent clinical implications during delayed-release dimethyl fumarate (DMF) treatment in patients with multiple sclerosis. METHODS:Using peripheral blood from several clinical trials of DMF, immune cell subsets were quantified using flow cytometry. For some patients, lymphocyte counts were assessed after DMF discontinuation. Incidence of adverse events, including serious and opportunistic infections, was assessed. RESULTS:In DMF-treated patients, absolute lymphocyte counts (ALCs) demonstrated a pattern of decline followed by stabilization, which also was reflected in the global reduction in numbers of circulating functional lymphocyte subsets. The relative frequencies of circulating memory T- and B-cell populations declined and naive cells increased. No increased incidence of serious infection or malignancy was observed for patients treated with DMF, even when stratified by ALC or T-cell subset frequencies. For patients who discontinued DMF due to lymphopenia, ALCs increased after DMF discontinuation; recovery time varied by ALC level at discontinuation. T-cell subsets closely correlated with ALCs in both longitudinal and cross-sectional analyses. CONCLUSIONS:DMF shifted the immunophenotype of circulating lymphocyte subsets. ALCs were closely correlated with CD4 and CD8 T-cell counts, indicating that lymphocyte subset monitoring is not required for safety vigilance. No increased risk of serious infection was observed in patients with low T-cell subset counts. Monitoring ALC remains the most effective way of identifying patients at risk of subsequently developing prolonged moderate to severe lymphopenia, a risk factor for progressive multifocal leukoencephalopathy in DMF-treated patients. TRIAL REGISTRATION NUMBERS:EUDRA CT 2015-001973-42, NCT00168701, NCT00420212, NCT00451451, and NCT00835770.
10.1212/WNL.0000000000007262
Dysregulation of the Adaptive Immune System in Patients With Early-Stage Parkinson Disease.
Yan Zhaoqi,Yang Wei,Wei Hairong,Dean Marissa N,Standaert David G,Cutter Gary R,Benveniste Etty N,Qin Hongwei
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To determine the activation status and cytokine profiles of CD4 T cells, CD8 T cells, and CD19 B cells from patients with early-stage Parkinson disease (PD) compared with healthy controls (HCs). METHODS:Peripheral blood samples from 41 patients with early-stage PD and 40 HCs were evaluated. Peripheral blood mononuclear cells were analyzed by flow cytometry for surface markers and intracellular cytokine production. Correlations of immunologic changes and clinical parameters were analyzed. RESULTS:Adaptive immunity plays a role in the pathogenesis of PD, yet the contribution of T cells and B cells, especially cytokine production by these cells, is poorly understood. We demonstrate that naive CD4 and naive CD8 T cells are significantly decreased in patients with PD, whereas central memory CD4 T cells are significantly increased in patients with PD. Furthermore, IL-17-producing CD4 Th17 cells, IL-4-producing CD4 Th2 cells, and IFN-γ-producing CD8 T cells are significantly increased in patients with PD. Regarding B cells, we observed a decrease in naive B cells and an increase in nonswitched memory and double-negative B cells. As well, TNF-α-producing CD19 B cells were significantly increased in patients with PD. Notably, some of the changes observed in CD4 T cells and B cells were associated with clinical motor disease severity. CONCLUSIONS:These findings suggest that alterations in the adaptive immune system may promote clinical disease in PD by skewing to a more proinflammatory state in the early-stage PD patient cohort. Our study may shed light on potential immunotherapies targeting dysregulated CD4 T cells, CD8 T cells, and CD19 B cells in patients with PD.
10.1212/NXI.0000000000001036
Contact-Dependent Granzyme B-Mediated Cytotoxicity of Th17-Polarized Cells Toward Human Oligodendrocytes.
Frontiers in immunology
Multiple sclerosis (MS) is characterized by the loss of myelin and of myelin-producing oligodendrocytes (OLs) in the central nervous system (CNS). Pro-inflammatory CD4 Th17 cells are considered pathogenic in MS and are harmful to OLs. We investigated the mechanisms driving human CD4 T cell-mediated OL cell death. Using fluorescent and brightfield live imaging, we found that compared to Th2-polarized cells, Th17-polarized cells show greater interactions with primary human OLs and human oligodendrocytic cell line MO3.13, displaying longer duration of contact, lower mean speed, and higher rate of vesicle-like structure formation at the sites of contact. Using single-cell RNA sequencing, we assessed the transcriptomic profile of primary human OLs and Th17-polarized cells in direct contact or separated by an insert. We showed that upon close interaction, OLs upregulate the expression of mRNA coding for chemokines and antioxidant/anti-apoptotic molecules, while Th17-polarized cells upregulate the expression of mRNA coding for chemokines and pro-inflammatory cytokines such as IL-17A, IFN-γ, and granzyme B. We found that secretion of CCL3, CXCL10, IFN-γ, TNFα, and granzyme B is induced upon direct contact in cocultures of human Th17-polarized cells with human OLs. In addition, we validated by flow cytometry and immunofluorescence that granzyme B levels are upregulated in Th17-polarized compared to Th2-polarized cells and are even higher in Th17-polarized cells upon direct contact with OLs or MO3.13 cells compared to Th17-polarized cells separated from OLs by an insert. Moreover, granzyme B is detected in OLs and MO3.13 cells following direct contact with Th17-polarized cells, suggesting the release of granzyme B from Th17-polarized cells into OLs/MO3.13 cells. To confirm granzyme B-mediated cytotoxicity toward OLs, we showed that recombinant human granzyme B can induce OLs and MO3.13 cell death. Furthermore, pretreatment of Th17-polarized cells with a reversible granzyme B blocker (Ac-IEPD-CHO) or a natural granzyme B blocker (serpina3N) improved survival of MO3.13 cells upon coculture with Th17 cells. In conclusion, we showed that human Th17-polarized cells form biologically significant contacts with human OLs and exert direct toxicity by releasing granzyme B.
10.3389/fimmu.2022.850616
Human dorsal root ganglion pulsed radiofrequency treatment modulates cerebrospinal fluid lymphocytes and neuroinflammatory markers in chronic radicular pain.
Das Basabjit,Conroy Melissa,Moore David,Lysaght Joanne,McCrory Connail
Brain, behavior, and immunity
Radicular pain is a common cause of disability. Traditionally treatment has been either epidural steroid injection providing short-term relief or surgery with associated complications. Pulsed radiofrequency (PRF) applied to the dorsal root ganglion (DRG) is a minimally invasive day-care treatment, which is gaining significant clinical acceptance in a selective group of patients with pure radicular pain. Greater insights into the immunomodulatory effects of this procedure may help to further optimise its application and find alternative treatment options. We have examined it's effect on lymphocyte frequencies and secreted inflammatory markers in the cerebrospinal fluid (CSF) and correlated this with clinical outcome to identify clinical markers of chronic radicular pain. Ten patients were recruited for the study. CSF lymphocyte frequencies and levels of cytokines, chemokines and growth factors were quantified using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Clinical assessment utilised Brief Pain Inventory scores. Nine out of ten patients (90%) demonstrated significant reduction in pain severity (p = 0.0007) and pain interference scores (p = 0.0015) three months post-treatment. Our data revealed significant reductions in CD56, CD3, NK cell frequencies (p = 0.03) and IFN-γ levels (p = 0.03) in treatment responders, while CD8 T cell frequencies (p = 0.02) and IL-6 levels were increased (p = 0.05). IL-17 inversely correlated with post-treatment pain severity score (p = 0.01) and pre and post-treatment pain interference scores (p = 0.03, p = 0.01). These results support the concept that chronic radicular pain is a centrally mediated neuroimmune phenomenon and the mechanism of action of DRG PRF treatment is immunomodulatory.
10.1016/j.bbi.2018.02.010
Changes in CD163+, CD11b+, and CCR2+ peripheral monocytes relate to Parkinson's disease and cognition.
Brain, behavior, and immunity
Alpha-synuclein pathology is associated with immune activation and neurodegeneration in Parkinson's disease. The immune activation involves not only microglia but also peripheral immune cells, such as mononuclear phagocytes found in blood and infiltrated in the brain. Understanding peripheral immune involvement is essential for developing immunomodulatory treatment. Therefore, we aimed to study circulating mononuclear phagocytes in early- and late-stage Parkinson's disease, defined by disease duration of less or more than five years, respectively, and analyze their association with clinical phenotypes. We performed a cross-sectional multi-color flow cytometry study on 78 sex-balanced individuals with sporadic Parkinson's disease, 28 controls, and longitudinal samples from seven patients and one control. Cell frequencies and surface marker expressions on natural killer cells, monocyte subtypes, and dendritic cells were compared between groups and correlated with standardized clinical scores. We found elevated frequencies and surface levels of migration- (CCR2, CD11b) and phagocytic- (CD163) markers, particularly on classical and intermediate monocytes in early Parkinson's disease. HLA-DR expression was increased in advanced stages of the disease, whereas TLR4 expression was decreased in women with Parkinson's Disease. The disease-associated immune changes of CCR2 and CD11b correlated with worse cognition. Increased TLR2 expression was related to worse motor symptoms. In conclusion, our data highlights the TLR2 relevance in the symptomatic motor presentation of the disease and a role for peripheral CD163 and migration-competent monocytes in Parkinson's disease cognitive defects. Our study suggests that the peripheral immune system is dynamically altered in Parkinson's disease stages and directly related to both symptoms and the sex bias of the disease.
10.1016/j.bbi.2022.01.005
MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.
Brain : a journal of neurology
The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.
10.1093/brain/awac389
Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.
Benvenuto Federica,Voci Adriana,Carminati Enrico,Gualandi Francesca,Mancardi Gianluigi,Uccelli Antonio,Vergani Laura
Stem cell research & therapy
INTRODUCTION:Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. METHODS:We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. RESULTS:Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. CONCLUSIONS:Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.
10.1186/s13287-015-0222-y
Identification of the regulatory role of lncRNA HCG18 in myasthenia gravis by integrated bioinformatics and experimental analyses.
Journal of translational medicine
BACKGROUND:Long non-coding RNAs (lncRNAs), functioning as competing endogenous RNAs (ceRNAs), have been reported to play important roles in the pathogenesis of autoimmune diseases. However, little is known about the regulatory roles of lncRNAs underlying the mechanism of myasthenia gravis (MG). The aim of the present study was to explore the roles of lncRNAs as ceRNAs associated with the progression of MG. METHODS:MG risk genes and miRNAs were obtained from public databases. Protein-protein interaction (PPI) network analysis and module analysis were performed. A lncRNA-mediated module-associated ceRNA (LMMAC) network, which integrated risk genes in modules, risk miRNAs and predicted lncRNAs, was constructed to systematically explore the regulatory roles of lncRNAs in MG. Through performing random walk with restart on the network, HCG18/miR-145-5p/CD28 ceRNA axis was found to play important roles in MG, potentially. The expression of HCG18 in MG patients was detected using RT-PCR. The effects of HCG18 knockdown on cell proliferation and apoptosis were determined by CCK-8 assay and flow cytometry. The interactions among HCG18, miR-145-5p and CD28 were explored by luciferase assay, RT-PCR and western blot assay. RESULTS:Based on PPI network, we identified 9 modules. Functional enrichment analyses revealed these modules were enriched in immune-related signaling pathways. We then constructed LMMAC network, containing 25 genes, 50 miRNAs, and 64 lncRNAs. Through bioinformatics algorithm, we found lncRNA HCG18 as a ceRNA, might play important roles in MG. Further experiments indicated that HCG18 was overexpressed in MG patients and was a target of miR-145-5p. Functional assays illustrated that HCG18 suppressed Jurkat cell apoptosis and promoted cell proliferation. Mechanistically, knockdown of HCG18 inhibited the CD28 mRNA and protein expression levels in Jurkat cells, while miR-145-5p inhibitor blocked the reduction of CD28 expression induced by HCG18 suppression. CONCLUSION:We have reported a novel HCG18/miR-145-5p/CD28 ceRNA axis in MG. Our findings will contribute to a deeper understanding of the molecular mechanism of and provide a novel potential therapeutic target for MG.
10.1186/s12967-021-03138-0
Functional differences between microglia and monocytes after ischemic stroke.
Ritzel Rodney M,Patel Anita R,Grenier Jeremy M,Crapser Joshua,Verma Rajkumar,Jellison Evan R,McCullough Louise D
Journal of neuroinflammation
BACKGROUND:The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models. METHODS:Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days. RESULTS:We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke. CONCLUSIONS:In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.
10.1186/s12974-015-0329-1
Evidence for the involvement of gamma delta T cells in the immune response in Rasmussen encephalitis.
Owens Geoffrey C,Erickson Kate L,Malone Colin C,Pan Calvin,Huynh My N,Chang Julia W,Chirwa Thabiso,Vinters Harry V,Mathern Gary W,Kruse Carol A
Journal of neuroinflammation
BACKGROUND:Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response. METHODS:Peripheral blood mononuclear cells (PBMCs) and brain-infiltrating lymphocytes (BILs) were isolated from 20 RE surgery specimens by standard methods, and CD3(+) T cell populations were analyzed by flow cytometry. Gamma delta T cell receptor spectratyping was carried out by nested PCR of reversed transcribed RNA extracted from RE brain tissue, followed by high resolution capillary electrophoresis. A MiSeq DNA sequencing platform was used to sequence the third complementarity determining region (CDR3) of δ1 chains. RESULTS:CD3(+) BILs from all of the RE brain specimens comprised both αβ and γδ T cells. The median αβ:γδ ratio was 1.9 (range 0.58-5.2) compared with a median ratio of 7.7 (range 2.7-40.8) in peripheral blood from the same patients. The αβ T cells isolated from brain tissue were predominantly CD8(+), and the majority of γδ T cells were CD4(-) CD8(-). Staining for the early activation marker CD69 showed that a fraction of the αβ and γδ T cells in the BILs were activated (median 42%; range 13-91%, and median 47%; range 14-99%, respectively). Spectratyping T cell receptor (TCR) Vδ1-3 chains from 14 of the RE brain tissue specimens indicated that the γδ T cell repertoire was relatively restricted. Sequencing δ1 chain PCR fragments revealed that the same prevalent CDR3 sequences were found in all of the brain specimens. These CDR3 sequences were also detected in brain tissue from 15 focal cortical dysplasia (FCD) cases. CONCLUSION:Neuroinflammation in RE involves both activated αβ and γδ T cells. The presence of γδ T cells with identical TCR δ1 chain CDR3 sequences in all of the brain specimens examined suggests that a non-major histocompatibility complex (MHC)-restricted immune response to the same antigen(s) is involved in the etiology of RE. The presence of the same δ1 clones in CD brain implies the involvement of a common inflammatory pathway in both diseases.
10.1186/s12974-015-0352-2
Regulatory impairment in untreated Parkinson's disease is not restricted to Tregs: other regulatory populations are also involved.
Journal of neuroinflammation
BACKGROUND:Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Various studies have suggested that the immune response plays a key role in this pathology. While a predominantly pro-inflammatory peripheral immune response has been reported in treated and untreated PD patients, the study of the role of the regulatory immune response has been restricted to regulatory T cells. Other immune suppressive populations have been described recently, but their role in PD is still unknown. This study was designed to analyze the pro and anti-inflammatory immune response in untreated PD patients, with emphasis on the regulatory response. METHODS:Thirty-two PD untreated patients and 20 healthy individuals were included in this study. Peripheral regulatory cells (CD4+Tregs, Bregs, CD8+Tregs, and tolerogenic dendritic cells), pro-inflammatory cells (Th1, Th2, and Th17 cells; active dendritic cells), and classical, intermediate, and non-classical monocytes were characterized by flow cytometry. Plasmatic levels of TNF-α, IFN-γ, IL-6, GM-CSF, IL-12p70, IL-4, IL-13, IL-17α, IL-1β, IL-10, TGF-β, and IL-35 were determined by ELISA. RESULTS:Decreased levels of suppressor Tregs, active Tregs, Tr1 cells, IL-10-producer CD8regs, and tolerogenic PD-L1+ dendritic cells were observed. With respect to the pro-inflammatory response, a decrease in IL-17-α and an increase in IL-13 levels were observed. CONCLUSION:A decrease in the levels of regulatory cell subpopulations in untreated PD patients is reported for the first time in this work. These results suggest that PD patients may exhibit a deficient suppression of the pro-inflammatory response, which could contribute to the pathophysiology of the disease.
10.1186/s12974-019-1606-1
CSF-Derived CD4 T-Cell Diversity Is Reduced in Patients With Alzheimer Clinical Syndrome.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Patients with Alzheimer dementia display evidence of amyloid-related neurodegeneration. Our focus was to determine whether such patients also display evidence of a disease-targeting adaptive immune response mediated by CD4 T cells. To test this hypothesis, we evaluated the CSF immune profiles of patients with Alzheimer clinical syndrome (ACS), who display clinically defined dementia. METHODS:Innate and adaptive immune profiles of patients with ACS were measured using multicolor flow cytometry. CSF-derived CD4 and CD8 T-cell receptor repertoire genetics were measured using next-generation sequencing. Brain-specific autoantibody signatures of CSF-derived antibody pools were measured using array technology or ELISA. CSF from similar-age healthy controls (HCs) was used as a comparator cohort. RESULTS:Innate cells were expanded in the CSF of patients with ACS in comparison to HCs, and innate cell expansion increased with age in the patients with ACS, but not HCs. Despite innate cell expansion in the CSF, the frequency of total CD4 T cells reduced with age in the patients with ACS. T-cell receptor repertoire genetics indicated that T-cell clonal expansion is enhanced, and diversity is reduced in the patients with ACS compared with similar-age HCs. DISCUSSION:Examination of CSF indicates that CD4 T cell-mediated adaptive immune responses are altered in patients with ACS. Understanding the underlying mechanisms affecting adaptive immunity will help move us toward the goal of slowing cognitive decline.
10.1212/NXI.0000000000001106
Increased circulating leukocyte-derived microparticles in ischemic cerebrovascular disease.
He Zhangping,Tang Yanyan,Qin Chao
Thrombosis research
OBJECTIVES:Circulating leukocyte-derived microparticles act as proinflammatory mediators that reflect vascular inflammation. In this study, we examined the hypothesis that the quantity of leukocyte-derived microparticles is increased in patients with ischemic cerebrovascular diseases, and investigated utility of various phenotypes of leukocyte-derived microparticles as specific biomarkers of vascular inflammation injury. Additionally we focused on identifying leukocyte-derived microparticles that may be correlated with stroke severity in acute ischemic stroke patients. METHODS:The plasma concentration of leukocyte-derived microparticles obtained by a series of centrifugations of 76 consecutive patients with ischemic cerebrovascular diseases and 70 age-, sex-, and race-matched healthy controls were determined by flow cytometry. RESULTS:Significantly elevated numbers of leukocyte (CD45+), monocyte (CD14+), lymphocyte (CD4+), granulocyte (CD15+) derived microparticles were found in the plasma samples of patients ischemic cerebrovascular diseases, compared to healthy controls (p<0.05). Furthermore, the plasma levels of CD14+ microparticles were significantly correlated with stroke severity (r=0.355, p=0.019), cerebral vascular stenosis severity (r=0.255, p=0.025) and stroke subtype (r=0.242, p=0.036). No association with stroke was observed for other leukocyte-derived phenotypes. CONCLUSIONS:These results demonstrate that circulating leukocyte-derived microparticles amounts are increased in patients with ischemic cerebrovascular diseases, compared with healthy controls. As proinflammatory mediators, leukocyte-derived microparticles may contribute to vascular inflammatory and the inflammatory process in acute ischemic stroke. Levels of CD14+ microparticles may be a promising biomarker of ischemic severity and outcome of stroke in the clinic.
10.1016/j.thromres.2017.03.025
Siponimod enriches regulatory T and B lymphocytes in secondary progressive multiple sclerosis.
Wu Qi,Mills Elizabeth A,Wang Qin,Dowling Catherine A,Fisher Caitlyn,Kirch Britany,Lundy Steven K,Fox David A,Mao-Draayer Yang,
JCI insight
BACKGROUNDSiponimod (BAF312) is a selective sphingosine-1-phosphate receptor 1 and 5 (S1PR1, S1PR5) modulator recently approved for active secondary progressive multiple sclerosis (SPMS). The immunomodulatory effects of siponimod in SPMS have not been previously described.METHODSWe conducted a multicentered, randomized, double-blind, placebo-controlled AMS04 mechanistic study with 36 SPMS participants enrolled in the EXPAND trial. Gene expression profiles were analyzed using RNA derived from whole blood with Affymetrix Human Gene ST 2.1 microarray technology. We performed flow cytometry-based assays to analyze the immune cell composition and microarray gene expression analysis on peripheral blood from siponimod-treated participants with SPMS relative to baseline and placebo during the first-year randomization phase.RESULTSMicroarray analysis showed that immune-associated genes involved in T and B cell activation and receptor signaling were largely decreased by siponimod, which is consistent with the reduction in CD4+ T cells, CD8+ T cells, and B cells. Flow cytometric analysis showed that within the remaining lymphocyte subsets there was a reduction in the frequencies of CD4+ and CD8+ naive T cells and central memory cells, while T effector memory cells, antiinflammatory Th2, and T regulatory cells (Tregs) were enriched. Transitional regulatory B cells (CD24hiCD38hi) and B1 cell subsets (CD43+CD27+) were enriched, shifting the balance in favor of regulatory B cells over memory B cells. The proregulatory shift driven by siponimod treatment included a higher proliferative potential of Tregs compared with non-Tregs, and upregulated expression of PD-1 on Tregs. Additionally, a positive correlation was found between Tregs and regulatory B cells in siponimod-treated participants.CONCLUSIONThe shift toward an antiinflammatory and suppressive homeostatic immune system may contribute to the clinical efficacy of siponimod in SPMS.TRIAL REGISTRATIONNCT02330965.
10.1172/jci.insight.134251
Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study.
Jiguet-Jiglaire Carine,Boissonneau Sebastien,Denicolai Emilie,Hein Victoria,Lasseur Romain,Garcia Josep,Romain Sylvie,Appay Romain,Graillon Thomas,Mason Warren,Carpentier Antoine F,Brandes Alba A,Ouafik L 'Houcine,Wick Wolfgang,Baaziz Ania,Gigan Julien P,Argüello Rafael J,Figarella-Branger Dominique,Chinot Olivier,Tabouret Emeline
Acta neuropathologica communications
We previously identified matrix metalloproteinase 2 (MMP2) and MMP9 plasma levels as candidate biomarkers of bevacizumab activity in patients with recurrent glioblastoma. The aim of this study was to assess the predictive value of MMP2 and MMP9 in a randomized phase III trial in patients with newly diagnosed glioblastoma and to explore their tumor source. In this post hoc analysis of the AVAglio trial (AVAGlio/NCT00943826), plasma samples from 577 patients (bevacizumab, n = 283; placebo, n = 294) were analyzed for plasma MMP9 and MMP2 levels by enzyme-linked immunosorbent assay. A prospective local cohort of 38 patients with newly diagnosed glioblastoma was developed for analysis of tumor characteristics by magnetic resonance imaging and measurement of plasma and tumor levels of MMP9 and MMP2. In this AVAglio study, MMP9, but not MMP2, was correlated with bevacizumab efficacy. Patients with low MMP9 derived a significant 5.2-month overall survival (OS) benefit with bevacizumab (HR 0.51, 95% CI 0.34-0.76, p = 0.0009; median 13.6 vs. 18.8 months). In multivariate analysis, a significant interaction was seen between treatment and MMP9 (p = 0.03) for OS. In the local cohort, we showed that preoperative MMP9 plasma levels decreased after tumor resection and were correlated with tumor levels of MMP9 mRNA (p = 0.03). However, plasma MMP9 was not correlated with tumor size, invasive pattern, or angiogenesis. Using immunohistochemistry, we showed that MMP9 was expressed by inflammatory cells but not by tumor cells. After cell sorting, we showed that MMP9 was expressed by CD45+ immune cells. Finally, using flow cytometry, we showed that MMP9 was expressed by tumor-infiltrating neutrophils. In conclusion, circulating MMP9 is predictive of bevacizumab efficacy and is released by tumor-infiltrating neutrophils.
10.1186/s40478-021-01305-4
Relationship between Multiple Sclerosis-Associated Risk Allele Variants and Circulating T Cell Phenotypes in Healthy Genotype-Selected Controls.
Buhelt Sophie,Søndergaard Helle Bach,Oturai Annette,Ullum Henrik,von Essen Marina Rode,Sellebjerg Finn
Cells
Single nucleotide polymorphisms (SNPs) in or near the gene, that encodes the interleukin-2 (IL-2) receptor α (CD25), are associated with increased risk of immune-mediated diseases including multiple sclerosis (MS). We investigated how the MS-associated SNPs rs2104286 and rs11256593 are associated with CD25 expression on T cells ex vivo by multiparameter flow cytometry in paired genotype-selected healthy controls. We observed that MS-associated SNPs rs2104286 and rs11256593 are associated with expression of CD25 in CD4 but not CD8 T cells. In CD4 T cells, carriers of the risk genotype had a reduced frequency of CD25 T1 cells ( = 0.001) and an increased frequency of CD25 recent thymic emigrant cells ( = 0.006). Furthermore, carriers of the risk genotype had a reduced surface expression of CD25 in post-thymic expanded CD4 T cells (CD31CD45RA), CD39 T cells and in several non-follicular memory subsets. Our study found novel associations of MS-associated SNPs on expression of CD25 in CD4 T cell subsets. Insight into the associations of MS-associated SNPs, as these new findings provide, offers a better understanding of CD25 variation in the immune system and can lead to new insights into how MS-associated SNPs contribute to development of MS.
10.3390/cells8060634
Immune aging in multiple sclerosis is characterized by abnormal CD4 T cell activation and increased frequencies of cytotoxic CD4 T cells with advancing age.
EBioMedicine
BACKGROUND:Immunosenescence (ISC) describes age-related changes in immune-system composition and function. Multiple sclerosis (MS) is a lifelong inflammatory condition involving effector and regulatory T-cell imbalance, yet little is known about T-cell ISC in MS. We examined age-associated changes in circulating T cells in MS compared to normal controls (NC). METHODS:Forty untreated MS (Mean Age 43·3, Range 18-72) and 49 NC (Mean Age 48·6, Range 20-84) without inflammatory conditions were included in cross-sectional design. T-cell subsets were phenotypically and functionally characterized using validated multiparametric flow cytometry. Their aging trajectories, and differences between MS and NC, were determined using linear mixed-effects models. FINDINGS:MS patients demonstrated early and persistent redistribution of naïve and memory CD4 T-cell compartments. While most CD4 and CD8 T-cell aging trajectories were similar between groups, MS patients exhibited abnormal age-associated increases of activated (HLA-DRCD38; (P = 0·013) and cytotoxic CD4 T cells, particularly in patients >60 (EOMES: P < 0·001). Aging MS patients also failed to upregulate CTLA-4 expression on both CD4 (P = 0·014) and CD8 (P = 0·009) T cells, coupled with abnormal age-associated increases in frequencies of B cells expressing costimulatory molecules. INTERPRETATION:While many aspects of T-cell aging in MS are conserved, the older MS patients harbour abnormally increased frequencies of CD4 T cells with activated and cytotoxic effector profiles. Age-related decreased expression of T-cell co-inhibitory receptor CTLA-4, and increased B-cell costimulatory molecule expression, may provide a mechanism that drives aberrant activation of effector CD4 T cells that have been implicated in progressive disease. FUNDING:Stated in Acknowledgements section of manuscript.
10.1016/j.ebiom.2022.104179
B-Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Autologous hematopoietic stem cell transplantation (aHSCT) is increasingly used to treat aggressive forms of multiple sclerosis (MS). This procedure is believed to result in an immune reset and restoration of a self-tolerant immune system. Immune reconstitution has been extensively studied for T cells, but only to a limited extent for B cells. As increasing evidence suggests an important role of B cells in MS pathogenesis, we sought here to better understand reconstitution and the extent of renewal of the B-cell system after aHSCT in MS. METHODS:Using longitudinal multidimensional flow cytometry and immunoglobulin heavy chain (IgH) repertoire sequencing following aHSCT with BCNU + Etoposide + Ara-C + Melphalan anti-thymocyte globulin, we analyzed the B-cell compartment in a cohort of 20 patients with MS in defined intervals before and up to 1 year after aHSCT and compared these findings with data from healthy controls. RESULTS:Total B-cell numbers recovered within 3 months and increased above normal levels 1 year after transplantation, successively shifting from a predominantly transitional to a naive immune phenotype. Memory subpopulations recovered slowly and remained below normal levels with reduced repertoire diversity 1 year after transplantation. Isotype subclass analysis revealed a proportional shift toward IgG1-expressing cells and a reduction in IgG2 cells. Mutation analysis of IgH sequences showed that highly mutated memory B cells and plasma cells may transiently survive conditioning while the analysis of sequence cluster overlap, variable (IGHV) and joining (IGHJ) gene usage and repertoire diversity suggested a renewal of the late posttransplant repertoire. In patients with early cytomegalovirus reactivation, reconstitution of naive and memory B cells was delayed. DISCUSSION:Our detailed characterization of B-cell reconstitution after aHSCT in MS indicates a reduced reactivation potential of memory B cells up to 1 year after transplantation, which may leave patients susceptible to infection, but may also be an important aspect of its mechanism of action.
10.1212/NXI.0000000000200027
Ocrelizumab Depletes CD20⁺ T Cells in Multiple Sclerosis Patients.
Gingele Stefan,Jacobus Thais Langer,Konen Franz Felix,Hümmert Martin W,Sühs Kurt-Wolfram,Schwenkenbecher Philipp,Ahlbrecht Jonas,Möhn Nora,Müschen Lars H,Bönig Lena,Alvermann Sascha,Schmidt Reinhold E,Stangel Martin,Jacobs Roland,Skripuletz Thomas
Cells
Ocrelizumab, a humanized monoclonal anti-CD20 antibody, has shown pronounced effects in reduction of disease activity in multiple sclerosis (MS) patients and has recently been approved for the treatment of patients with relapsing MS (RMS) and primary progressive MS (PPMS). CD20 is mainly expressed by B cells, but a subset of T cells (CD3⁺CD20⁺ T cells) also expresses CD20, and these CD20⁺ T cells are known to be a highly activated cell population. The blood of MS patients was analyzed with multicolor flow cytometry before and two weeks after treatment with ocrelizumab regarding the phenotype of peripheral blood mononuclear cells. CD20-expressing CD3⁺ T cells were found in blood samples of all MS patients, accounted for 2.4% of CD45⁺ lymphocytes, and constituted a significant proportion (18.4%) of all CD20⁺ cells. CD3⁺CD20⁺ T cells and CD19⁺CD20⁺ B cells were effectively depleted two weeks after a single administration of 300 mg ocrelizumab. Our results demonstrate that treatment with ocrelizumab does not exclusively target B cells, but also CD20⁺ T cells, which account for a substantial amount of CD20-expressing cells. Thus, we speculate that the efficacy of ocrelizumab might also be mediated by the depletion of CD20-expressing T cells.
10.3390/cells8010012
Peripheral innate immune and bacterial signals relate to clinical heterogeneity in Parkinson's disease.
Brain, behavior, and immunity
The innate immune system is implicated in Parkinson's disease (PD), but peripheral in-vivo clinical evidence of the components and driving mechanisms involved and their relationship with clinical heterogeneity and progression to dementia remain poorly explored. We examined changes in peripheral innate immune-related markers in PD cases (n = 41) stratified according to risk of developing early dementia. 'Higher Risk'(HR) (n = 23) and 'Lower Risk' (LR) (n = 18) groups were defined according to neuropsychological predictors and MAPT H1/H2 genotype, and compared to age, gender and genotype-matched controls. Monocyte subsets and expression of key surface markers were measured using flow cytometry. Serum markers including alpha-synuclein, inflammasome-related caspase-1 and bacterial translocation-related endotoxin were measured using quantitative immuno-based assays. Specific markers were further investigated using monocyte assays and validated in plasma samples from a larger incident PD cohort (n = 95). We found that classical monocyte frequency was elevated in PD cases compared to controls, driven predominantly by the HR group, in whom Toll-Like Receptor (TLR)4+ monocytes and monocyte Triggering Receptor Expressed on Myeloid cells-2 (TREM2) expression were also increased. Monocyte Human Leukocyte Antigen (HLA)-DR expression correlated with clinical variables, with lower levels associated with worse cognitive/motor performance. Notably, monocyte changes were accompanied by elevated serum bacterial endotoxin, again predominantly in the HR group. Serum alpha-synuclein and inflammasome-related caspase-1 were decreased in PD cases compared to controls regardless of group, with decreased monocyte alpha-synuclein secretion in HR cases. Further, alpha-synuclein and caspase-1 correlated positively in serum and monocyte lysates, and in plasma from the larger cohort, though no associations were seen with baseline or 36-month longitudinal clinical data. Principal Components Analysis of all monocyte and significant serum markers indicated 3 major components. Component 1 (alpha-synuclein, caspase-1, TLR2+ monocytes) differentiated PD cases and controls in both groups, while Component 2 (endotoxin, monocyte TREM2, alpha-synuclein) did so predominantly in the HR group. Component 3 (classical monocytes, alpha-synuclein) also differentiated cases and controls overall in both groups. These findings demonstrate that systemic innate immune changes are present in PD and are greatest in those at higher risk of rapid progression to dementia. Markers associated with PD per-se (alpha-synuclein, caspase-1), differ from those related to cognitive progression and clinical heterogeneity (endotoxin, TREM2, TLR4, classical monocytes, HLA-DR), with mechanistic and therapeutic implications. Alpha-synuclein and caspase-1 are associated, suggesting inflammasome involvement common to all PD, while bacterial translocation associated changes may contribute towards progression to Parkinson's dementia. Additionally, HLA-DR-associated variations in antigen presentation/clearance may modulate existing clinical disease.
10.1016/j.bbi.2020.01.018
Increased Percentage of CD8CD28 Regulatory T Cells With Fingolimod Therapy in Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Fingolimod, an oral therapy for MS, decreases expression of membrane S1P1 receptors on CD4 memory cells, causing their retention and deactivation in lymph nodes. We determined fingolimod effects on the number and proportion of potentially CNS-damaging CD8CD28 cytolytic T lymphocyte cells (CTLs) and on MS-depleted and dysfunctional CD8CD28 anti-inflammatory suppressor/regulatory T cells (Treg) and on CD8 T-cell expression of the CD69 activation/lymph node retention protein in MS. METHODS:CD8, CD28, CD4, and CD69 expression on peripheral blood mononuclear cells was measured with flow cytometry. In vitro concanavalin A (ConA) activation of T cells, including CD8CD28 cells, was used to mimic inflammation. RESULTS:Fifty-nine patients with MS, 35 therapy-naive (16 clinically stable; 19 exacerbating) and 24 fingolimod-treated (19 clinically stable; 5 exacerbating), and 26 matched healthy controls (HCs) were compared. In therapy-naive patients, the CD8 Treg percent of total lymphocytes was only 1/4 of HC levels. In fingolimod-treated patients, however, CD8 Treg percentages rose to 2.5-fold higher than in HC and 10-fold higher than in therapy-naive MS. With fingolimod therapy, in contrast, CD8 CTL levels were less than half of levels in HCs and therapy-naive patients. In HCs and all MS, activation with ConA strongly induced CD69 expression on CD4 cells and induced 3-fold higher CD69 levels on CD8 CTL than on CD8 Treg. Fingolimod and analogs in vitro did not modify lymphocyte CD69 expression. Lower levels of CD69 on CD8 Treg than on CTL may allow easier Treg egress from lymph nodes and enhance control of peripheral inflammation. In vitro activation reduced the already low CD8 Treg population in therapy-naive MS, but only slightly altered Treg levels in fingolimod-treated MS. DISCUSSION:Fingolimod therapy markedly increases the percentage of CD8 Treg in MS, reversing the low CD8 Treg:CTL ratio seen in untreated MS. The increase in immune regulatory cells has potential therapeutic benefit in MS. Activation in vitro depletes CD8CD28CTL in patients with MS; the loss is more pronounced in older patients with MS. This suggests that inflammation can disrupt the tenuous immune regulation in MS, especially in older patients.
10.1212/NXI.0000000000200075
Early Intrathecal T Helper 17.1 Cell Activity in Huntington Disease.
von Essen Marina R,Hellem Marie N N,Vinther-Jensen Tua,Ammitzbøll Cecilie,Hansen Rikke H,Hjermind Lena E,Nielsen Troels T,Nielsen Jørgen E,Sellebjerg Finn
Annals of neurology
OBJECTIVE:Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. No disease-modifying therapy exists for the treatment of patients with HD. The purpose of this study was therefore to investigate early disease mechanisms that potentially could be used as a target therapeutically. METHODS:Lymphocyte activity in cerebrospinal fluid (CSF) from 4 cohorts of HTT gene expansion carriers (n = 121 in total) and controls was analyzed by techniques based on flow cytometry and enzyme-linked immunosorbent assays. RESULTS:The data of this study provide evidence of immune abnormalities before motor onset of disease. In CSF of HTT gene expansion carriers, we found increased levels of proinflammatory cytokines, including IL-17, and increased consumption of the lymphocyte growth factor IL-7 before motor onset of HD. In concordance, we observed an increased prevalence of IL-17-producing Th17.1 cells in the CSF of HTT gene expansion carriers, predominantly in pre-motor manifest individuals. The frequency of intrathecal Th17.1 cells correlated negatively with progression of HD and the level of neurodegeneration, suggesting a role of Th17.1 cells in the early disease stage. We also observed a skewing in the balance between proinflammatory and regulatory T cells potentially favoring a proinflammatory intrathecal environment in HTT gene expansion carriers. INTERPRETATION:These data suggest that Th17.1 cells are implicated in the earliest pathogenic phases of HD and suggest that treatment to dampen T -cell-driven inflammation before motor onset might be of benefit in HTT gene expansion carriers. ANN NEUROL 2020;87:246-255.
10.1002/ana.25647
Selective Brain Network and Cellular Responses Upon Dimethyl Fumarate Immunomodulation in Multiple Sclerosis.
Ciolac Dumitru,Luessi Felix,Gonzalez-Escamilla Gabriel,Koirala Nabin,Riedel Christian,Fleischer Vinzenz,Bittner Stefan,Krämer Julia,Meuth Sven G,Muthuraman Muthuraman,Groppa Sergiu
Frontiers in immunology
Efficient personalized therapy paradigms are needed to modify the disease course and halt gray (GM) and white matter (WM) damage in patients with multiple sclerosis (MS). Presently, promising disease-modifying drugs show impressive efficiency, however, tailored markers of therapy responses are required. Here, we aimed to detect in a real-world setting patients with a more favorable brain network response and immune cell dynamics upon dimethyl fumarate (DMF) treatment. In a cohort of 78 MS patients we identified two thoroughly matched groups, based on age, disease duration, disability status and lesion volume, receiving DMF ( = 42) and NAT ( = 36) and followed them over 16 months. The rate of cortical atrophy and deep GM volumes were quantified. GM and WM network responses were characterized by brain modularization as a marker of regional and global structural alterations. In the DMF group, lymphocyte subsets were analyzed by flow cytometry and related to clinical and MRI parameters. Sixty percent (25 patients) of the DMF and 36% (13 patients) of the NAT group had disease activity during the study period. The rate of cortical atrophy was higher in the DMF group (-2.4%) compared to NAT (-2.1%, < 0.05) group. GM and WM network dynamics presented increased modularization in both groups. When dividing the DMF-treated cohort into patients free of disease activity ( = 17, DMF) and patients with disease activity ( = 25, DMF) these groups differed significantly in CD8+ cell depletion counts (DMF: 197.7 ± 97.1/μl; DMF: 298.4 ± 190.6/μl, = 0.03) and also in cortical atrophy (DMF: -1.7%; DMF: -3.2%, = 0.01). DMF presented reduced longitudinal GM and WM modularization and less atrophy as markers of preserved structural global network integrity in comparison to DMF and even NAT patients. NAT treatment contributes to a reduced rate of cortical atrophy compared to DMF therapy. However, patients under DMF treatment with a stronger CD8+ T cell depletion present a more favorable response in terms of cortical integrity and GM and WM network responses. Our findings may serve as basis for the development of personalized treatment paradigms.
10.3389/fimmu.2019.01779
Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients.
Adriani Marsilio,Nytrova Petra,Mbogning Cyprien,Hässler Signe,Medek Karel,Jensen Poul Erik H,Creeke Paul,Warnke Clemens,Ingenhoven Kathleen,Hemmer Bernhard,Sievers Claudia,Lindberg Gasser Raija Lp,Fissolo Nicolas,Deisenhammer Florian,Bocskei Zsolt,Mikol Vincent,Fogdell-Hahn Anna,Kubala Havrdova Eva,Broët Philippe,Dönnes Pierre,Mauri Claudia,Jury Elizabeth C,
JCI insight
Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.
10.1172/jci.insight.99274
Immunophenotyping of Inclusion Body Myositis Blood T and NK Cells.
Neurology
BACKGROUND AND OBJECTIVES:To evaluate the therapeutic potential of targeting highly differentiated T cells in patients with inclusion body myositis (IBM) by establishing high-resolution mapping of killer cell lectin-like receptor subfamily G member 1 (KLRG1) within the T and natural killer (NK) cell compartments. METHODS:Blood was collected from 51 patients with IBM and 19 healthy age-matched donors. Peripheral blood mononuclear cells were interrogated by flow cytometry using a 12-marker antibody panel. The panel allowed the delineation of naive T cells (Tn), central memory T cells (Tcm), 4 stages of effector memory differentiation T cells (Tem 1-4), and effector memory re-expressing CD45RA T cells (TemRA), as well as total and subpopulations of NK cells based on the differential expression of CD16 and C56. RESULTS:We found that a population of KLRG1 Tem and TemRA were expanded in both the CD4 and CD8 T-cell subpopulations in patients with IBM. KLRG1 expression in CD8 T cells increased with T-cell differentiation with the lowest levels of expression in Tn and highest in highly differentiated TemRA and CD56CD8 T cells. The frequency of KLRG1 total NK cells and subpopulations did not differ between patients with IBM and healthy donors. IBM disease duration correlated with increased CD8 T-cell differentiation. DISCUSSION:Our findings reveal that the selective expansion of blood KLRG1 T cells in patients with IBM is confined to the TemRA and Tem cellular compartments.
10.1212/WNL.0000000000200013
Neuropathologic, phenotypic and functional analyses of Mucosal Associated Invariant T cells in Multiple Sclerosis.
Salou Marion,Nicol Bryan,Garcia Alexandra,Baron Daniel,Michel Laure,Elong-Ngono Annie,Hulin Philippe,Nedellec Steven,Jacq-Foucher Marylène,Le Frère Fabienne,Jousset Natacha,Bourreille Arnaud,Wiertlewski Sandrine,Soulillou Jean-Paul,Brouard Sophie,Nicot Arnaud B,Degauque Nicolas,Laplaud David-Axel
Clinical immunology (Orlando, Fla.)
BACKGROUND:The involvement of Mucosal Associated Invariant T (MAIT) cells, which are anti-microbial semi-invariant T cells, remains elusive in Multiple Sclerosis (MS). OBJECTIVE:Deciphering the potential involvement of MAIT cells in the MS inflammatory process. METHODS:By flow cytometry, blood MAIT cells from similar cohorts of MS patients and healthy volunteers (HV) were compared for frequency, phenotype, activation potential after in vitro TCR engagement by bacterial ligands and transmigration abilities through an in vitro model of blood-brain barrier. MS CNS samples were also studied by immunofluorescent staining and quantitative PCR. RESULTS AND CONCLUSION:Blood MAIT cells from relapsing-remitting MS patients and HV presented similar frequency, ex vivo effector phenotype and activation abilities. MAIT cells represented 0.5% of the total infiltrating T cells on 39 MS CNS lesions. This is low as compared to blood frequency (p<0.001), but consistent with their low transmigration rate. Finally, transcriptional over-expression of MR1 - which presents cognate antigens to MAIT cells - and of the activating cytokines IL-18 and IL-23 was evidenced in MS lesions, suggesting that the CNS microenvironment is suited to activate the few infiltrating MAIT cells. Taken together, these data place MAIT cells from MS patients as minor components of the inflammatory pathological process.
10.1016/j.clim.2016.03.014
Transient receptor potential melastatin 2 channels are overexpressed in myalgic encephalomyelitis/chronic fatigue syndrome patients.
Balinas Cassandra,Cabanas Helene,Staines Donald,Marshall-Gradisnik Sonya
Journal of translational medicine
BACKGROUND:Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N-Benzoyladenosine-3',5'-cyclic monophosphate (N-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants. METHODS:Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N-Bnz-cAMP drug treatments by flow cytometry. RESULTS:Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56CD16 subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56CD16 subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group. CONCLUSION:Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca] mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.
10.1186/s12967-019-02155-4
Selective emergence of antibody-secreting cells in the multiple sclerosis brain.
EBioMedicine
BACKGROUND:Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS:Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS:ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45 phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4 memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION:These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4 memory T cells. FUNDING:Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).
10.1016/j.ebiom.2023.104465
circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma.
Chen Jiansheng,Chen Taoliang,Zhu Yubo,Li Yan,Zhang Yuxuan,Wang Yun,Li Xiao,Xie Xiaomi,Wang Jihui,Huang Min,Sun Xinlin,Ke Yiquan
Journal of experimental & clinical cancer research : CR
BACKGROUND:Growing evidences indicate that circular RNAs (circRNAs) play an important role in the regulation of biological behavior of tumor. We aim to explore the role of circRNA in glioma and elucidate how circRNA acts. METHODS:Real-time PCR was used to examine the expression of circPTN in glioma tissues and normal brain tissues (NBT). Assays of dual- luciferase reporter system, biotin label RNA pull-down and FISH were used to determine that circPTN could sponge miR-145-5p and miR-330-5p. Tumor sphere formation assay was performed to determine self- renewal of glioma stem cell (GSCs). Cell counting Kit-8 (CCK8), EdU assay and flow cytometry were used to investigate proliferation and cell cycle. Intracranial xenograft was established to determine how circPTN impacts in vivo. Tumor sphere formation assay was performed to determine self- renewal of glioma stem cell (GSCs). RESULTS:We demonstrated circPTN was significantly higher expression in glioma tissues and glioma cell lines, compared with NBT and HEB (human astrocyte). In gain- and loss-of-function experiments, circPTN significantly promoted glioma growth in vitro and in vivo. Furthermore, we performed dual-luciferase reporter assays and RNA pull-down assays to verify that circPTN acts through sponging miR-145-5p and miR-330-5p. Increasing expression of circPTN rescued the inhibition of proliferation and downregulation of SOX9/ITGA5 in glioma cells by miR-145-5p/miR-330-5p. In addition, we found that circPTN promoted self-renewal and increased the expression of stemness markers (Nestin, CD133, SOX9, and SOX2) via sponging miR-145-5p. Moreover, this regulation was disappeared when circPTN binding sites in miR-145-5p were mutated. CONCLUSIONS:Our results suggest that circPTN is an oncogenic factor that acts by sponging miR-145-5p/miR-330-5p in glioma.
10.1186/s13046-019-1376-8
Elevated levels of polymorphonuclear myeloid-derived suppressor cells in patients with glioblastoma highly express S100A8/9 and arginase and suppress T cell function.
Gielen Paul R,Schulte Barbara M,Kers-Rebel Esther D,Verrijp Kiek,Bossman Sandra A J F H,Ter Laan Mark,Wesseling Pieter,Adema Gosse J
Neuro-oncology
BACKGROUND:Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). METHODS:We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. RESULTS:We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. CONCLUSIONS:These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade.
10.1093/neuonc/now034
Senescent-like Blood Lymphocytes and Disease Progression in Amyotrophic Lateral Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Aging is known to exacerbate neuroinflammation, and in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS), an older age is associated with a worse prognosis. We have previously shown the activation of cell senescence pathways in the proteome of peripheral blood mononuclear cells and the increase of proinflammatory cytokines in blood from individuals living with ALS. In this single-center, retrospective study, we investigated the expression of senescent-like blood mononuclear cells in ALS. METHODS:We first applied multidimensional cytometry by time-of-flight (CyTOF) to study the senescent immunophenotype of blood mononuclear cells from 21 patients with ALS and 10 healthy controls (HCs). We then used targeted flow cytometry (FC) to investigate frequencies of senescent blood lymphocytes in 40 patients with ALS and 20 HCs. Longitudinal analysis included 2 additional time points in 17 patients with ALS. Frequencies of senescent-like lymphocytes were analyzed in relation to survival. RESULTS:Unsupervised clustering of CyTOF data showed higher frequencies of senescent CD4CD27CD57 T cells in patients with ALS compared with those in HCs ( = 0.0017, false discovery (FDR)-adjusted = 0.029). Moderate to strong negative correlations were identified between CD4 T central memory-cell frequencies and survival (R = -061, = 0.01; FDR-adjusted < 0.1) and between CD95 CD8 cells and ALS functional rating scale revised at baseline (R = -0.72, = 0.001; FDR-adjusted < 0.1).Targeted FC analysis showed higher memory T regulatory cells ( = 0.0052) and memory CD8 T cell (M-Tc; = 0.0006) in bulbar ALS (A-B) compared with those in limb ALS (A-L), while late memory B cells (LM-B) were also elevated in A-B and fast-progressing ALS ( = 0.0059). Higher M-Tc levels separated A-B from A-L (AUC: 0.887; < 0.0001). A linear regression model with prespecified clinical independent variables and neurofilament light chain plasma concentration showed that higher frequencies of LM-B predicted a shorter survival (hazard ratio: 1.094, CI: 1.026-1.167; = 0.006). DISCUSSION:Our data suggest that a systemic elevation of senescent and late memory T and B lymphocytes is a feature of faster progressing ALS and of ALS individuals with bulbar involvement. Lymphocyte senescence and their memory state may be central to the immune dysregulation known to drive disease progression in ALS and a target for biomarkers and therapeutics discovery.
10.1212/NXI.0000000000200042
Cytokine Profiling in Aqueous Humor Samples From Patients With Non-Infectious Uveitis Associated With Systemic Inflammatory Diseases.
Bonacini Martina,Soriano Alessandra,Cimino Luca,De Simone Luca,Bolletta Elena,Gozzi Fabrizio,Muratore Francesco,Nicastro Maria,Belloni Lucia,Zerbini Alessandro,Fontana Luigi,Salvarani Carlo,Croci Stefania
Frontiers in immunology
Non-infectious uveitis are intraocular inflammatory conditions caused by dysregulated activation of the immune response without any detectable infectious agents. The aim of this study was to explore potential markers and therapeutic targets for two distinct types of non-infectious uveitis associated with Behçet's disease (BD) and Vogt Koyanagi Harada (VKH) disease. Concentrations of 27 cytokines were investigated in aqueous humor (AH) samples from patients with active uveitis vs. healthy controls (HC) ( = 10 patients with BD-associated uveitis; = 10 patients with VKH-associated uveitis; = 10 HC) using the Bio-Plex Pro human cytokine group I panel. Additionally, leukocytes in AH samples were counted with hemocytometers and characterized by flow cytometry. Eleven cytokines were differentially expressed between patients with uveitis and HC with a median concentration greater than 10 pg/ml. IL-6, IP-10, G-CSF, and IFNγ showed higher concentrations in AH samples from both BD and VKH patients while IL-2, IL-8, IL-13, TNFα, eotaxin, IL-1ra showed statistically significant higher concentrations only in AH samples from BD patients. GM-CSF was the sole cytokine with an opposite profile showing decreased levels in AH samples from BD patients. IL-1ra and IL-6 were detected at higher frequencies in AH samples from BD and VKH patients compared with those from HC while IFNγ and TNFα were not detected in HC. The concentrations of IL-6, IL-8, IP-10, G-CSF, IFNγ, TNFα, eotaxin, IL-1ra positively correlated with the concentrations of leukocytes in AH, suggesting that such cytokines can be produced by immune cells and/or attract and/or promote proliferation and survival of immune cells in these types of uveitis. The correlation matrix of cytokine concentrations in AH samples revealed that IFNγ, TNFα, eotaxin, IL-6, G-CSF highly correlated each other. The ratios of cytokine concentrations between AH and plasma intra-individuals showed that IL-2, IL-6, IP-10, GM-CSF were increased intraocularly. In conclusion, AH sampling followed by multiplex analysis of cytokines should be fostered in non-infectious uveitis to identify cytokines dysregulated intraocularly in each individual laying the groundwork for precision medicine.
10.3389/fimmu.2020.00358
Redox Imbalance in CD4+ T Cells of Relapsing-Remitting Multiple Sclerosis Patients.
Tavassolifar Mohammad Javad,Moghadasi Abdorreza Naser,Esmaeili Behnaz,Sadatpour Omid,Vodjgani Mohammad,Izad Maryam
Oxidative medicine and cellular longevity
As a prevalent autoimmune disease of the central nervous system in young adults, multiple sclerosis (MS) is mediated by T cells, particularly CD4+ subsets. Given the evidence that the perturbation in reactive oxygen species (ROS) production has a pivotal role in the onset and progression of MS, its regulation through the antioxidant molecules is too important. Here, we investigated the level of the redox system components in lymphocytes and CD4+ T cells of MS patients. The study was performed on relapsing-remitting MS (RRMS) patients ( = 29) and age- and sex-matched healthy controls ( = 15). Peripheral blood mononuclear cells (PBMCs) were cultured and stimulated by anti-CD3/CD28. The level of ROS, anion superoxide (O ), and L-𝛾-glutamyl-Lcysteinylglycine (GSH) was measured by flow cytometry in lymphocytes/CD4+ T cells. The gene expression level of gp91phox, catalase, superoxide dismutase 1/2 (SOD), and nuclear factor-E2-related factor (Nrf2) was also measured by real-time PCR. We found that lymphocytes/CD4+ T cells of RRMS patients at the relapse phase significantly produced higher levels of ROS and O compared to patients at the remission phase ( value < 0.001) and healthy controls ( value < 0.001 and value < 0.05, respectively). Interestingly, the gene expression level of gp91phox, known as the catalytic subunit of the NADPH oxidase, significantly increased in MS patients at the relapse phase ( value < 0.05). Furthermore, the catalase expression augmented in patients at the acute phase ( value < 0.05), while an increased expression of SOD1 and Nrf2 was found in RRMS patients at relapse and remission phases ( value < 0.05). The increased production of ROS in CD4+ T cells of RRMS patients highlights the importance of amplifying antioxidant components as an efficient approach to ameliorate disease activity in MS patients.
10.1155/2020/8860813
Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Live cell-based assay (CBA) can detect acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK) antibodies (Abs) in a proportion of patients with radioimmunoassay (RIA)-double seronegative myasthenia gravis (dSN-MG). A commercial fixed CBA for AChR and MuSK Abs has recently become available; however, comparative studies on fixed and live CBAs are lacking. In this study, we compared the performance of fixed and live CBAs in patients with RIA-dSN MG and assessed their sensitivity in RIA-positive MG samples and their specificity. METHODS:AChR and MuSK Abs were tested in 292 serum samples from 2 Italian MG referral centers by live and fixed CBAs: 192 from patients with MG and 100 from controls. All samples had been previously assessed by RIA: 66 were AChR positive, 40 MuSK positive, and 86 dSN. All controls were negative. Two independent raters assessed the CBA results. Fixed and live CBAs were compared with the McNemar test; interrater and interlaboratory agreement were assessed with Cohen's kappa or interclass correlation coefficient (ICC), as appropriate. RESULTS:In 86 RIA-dSN samples, fixed CBA detected Abs in 10 cases (11.6%, 95% CI 5.7-20.3), whereas live CBA detected Abs in 16 (18.6%, 95% CI 11.0-28.5) ( = 0.0143). Of these sera, those positive by fixed CBA were also positive by live CBA. In addition, live CBA could detect MuSK Abs in 4 and AChR Abs in 2 samples that were negative by fixed CBA, providing an 8% (95% CI 2.9-16.6) further increase in the Ab detection rate. These results were confirmed by flow cytometry. In the RIA-positive cohort, the sensitivity for AChR Abs was 98.5% (95% CI 91.9%-99.9%) for fixed CBA and 100% (95% CI 94.6-100) for live CBA ( = 0.1573). For both assays, the sensitivity for MuSK Abs was 100% (95% CI 91.2-100), and the specificity was 100% (95% CI 96.4-100). Interrater agreement was almost perfect for live and fixed CBAs (Cohen's kappa 0.972 and 0.978, respectively), alike interlaboratory agreement. Interrater agreement for the CBA score ranged from good to excellent (ICC: 0.832-0.973). DISCUSSION:Fixed CBA represents a valuable alternative to RIA for AChR and MuSK Ab detection in patients with MG and could be considered as a first-step diagnostic test. Live CBA can be useful in the serologic evaluation of RIA- and fixed CBA-negative samples.
10.1212/NXI.0000000000200038
Measuring the lactate-to-creatine ratio via H NMR spectroscopy can be used to noninvasively evaluate apoptosis in glioma cells after X-ray irradiation.
Cellular & molecular biology letters
BACKGROUND:Radiotherapy is among the commonly applied treatment options for glioma, which is one of the most common types of primary brain tumor. To evaluate the effect of radiotherapy noninvasively, it is vital for oncologists to monitor the effects of X-ray irradiation on glioma cells. Preliminary research had showed that PKC-ι expression correlates with tumor cell apoptosis induced by X-ray irradiation. It is also believed that the lactate-to-creatine (Lac/Cr) ratio can be used as a biomarker to evaluate apoptosis in glioma cells after X-ray irradiation. In this study, we evaluated the relationships between the Lac/Cr ratio, apoptotic rate, and protein kinase C iota (PKC-ι) expression in glioma cells. METHODS:Cells of the glioma cell lines C6 and U251 were randomly divided into 4 groups, with every group exposed to X-ray irradiation at 0, 1, 5, 10 and 15 Gy. Single cell gel electrophoresis (SCGE) was conducted to evaluate the DNA damage. Flow cytometry was performed to measure the cell cycle blockage and apoptotic rates. Western blot analysis was used to detect the phosphorylated PKC-ι (p-PKC-ι) level. H NMR spectroscopy was employed to determine the Lac/Cr ratio. RESULTS:The DNA damage increased in a radiation dose-dependent manner ( < 0.05). With the increase in X-ray irradiation, the apoptotic rate also increased (C6, < 0.01; U251, < 0.05), and the p-PKC-ι level decreased (C6, < 0.01; U251, < 0.05). The p-PKC-ι level negatively correlated with apoptosis, whereas the Lac/Cr ratio positively correlated with the p-PKC-ι level. CONCLUSION:The Lac/Cr ratio decreases with an increase in X-ray irradiation and thus can be used as a biomarker to reflect the effects of X-ray irradiation in glioma cells.
10.1186/s11658-018-0092-2
Endothelial and Leukocyte-Derived Microvesicles and Cardiovascular Risk After Stroke: PROSCIS-B.
Huo Shufan,Kränkel Nicolle,Nave Alexander Heinrich,Sperber Pia Sophie,Rohmann Jessica Lee,Piper Sophie Käthe,Heuschmann Peter Ulrich,Landmesser Ulf,Endres Matthias,Siegerink Bob,Liman Thomas Günter
Neurology
OBJECTIVE:To determine the role of circulating microvesicles (MV) on long-term cardiovascular outcomes after stroke, we measured them in patients with first-ever stroke with a 3-year follow-up. METHODS:In the Prospective Cohort With Incident Stroke Berlin (PROSCIS-B), patients with first-ever ischemic stroke were followed up for 3 years. The primary combined endpoint consisted of recurrent stroke, myocardial infarction, and all-cause mortality. Citrate-blood levels of endothelial MV (EMV), leukocyte-derived MV (LMV), monocytic MV (MMV), and platelet-derived MV (PMV) were measured with flow cytometry. Kaplan-Meier curves and adjusted Cox proportional hazards models were used to estimate the effect of MV levels on the combined endpoint. RESULTS:Five hundred seventy-one patients were recruited (median age 69 years, 39% female, median NIH Stroke Scale score 2, interquartile range 1-4), and 95 endpoints occurred. Patients with levels of EMV (adjusted hazard ratio [HR] 2.5, 95% confidence interval [CI] 1.2-4.9) or LMV (HR 3.1, 95% CI 1.4-6.8) in the highest quartile were more likely to experience an event than participants with lower levels with the lowest quartile used as the reference category. The association was less pronounced for PMV (HR 1.7, 95% CI 0.9-3.2) and absent for MMV (HR 1.1, 95% CI 0.6-1.8). CONCLUSION:High levels of EMV and LMV after stroke were associated with worse cardiovascular outcome within 3 years. These results reinforce that endothelial dysfunction and vascular inflammation affect the long-term prognosis after stroke. EMV and LMV might play a role in risk prediction for stroke patients. CLINICALTRIALSGOV IDENTIFIER:NCT01363856. CLASSIFICATION OF EVIDENCE:This study provides Class II evidence of the effect of MV levels on subsequent stroke, myocardial infarction, or all-cause mortality in survivors of mild stroke.
10.1212/WNL.0000000000011223
A fructan from Anemarrhena asphodeloides Bunge showing neuroprotective and immunoregulatory effects.
Zhang Shaojie,Zhang Qi,An Lijun,Zhang Jiaojiao,Li Zhengguo,Zhang Jie,Li Yuhao,Tuerhong Muhetaer,Ohizumi Yasushi,Jin Jin,Xu Jing,Guo Yuanqiang
Carbohydrate polymers
A novel polysaccharide, AAP70-1, was isolated from Anemarrhena asphodeloides for the first time. The primary structural analysis revealed that AAP70-1 was composed of glucose and fructose, had an absolute molecular weight of 2720 Da, and contained a (2→6)-linked β-D-fructofuranose (Fruf) backbone and a (2→1,6)-linked β-D-Fruf side chain with an internal α-D-glucopyranose (Glcp) in the form of a neokestose. To explore the potential factors responsible for the medicinally relevant bioactivities of A. asphodeloides, a biological assay was performed. Using flow cytometry analysis, AAP70-1 was experimentally shown to have neuroprotective effects, and it can prevent and ameliorate neurological damage via reducing apoptosis. The immunomodulation assay further revealed that AAP70-1 can significantly improve immune function by promoting phagocytic capacity and the secretion of cytokines (IL-6, IL-1β and TNF-α) in RAW264.7 cells. These results suggest that AAP70-1 has potential as a therapeutic agent for central nervous system diseases or as an immunomodulatory agent.
10.1016/j.carbpol.2019.115477
Effects of pro-inflammatory cytokines on cannabinoid CB1 and CB2 receptors in immune cells.
Jean-Gilles L,Braitch M,Latif M L,Aram J,Fahey A J,Edwards L J,Robins R A,Tanasescu R,Tighe P J,Gran B,Showe L C,Alexander S P,Chapman V,Kendall D A,Constantinescu C S
Acta physiologica (Oxford, England)
AIMS:To investigate the regulation of cannabinoid receptors CB1 and CB2 on immune cells by pro-inflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS). CB1 and CB2 signalling may be anti-inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB1 and CB2 expression and function are unknown. METHODS:Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB1 and CB2 mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression of CB1 and CB2 protein was determined by flow cytometry. CB1 and CB2 signalling in PBMC was determined by Western blotting for Erk1/2. RESULTS:Pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (the latter likely NF-κB dependently) can upregulate CB1 and CB2 on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate upregulation of CB1 and CB2 and increased IL-1β, IL-6 and TNF-α mRNA in blood of patients with MS compared with controls. CONCLUSION:The levels of CB1 and CB2 can be upregulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.
10.1111/apha.12474
Immune Profiling Reveals the T-Cell Effect of Ocrelizumab in Early Relapsing-Remitting Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Ocrelizumab (OCR), a humanized anti-CD20 monoclonal antibody, is highly efficient in patients with relapsing-remitting multiple sclerosis (RR-MS). We assessed early cellular immune profiles and their association with disease activity at treatment start and under therapy, which may provide new clues on the mechanisms of action of OCR and on the disease pathophysiology. METHODS:A first group of 42 patients with an early RR-MS, never exposed to disease-modifying therapy, was included in 11 centers participating to an ancillary study of the ENSEMBLE trial (NCT03085810) to evaluate the effectiveness and safety of OCR. The phenotypic immune profile was comprehensively assessed by multiparametric spectral flow cytometry at baseline and after 24 and 48 weeks of OCR treatment on cryopreserved peripheral blood mononuclear cells and analyzed in relation to disease clinical activity. A second group of 13 untreated patients with RR-MS was included for comparative analysis of peripheral blood and CSF. The transcriptomic profile was assessed by single-cell qPCRs of 96 genes of immunologic interest. RESULTS:Using an unbiased analysis, we found that OCR as an effect on 4 clusters of CD4 T cells: one corresponding to naive CD4 T cells was increased, the other clusters corresponded to effector memory (EM) CD4CCR6 T cells expressing homing and migration markers, 2 of them also expressing CCR5 and were decreased by the treatment. Of interest, one CD8 T-cell cluster was decreased by OCR corresponding to EM CCR5-expressing T cells with high expression of the brain homing markers CD49d and CD11a and correlated with the time elapsed since the last relapse. These EM CD8CCR5 T cells were enriched in the CSF of patients with RR-MS and corresponded to activated and cytotoxic cells. DISCUSSION:Our study provides novel insights into the mode of action of anti-CD20, pointing toward the role of EM T cells, particularly a subset of CD8 T cells expressing CCR5.
10.1212/NXI.0000000000200091
Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFβ in patients with Multiple Sclerosis.
Blumenfeld Shiri,Staun-Ram Elsebeth,Miller Ariel
Journal of autoimmunity
Fingolimod, an oral therapeutic agent approved for patients with relapsing-remitting Multiple Sclerosis (MS), has been shown to prevent lymphocyte egress from secondary lymphoid tissues; however the specific drug effect on B cells in fingolimod-treated patients remains to be fully elucidated. We present here a comprehensive analysis on the proportions of B cell subsets in the periphery, and the levels of activation, functional surface markers and cytokine profile of B cells in MS patients, following initiation of fingolimod therapy, using flow cytometry and cytokine bead array. Fingolimod therapy increased the ratio of naïve to memory cells, elevated the percentage of plasma cells and highly increased the proportion of transitional B cells as well as additional regulatory subsets, including: IL10(+), CD25(+) and CD5(+) B cells. The percentage of activated CD69(+) cells was highly elevated in the remaining circulating B cells, which produced increased levels of IL10, TGFβ, IL6, IL4, LTα, TNFα and IFNγ cytokines, with an overall increased ratio of TGFβ to pro-inflammatory cytokines. Furthermore, fingolimod therapy reduced ICAM-1(+) cells, suggesting a possible reduction in antigen-presenting capacity. Phosphorylated-fingolimod was shown in vitro to reduce S1PR1 RNA and protein, to slightly increase viability and to activate anti-apoptotic Bcl2 in transformed B cells of patients with MS. In conclusion, fingolimod therapy modulates significantly the composition of circulating B cells, promoting regulatory subsets and an anti-inflammatory cytokine repertoire.
10.1016/j.jaut.2016.03.012
Autologous nonmyeloablative hematopoietic stem cell transplantation for neuromyelitis optica.
Burt Richard K,Balabanov Roumen,Han Xiaoqiang,Burns Carol,Gastala Joseph,Jovanovic Borko,Helenowski Irene,Jitprapaikulsan Jiraporn,Fryer James P,Pittock Sean J
Neurology
OBJECTIVE:To determine if autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) could be a salvage therapy for neuromyelitis optica spectrum disorder (NMOSD). METHODS:Thirteen patients were enrolled in a prospective open-label cohort study (11 NMOSD aquaporin-4-immunoglobulin G [AQP4-IgG]-positive, 1 NMOSD without AQP4, and 1 NMOSD AQP4-IgG-positive with neuropsychiatric systemic lupus erythematosus [SLE]). Following stem cell mobilization with cyclophosphamide (2 g/m) and filgrastim, patients were treated with cyclophosphamide (200 mg/kg) divided as 50 mg/kg IV on day -5 to day -2, rATG (thymoglobulin) given IV at 0.5 mg/kg on day -5, 1 mg/kg on day -4, and 1.5 mg/kg on days -3, -2, and -1 (total dose 6 mg/kg), and rituximab 500 mg IV on days -6 and +1. Unselected peripheral blood stem cells were infused on day 0. AQP4-IgG antibody status was determined by Clinical Laboratory Improvement Amendments-validated ELISA or flow cytometry assays. Cell-killing activity was measured using a flow cytometry-based complement assay. RESULTS:Median follow-up was 57 months. The patient with coexistent SLE died of complications of active lupus 10 months after HSCT. For the 12 patients with NMOSD without other active coexisting autoimmune diseases, 11 patients are more than 5 years post-transplant, and 80% are relapse-free off all immunosuppression ( < 0.001). At 1 and 5 years after HSCT, Expanded Disability Status Scale score improved from a baseline mean of 4.4 to 3.3 ( < 0.01) at 5 years. The Neurologic Rating Scale score improved after HSCT from a baseline mean of 69.5 to 85.7 at 5 years ( < 0.01). The Short Form-36 health survey for quality of life total score improved from mean 34.2 to 62.1 ( = 0.001) at 5 years. In the 11 patients whose baseline AQP4-IgG serostatus was positive, 9 patients became seronegative by the immunofluorescence or cell-binding assays available at the time; complement activating and cell-killing ability of patient serum was switched off in 6 of 7 patients with before and after HSCT testing. Two patients remained AQP4-IgG-seropositive (with persistent complement activating and cell-killing ability) and relapsed within 2 years of HSCT. No patient with seronegative conversion relapsed. CONCLUSION:Prolonged drug-free remission with AQP4-IgG seroconversion to negative following nonmyeloablative autologous HSCT warrants further investigation.
10.1212/WNL.0000000000008394
Association of Decreased Percentage of Vδ2Vγ9 γδ T Cells With Disease Severity in Multiple Sclerosis.
Maimaitijiang Guzailiayi,Shinoda Koji,Nakamura Yuri,Masaki Katsuhisa,Matsushita Takuya,Isobe Noriko,Yamasaki Ryo,Yoshikai Yasunobu,Kira Jun-Ichi
Frontiers in immunology
We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2 and Vδ2Vγ9 cells in γδ T cells ( = 0.0297 and = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs ( = 0.0033). The percentages of interferon (IFN)-γVδ2 and interleukin (IL)-17AIFN-γVδ2 cells in γδ T cells, as well as IFN-γ cells in Vδ2 γδ T cells, were significantly lower in MS patients than in HCs ( < 0.0009, = 0.0135, and = 0.0054, respectively). The percentages of Vδ2 and Vδ2Vγ9 cells in γδ T cells were negatively correlated with both the Expanded Disability Status Scale score ( = -0.5006, = 0.0048; and = -0.5040, = 0.0045, respectively) and Multiple Sclerosis Severity Score ( = -0.4682, = 0.0091; and = -0.4706, = 0.0087, respectively), but not with age at disease onset, disease duration, or annualized relapse rate. In HCs, the percentages of Vδ2 and Vδ2Vγ9 cells of total CD3 T cells had strong positive correlations with the percentage of CD25CD127 cells in CD4 T cells ( = 0.7826, < 0.0001; and = 0.7848, < 0.0001, respectively), whereas such correlations were totally absent in MS patients. These findings suggest that decreased Vδ2Vγ9 γδ T cells are associated with disability in MS. Therefore, the Vδ1/Vδ2 ratio might be a candidate biomarker for predicting disease severity in MS.
10.3389/fimmu.2018.00748
Abnormal B-Cell and Tfh-Cell Profiles in Patients With Parkinson Disease: A Cross-sectional Study.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:There has been growing interest in potential roles of the immune system in the pathogenesis of Parkinson disease (PD). The aim of the current study was to comprehensively characterize phenotypic and functional profiles of circulating immune cells in patients with PD vs controls. METHODS:Peripheral blood was collected from patients with PD and age- and sex-matched neurologically normal controls (NCs) in 2 independent cohorts (discovery and validation). Comprehensive multicolor flow cytometry was performed on whole blood leukocytes and peripheral blood mononuclear cells to characterize different immune subsets and their ex vivo responses. RESULTS:The discovery cohort included 17 NCs and 12 participants with PD, and the validation cohort included 18 NCs and 18 participants with PD. Among major immune cell types, B cells appeared to be preferentially affected in PD. Proliferating B cell counts were decreased in patients with PD compared with controls. Proportions of B-cell subsets with regulatory capacity such as transitional B cells were preferentially reduced in the patients with PD, whereas proportions of proinflammatory cytokine-producing B cells increased, resulting in a proinflammatory shift of their B-cell functional cytokine responses. Unsupervised principal component analysis revealed increased expression of TNFα and GM-CSF by both B cells and T cells of patients with PD. In addition, levels of follicular T cells, an important B-cell helper T-cell population, decreased in the patients with PD, correlating with their B-cell abnormality. DISCUSSION:Our findings define a novel signature of peripheral immune cells and implicate aberrant Tfh:B-cell interactions in patients with PD.
10.1212/NXI.0000000000001125
Methylation and Gene Expression in Relation to the Multiple Sclerosis-Associated Gene Variant rs2104286 and Soluble IL-2Rα in CD8 T Cells.
Buhelt Sophie,Laigaard Hannah-Marie,von Essen Marina Rode,Ullum Henrik,Oturai Annette,Sellebjerg Finn,Søndergaard Helle Bach
Frontiers in immunology
CD8 T cells are involved in the pathogenesis of multiple sclerosis (MS). The interleukin-2 receptor α (IL-2Rα) is important for CD8 T cell function, and single nucleotide polymorphisms (SNPs) in the gene encoding IL-2Rα increase the risk of MS. Therefore, in isolated CD8 T cells we investigated gene methylation and gene expression in relation to the MS-associated SNP rs2104286 and soluble IL-2Rα (sIL-2Rα). We have identified allele specific methylation of the CpG-site located in intron 1 that is perturbed by the rs2104286 SNP in CD8 T cells from genotype-selected healthy subjects (HS). However, methylation of selected CpG-sites in the promotor or 5'UTR region of the gene was neither associated with the rs2104286 SNP nor significantly correlated with gene expression in HS. In CD8 T cells from HS, we explored expression of immune relevant genes but observed only few associations with the rs2104286 SNP. However, we found that sIL-2Rα correlated negatively with expression of 55 immune relevant genes, including the IL-7 receptor gene, with Spearman's rho between -0.49 and -0.32. Additionally, in HS by use of flow cytometry we observed that the IL-7 receptor on naïve CD8 T cells correlated negatively with sIL-2Rα and was downregulated in carriers of the rs2104286 MS-associated risk genotype. Collectively, our study of resting CD8 T cells indicates that the rs2104286 SNP has a minor effect and sIL-2Rα may negatively regulate the CD8 T cell response.
10.3389/fimmu.2021.676141
Immune response and pathogen invasion at the choroid plexus in the onset of cerebral toxoplasmosis.
Figueiredo Caio Andreeta,Steffen Johannes,Morton Lorena,Arumugam Sushmitha,Liesenfeld Oliver,Deli Mária A,Kröger Andrea,Schüler Thomas,Dunay Ildiko Rita
Journal of neuroinflammation
BACKGROUND:Toxoplasma gondii (T. gondii) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood-brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood-cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. METHODS:To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. RESULTS:Here we revealed that endothelial cells in the CP are initially infected by T. gondii, and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. CONCLUSIONS:Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.
10.1186/s12974-021-02370-1
Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells.
Klemm Florian,Maas Roeltje R,Bowman Robert L,Kornete Mara,Soukup Klara,Nassiri Sina,Brouland Jean-Philippe,Iacobuzio-Donahue Christine A,Brennan Cameron,Tabar Viviane,Gutin Philip H,Daniel Roy T,Hegi Monika E,Joyce Johanna A
Cell
Brain malignancies encompass a range of primary and metastatic cancers, including low-grade and high-grade gliomas and brain metastases (BrMs) originating from diverse extracranial tumors. Our understanding of the brain tumor microenvironment (TME) remains limited, and it is unknown whether it is sculpted differentially by primary versus metastatic disease. We therefore comprehensively analyzed the brain TME landscape via flow cytometry, RNA sequencing, protein arrays, culture assays, and spatial tissue characterization. This revealed disease-specific enrichment of immune cells with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and T cells. These integrated analyses also uncovered multifaceted immune cell activation within brain malignancies entailing converging transcriptional trajectories while maintaining disease- and cell-type-specific programs. Given the interest in developing TME-targeted therapies for brain malignancies, this comprehensive resource of the immune landscape offers insights into possible strategies to overcome tumor-supporting TME properties and instead harness the TME to fight cancer.
10.1016/j.cell.2020.05.007
Spinal Fluid Myeloid Microvesicles Predict Disease Course in Multiple Sclerosis.
Gelibter Stefano,Pisa Marco,Croese Tommaso,Finardi Annamaria,Mandelli Alessandra,Sangalli Francesca,Colombo Bruno,Martinelli Vittorio,Comi Giancarlo,Filippi Massimo,Furlan Roberto
Annals of neurology
OBJECTIVE:In vivo measures of myeloid activity are promising biomarkers in multiple sclerosis. We previously demonstrated that cerebrospinal fluid (CSF) myeloid microvesicles are markers of microglial/macrophage activity and neuroinflammation in multiple sclerosis. Here, we aimed at investigating the diagnostic and prognostic value of myeloid microvesicles in a clinical setting. METHODS:Six hundred one patients discharged with a diagnosis of neuroinflammatory, neurodegenerative, or no neurological disease were enrolled. Myeloid microvesicles were measured with flow cytometry as isolectin B4-positive events in fresh CSF. Clinical, demographical, and magnetic resonance imaging (MRI) data were collected at diagnosis (all patients) and during follow-up (n = 176). RESULTS:CSF myeloid microvesicles were elevated in neuroinflammatory patients compared to the neurodegenerative and control groups. In multiple sclerosis, microvesicles were higher in patients with MRI disease activity and their concentration increased along with the number of enhancing lesions (p < 0.0001, Jonckheere-Terpstra test). CSF myeloid microvesicles were also higher in patients with higher disease activity in the month and year preceding diagnosis. Microvesicles excellently discriminated between the relapsing-remitting and control groups (receiver operator characteristic curve, area under the curve = 0.939, p < 0.0001) and between radiologically isolated syndrome and unspecific brain lesions (0.942, p < 0.0001). Furthermore, microvesicles were independent predictors of prognosis for both the relapsing-remitting and progressive groups. Microvesicles independently predicted future disease activity in relapsing-remitting patients (hazard ratio [HR] = 1.967, 95% confidence interval [CI] = 1.147-3.372), correcting for prognostic factors of standard clinical use. In the progressive group, microvesicles were independent predictors of disability accrual (HR = 10.767, 95% CI = 1.335-86.812). INTERPRETATION:Our results confirm that CSF myeloid microvesicles are a clinically meaningful biomarker of neuroinflammation and microglial/macrophage activity in vivo. These findings may support a possible use in clinical practice during diagnostic workup and prognostic assessment. ANN NEUROL 2021;90:253-265.
10.1002/ana.26154
Increased NK Cell Count in Multiple Sclerosis Patients Treated With Dimethyl Fumarate: A 2-Year Longitudinal Study.
Marastoni Damiano,Buriani Alessandro,Pisani Anna Isabella,Crescenzo Francesco,Zuco Carmela,Fortinguerra Stefano,Sorrenti Vincenzo,Marenda Bruno,Romualdi Chiara,Magliozzi Roberta,Monaco Salvatore,Calabrese Massimiliano
Frontiers in immunology
Dimethyl fumarate (DMF) is a disease-modifying drug for relapsing-remitting multiple sclerosis. Among others, DMF impedes immune activation by shifting the balance between inflammatory and regulatory cell types and by inducing apoptosis-triggered lymphopenia. Although the decrease in lymphocyte count is an early effect of the drug in several patients, the long-term impact on lymphocyte subsets is largely unknown. We performed a 2-years observational study on total lymphocyte count and subsets thereof by flow cytometry of peripheral blood of 38 multiple sclerosis patients in treatment with DMF. Data were collected at the beginning and after 3, 6, 12, and 24 months of therapy. Total lymphocyte count decreased in relation to time of exposure to DMF. Mean absolute B cell count decreased by 34.1% ( < 0.001) within the first 3 months of therapy and then remained stable over time. Mean absolute CD3 T cells count decrement reached 47.5% after 12 months of treatment ( < 0.001). NK cells count showed a heterogeneous trend, increasing by 85.9% ( < 0.001) after 2 years of treatment. CD4 T cells and CD8 T cells substantially decreased, with a significant increase of CD4/CD8 ratio during the first year of therapy. NK cells showed a heterogeneous behavior during DMF treatment with a significant increase over time. Since NK cells may also have a regulatory effect on immune system modulation, their increase during DMF treatment might play a role in the efficacy and safety of the drug.
10.3389/fimmu.2019.01666
Soluble ST2 links inflammation to outcome after subarachnoid hemorrhage.
Annals of neurology
OBJECTIVE:To investigate whether soluble growth stimulation expressed gene 2 (sST2), a prognostic marker in cardiovascular and inflammatory disorders, is associated with neurological injury after aneurysmal subarachnoid hemorrhage (SAH). METHODS:We studied SAH patients from 2 independent cohorts. Outcome assessments included functional status at 90 days using the modified Rankin Scale (mRS), mortality, and delayed cerebral ischemia (DCI). The relationships between sST2 plasma level and outcome measures were assessed in both cross-sectional and longitudinal analysis. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry. RESULTS:In the discovery cohort, sST2 predicted 90-day mRS 3-6 (C index = 0.724, p < 0.001) and mortality in Kaplan-Meier analysis (p < 0.001). The association with functional status was independent of age, sex, World Federation of Neurosurgical Societies score, modified Fisher score, treatment modality, and cardiac comorbidities (adjusted odds ratio = 2.28, 95% confidence interval = 1.04-5.00, p = 0.039). Higher sST2 concentration was observed in those patients with DCI (90.8 vs 53.7ng/ml, p = 0.003). These associations were confirmed in a replication cohort. In patients with high sST2, flow cytometry identified decreased expression of CD14 (4.27 × 10 ± 2,950 arbitrary unit (AU) vs 5.64 × 10 ± 1,290 AU, p < 0.001), and increased expression of CD16 (39,960 ± 272 AU vs 34,869 ± 183 AU, p < 0.001). INTERPRETATION:Plasma sST2 predicts DCI, functional outcome, and mortality after SAH, independent of clinical and radiographic markers. Elevated sST2 is also associated with changes in CD14 CD16 monocytes. ANN NEUROL 2019;86:384-394.
10.1002/ana.25545
Vitamin D enhances responses to interferon-β in MS.
Feng Xuan,Wang Zhe,Howlett-Prieto Quentin,Einhorn Nathan,Causevic Suad,Reder Anthony T
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To determine the effect of vitamin D3 on interferon-β (IFN-β) response and immune regulation in MS mononuclear cells (MNCs). METHODS:MNCs from 126 subjects, including therapy-naive patients with different forms of MS, plus patients with MS receiving IFN-β or glatiramer treatment, plus healthy controls were incubated in vitro with IFN-β-1b ± vitamin D3 (calcitriol). Activation of the IFN-β-induced transcription factor, p-Y-STAT1, and antiviral myxovirus A (MxA) protein was measured with flow cytometry and Western blots; serum proteins were measured with a customized 31-protein multiplex assay. RESULTS:Vitamin D enhanced in vitro IFN responses, as measured by induction of p-Y-STAT1 and MxA in MNCs, T cells, and monocytes. Vitamin D augmentation of IFN responses was seen in untreated and in IFN-β-1b-treated MS. The combination of vitamin D plus IFN-β reduced Th1 and Th17 cytokines, and increased Th2 responses, reversing the effect of IFN-β alone. Exacerbations and progression in untreated patients reduced the vitamin D enhancement of IFN responses. Vitamin D had less effect on IFN response in clinically stable glatiramer-treated than in IFN-β-treated patients. CONCLUSION:Vitamin D enhances IFN-β induction of multiple proteins and also reverses the Th1/Th2 bias in MS seen with IFN-β alone. The combination of vitamin D and IFN-β has potential benefit in ameliorating MS.
10.1212/NXI.0000000000000622
EBV-specific CD8 T lymphocytes and B cells during glatiramer acetate therapy in patients with MS.
Guerrera Gisella,Ruggieri Serena,Picozza Mario,Piras Eleonora,Gargano Francesca,Placido Roberta,Gasperini Claudio,Salvetti Marco,Buscarinu Maria Chiara,Battistini Luca,Borsellino Giovanna,Angelini Daniela F
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:Infection with Epstein-Barr virus (EBV) has been associated with clinical activity and risk of developing MS. The purpose of this study is to investigate the impact of glatiramer acetate (GA) therapy on EBV-specific immune responses and disease course. METHODS:We characterized EBV-specific CD8 T lymphocytes and B cells during disease-modifying treatments in 2 groups of patients with MS. We designed a 2-pronged approach consisting of a cross-sectional study (39 untreated patients, 38 patients who had undergone 12 months of GA treatment, and 48 healthy donors compatible for age and sex with the patients with MS) and a 12-month longitudinal study (35 patients treated with GA). CD8 EBV-specific T cells and B lymphocytes were studied using pentamers and multiparametric flow cytometry. RESULTS:We find that treatment with GA enhances viral recognition by inducing an increased number of circulating virus-specific CD8 T cells ( = 0.0043) and by relieving their features of exhaustion ( = 0.0053) and senescence ( < 0.0001, = 0.0001). B cells, phenotypically and numerically tracked along the 1-year follow-up study, show a steady decrease in memory B-cell frequencies ( = 0.025), paralleled by an increase of the naive B subset. CONCLUSION:GA therapy acts as a disease-modifying therapy restoring homeostasis in the immune system, including anti-EBV responses.
10.1212/NXI.0000000000000876
High-dimensional immune profiling identifies a biomarker to monitor dimethyl fumarate response in multiple sclerosis.
Proceedings of the National Academy of Sciences of the United States of America
Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS). Despite its wide clinical use, the mechanisms underlying clinical response are not understood. This study aimed to reveal immune markers of therapeutic response to DMF treatment in MS. For this purpose, we prospectively collected peripheral blood mononuclear cells (PBMCs) from a highly characterized cohort of 44 individuals with MS before and at 12 and 48 wk of DMF treatment. Single cells were profiled using high-dimensional mass cytometry. To capture the heterogeneity of different immune subsets, we adopted a bioinformatic multipanel approach that allowed cell population-cluster assignment of more than 50 different parameters, including lineage and activation markers as well as chemokine receptors and cytokines. Data were further analyzed in a semiunbiased fashion implementing a supervised representation learning approach to capture subtle longitudinal immune changes characteristic for therapy response. With this approach, we identified a population of memory T helper cells expressing high levels of neuroinflammatory cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFNγ]) as well as CXCR3, whose abundance correlated with treatment response. Using spectral flow cytometry, we confirmed these findings in a second cohort of patients. Serum neurofilament light-chain levels confirmed the correlation of this immune cell signature with axonal damage. The identified cell population is expanded in peripheral blood under natalizumab treatment, substantiating a specific role in treatment response. We propose that depletion of GM-CSF-, IFNγ-, and CXCR3-expressing T helper cells is the main mechanism of action of DMF and allows monitoring of treatment response.
10.1073/pnas.2205042119
Tetracyclines Diminish IFN-γ and IL-17-Producing Adaptive and Innate Immune Cells in Multiple Sclerosis.
Florou Despoina T,Mavropoulos Athanasios,Dardiotis Efthymios,Tsimourtou Vana,Siokas Vasileios,Aloizou Athina-Maria,Liaskos Christos,Tsigalou Christina,Katsiari Christina,Sakkas Lazaros I,Hadjigeorgiou Georgios,Bogdanos Dimitrios P
Frontiers in immunology
Introduction:Limited data from clinical trials in multiple sclerosis (MS) reported that minocycline, a widely used antibiotic belonging to the family of tetracyclines (TCs), exerts a beneficial short-lived clinical effect A similar anti-inflammatory effect of minocycline attributed to a deviation from Th1 to Th2 immune response has been reported in experimental models of MS. Whether such an immunomodulatory mechanism is operated in the human disease remains largely unknown. Aim:To assess the immunomodulatory effect of tetracyclines, and in particular minocycline and doxycycline, in naïve and treated patients with MS. Material and Methods:Peripheral blood mononuclear cells from 45 individuals (35 MS patients, amongst which 15 naïve patients and 10 healthy controls, HCs) were cultured with minocycline or doxycycline and conventional stimulants (PMA/Ionomycin or IL-12/IL-18). IFN-γ and IL-17 producing T-, NK- and NKT cells were assessed by flow cytometry. The effect of TCs on cell viability and apoptosis was further assessed by flow cytometry with Annexin V staining. Results:Both tetracyclines significantly decreased, in a dose dependent manner, IFN-γ production in NKT and CD4 T lymphocytes from MS patients (naïve or treated) stimulated with IL-12/IL-18 but did not decrease IFN-γ producing CD8 T cells from naive MS or treated RRMS patients. They also decreased IL-17 T and NKT cells following PMA and Ionomycin-stimulation. Tetracyclines did not affect the viability of cell subsets. Conclusion:Tetracyclines can suppress IFN-γ and IL-17- producing cells from MS patients, and this may explain their potential therapeutic effect .
10.3389/fimmu.2021.739186
Alterations in Blood Monocyte Functions in Parkinson's Disease.
Nissen Sara Konstantin,Shrivastava Kalpana,Schulte Claudia,Otzen Daniel Erik,Goldeck David,Berg Daniela,Møller Holger Jon,Maetzler Walter,Romero-Ramos Marina
Movement disorders : official journal of the Movement Disorder Society
BACKGROUND:PD is a multisystem disease where both central and peripheral nervous systems are affected. This systemic involvement also includes the immune response in PD, which implicates not only microglia in the brain, but also peripheral immune cells, such as monocytes; however, this aspect has been understudied. OBJECTIVES:The purpose of this study was to investigate the PD-related changes in peripheral immune cells, their responsiveness to stimulation, and their ability to release immunomodulatory molecules that might have consequences for the disease progression. METHODS:Using flow cytometry, we investigated the monocytic population in peripheral blood mononuclear cells from PD patients and healthy individuals. We also evaluated the in vitro response to inflammogen lipopolysaccharides and to fibrillar α-synuclein by measuring the expression of CD14, CD163, and HLA-DR and by analysis of soluble immune-related molecules in the supernatant. RESULTS:Peripheral blood immune cells from PD patients had lower survival in culture, but showed a higher monocytic proliferative ability than control cells, which was correlated with shorter disease duration and late disease onset. In addition, PD patients' cells were less responsive to stimulation, as shown by the lack of changes in CD163 and CD14 expression, and by the absence of significant upregulation of anti-inflammatory cytokines in culture. Moreover, PD peripheral immune cells shed lower in vitro levels of soluble CD163, which suggests a less responsive monocytic population and/or an activation status different from control cells. Interestingly, some of the results were sex associated, supporting a differential immune response in females versus males. CONCLUSIONS:Our data suggest that PD involves monocytic changes in blood. These cells show reduced viability and are unresponsive to specific stimuli, which might have a relevant consequence for disease progression. © 2019 International Parkinson and Movement Disorder Society.
10.1002/mds.27815
Cytotoxic B Cells in Relapsing-Remitting Multiple Sclerosis Patients.
Boldrini Vinícius O,Marques Ana M,Quintiliano Raphael P S,Moraes Adriel S,Stella Carla R A V,Longhini Ana Leda F,Santos Irene,Andrade Marília,Ferrari Breno,Damasceno Alfredo,Carneiro Rafael P D,Brandão Carlos Otávio,Farias Alessandro S,Santos Leonilda M B
Frontiers in immunology
Background:Emerging evidence of antibody-independent functions, as well as the clinical efficacy of anti-CD20 depleting therapies, helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. Objective:To investigate whether CD19 B cells may share expression of the serine-protease granzyme-B (GzmB), resembling classical cytotoxic CD8 T lymphocytes, in the peripheral blood from relapsing-remitting MS (RRMS) patients. Methods:In this study, 104 RRMS patients during different treatments and 58 healthy donors were included. CD8, CD19, Runx3, and GzmB expression was assessed by flow cytometry analyses. Results:RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8GzmB T lymphocytes when compared to healthy volunteers. An increase in circulating CD19GzmB B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA), untreated RRMS patients, and healthy donors but not when compared to interferon-β (IFN). Moreover, regarding Runx3, the transcriptional factor classically associated with cytotoxicity in CD8 T lymphocytes, the expression of GzmB was significantly higher in CD19Runx3-expressing B cells when compared to CD19Runx3 counterparts in RRMS patients. Conclusions:CD19 B cells may exhibit cytotoxic behavior resembling CD8 T lymphocytes in MS patients during different treatments. In the future, monitoring "cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions.
10.3389/fimmu.2022.750660
Differential expression of CD8 defines phenotypically distinct cytotoxic T cells in cancer and multiple sclerosis.
Clinical and translational medicine
BACKGROUND:Cytotoxic T lymphocytes take on a leading role in many immune-related diseases. They function as key effector immune cells fighting cancer cells, but they are also considerably involved in autoimmune diseases. Common to both situations, CD8 T cells need to adapt their metabolism and effector function to the harsh and nutrient-deprived conditions of the disease-associated microenvironment. METHODS:We used an in vitro starvation as well as rapamycin treatment protocol mimicking nutrient deprivation to generate CD8 versus CD8 T cells and performed FACS-Sorting followed by transcriptomic profiling of the cytotoxic T cell subsets. Prominent markers identified in the CD8 versus the CD8 T cells were then used to investigate the presence of these cell subsets in immune-related human diseases. Employing cancer tissue microarrays and PhenOptics multispectral imaging as well as flow cytometry, we studied these CD8 T cell subsets in cancer and relapsing-remitting multiple sclerosis patients. RESULTS:Starvation induced a decreased expression of CD8, yielding a CD8 T cell subpopulation with an altered transcriptomic signature and reduced effector function. CD8 T cell showed enhanced ST2L and IL6ST (CD130) expression compared to CD8 T cells which expressed elevated KLRD1 (CD94) and granzyme B levels within the tumour microenvironment (TME). Spatial analysis revealed the presence of CD8 T cells in close proximity to tumour cells, while the CD8 T cells resided at the tumour boundaries. Importantly, the number of tumour-infiltrating CD8 T lymphocytes correlated with a poor prognosis as well as with enhanced cancer progression in human mammary carcinoma. We also found a reduced frequency of CD8 T lymphocytes in a cohort of relapse (disease active) multiple sclerosis patients compared to healthy subjects during immune cell starvation in vitro. CONCLUSIONS:In summary, our data show that functionally distinct cytotoxic T lymphocytes can be identified based on their expression of CD8. Indicating a more general role in CD8 T cell immunity, these cells may play opposing roles in the TME, and also in the pathophysiology of autoimmune diseases such as multiple sclerosis.
10.1002/ctm2.1068
MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis.
Kotelnikova Ekaterina,Kiani Narsis A,Messinis Dimitris,Pertsovskaya Inna,Pliaka Vicky,Bernardo-Faura Marti,Rinas Melanie,Vila Gemma,Zubizarreta Irati,Pulido-Valdeolivas Irene,Sakellaropoulos Theodore,Faigle Wolfgang,Silberberg Gilad,Masso Mar,Stridh Pernilla,Behrens Janina,Olsson Tomas,Martin Roland,Paul Friedemann,Alexopoulos Leonidas G,Saez-Rodriguez Julio,Tegner Jesper,Villoslada Pablo
Proceedings of the National Academy of Sciences of the United States of America
Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor's downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19 cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.
10.1073/pnas.1818347116
Immune Skew of Circulating Follicular Helper T Cells Associates With Myasthenia Gravis Severity.
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To clarify functional alterations of follicular helper T cells (Tfh) in myasthenia gravis (MG) because Tfh play important roles in helping B cells generate antibody-producing cells. METHODS:A total of 24 immunotherapy-naive patients with anti-acetylcholine receptor (AchR) antibody-positive MG and 18 age-matched healthy subjects (HS) were enrolled. Samples from 6 patients were available for posttreatment analysis. Subsets of circulating Tfh (cTfh) and B cells were identified by flow cytometry analysis of surface molecules. Cytokine production by isolated cTfh subsets from 5 patients with MG and 5 HS was measured in vitro. Analysis was performed to examine the correlation between the frequency of cTfh subsets and that of plasmablasts and between cTfh subsets and the quantitative MG score. RESULTS:cTfh increased with elevated expression of inducible T-cell costimulator (ICOS) in patients with MG. cTfh shifted to Th2 and Th17 over Th1 in MG. ICOScTfh produced significantly higher levels of interleukin (IL)-21, IL-4, and IL-17A than ICOS cTfh only in patients with MG. The frequency of cTfh within CD4 T cells was more closely associated with disease severity than the serum anti-AchR antibody titer and frequency of plasmablasts within B cells. Abnormalities of cTfh were improved after immunotherapy in parallel with clinical improvement. CONCLUSIONS:Alternation of cTfh is a key feature in the development of MG and may become a biomarker for disease severity and therapeutic efficacy. CLASSIFICATION OF EVIDENCE:This study provides Class II evidence that the level of cTfh is associated with disease severity in patients with MG.
10.1212/NXI.0000000000000945
Selective BCL-XL inhibition promotes apoptosis in combination with MLN8237 in medulloblastoma and pediatric glioblastoma cells.
Levesley Jane,Steele Lynette,Brüning-Richardson Anke,Davison Adam,Zhou Jia,Ding Chunyong,Lawler Sean,Short Susan C
Neuro-oncology
Background:CNS tumors, including medulloblastoma and pediatric glioblastoma (pGBM) account for the majority of solid pediatric malignancies. There remains an unmet need to identify novel treatment approaches in poor prognosis and relapsed pediatric brain tumors, where therapeutic options are limited. Small-molecule B-cell lymphoma 2 (BCL-2) family inhibitors may enhance tumor cell killing when combined with conventional and targeted chemotherapeutic agents. We investigated the effect of disrupting BCL-2 and B cell lymphoma-extra large (BCL-XL) protein function using ABT-263, ABT-199 and WEHI-539 in medulloblastoma and pGBM cells following treatment with MLN8237, an Aurora kinase inhibitor under investigation as a novel agent for the treatment of malignant brain tumors. Methods:Tumor cell growth and viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression, and ploidy were determined by live cell imaging and DNA content analysis. Apoptosis was determined by annexin V/propidium iodide staining and time-lapse microscopy and confirmed by measuring caspase-3/7 activity and western blotting and by short interfering RNA (siRNA) knockdown of BCL-2 associated X protein/BCL-2 antagonist killer (BAX/BAK). Results:ABT-263, in combination with MLN8237, reduced mitotic slippage and polyploidy and promoted the elimination of mitotically defective cells via a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-XL antagonist, WEHI-539, significantly augmented tumor cell killing when used in combination with MLN8237, as well as sensitized resistant brain tumor cells to a novel BAX activator, SMBA1. In addition, siRNA-mediated knockdown of BCL-XL sensitized pGBM and medulloblastoma cells to MLN8237 and mimicked the effect of combination drug treatment. Conclusion:Selective small-molecule inhibitors of BCL-XL may enhance the efficacy of MLN8237 and other targeted chemotherapeutic agents.
10.1093/neuonc/nox134
The Restorative Effect of Human Amniotic Fluid Stem Cells on Spinal Cord Injury.
Lale Ataei Maryam,Karimipour Mohammad,Shahabi Parviz,Pashaei-Asl Roghiyeh,Ebrahimie Esmaeil,Pashaiasl Maryam
Cells
Spinal cord injury (SCI) is a debilitating condition within the neural system which is clinically manifested by sensory-motor dysfunction, leading, in some cases, to neural paralysis for the rest of the patient's life. In the current study, mesenchymal stem cells (MSCs) were isolated from the human amniotic fluid, in order to study their juxtacrine and paracrine activities. Flow cytometry analysis was performed to identify the MSCs. A conditioned medium (CM) was collected to measure the level of BDNF, IL-1β, and IL-6 proteins using the ELISA assay. Following the SCI induction, MSCs and CM were injected into the lesion site, and also CM was infused intraperitoneally in the different groups. Two weeks after SCI induction, the spinal cord samples were examined to evaluate the expression of the doublecortin () and glial fibrillary acid protein () markers using immunofluorescence staining. The MSCs' phenotype was confirmed upon the expression and un-expression of the related CD markers. Our results show that MSCs increased the expression level of the and decreased the level of the relative to the injury group ( < 0.001). Additionally, the CM promoted the expression rate ( < 0.001) and decreased the expression rate ( < 0.01) as compared with the injury group. Noteworthily, the restorative potential of the MSCs was higher than that of the CM ( < 0.01). Large-scale meta-analysis of transcriptomic data highlighted , , and as positively coexpressed genes with . These genes are involved in neuroactive ligand-receptor interaction. Overall, our data revealed that both therapeutic interventions could promote the regeneration and restoration of the damaged neural tissue by increasing the rate of neuroblasts and decreasing the astrocytes.
10.3390/cells10102565
Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS Patients - An Study.
Dobreanu Minodora,Manu Doina Ramona,Mănescu Ion Bogdan,Gabor Manuela Rozalia,Huţanu Adina,Bărcuţean Laura,Bălaşa Rodica
Frontiers in immunology
Background:Multiple sclerosis (MS) is an incurable autoimmune disease mediated by a heterogeneous T cell population (CD3+CD161+CXCR3-CCR6+IFNγ-IL17+, CD3+CXCR3+CCR6+IFNγ+IL17+, and CD3+CXCR3+IFNγ+IL17- phenotypes) that infiltrates the central nervous system, eliciting local inflammation, demyelination and neurodegeneration. Cladribine is a lymphocyte-depleting deoxyadenosine analogue recently introduced for MS therapy as a Disease Modifying Drug (DMD). Our aim was to establish a method for the early identification and prediction of cladribine responsiveness among MS patients. Methods:An experimental model was designed to study the cytotoxic and immunomodulatory effect of cladribine. T cell subsets of naïve relapsing-remitting MS (RRMS) patients were analyzed and comparatively to healthy controls (HC). Surviving cells were stimulated with rh-interleukin-2 for up to 14days. Cell proliferation and immunophenotype changes were analyzed after maximal (phorbol myristate acetate/ionomycin/monensin) and physiological T-cell receptor (CD3/CD28) activation, using multiparametric flow cytometry and xMAP technology. Results: CD161+Th17 cells were increased in RRMS patients. to phenotype shifts included: decreased CD3+CCR6+ and CD3+CD161+ in all subjects and increased CD3+CXCR3+ in RRMS patients only; Th17.1 showed increased proliferation vs Th17 in all subjects; CD3+IL17+ and CD3+IFNγ+IL17+ continued to proliferate till day 14, CD3+IFNγ+ only till day 7. Regarding cladribine exposure: RRMS CD3+ cells were more resistant compared to HC; treated CD3+ cells proliferated continuously for up to 14 days, while untreated cells only up to 7 days; both HC/RRMS CD3+CXCR3+ populations increased from baseline till day 14; in RRMS patients vs HC, IL17 secretion from cladribine-treated cells increased significantly, in line with the observed proliferation of CD3+IL17+ and CD3+IFNγ+IL17+ cells; in both HC/RRMS, cladribine led to a significant increase in CD3+IFNγ+ cells at day 7 only, having no further effect at day14. IFNγ and IL17 secreted in culture media decreased significantly from to . Conclusions:CD3+ subtypes showed different responsiveness due to selectivity of cladribine action, in most patients leading to survival/proliferation of lymphocyte subsets known as pathogenic in MS. This experimental model is a promising tool for the prediction of individual responsiveness of MS patients to cladribine and other DMDs.
10.3389/fimmu.2021.743010
Amyotrophic Lateral Sclerosis Survival Associates With Neutrophils in a Sex-specific Manner.
Murdock Benjamin J,Goutman Stephen A,Boss Jonathan,Kim Sehee,Feldman Eva L
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To determine whether neutrophils contribute to amyotrophic lateral sclerosis (ALS) progression, we tested the association of baseline neutrophil count on ALS survival, whether the effect was sex specific, and whether neutrophils accumulate in the spinal cord. METHODS:A prospective cohort study was conducted between June 22, 2011, and October 30, 2019. Blood leukocytes were isolated from ALS participants and neutrophil levels assessed by flow cytometry. Participant survival outcomes were analyzed by groups (<2 × 10, 2-4 × 10, and >4 × 10 neutrophils/mL) with adjustments for relevant ALS covariates and by sex. Neutrophil levels were assessed from CNS tissue from a subset of participants. RESULTS:A total of 269 participants with ALS within 2 years of an ALS diagnosis were included. Participants with baseline neutrophil counts over 4 × 10/mL had a 2.1 times higher mortality rate than those with a neutrophil count lower than 2 × 10/mL (95% CI: 1.3-3.5, = 0.004) when adjusting for age, sex, and other covariates. This effect was more pronounced in females, with a hazard ratio of 3.8 (95% CI: 1.8-8.2, = 0.001) in the >4 × 10/mL vs <2 × 10/mL group. Furthermore, ALS participants (n = 8) had increased neutrophils in cervical ( = 0.049) and thoracic ( = 0.022) spinal cord segments compared with control participants (n = 8). CONCLUSIONS:Higher neutrophil counts early in ALS associate with a shorter survival in female participants. Furthermore, neutrophils accumulate in ALS spinal cord supporting a pathophysiologic correlate. These data justify the consideration of immunity and sex for personalized therapeutic development in ALS. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that in female participants with ALS, higher baseline neutrophil counts are associated with shorter survival.
10.1212/NXI.0000000000000953
Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Autoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. METHODS:An HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry. RESULTS:Serum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset. DISCUSSION:A novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.
10.1212/NXI.0000000000001169
Bi-allelic LoF NRROS Variants Impairing Active TGF-β1 Delivery Cause a Severe Infantile-Onset Neurodegenerative Condition with Intracranial Calcification.
American journal of human genetics
Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat-containing protein that uniquely associates with latent transforming growth factor beta-1 (TGF- β1) and anchors it on the cell surface; this anchoring is required for activation of TGF-β1 in macrophages and microglia. We report six individuals from four families with bi-allelic variants in NRROS. All affected individuals had neurodegenerative disease with refractory epilepsy, developmental regression, and reduced white matter volume with delayed myelination. The clinical course in affected individuals began with normal development or mild developmental delay, and the onset of seizures occurred within the first year of life, followed by developmental regression. Intracranial calcification was detected in three individuals. The phenotypic features in affected individuals are consistent with those observed in the Nrros knockout mouse, and they overlap with those seen in the human condition associated with TGF-β1 deficiency. The disease-causing NRROS variants involve two significant functional NRROS domains. These variants result in aberrant NRROS proteins with impaired ability to anchor latent TGF-β1 on the cell surface. Using confocal microscopy in HEK293T cells, we demonstrate that wild-type and mutant NRROS proteins co-localize with latent TGF-β1 intracellularly. However, using flow cytometry, we show that our mutant NRROS proteins fail to anchor latent TGF-β1 at the cell surface in comparison to wild-type NRROS. Moreover, wild-type NRROS rescues the defect of our disease-associated mutants in presenting latent TGF-β1 to the cell surface. Taken together, our findings suggest that loss of NRROS function causes a severe childhood-onset neurodegenerative condition with features suggestive of a disordered response to inflammation.
10.1016/j.ajhg.2020.02.014
Serum of Post-COVID-19 Syndrome Patients with or without ME/CFS Differentially Affects Endothelial Cell Function In Vitro.
Cells
A proportion of COVID-19 reconvalescent patients develop post-COVID-19 syndrome (PCS) including a subgroup fulfilling diagnostic criteria of Myalgic encephalomyelitis/Chronic Fatigue Syndrome (PCS/CFS). Recently, endothelial dysfunction (ED) has been demonstrated in these patients, but the mechanisms remain elusive. Therefore, we investigated the effects of patients' sera on endothelia cells (ECs) in vitro. PCS (n = 17), PCS/CFS (n = 13), and healthy controls (HC, n = 14) were screened for serum anti-endothelial cell autoantibodies (AECAs) and dysregulated cytokines. Serum-treated ECs were analysed for the induction of activation markers and the release of small molecules by flow cytometry. Moreover, the angiogenic potential of sera was measured in a tube formation assay. While only marginal differences between patient groups were observed for serum cytokines, AECA binding to ECs was significantly increased in PCS/CFS patients. Surprisingly, PCS and PCS/CFS sera reduced surface levels of several EC activation markers. PCS sera enhanced the release of molecules associated with vascular remodelling and significantly promoted angiogenesis in vitro compared to the PCS/CFS and HC groups. Additionally, sera from both patient cohorts induced the release of molecules involved in inhibition of nitric oxide-mediated endothelial relaxation. Overall, PCS and PCS/CFS patients' sera differed in their AECA content and their functional effects on ECs, i.e., secretion profiles and angiogenic potential. We hypothesise a pro-angiogenic effect of PCS sera as a compensatory mechanism to ED which is absent in PCS/CFS patients.
10.3390/cells11152376
Changes in Peripheral Blood Regulatory T Cells and IL-6 and IL-10 Levels Predict Response of Pediatric Medulloblastoma and Germ Cell Tumors With Residual or Disseminated Disease to Craniospinal Irradiation.
Song Linan,Wang Shuo,Fang Tong,Qiu Xiaoguang,Wang Xiaoli,Zhou Xinna,Morse Michael A,Hobeika Amy,Wu Wanshui,Yang Huabing,Ren Jun,Lyerly Herbert Kim
International journal of radiation oncology, biology, physics
PURPOSE:Radiation therapy (RT) modulates immune cells and cytokines, resulting in both clinically beneficial and detrimental effects. The changes in peripheral blood T lymphocyte subsets and cytokines during RT for pediatric brain tumors and the association of these changes with therapeutic outcomes have not been well described. METHODS AND MATERIALS:The study population consisted of children (n = 83, aged 3~18) with primary brain tumors (medulloblastoma, glioma, germ cell tumors (GCT), and central nervous system embryonal tumor-not otherwise specified), with or without residual or disseminated (R/D) diseases who were starting standard postoperative focal or craniospinal irradiation (CSI). Peripheral blood T lymphocyte subsets collected before and 4 weeks after RT were enumerated by flow cytometry. Plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, and IL-17A were measured by cytometric bead array. RESULTS:Patients with R/D lesions receiving CSI (n = 32) had a post-RT increase in the frequency of CD3+T and CD8+T cells, a decrease in CD4+T cells, and an increase in regulatory T cells (Tregs) and CD8+CD28- suppressor cells, which was more predominantly seen in these patients than in other groups. In the CSI group with such R/D lesions, consisting of patients with medulloblastoma and germ cell tumors, 19 experienced a complete response (CR) and 13 experienced a partial response (PR) on imaging at 4 weeks after RT. The post/pre-RT ratio of Tregs (P = .0493), IL-6 (P = .0111), and IL-10 (P = .0070) was lower in the CR group than in the PR group. Multivariate analysis revealed that the post/pre-RT ratios of Treg, IL-6, and IL-10 were independent predictors of CR (P < .0001, P = .018, P < .0001, respectively). The areas under the receiver operating curves and confidence intervals were 0.7652 (0.5831-0.8964), 0.7794 (0.5980-0.9067), and 0.7085 (0.5223-0.8552) for IL-6, IL-10, and Treg, respectively. The sensitivities of IL-6, IL-10, and Treg to predict radiotherapeutic responses were 100%, 92.3%, and 61.5%, and specificity was 52.6%, 57.9%, and 84.2%, respectively. CONCLUSIONS:CSI treatment to those with R/D lesions predominantly exerted an effect on antitumor immune response compared with both R/D lesion-free but exposed to focal or CSI RT and with R/D lesions and exposed to focal RT. Such CSI with R/D lesions group experiencing CR is more likely to have a decrease in immunoinhibitory molecules and cells than patients who only achieve PR. Measuring peripheral blood Treg, IL-6, and IL-10 levels could be valuable for predicting radiotherapeutic responses of pediatric brain tumors with R/D lesions to CSI for medulloblastoma and intracranial germ cell tumors.
10.1016/j.ijrobp.2021.04.041
Natalizumab promotes activation and pro-inflammatory differentiation of peripheral B cells in multiple sclerosis patients.
Traub Jan W,Pellkofer Hannah L,Grondey Katja,Seeger Ira,Rowold Christoph,Brück Wolfgang,Husseini Leila,Häusser-Kinzel Silke,Weber Martin S
Journal of neuroinflammation
BACKGROUND:In the past, multiple sclerosis (MS) medications have been primarily designed to modulate T cell properties. Based on the emerging concept that B cells are equally important for the propagation of MS, we compared the effect of four commonly used, primarily T cell-targeting MS medications on B cells. METHODS:Using flow cytometry, we analyzed peripheral blood mononuclear cells (PBMC) of untreated (n = 19) and dimethyl fumarate (DMF; n = 21)-, fingolimod (FTY; n = 17)-, glatiramer acetate (GA; n = 18)-, and natalizumab (NAT; n = 20)-treated MS patients, focusing on B cell maturation, differentiation, and cytokine production. RESULTS:While GA exerted minor effects on the investigated B cell properties, DMF and FTY robustly inhibited pro-inflammatory B cell function. In contrast, NAT treatment enhanced B cell differentiation, activation, and pro-inflammatory cytokine production when compared to both intraindividual samples collected before NAT treatment initiation as well as untreated MS controls. Our mechanistic in vitro studies confirm this observation. CONCLUSION:Our data indicate that common MS medications have differential, in part opposing effects on B cells. The observed activation of peripheral B cells upon NAT treatment may be instructive to interpret its unfavorable effect in certain B cell-mediated inflammatory conditions and to elucidate the immunological basis of MS relapses after NAT withdrawal. TRIAL REGISTRATION:Protocols were approved by the ethical review committee of the University Medical Center Göttingen (#3/4/14).
10.1186/s12974-019-1593-2
IgA natural antibodies are produced following T-cell independent B-cell activation following stroke.
Zbesko Jacob C,Frye Jennifer Beischel,Becktel Danielle A,Gerardo Diana K,Stokes Jessica,Calderon Kylie,Nguyen Thuy-Vi V,Bhattacharya Deepta,Doyle Kristian P
Brain, behavior, and immunity
Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII, CD4, and MyD88 mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.
10.1016/j.bbi.2020.09.014
The INTUIT Study: Investigating Neuroinflammation Underlying Postoperative Cognitive Dysfunction.
Berger Miles,Oyeyemi Deborah,Olurinde Mobolaji O,Whitson Heather E,Weinhold Kent J,Woldorff Marty G,Lipsitz Lewis A,Moretti Eugene,Giattino Charles M,Roberts Kenneth C,Zhou Junhong,Bunning Thomas,Ferrandino Michael,Scheri Randall P,Cooter Mary,Chan Cliburn,Cabeza Roberto,Browndyke Jeffrey N,Murdoch David M,Devinney Michael J,Shaw Leslie M,Cohen Harvey Jay,Mathew Joseph P,
Journal of the American Geriatrics Society
BACKGROUND/OBJECTIVES:Every year, up to 40% of the more than 16 million older Americans who undergo anesthesia/surgery develop postoperative cognitive dysfunction (POCD) or delirium. Each of these distinct syndromes is associated with decreased quality of life, increased mortality, and a possible increased risk of Alzheimer's disease. One pathologic process hypothesized to underlie both delirium and POCD is neuroinflammation. The INTUIT study described here will determine the extent to which postoperative increases in cerebrospinal fluid (CSF) monocyte chemoattractant protein 1 (MCP-1) levels and monocyte numbers are associated with delirium and/or POCD and their underlying brain connectivity changes. DESIGN:Observational prospective cohort. SETTING:Duke University Medical Center, Duke Regional Hospital, and Duke Raleigh Hospital. PARTICIPANTS:Patients 60 years of age or older (N = 200) undergoing noncardiac/nonneurologic surgery. MEASUREMENTS:Participants will undergo cognitive testing before, 6 weeks, and 1 year after surgery. Delirium screening will be performed on postoperative days 1 to 5. Blood and CSF samples are obtained before surgery, and 24 hours, 6 weeks, and 1 year after surgery. CSF MCP-1 levels are measured by enzyme-linked immunosorbent assay, and CSF monocytes are assessed by flow cytometry. Half the patients will also undergo pre- and postoperative functional magnetic resonance imaging scans. 32-channel intraoperative electroencephalogram (EEG) recordings will be performed to identify intraoperative EEG correlates of neuroinflammation and/or postoperative cognitive resilience. Eighty patients will also undergo home sleep apnea testing to determine the relationships between sleep apnea severity, neuroinflammation, and impaired postoperative cognition. Additional assessments will help evaluate relationships between delirium, POCD, and other geriatric syndromes. CONCLUSION:INTUIT will use a transdisciplinary approach to study the role of neuroinflammation in postoperative delirium and cognitive dysfunction and their associated functional brain connectivity changes, and it may identify novel targets for treating and/or preventing delirium and POCD and their sequelae. J Am Geriatr Soc 67:794-798, 2019.
10.1111/jgs.15770
GPR15 T cells are Th17 like, increased in smokers and associated with multiple sclerosis.
Ammitzbøll Cecilie,von Essen Marina R,Börnsen Lars,Petersen Eva Rosa,McWilliam Oskar,Ratzer Rikke,Romme Christensen Jeppe,Oturai Annette B,Søndergaard Helle B,Sellebjerg Finn
Journal of autoimmunity
Smoking is a risk factor for the development and progression of multiple sclerosis (MS); however, the pathogenic effects of smoking are poorly understood. We studied the smoking-associated chemokine receptor-like molecule GPR15 in relation to relapsing-remitting MS (RRMS). Using microarray analyses and qPCR we found elevated GPR15 in blood cells from smokers, and increased GPR15 expression in RRMS. By flow cytometry we detected increased frequencies of GPR15 expressing T and B cells in smokers, but no difference between patients with RRMS and healthy controls. However, after cell culture with the autoantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein, frequencies of MBP-reactive and non-proliferating GPR15CD4 T cells were increased in patients with RRMS compared with healthy controls. GPR15CD4 T cells produced IL-17 and were enriched in the cerebrospinal fluid (CSF). Furthermore, in the CSF of patients with RRMS, GPR15 T cells were associated with CCR6CXCR3/CCR6CXCR3 phenotypes and correlated positively with concentrations of the newly identified GPR15-ligand (GPR15L), myelin degradation and disability. In conclusion, we have identified a proinflammatory cell type linking smoking with pathogenic immune cell functions in RRMS.
10.1016/j.jaut.2018.09.005
Harmful neutrophil subsets in patients with ischemic stroke: Association with disease severity.
Weisenburger-Lile David,Dong Yuan,Yger Marion,Weisenburger Gaëlle,Polara Giulia Frasca,Chaigneau Thomas,Ochoa Riccardo Zapata,Marro Beatrice,Lapergue Bertrand,Alamowitch Sonia,Elbim Carole
Neurology(R) neuroimmunology & neuroinflammation
Objective:To better understand the functional state of circulating neutrophils in patients with ischemic stroke (IS) for planning future clinical trials. Methods:We analyzed by flow cytometry activation state of circulating neutrophils and the distribution of neutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission and compared them with 22 age-matched healthy controls. Results:Our results demonstrated continuous basal hyperactivation of circulating neutrophils during acute IS, characterized by lower l-selectin expression and higher CD11b expression at the cell surface, increased ROS production by neutrophils, and greater circulating levels of neutrophil elastase. Neutrophil hyperactivation was associated with deregulation of the equilibrium between apoptotic and necrotic. Patients also had higher percentages than controls of the overactive senescent (CXCR4/CD62L) neutrophil subset and increased percentage of neutrophils with a reverse transendothelial migration (CD54CXCR1) phenotype. Importantly, neutrophil alterations were associated with the clinical severity of the stroke, evaluated by its NIH Stroke Scale score. Conclusion:Altogether, our results indicate that during acute IS, the inflammatory properties of circulating neutrophils rise, associated with the expansion of harmful neutrophil subsets. These changes in neutrophil homeostasis, associated with disease severity, may play an instrumental role by contributing to systemic inflammation and to the blood-brain barrier breakdown. Our findings highlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescent to immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both.
10.1212/NXI.0000000000000571
Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.
Zhou Peng,Li Tao,Jin Jiaqi,Liu Yingmiao,Li Baorong,Sun Quanye,Tian Jiawei,Zhao Hongtao,Liu Zhihui,Ma Shuai,Zhang Shuoqi,Novakovic Valerie A,Shi Jialan,Hu Shaoshan
EBioMedicine
BACKGROUND:The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. METHODS:The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. FINDINGS:The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. INTERPRETATION:NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. FUNDING:This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).
10.1016/j.ebiom.2020.102671
Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.
Dominguez-Mozo Maria Inmaculada,Perez-Perez Silvia,Villarrubia Noelia,Costa-Frossard Lucienne,Fernandez-Velasco Jose Ignacio,Ortega-Madueño Isabel,Garcia-Martinez Maria Angel,Garcia-Calvo Estefania,Estevez Hector,Luque Garcia Jose Luis,Torrejon Maria Josefa,Arroyo Rafael,Villar Luisa Maria,Alvarez-Lafuente Roberto
Cells
Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.
10.3390/cells10010119
Hypertonic saline for prevention of delirium in geriatric patients who underwent hip surgery.
Xin Xi,Xin Fei,Chen Xuguang,Zhang Qi,Li Yanan,Huo Shuping,Chang Chongfu,Wang Qiujun
Journal of neuroinflammation
BACKGROUND:Postoperative delirium (POD) is a common disorder in the elderly patients, and neuroinflammation is the possible underlying mechanism. This study is designed to determine whether or not hypertonic saline (HS) pre-injection can alleviate POD in aged patients. METHODS:This prospective study recruited 120 geriatric patients who underwent hip surgery. The patients were randomly divided into two groups: control group (NS group) and HS group. Patients in the NS group were pre-injected with 4 mL/kg isotonic saline, and those in the HS group were pre-injected with 4 mL/kg 7.5% HS. All 120 patients were then subjected to general anesthesia. Blood samples were extracted to detect the concentration of inflammatory factors, namely, IL-1β, IL-6, IL-10, and TNF-α, and the nerve injury factor S100β. Flow cytometry was used to detect the number of monocytes in peripheral venous blood and evaluate the relationship of inflammation to delirium. The nursing delirium screening scale (Nu-DESC) was used to determine cognitive function 1 to 3 days postoperatively. RESULTS:Analysis using random-effect multivariable logistic regression indicated that HS administration before anesthesia was associated with a low risk of POD (odds ratio [OR], 0.13; 95% CI, 0.04 to 0.41; P = 0.001) and few CD14 + CD16+ monocytes (β = - 0.61; 95% CI, - 0.74 to - 0.48; P = 0.000) the following day. When the association between HS and delirium was controlled for CD14 + CD16+ monocytes, the effect size became nonsignificant (odds ratio [OR], 0.86; 95% CI, 0.14 to 5.33; P = 0.874). TNF-α was significantly associated with POD (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.16; P = 0.000). However, IL-1β, IL-6, IL-10, and S100β were not significantly related to POD. CONCLUSION:HS can alleviate POD in geriatric patients and may inhibit the secretion of inflammatory factors by monocytes.
10.1186/s12974-017-0999-y
Intermittent calorie restriction alters T cell subsets and metabolic markers in people with multiple sclerosis.
EBioMedicine
BACKGROUND:Intermittent fasting or calorie restriction (CR) diets provide anti-inflammatory and neuroprotective advantages in models of multiple sclerosis (MS); data in humans are sparse. METHODS:We conducted a randomised-controlled feeding study of different CR diets in 36 people with MS over 8 weeks. Participants were randomised to 1 of 3 diets: 1) a control diet, in which the participant received 100% of his or her calorie needs 7 days per week, 2) a daily CR diet, in which the participant received 78% of his or her calorie needs 7 days per week, or 3) an intermittent CR diet, in which the participant received 100% of his or her calorie needs on 5 days per week and 25% of his or her calorie needs 2 days per week (i.e., a "5:2" style diet). Untargeted metabolomics was performed on plasma samples at weeks 0, 4 and 8 at Metabolon Inc (Durham, NC). Flow cytometry of cryopreserved peripheral blood mononuclear cells at weeks 0 and 8 were used to identify CD3;CD4 (CD4) and CD3;CD4 (as a proxy for CD8) T cell subsets including effector memory, central memory, and naïve cells. FINDINGS:31 (86%) completed the trial. Over time, individuals randomised to intermittent CR had significant reductions in effector memory (for CD4: -3.82%; 95%CI: -7.44, -0.21; for CD4: -6.96%; 95%CI: -11.96, -1.97) and Th1 subsets (-4.26%; 95% CI: -7.11, -1.40) and proportional increases in naïve subsets (for CD4: 10.11%; 95%CI: 3.30, 16.92%). No changes were observed for daily CR or weight-stable diets. Larger within-person changes in lysophospholipid and lysoplasmalogen metabolites in intermittent CR were associated with larger reductions in memory T cell subsets and larger increases in naïve T cell subsets. INTERPRETATION:In people with MS, an intermittent CR diet was associated with reduction in memory T cell subsets and certain biologically-relevant lipid markers. FUNDING:National MS Society, NIH, Johns Hopkins Catalyst Award.
10.1016/j.ebiom.2022.104124
Astrocytic Changes in Mitochondrial Oxidative Phosphorylation Protein Levels in Parkinson's Disease.
Chen Chun,Mossman Emily,Malko Philippa,McDonald David,Blain Alasdair P,Bone Laura,Erskine Daniel,Filby Andrew,Vincent Amy E,Hudson Gavin,Reeve Amy K
Movement disorders : official journal of the Movement Disorder Society
BACKGROUND:Mitochondrial dysfunction within neurons, particularly those of the substantia nigra, has been well characterized in Parkinson's disease and is considered to be related to the pathogenesis of this disorder. Dysfunction within this important organelle has been suggested to impair neuronal communication and survival; however, the reliance of astrocytes on mitochondria and the impact of their dysfunction on this essential cell type are less well characterized. OBJECTIVE:This study aimed to uncover whether astrocytes harbor oxidative phosphorylation (OXPHOS) deficiencies in Parkinson's disease and whether these deficiencies are more likely to occur in astrocytes closely associated with neurons or those more distant from them. METHODS:Postmortem human brain sections from patients with Parkinson's disease were subjected to imaging mass cytometry for individual astrocyte analysis of key OXPHOS proteins across all five complexes. RESULTS:We show the variability in the astrocytic expression of mitochondrial proteins between individuals. In addition, we found that there is evidence of deficiencies in respiratory chain subunit expression within these important glia and changes, particularly in mitochondrial mass, associated with Parkinson's disease and that are not simply a consequence of advancing age. CONCLUSION:Our data show that astrocytes, like neurons, are susceptible to mitochondrial defects and that these could have an impact on their reactivity and ability to support neurons in Parkinson's disease.
10.1002/mds.28849
High leukocyte mitochondrial DNA content contributes to poor prognosis in glioma patients through its immunosuppressive effect.
Chen Y,Zhang J,Huang X,Zhang J,Zhou X,Hu J,Li G,He S,Xing J
British journal of cancer
BACKGROUND:Epidemiological studies have indicated significant associations of leukocyte mitochondrial DNA (mtDNA) copy number with risk of several malignancies, including glioma. However, whether mtDNA content can predict the clinical outcome of glioma patients has not been investigated. METHODS:The mtDNA content of peripheral blood leukocytes from 336 glioma patients was examined using a real-time PCR-based method. Kaplan-Meier curves and Cox proportional hazards regression model were used to examine the association of mtDNA content with overall survival (OS) and progression-free survival (PFS) of patients. To explore the potential mechanism, the immune phenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines from another 20 glioma patients were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS:Patients with high mtDNA content showed both poorer OS and PFS than those with low mtDNA content. Multivariate Cox regression analysis demonstrated that mtDNA content was an independent prognostic factor for both OS and PFS. Stratified analyses showed that high mtDNA content was significantly associated with poor prognosis of patients with younger age, high-grade glioma or adjuvant radiochemotherapy. Immunological analysis indicated that patients with high mtDNA content had significantly lower frequency of natural killer cells in PBMCs and higher plasma concentrations of interleukin-2 and tumour necrosis factor-α, suggesting an immunosuppression-related mechanism involved in mtDNA-mediated prognosis. CONCLUSIONS:Our study for the first time demonstrated that leukocyte mtDNA content could serve as an independent prognostic marker and an indicator of immune functions in glioma patients.
10.1038/bjc.2015.184
Immunological risk factors for sepsis-associated delirium and mortality in ICU patients.
Frontiers in immunology
Background:A major challenge in intervention of critical patients, especially sepsis-associated delirium (SAD) intervention, is the lack of predictive risk factors. As sepsis and SAD are heavily entangled with inflammatory and immunological processes, to identify the risk factors of SAD and mortality in the intensive care unit (ICU) and determine the underlying molecular mechanisms, the peripheral immune profiles of patients in the ICU were characterized. Methods:This study contains a cohort of 52 critical patients who were admitted to the ICU of the First Affiliated Hospital of Jinan University. Comorbidity, including sepsis and SAD, of this cohort was diagnosed and recorded. Furthermore, peripheral blood samples were collected on days 1, 3, and 5 of admission for peripheral immune profiling with blood routine examination, flow cytometry, ELISA, RNA-seq, and qPCR. Results:The patients with SAD had higher mortality during ICU admission and within 28 days of discharge. Compared with survivors, nonsurvivors had higher neutrophilic granulocyte percentage, higher CRP concentration, lower monocyte count, lower monocyte percentage, lower C3 complement level, higher CD14CD16 monocytes percentage, and higher levels of IL-6 and TNFα. The CD14CD16 monocyte percentage manifested favorable prediction values for the occurrence of SAD. Differentially expressed genes between the nonsurvival and survival groups were mainly associated with immune response and metabolism process. The longitudinal expression pattern of SLC2A1 and STIMATE were different between nonsurvivors and survivors, which were validated by qPCR. Conclusions:Nonsurvival critical patients have a distinct immune profile when compared with survival patients. CD14CD16 monocyte prevalence and expression levels of SLC2A1 and STIMATE may be predictors of SAD and 28-day mortality in ICU patients.
10.3389/fimmu.2022.940779
Transforming Growth Factor-β Signaling in Regulatory T Cells Controls T Helper-17 Cells and Tissue-Specific Immune Responses.
Konkel Joanne E,Zhang Dunfang,Zanvit Peter,Chia Cheryl,Zangarle-Murray Tamsin,Jin Wenwen,Wang Songlin,Chen WanJun
Immunity
Regulatory T cells (Treg cells) perform suppressive functions in disparate tissue environments and against many inflammatory insults, yet the tissue-enriched factor(s) that influence Treg cell phenotype and function remain largely unknown. We have shown a vital role for transforming growth factor-β (TGF-β) signals in safe-guarding specific Treg cell functions. TGF-β signals were dispensable for steady-state Treg cell homeostasis and for Treg cell suppression of T cell proliferation and T helper-1 (Th1) cell differentiation. However, Treg cells require TGF-β signals to appropriately dampen Th17 cells and regulate responses in the gastrointestinal tract. TGF-β signaling maintains CD103 expression, promotes expression of the colon-specific trafficking molecule GPR15, and inhibits expression of GPR174, a receptor for lysophosphatidylserine, on Treg cells, collectively supporting the accumulation and retention of Treg cells in the colon and control of colitogenic responses. Thus, we reveal an unrecognized function for TGF-β signaling as an upstream factor controlling Treg cell activity in specific tissue environments.
10.1016/j.immuni.2017.03.015
Evaluation of Age-Dependent Immune Signatures in Patients With Multiple Sclerosis.
Eschborn Melanie,Pawlitzki Marc,Wirth Timo,Nelke Christopher,Pfeuffer Steffen,Schulte-Mecklenbeck Andreas,Lohmann Lisa,Rolfes Leoni,Pape Katrin,Eveslage Maria,Bittner Stefan,Gross Catharina C,Ruck Tobias,Wiendl Heinz,Meuth Sven G,Klotz Luisa
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:In MS, an age-related decline in disease activity and a decreased efficacy of disease-modifying treatment have been linked to immunosenescence, a state of cellular dysfunction associated with chronic inflammation. METHODS:To evaluate age-related immunologic alterations in MS, we compared immune signatures in peripheral blood (PB) and CSF by flow cytometry in patients with relapsing-remitting (RR) (PB n = 38; CSF n = 51) and primary progressive (PP) MS (PB n = 40; CSF n = 36) and respective controls (PB n = 40; CSF n = 85). RESULTS:Analysis revealed significant age-related changes in blood immune cell composition, especially in the CD8 T-cell compartment of healthy donors (HDs) and patients with MS. However, HDs displayed a strong age-dependent decline in the expression of the immunoregulatory molecules KLRG1, LAG3, and CTLA-4 on memory CD8 T cells, whereas this age-dependent reduction was completely abrogated in patients with MS. An age-dependent increase in the expression of the costimulatory molecule CD226 on memory CD8 T cells was absent in patients with MS. CD226 expression correlated with disability in younger (≤50 years) patients with MS. CSF analysis revealed a significant age-dependent decline in various immune cell populations in PPMS but not RRMS, suggesting a differential effect of aging on the intrathecal compartment in PPMS. DISCUSSION:Our data illustrate that aging in MS is associated with a dysbalance between costimulatory and immunoregulatory signals provided by CD8 T cells favoring a proinflammatory phenotype and, more importantly, a pattern of premature immune aging in the CD8 T-cell compartment of young patients with MS with potential implications for disease severity.
10.1212/NXI.0000000000001094
CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:In multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor-like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS. METHODS:Expression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP. RESULTS:Using bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS. DISCUSSION:Collectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.
10.1212/NXI.0000000000200022
Impact of cytomegalovirus infection on B cell differentiation and cytokine production in multiple sclerosis.
Zabalza Ana,Vera Andrea,Alari-Pahissa Elisenda,Munteis Elvira,Moreira Antía,Yélamos Jose,Llop Mireia,López-Botet Miguel,Martínez-Rodríguez Jose E
Journal of neuroinflammation
BACKGROUND:Human cytomegalovirus (HCMV) infection has been recently associated with a low risk of multiple sclerosis (MS), yet the basis behind this observation remains uncertain. In this study, we aimed to determine in MS patients whether HCMV induces modifications in the peripheral B cell compartment. METHODS:HCMV serostatus was determined in 73 MS patients (55 relapsing-remitting MS (RRMS); 18 progressive MS (PMS)) and 30 healthy controls, assessing their B cell immunophenotype and cytokine production (GM-CSF, IL-6, IL-10, and TNFα) by flow cytometry. RESULTS:HCMV seropositivity in untreated MS patients (n = 45) was associated with reduced switched memory B cells, contrasting with an opposite effect in PMS. Expansions of transitional B cells were observed in HCMV(+) IFNβ-treated RRMS patients but not in HCMV(-) cases (p < 0.01), suggesting that HCMV may influence the distribution of B cell subsets modulating the effects of IFNβ. Considering the B cell functional profile, HCMV(-) PMS displayed an increased secretion of proinflammatory cytokines (IL-6, TNFα) as compared to HCMV(+) PMS and RRMS cases (p < 0.001). CONCLUSIONS:Our study reveals an influence of HCMV infection on the phenotype and function of B cells, promoting early differentiation stages in RRMS and reducing the proinflammatory cytokine profile in advanced MS forms, which might be related with the putative protective role of this virus in MS.
10.1186/s12974-020-01840-2
CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patients.
Lieberman Linda A,Zeng Wanyong,Singh Carol,Wang Wenting,Otipoby Kevin L,Loh Christine,Plavina Tatiana,Gorelik Leonid,Ransohoff Richard M,Cahir-McFarland Ellen
Neurology
OBJECTIVE:To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. METHODS:Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. RESULTS:In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. CONCLUSION:Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk.
10.1212/WNL.0000000000002314
Frequency and immunophenotype of IL10-producing regulatory B cells in optic neuritis.
Lundqvist Sara,Modvig Signe,Fischer Emilie A,Frederiksen Jette L,Degn Matilda
Immunology
Mouse models of multiple sclerosis (MS) have shown the importance of interleukin-10 (IL-10) -producing regulatory B (Breg) cells in dampening disease activity and inhibiting disease initiation and progression. In MS and other autoimmune diseases decreased frequency and functionality of Breg cells correlate with disease activity and the percentage of IL-10-producing Breg cells decreases during relapse and normalizes in remission. Optic neuritis (ON) is a common first clinical manifestation of MS and IL-10-producing Breg cells may be crucial in the transition from ON to MS, we therefore investigate the frequency and function of Breg cells in ON as a clinical model of early demyelinating disease. B cells were purified from 27 patients with ON sampled close to symptom onset (median 23 days, range 7-41 days) and 13 healthy controls. The B cells were stimulated and cultured for 48 hr with CD40 ligand and CpG before measurement of intracellular IL-10 and the surface markers CD19, CD1d, CD5, CD24, CD38 and CD27 by flow cytometry. The frequency of B-cell subsets was analysed in peripheral blood and cerebral spinal fluid (CSF) of patients. Sixty-five per cent of the IL-10-producing Breg cells co-expressed CD24 and CD38, and only 14% were CD24 CD27 , suggesting that the naive B cells are the primary source of IL-10 in the B-cell culture, followed by memory cells in both healthy controls and patients. The frequency of naive CD19 CD24 CD38 Breg cells was higher in patients with ON compared with controls. The ability of Breg cells to produce IL-10 was at normal levels in both ON patients with high risk and those with low risk of progression to MS. We found no correlation between Breg cell function and the presence of brain white matter lesions by magnetic resonance imaging or CSF oligoclonal bands indicative of ON patients carrying a higher risk of conversion to MS. The frequencies of IL-10-producing B cells did not correlate with the conversion to MS at 2-year follow up. Interleukin-10 was primarily produced by naive and memory B cells. The frequency of IL-10-secreting B cells did not correlate with risk factors of MS. Breg cell function at clinical onset of ON is not a determining factor for conversion to MS.
10.1111/imm.13024
Black African and Latino/a identity correlates with increased plasmablasts in MS.
Telesford Kiel M,Kaunzner Ulrike W,Perumal Jai,Gauthier Susan A,Wu Xian,Diaz Ivan,Kruse-Hoyer Mason,Engel Casey,Marcille Melanie,Vartanian Timothy
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To determine the influence of self-reported Black African and Latin American identity on peripheral blood antibody-secreting cell (ASC) frequency in the context of relapsing-remitting MS. METHODS:In this cross-sectional study, we recruited 74 subjects with relapsing-remitting MS and 24 age-, and self-reported ethno-ancestral identity-matched healthy donors (HDs) to provide peripheral blood study samples. Subjects with MS were either off therapy at the time of study draw or on monthly natalizumab therapy infusions. Using flow cytometry, we assessed peripheral blood mononuclear cells for antibody-secreting B-cell subsets. RESULTS:When stratified by self-reported ethno-ancestry, we identified significantly elevated frequencies of circulating plasmablasts among individuals with MS identifying as Black African or Latin American relative to those of Caucasian ancestry. Ethno-ancestry-specific differences in ASC frequency were observed only among individuals with MS. By contrast, this differential was not observed among HDs. ASCs linked with poorer MS prognosis and active disease, including IgM- and class-switched CD138 subsets, were among those significantly increased. CONCLUSION:The enhanced peripheral blood plasmablast signature revealed among Black African or Latin American subjects with MS points to distinct underlying mechanisms associated with MS immunopathogenesis. This dysregulation may contribute to the disease disparity experienced by patient populations of Black African or Latin American ethno-ancestry.
10.1212/NXI.0000000000000634
Liquid Biopsy of Extracellular Microvesicles Predicts Future Major Ischemic Events in Genetically Characterized Familial Hypercholesterolemia Patients.
Suades Rosa,Padró Teresa,Crespo Javier,Sionis Alessandro,Alonso Rodrigo,Mata Pedro,Badimon Lina
Arteriosclerosis, thrombosis, and vascular biology
Objective- Circulating microvesicles (cMVs) exert regulatory roles in atherothrombosis. Patients with familial hypercholesterolemia (FH) that are at high risk for premature cardiovascular events (CVEs) have previously shown high levels of cMVs related to disease severity. However, much remains unknown about their value as markers of CVE. We sought to investigate the prognostic cMV signature for future major CVE presentation in patients with FH. Approach and Results- Liquid biopsies from genetically characterized patients with FH from the SAFEHEART (Spanish Familial Hypercholesterolemia Cohort Study)-cohort without clinical manifestation of disease at entry that were going to suffer a CVE within a mean period of 3.3±2.6 years postsampling (CVE, N=92) and from age/cardiovascular risk factor/treatment-matched patients with FH that did not suffer an event within the same time-period (non-CVE, N=48) were investigated. cMVs were phenotyped by flow cytometry to identify activated parental cells. Patients with CVE had higher number of overall procoagulant annexin V-cMVs than non-CVE ( P<0.05). Pan-leukocyte-derived and neutrophil-derived cMVs, as well as activated platelet-derived cMVs, were significantly higher in patients with CVE. Baseline number of cMVs derived from lymphocytes, neutrophils, and activated platelets were positively associated with mortality at follow-up ( P<0.05). Patient-risk calculated by classical cardiovascular risk-factor scores did not correlate with cMVs. Inclusion of the cMV signature into the SAFEHEART risk model for patients with FH for the prediction of ischemic events increased the area under the curve from 0.603±0.050 to 0.768±0.042 ( P<0.005). Conclusions- Patients with FH who are going to suffer a CVE within a mean period of 3.3 years, despite being treated according to guidelines, have ongoing innate immune cell and platelet activation. The proposed cMV signature is a prognostic marker for accelerated atherosclerosis and clinical event presentation in patients with FH.
10.1161/ATVBAHA.119.312420
Hematopoietic mobilization: Potential biomarker of response to natalizumab in multiple sclerosis.
Mattoscio Miriam,Nicholas Richard,Sormani Maria P,Malik Omar,Lee Jean S,Waldman Adam D,Dazzi Francesco,Muraro Paolo A
Neurology
OBJECTIVE:To ascertain the mobilization from the bone marrow and the functional relevance of the increased number of circulating hematopoietic stem and progenitor cells (HSPC) induced by the anti-α-4 integrin antibody natalizumab in patients with multiple sclerosis (MS). METHODS:We evaluated CD45(low)CD34+ HSPC frequency by flow cytometry in blood from 45 natalizumab-treated patients (12 of whom were prospectively followed during the first year of treatment as part of a pilot cohort and 16 prospectively followed for validation), 10 untreated patients with MS, and 24 healthy donors. In the natalizumab-treated group, we also assessed sorted HSPC cell cycle status, T- and B-lymphocyte subpopulation frequencies (n = 29), and HSPC differentiation potential (n = 10). RESULTS:Natalizumab-induced circulating HSPC were predominantly quiescent, suggesting recent mobilization from the bone marrow, and were capable of differentiating ex vivo. Circulating HSPC numbers were significantly increased during natalizumab, but heterogeneously, allowing the stratification of mobilizer and nonmobilizer subgroups. Nonmobilizer status was associated with persistence of disease activity during treatment. The frequency of B cells and CD103+CD8+ regulatory T cells persistently increased, more significantly in mobilizer patients, who also showed a specific naive/memory B-cell profile. CONCLUSIONS:The data suggest that natalizumab-induced circulating HSPC increase is the result of true mobilization from the bone marrow and has clinical and immunologic relevance. HSPC mobilization, associated with clinical remission and increased proportion of circulating B and regulatory T cells, may contribute to the treatment's mode of action; thus, HSPC blood counts could represent an early biomarker of responsiveness to natalizumab.
10.1212/WNL.0000000000001454
Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma.
Theranostics
: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. : To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and and mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. : We demonstrated that EGFR EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of and mRNA in EVs for detecting glioma patients. : We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma.
10.7150/thno.33114
Multiple sclerosis risk variants alter expression of co-stimulatory genes in B cells.
Smets Ide,Fiddes Barnaby,Garcia-Perez Josselyn E,He Di,Mallants Klara,Liao Wenjia,Dooley James,Wang George,Humblet-Baron Stephanie,Dubois Bénédicte,Compston Alastair,Jones Joanne,Coles Alasdair,Liston Adrian,Ban Maria,Goris An,Sawcer Stephen
Brain : a journal of neurology
The increasing evidence supporting a role for B cells in the pathogenesis of multiple sclerosis prompted us to investigate the influence of known susceptibility variants on the surface expression of co-stimulatory molecules in these cells. Using flow cytometry we measured surface expression of CD40 and CD86 in B cells from 68 patients and 162 healthy controls that were genotyped for the multiple sclerosis associated single nucleotide polymorphisms (SNPs) rs4810485, which maps within the CD40 gene, and rs9282641, which maps within the CD86 gene. We found that carrying the risk allele rs4810485*T lowered the cell-surface expression of CD40 in all tested B cell subtypes (in total B cells P ≤ 5.10 × 10-5 in patients and ≤4.09 × 10-6 in controls), while carrying the risk allele rs9282641*G increased the expression of CD86, with this effect primarily seen in the naïve B cell subset (P = 0.048 in patients and 5.38 × 10-5 in controls). In concordance with these results, analysis of RNA expression demonstrated that the risk allele rs4810485*T resulted in lower total CD40 expression (P = 0.057) but with an increased proportion of alternative splice-forms leading to decoy receptors (P = 4.00 × 10-7). Finally, we also observed that the risk allele rs4810485*T was associated with decreased levels of interleukin-10 (P = 0.020), which is considered to have an immunoregulatory function downstream of CD40. Given the importance of these co-stimulatory molecules in determining the immune reaction that appears in response to antigen our data suggest that B cells might have an important antigen presentation and immunoregulatory role in the pathogenesis of multiple sclerosis.
10.1093/brain/awx372
Oncolytic virotherapy in glioblastoma patients induces a tumor macrophage phenotypic shift leading to an altered glioblastoma microenvironment.
van den Bossche Wouter B L,Kleijn Anne,Teunissen Charlotte E,Voerman Jane S A,Teodosio Cristina,Noske David P,van Dongen Jacques J M,Dirven Clemens M F,Lamfers Martine L M
Neuro-oncology
Background:Immunosuppressive protumoral M2 macrophages are important in pathogenesis, progression, and therapy resistance in glioblastoma (GBM) and provide a target for therapy. Recently oncolytic virotherapy in murine models was shown to change these M2 macrophages toward the pro-inflammatory and antitumoral M1 phenotype. Here we study the effects of the oncolytic virotherapy Delta24-RGD in humans, using both in vitro models and patient material. Methods:Human monocyte-derived macrophages were co-cultured with Delta24-RGD-infected primary glioma stem-like cells (GSCs) and were analyzed for their immunophenotype, cytokine expression, and secretion profiles. Cerebrospinal fluid (CSF) from 18 Delta24-RGD-treated patients was analyzed for inflammatory cytokine levels, and the effects of these CSF samples on macrophage phenotype in vitro were determined. In addition, tumor macrophages in resected material from a Delta24-RGD-treated GBM patient were compared with 5 control GBM patient samples by flow cytometry. Results:Human monocyte-derived M2 macrophages co-cultured with Delta24-RGD-infected GSCs shifted toward an M1-immunophenotype, coinciding with pro-inflammatory gene expression and cytokine production. This phenotypic switch was induced by the concerted effects of a change in tumor-produced soluble factors and the presence of viral particles. CSF samples from Delta24-RGD-treated GBM patients revealed cytokine levels indicative of a pro-inflammatory microenvironment. Furthermore, tumoral macrophages in a Delta24-RGD-treated patient showed significantly greater M1 characteristics than in control GBM tissue. Conclusion:Together these in vitro and patient studies demonstrate that local Delta24-RGD therapy may provide a therapeutic tool to promote a prolonged shift in the protumoral M2 macrophages toward M1 in human GBM, inducing a pro-inflammatory and potentially tumor-detrimental microenvironment.
10.1093/neuonc/noy082
Induction of brain-infiltrating T-bet-expressing B cells in multiple sclerosis.
van Langelaar Jamie,Rijvers Liza,Janssen Malou,Wierenga-Wolf Annet F,Melief Marie-José,Siepman Theodora A,de Vries Helga E,Unger Peter-Paul A,van Ham S Marieke,Hintzen Rogier Q,van Luijn Marvin M
Annals of neurology
OBJECTIVE:Results from anti-CD20 therapies demonstrate that B- and T-cell interaction is a major driver of multiple sclerosis (MS). The local presence of B-cell follicle-like structures and oligoclonal bands in MS patients indicates that certain B cells infiltrate the central nervous system (CNS) to mediate pathology. Which peripheral triggers underlie the development of CNS-infiltrating B cells is not fully understood. METHODS:Ex vivo flow cytometry was used to assess chemokine receptor profiles of B cells in blood, cerebrospinal fluid, meningeal, and brain tissues of MS patients (n = 10). Similar analyses were performed for distinct memory subsets in the blood of untreated and natalizumab-treated MS patients (n = 38). To assess T-bet(CXCR3) B-cell differentiation, we cultured B cells from MS patients (n = 21) and healthy individuals (n = 34) under T helper 1- and TLR9-inducing conditions. Their CNS transmigration capacity was confirmed using brain endothelial monolayers. RESULTS:CXC chemokine receptor 3 (CXCR3)-expressing B cells were enriched in different CNS compartments of MS patients. Treatment with the clinically effective drug natalizumab prevented the recruitment of CXCR3 IgG1 subsets, corresponding to their increased ability to cross CNS barriers in vitro. Blocking of interferon-γ (IFNγ) reduced the transmigration potential and antigen-presenting function of these cells. IFNγ-induced B cells from MS patients showed increased T-bet expression and plasmablast development. Additional TLR9 triggering further upregulated T-bet and CXCR3, and was essential for IgG1 switching. INTERPRETATION:This study demonstrates that T-bet IgG1 B cells are triggered by IFNγ and TLR9 signals, likely contributing to enhanced CXCR3-mediated recruitment and local reactivity in the CNS of MS patients. ANN NEUROL 2019;86:264-278.
10.1002/ana.25508
Enhanced T-Cell Maturation and Monocyte Aggregation Are Features of Cellular Inflammation in Human T-Lymphotropic Virus Type 1-Associated Myelopathy.
Saeed Zainab,Rowan Aileen,Greiller Claire,Taylor Graham P,Pollock Katrina M
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
BACKGROUND:Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM) is an inflammatory condition characterized by severe disability and high levels of infected white blood cells. The circulating cellular inflammatory changes that distinguish this condition from asymptomatic infection are not well understood. METHODS:To investigate the immune characteristics of individuals with low or high HTLV-1 proviral load (pVL), symptomatic disease, and the impact of immunosuppressive therapy, 38 women living with HTLV-1 infection, at a median age of 59 (52-68) years, were studied. Nineteen were asymptomatic carriers with low or high pVL; 19 were diagnosed with HAM, with 10 receiving anti-inflammatory therapy. Peripheral blood mononuclear cells were stained and analyzed for frequency distribution and activation of innate and adaptive immune cell subsets using multiparameter flow cytometry. RESULTS:Inflation of the CD4:CD8 ratio (>2) was observed among all groups irrespective of pVL. The frequency of naive CD4+ T cells correlated inversely with HTLV-1 pVL (rs = -0.344, P = .026). Mature T effector memory TEM CD4+ T cells were expanded in patients with untreated HAM compared with asymptomatic carriers (P < .001) but less so in those on therapy. High levels of exhausted (PD-1+) and senescent (CD28null) CD4+ and CD8+ T cells were observed in all individuals, particularly in those with HAM, while monocytes showed increased aggregation and CD14+CD56- monocytes were less frequent. CONCLUSIONS:CD4:CD8 ratio inflation is a feature of HTLV-1 infection, whereas enhanced CD4+ T cell maturation and monocyte aggregation are features of HAM, reflecting widespread inflammatory change, which may be detectable presymptomatically and be amenable to anti-inflammatory treatment.
10.1093/cid/ciz369
Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury.
Cell death & disease
Following acute sciatic nerve crush injury (SNCI), inflammation and the improper phagocytic clearance of dying Schwann cells (SCs) has effects on remodeling that lead to morbidity and incomplete functional recovery. Therapeutic strategies like the use of erythropoietin (EPO) for peripheral nerve trauma may serve to bring immune cell phagocytotic clearance under control to support debris clearance. We evaluated EPO's effect on SNCI and found EPO treatment increased myelination and sciatic functional index (SFI) and bolstered anti-apoptosis and phagocytosis of myelin debris via CD206 macrophages when compared to saline treatment. EPO enhanced M2 phenotype activity, both in bone marrow-derived macrophages (BMMØs) and peritoneal-derived macrophages (PMØs) in vitro, as well as in PMØs in vivo. EPO increased efferocytosis of apoptotic sciatic nerve derived Schwann cells (SNSCs) in both settings as demonstrated using immunofluorescence (IF) and flow cytometry. EPO treatment significantly attenuated pro-inflammatory genes (IL1β, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) and the cell-surface marker CD206. EPO also increased anti-apoptotic (Annexin V/7AAD) effects after lipopolysaccharide (LPS) induction in macrophages. Our data demonstrate EPO promotes the M2 phenotype macrophages to ameliorate apoptosis and efferocytosis of dying SCs and myelin debris and improves SN functional recovery following SNCI.
10.1038/s41419-022-04671-6
Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas.
Molecular cell
Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.
10.1016/j.molcel.2022.05.023
Effect of the sphingosine-1-phosphate receptor modulator ozanimod on leukocyte subtypes in relapsing MS.
Harris Sarah,Tran Jonathan Q,Southworth Harry,Spencer Collin M,Cree Bruce A C,Zamvil Scott S
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To better understand ozanimod's mechanism of action (MOA), we conducted exploratory analyses from a phase 1 study to characterize ozanimod's effect on circulating leukocyte subsets in patients with relapsing multiple sclerosis. METHODS:An open-label pharmacodynamic study randomized patients to oral ozanimod hydrochloride (HCl) 0.5 (n = 13) or 1 mg/d (n = 11) for ∼12 weeks (including 7-day dose escalation). Circulating leukocyte subsets were quantified using flow cytometry (days 28, 56, and 85) and epigenetic cell counting (days 2, 5, 28, 56, and 85) and compared with baseline (day 1) using descriptive statistics. RESULTS:Ozanimod caused dose-dependent reductions in absolute lymphocyte counts. Observed by both methodologies, circulating CD19 B- and CD3 T-cell counts were reduced by >50% with ozanimod HCl 0.5 mg and >75% with 1 mg at day 85. Based on flow cytometry, ozanimod HCl 1 mg showed greater decreases in CD4 than CD8 T cells, greater decreases in both CD4 and CD8 central memory vs effector memory T cells, and reductions in mean CD4 and CD8 naive T cells by ≥90% at day 85. In the flow cytometry analysis, changes in monocytes, natural killer, and natural killer T cells were minimal. Using epigenetic cell counting, greater reductions for Th17 than T regulatory cells were determined. CONCLUSION:Ozanimod induced dose-dependent reductions in circulating B- and T-cell counts and differential effects on naive and memory CD4 and CD8 T cells and CD19 B cells. Data characterized with both a novel epigenetic cell-counting method and flow cytometry support ozanimod's MOA. CLINICAL TRIAL REGISTRATION:clinicaltrials.gov NCT02797015.
10.1212/NXI.0000000000000839
Longitudinal brain structural alterations and systemic inflammation in obstructive sleep apnea before and after surgical treatment.
Lin Wei-Che,Huang Chih-Cheng,Chen Hsiu-Ling,Chou Kun-Hsien,Chen Pei-Chin,Tsai Nai-Wen,Chen Meng-Hsiang,Friedman Michael,Lin Hsin-Ching,Lu Cheng-Hsien
Journal of translational medicine
BACKGROUND:Systemic inflammation, neurocognitive impairments, and morphologic brain changes are associated with obstructive sleep apnea (OSA). Understanding their longitudinal evolution and interactions after surgical treatment provides clues to the pathogenesis of cognitive impairment and its reversibility. In the present study, we investigate clinical disease severity, systemic inflammation, cognitive deficits, and corresponding gray matter volume (GMV) changes in OSA, and the modifications following surgery. METHODS:Twenty-one patients with OSA (apnea-hypopnea index, AHI > 5) and 15 healthy volunteers (AHI < 5) underwent serial evaluation, including polysomnography, flow cytometry for leukocyte apoptosis categorization, cognitive function evaluation, and high-resolution brain scan. Disease severity, leukocyte apoptosis, cognitive function, and imaging data were collected to assess therapeutic efficacy 3 months after surgery. RESULTS:Pre-operatively, patients presented with worse cognitive function, worse polysomnography scores, and higher early leukocyte apoptosis associated with increased insular GMV. There was reduced GMV in the anterior cingulate gyrus before and after surgery in the cases compared to that in controls, suggesting an irreversible structural deficit. Post-operatively, there were significant improvements in different cognitive domains, including attention, executive and visuospatial function, and depression, and in early leukocyte apoptosis. There was also a significant decrease in GMVs after treatment, suggesting recovery from vasogenic edema in the precuneus, insula, and cerebellum. Improvement in early leukocyte apoptosis post-surgery predicted better recovery of precuneus GMV. CONCLUSIONS:In OSA, increased disease severity and systemic inflammation can alter GMV in vulnerable regions. Surgical treatment may improve disease severity and systemic inflammation, with subsequent recovery in brain structures and functions.
10.1186/s12967-016-0887-8
Dysregulated B cell differentiation towards antibody-secreting cells in neuromyelitis optica spectrum disorder.
Hoshino Yasunobu,Noto Daisuke,Sano Shuhei,Tomizawa Yuji,Yokoyama Kazumasa,Hattori Nobutaka,Miyake Sachiko
Journal of neuroinflammation
BACKGROUND:Anti-aquaporin 4 (AQP4) antibody (AQP4-Ab) is involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism involved in AQP4-Ab production remains unclear. METHODS:We analyzed the immunophenotypes of patients with NMOSD and other neuroinflammatory diseases as well as healthy controls (HC) using flow cytometry. Transcriptome analysis of B cell subsets obtained from NMOSD patients and HCs was performed. The differentiation capacity of B cell subsets into antibody-secreting cells was analyzed. RESULTS:The frequencies of switched memory B (SMB) cells and plasmablasts were increased and that of naïve B cells was decreased in NMOSD patients compared with relapsing-remitting multiple sclerosis patients and HC. SMB cells from NMOSD patients had an enhanced potential to differentiate into antibody-secreting cells when cocultured with T peripheral helper cells. Transcriptome analysis revealed that the profiles of B cell lineage transcription factors in NMOSD were skewed towards antibody-secreting cells and that IL-2 signaling was upregulated, particularly in naïve B cells. Naïve B cells expressing CD25, a receptor of IL-2, were increased in NMOSD patients and had a higher potential to differentiate into antibody-secreting cells, suggesting CD25 naïve B cells are committed to differentiate into antibody-secreting cells. CONCLUSIONS:To the best of our knowledge, this is the first study to demonstrate that B cells in NMOSD patients are abnormally skewed towards antibody-secreting cells at the transcriptome level during the early differentiation phase, and that IL-2 might participate in this pathogenic process. Our study indicates that CD25 naïve B cells are a novel candidate precursor of antibody-secreting cells in autoimmune diseases.
10.1186/s12974-021-02375-w
Prospective Evaluation of CD45RA+/CCR7- Effector Memory T (T) Cell Subsets in Patients with Primary and Secondary Brain Tumors during Radiotherapy of the Brain within the Scope of the Prospective Glio-CMV-01 Clinical Trial.
Cells
Radiotherapy (RT) of the brain is a common treatment for patients with high-grade gliomas and brain metastases. It has previously been shown that reactivation of cytomegalovirus (CMV) frequently occurs during RT of the brain. This causes neurological decline, demands antiviral treatment, and is associated with a worse prognosis. CMV-specific T cells are characterized by a differentiated effector memory phenotype and CD45RA+ CCR7- effector memory T (T) cells were shown to be enriched in CMV seropositive individuals. In this study, we investigated the distribution of T cells and their subsets in the peripheral blood of healthy donors and, for the first time, prospectively within the scope of the prospective Glio-CMV-01 clinical trial of patients with high-grade glioma and brain metastases during radiation therapy as a potential predictive marker. First, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of T cells in a longitudinal manner. The CMV serostatus and age were considered as influencing factors. We revealed that patients who had a reactivation of CMV have significantly higher amounts of CD8+ T cells. Further, the distribution of the subsets of T cells based on the expression of CD27, CD28, and CD57 is highly dependent on the CMV serostatus. We conclude that the percentage of CD8+ T cells out of all CD8+ T cells has the potential to serve as a biomarker for predicting the risk of CMV reactivation during RT of the brain. Furthermore, this study highlights the importance of taking the CMV serostatus into account when analyzing T cells and their subsets.
10.3390/cells12040516
Simultaneous quantification of natural and inducible regulatory T-cell subsets during interferon-β therapy of multiple sclerosis patients.
Chiarini Marco,Capra Ruggero,Serana Federico,Bertoli Diego,Sottini Alessandra,Giustini Viviana,Scarpazza Cristina,Rovaris Marco,Torri Clerici Valentina,Ferraro Diana,Galgani Simonetta,Solaro Claudio,Conti Marta Zaffira,Visconti Andrea,Imberti Luisa,
Journal of translational medicine
BACKGROUND:The mechanisms underlying the therapeutic activity of interferon-β in multiple sclerosis are still not completely understood. In the present study, we evaluated the short and long-term effects of interferon-β treatment on different subsets of regulatory T cells in relapsing-remitting multiple sclerosis patients biologically responsive to treatment because of mixovirus resistance protein A inducibility. METHODS:In this prospective longitudinal study, subsets of natural regulatory T cells (naïve, central memory and effector memory) and inducible regulatory T cells (Tr1), as well as in vitro-induced regulatory T cells (Tr1-like cells), were simultaneously quantified by flow cytometry in samples prepared from 148 therapy-naïve multiple sclerosis patients obtained before and after 6, 12, 18, and 24 months of interferon-β-1a treatment. mRNA for interleukin-10 and Tr1-related genes (CD18, CD49b, and CD46, together with Cyt-1 and Cyt-2 CD46-associated isoforms) were quantified in Tr1-like cells. RESULTS:Despite profound inter-individual variations in the modulation of all regulatory T-cell subsets, the percentage of natural regulatory T cells increased after 6, 12, and 24 months of interferon-β treatment. This increase was characterized by the expansion of central and effector memory regulatory T-cell subsets. The percentage of Tr1 significantly enhanced at 12 months of therapy and continued to be high at the subsequent evaluation points. Patients experiencing relapses displayed a higher percentage of naïve regulatory T cells and a lower percentage of central memory regulatory T cells and of Tr1 before starting interferon-β therapy. In addition, an increase over time of central memory and of Tr1 was observed only in patients with stable disease. However, in vitro-induced Tr1-like cells, prepared from patients treated for 24 months, produced less amount of interleukin-10 mRNA compared with pre-treatment Tr1-like cells. CONCLUSION:Interferon-β induces the expansion of T regulatory subsets endowed with a high suppressive activity, especially in clinically stable patients. The overall concurrent modulation of natural and inducible regulatory T-cell subsets might explain the therapeutic effects of interferon-β in multiple sclerosis patients.
10.1186/s12967-020-02329-5
Air pollution as a contributor to the inflammatory activity of multiple sclerosis.
Cortese Andrea,Lova Luca,Comoli Patrizia,Volpe Elisabetta,Villa Silvia,Mallucci Giulia,La Salvia Sabrina,Romani Alfredo,Franciotta Diego,Bollati Valentina,Basso Sabrina,Guido Ilaria,Quartuccio Giuseppe,Battistini Luca,Cereda Cristina,Bergamaschi Roberto
Journal of neuroinflammation
OBJECTIVE:Air pollution has been recently identified as a risk factor for multiple sclerosis. Aim of this study was to investigate the immunological mechanism underlying the clinical association between air pollution, namely exposure to particulate matter 10 (PM10), and inflammatory activity of multiple sclerosis (MS) METHODS: Daily recording of PM10 was obtained by monitors depending on the residence of subjects. Expression of molecules involved in activation, adhesion, and migration of T lymphocytes were tested by flow cytometry in 57 MS patients and 19 healthy controls. We next assessed in vitro the effect of PM10 on expression of C-C chemokine receptors 6 (CCR6) by peripheral blood mononuclear cells (PBMCs), on cytokine production by monocyte-derived dendritic cells (mdDC), and on T cell polarization in PBMC/mdDC mixed cultures. RESULTS:We identified a significant correlation between mean PM10 levels and expression of CCR6 CD4+ T circulating cells in MS patients. This was paralleled by the observation in vitro of a higher level of CCR6 expression on PBMC following treatment with increased doses of particulate matter. Moreover, in mdDC cultures, particulate matter induced the secretion by mdDC of Th17 polarizing IL1 beta, IL6, and IL23 and, in mdDC/PBMC mixed cultures, enhanced generation of IL17-producing T cells. CONCLUSIONS:Ex vivo and in vitro studies support the pro-inflammatory role of PM in MS, by upregulating expression of CCR6 on circulating CD4+ T cells and inducing in innate immune cells the production of Th17 polarizing cytokines. Therefore, we speculate that in MS respiratory exposure to PM10 may induce the production in the lung of autoreactive Th17 lymphocytes and boost their migratory properties through the blood-brain barrier.
10.1186/s12974-020-01977-0
Morphine increases macrophages at the lesion site following spinal cord injury: Protective effects of minocycline.
Aceves Miriam,Terminel Mabel N,Okoreeh Andre,Aceves Alejandro R,Gong Yan Ming,Polanco Alan,Sohrabji Farida,Hook Michelle A
Brain, behavior, and immunity
Opioids are among the most effective and widely prescribed medications for the treatment of pain following spinal cord injury (SCI). Spinally-injured patients receive opioids within hours of arrival at the emergency room, and prolonged opioid regimens are often employed for the management of post-SCI chronic pain. However, previous studies in our laboratory suggest that the effects of opioids such as morphine may be altered in the pathophysiological context of neurotrauma. Specifically, we have shown that morphine administration in a rodent model of SCI increases mortality and tissue loss at the injury site, and decreases recovery of motor and sensory function, and overall health, even weeks after treatment. The literature suggests that opioids may produce these adverse effects by acting as endotoxins and increasing glial activation and inflammation. To better understand the effects of morphine following SCI, in this study we used flow cytometry to assess immune-competent cells at the lesion site. We observed a morphine-induced increase in the overall number of CD11b+ cells, with marked effects on microglia, in SCI subjects. Next, to investigate whether this increase in the inflammatory profile is necessary to produce morphine's effects, we challenged morphine treatment with minocycline. We found that pre-treatment with minocycline reduced the morphine-induced increase in microglia at the lesion site. More importantly, minocycline also blocked the adverse effects of morphine on recovery of function without disrupting the analgesic efficacy of this opioid. Together, our findings suggest that following SCI, morphine may exacerbate the inflammatory response, increasing cell death at the lesion site and negatively affecting functional recovery.
10.1016/j.bbi.2019.01.023
Dynamics of T cell repertoire renewal following autologous hematopoietic stem cell transplantation in multiple sclerosis.
Science translational medicine
Autologous hematopoietic stem cell transplantation (aHSCT) is a highly effective treatment of multiple sclerosis (MS). It depletes autoreactive cells and subsequently renews adaptive immune cells. The possible proinflammatory potential of surviving T cells early after aHSCT has not been studied. Here, we examined the dynamics of new and surviving T cells in 27 patients after aHSCT by multidimensional flow cytometry, T cell receptor (TCR) sequencing, specificity testing, telomere length profiling, and HLA genotyping. Early after aHSCT, naïve T cells are barely detectable, whereas effector memory (EM) T cells quickly reconstitute to pre-aHSCT values. EM CD4 T cells early after aHSCT have shorter telomeres, have higher expression of senescence and exhaustion markers, and proliferate less than those before aHSCT. We find a median TCR repertoire overlap of 26% between the early post-aHSCT EM CD4 T cells and pre-aHSCT, indicating persistence of EM CD4 T cells early after transplantation. The EM CD4 TCR repertoire overlap declines to 15% at 12 months after aHSCT, whereas the naïve TCR repertoire entirely renews. HLA-DR-associated EM CD4 T cell reactivity toward MS-related antigens decreased after aHSCT, whereas reactivity toward EBV increased. Our data show substantial survival of pre-aHSCT EM CD4 T cells early after transplantation but complete renewal of the T cell repertoire by nascent T cells later.
10.1126/scitranslmed.abq1693
Increased Intrathecal Activity of Follicular Helper T Cells in Patients With Relapsing-Remitting Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:Follicular helper T (Tfh) cells play a critical role in protective immunity helping B cells produce antibodies against foreign pathogens and are likely implicated in the pathogenesis of various autoimmune diseases. The purpose of this study was to investigate the role of Tfh cells in the pathogenesis of multiple sclerosis (MS). METHODS:Using flow cytometry, we investigated phenotype, prevalence, and function of Tfh cells in blood and CSF from controls and patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS). In addition, an in vitro blood-brain barrier coculture assay of primary human astrocytes and brain microvascular endothelial cells grown in a Boyden chamber was used to assess the migratory capacity of peripheral Tfh cells. RESULTS:This study identified 2 phenotypically and functionally distinct Tfh cell populations: CD25 Tfh cells (Tfh1-like) and CD25 Tfh cells (Tfh17-like). Whereas minor differences in Tfh cell populations were found in blood between patients with MS and controls, we observed an increased frequency of CD25 Tfh cells in CSF of patients with RRMS and PPMS and CD25 Tfh cells in patients with RRMS, compared with controls. Increasing frequencies of CSF CD25 Tfh cells and the CD25 Tfh/Tfr ratio scaled with increasing IgG index in patients with RRMS. Despite an increased prevalence of intrathecal Tfh cells in patients with MS, no difference in the migratory capacity of circulating Tfh cells was observed between controls and patients with MS. Instead, CSF concentrations of CXCL13 scaled with total counts of Tfh and Tfr cell subsets in the CSF. DISCUSSION:Our study indicates substantial changes in intrathecal Tfh dynamics, particularly in patients with RRMS, and suggests that the intrathecal inflammatory environment in patients with RRMS promotes recruitment of peripheral Tfh cells rather than the Tfh cells having an increased capacity to migrate to CNS.
10.1212/NXI.0000000000200009
Variable Abnormalities in T and B Cell Subsets in Ataxia Telangiectasia.
Moeini Shad Tannaz,Yousefi Bahman,Amirifar Parisa,Delavari Samaneh,Rae William,Kokhaei Parviz,Abolhassani Hassan,Aghamohammadi Asghar,Yazdani Reza
Journal of clinical immunology
BACKGROUND:Ataxia-telangiectasia (AT) is a rare genetic condition, caused by biallelic deleterious variants in the ATM gene, and has variable immunological abnormalities. This study aimed to examine immunologic parameters reflecting cell development, activation, proliferation, and class switch recombination (CSR) and determine their relationship to the clinical phenotype in AT patients. METHODS:In this study, 40 patients with a confirmed diagnosis of AT from the Iranian immunodeficiency registry center and 28 age-sex matched healthy controls were enrolled. We compared peripheral B and T cell subsets and T cell proliferation response to CD3/CD28 stimulation in AT patients with and without CSR defects using flow cytometry. RESULTS:A significant decrease in naïve, transitional, switched memory, and IgM only memory B cells, along with a sharp increase in the marginal zone-like and CD21 B cells was observed in the patients. We also found CD4 and CD8 naïve, central memory, and terminally differentiated effector memory CD4 (T) T cells were decreased. CD4 and CD8 effector memory, CD8 T, and CD4 regulatory T cells were significantly elevated in our patients. CD4 T cell proliferation was markedly impaired compared to the healthy controls. Moreover, immunological investigations of 15 AT patients with CSR defect revealed a significant reduction in the marginal zone, switched memory, and more intense defects in IgM only memory B cells, CD4 naïve and central memory T cells. CONCLUSION:The present study revealed that patients with AT have a broad spectrum of cellular and humoral deficiencies. Therefore, a detailed evaluation of T and B cell subsets increases understanding of the disease in patients and the risk of infection.
10.1007/s10875-020-00881-9
Laquinimod dampens IL-1β signaling and Th17-polarizing capacity of monocytes in patients with MS.
Engel Sinah,Jolivel Valérie,Kraus Stefan H-P,Zayoud Morad,Rosenfeld Karolina,Tumani Hayrettin,Furlan Roberto,Kurschus Florian C,Waisman Ari,Luessi Felix
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To assess the impact of laquinimod treatment on monocytes and to investigate the underlying immunomodulatory mechanisms in MS. METHODS:In this cross-sectional study, we performed in vivo and in vitro analyses of cluster of differentiation (CD14) monocytes isolated from healthy donors (n = 15), untreated (n = 13), and laquinimod-treated patients with MS (n = 14). Their frequency and the expression of surface activation markers were assessed by flow cytometry and the viability by calcein staining. Cytokine concentrations in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes were determined by flow cytometry. The messenger ribonucleic acid (mRNA) expression level of genes involved in cytokine expression was measured by quantitative PCR. The LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation was determined by the quantification of the phosphorylation level of the p65 subunit. Laquinimod-treated monocytes were cocultured with CD4 T cells, and the resulting cytokine production was analyzed by flow cytometry after intracellular cytokine staining. The interleukin (IL)-17A concentration of the supernatant was assessed by ELISA. RESULTS:Laquinimod did not alter the frequency or viability of circulating monocytes, but led to an upregulation of CD86 expression. LPS-stimulated monocytes of laquinimod-treated patients with MS secreted less IL-1β following a downregulation of IL-1β gene expression. Phosphorylation levels of the NF-κB p65 subunit were reduced after laquinimod treatment, indicating a laquinimod-associated inhibition of the NF-κB pathway. T cells primed with laquinimod-treated monocytes differentiated significantly less into IL-17A-producing T helper (Th)-17 cells. CONCLUSIONS:Our findings suggest that inhibited NF-κB signaling and downregulation of IL-1β expression in monocytes contributes to the immunomodulatory effects of laquinimod and that the impairment of Th17 polarization might mediate its disease-modifying activity in MS.
10.1212/NXI.0000000000000908
Cytokine production pattern of T lymphocytes in neonatal arterial ischemic stroke during the first month of life-a case study.
Bajnok Anna,Berta László,Orbán Csaba,Tulassay Tivadar,Toldi Gergely
Journal of neuroinflammation
BACKGROUND:The perinatal period carries the highest risk for stroke in childhood; however, the pathophysiology is poorly understood and preventive, prognostic, and therapeutic strategies are not available. A new pathophysiological model describes the development of neonatal arterial ischemic stroke (NAIS) as the combined result of prenatal inflammation and hypoxic-ischemic insult. Neuroinflammation and a systemic inflammatory response are also important features of NAIS. Identifying key players of the inflammatory system is in the limelight of current research. CASE PRESENTATION:We present four NAIS cases, in whom detailed analysis of intracellular and plasma cytokine levels are available from the first month of life. All neonates were admitted with the initial diagnosis of hypoxic ischemic encephalopathy (HIE); however, early MRI examination revealed NAIS. Blood samples were collected between 3 and 6 h of life, at 24 h, 72 h, 1 week, and 1 month of life. Peripheral blood mononuclear cells were assessed with flow cytometry and plasma cytokine levels were measured. Pooled data from the cohort of four NAIS patients were compared to infants with HIE. At 6 and 72 h of age, the prevalence of IL10+ CD8+ lymphocytes remained lower in NAIS. At 6 h, CD8+ lymphocytes in NAIS produced more IL-17. At 72 h, CD8+ cells produced more IL-6 in severe HIE than in NAIS, but IL-6 production remained elevated in CD8 cells at 1 month in NAIS, while it decreased in HIE. At 1 week, the prevalence of TGF-β + lymphocytes prone to enter the CNS was elevated in NAIS. On the other hand, by 1 month of age, the prevalence of TGF-β + CD4+ lymphocytes decreased in NAIS compared to HIE. At 72 h, we found elevated plasma levels of IL-5, MCP-1, and IL-17 in NAIS. By 1 month, plasma levels of IL-4, IL-12, and IL-17 decreased in NAIS but remained elevated in HIE. CONCLUSIONS:Differences in the cytokine network are present between NAIS and HIE. CD8 lymphocytes appear to shift towards the pro-inflammatory direction in NAIS. The inflammatory response appears to be more pronounced at 72 h in NAIS but decreases faster, reaching lower plasma levels of inflammatory markers at 1 month.
10.1186/s12974-018-1229-y
T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis.
Martin Bradley N,Wang Chenhui,Zhang Cun-jin,Kang Zizhen,Gulen Muhammet Fatih,Zepp Jarod A,Zhao Junjie,Bian Guanglin,Do Jeong-su,Min Booki,Pavicic Paul G,El-Sanadi Caroline,Fox Paul L,Akitsu Aoi,Iwakura Yoichiro,Sarkar Anasuya,Wewers Mark D,Kaiser William J,Mocarski Edward S,Rothenberg Marc E,Hise Amy G,Dubyak George R,Ransohoff Richard M,Li Xiaoxia
Nature immunology
Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1β production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.
10.1038/ni.3389
NK cell markers predict the efficacy of IV immunoglobulins in CIDP.
Mausberg Anne K,Heininger Maximilian K,Meyer Zu Horste Gerd,Cordes Steffen,Fleischer Michael,Szepanowski Fabian,Kleinschnitz Christoph,Hartung Hans-Peter,Kieseier Bernd C,Stettner Mark
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:To assess whether IV immunoglobulins (IVIgs) as a first-line treatment for chronic inflammatory demyelinating polyneuropathy (CIDP) have a regulative effect on natural killer (NK) cells that is related to clinical responsiveness to IVIg. METHODS:In a prospective longitudinal study, we collected blood samples of 29 patients with CIDP before and after initiation of IVIg treatment for up to 6 months. We used semiquantitative PCR and flow cytometry in the peripheral blood to analyze the effects of IVIg on the NK cells. The results were correlated with clinical aspects encompassing responsiveness. RESULTS:We found a reduction in the expression of several typical NK cell genes 1 day after IVIg administration. Flow cytometry furthermore revealed a reduced cytotoxic CD56 NK cell population, whereas regulatory CD56 NK cells remained mostly unaffected or were even increased after IVIg treatment. Surprisingly, the observed effects on NK cells almost exclusively occurred in IVIg-responsive patients with CIDP. CONCLUSIONS:The correlation between the altered NK cell population and treatment efficiency suggests a crucial role for NK cells in the still speculative mode of action of IVIg treatment. Analyzing NK cell subsets after 24 hours of treatment initiation appeared as a predictive marker for IVIg responsiveness. Further studies are warranted investigating the potential of NK cell status as a routine parameter in patients with CIDP before IVIg therapy. CLASSIFICATION OF EVIDENCE:This study provides Class I evidence that NK cell markers predict clinical response to IVIg in patients with CIDP.
10.1212/NXI.0000000000000884
NK Cells and Innate-Like T Cells After Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.
Ruder Josefine,Rex Jordan,Obahor Simon,Docampo María José,Müller Antonia M S,Schanz Urs,Jelcic Ilijas,Martin Roland
Frontiers in immunology
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, in which autoreactive T and B cells play important roles. Other lymphocytes such as NK cells and innate-like T cells appear to be involved as well. To name a few examples, CD56 NK cells were described as an immunoregulatory NK cell subset in MS while innate-like T cells in MS were described in brain lesions and with proinflammatory signatures. Autologous hematopoietic stem cell transplantation (aHSCT) is a procedure used to treat MS. This procedure includes hematopoietic stem/progenitor cell (HSPC) mobilization, then high-dose chemotherapy combined with anti-thymocyte globulin (ATG) and subsequent infusion of the patients own HSPCs to reconstitute a functional immune system. aHSCT inhibits MS disease activity very effectively and for long time, presumably due to elimination of autoreactive T cells. Here, we performed multidimensional flow cytometry experiments in peripheral blood lymphocytes of 27 MS patients before and after aHSCT to address its potential influence on NK and innate-like T cells. After aHSCT, the relative frequency and absolute numbers of CD56 NK cells rise above pre-aHSCT levels while all studied innate-like T cell populations decrease. Hence, our data support an enhanced immune regulation by CD56 NK cells and the efficient reduction of proinflammatory innate-like T cells by aHSCT in MS. These observations contribute to our current understanding of the immunological effects of aHSCT in MS.
10.3389/fimmu.2021.794077
Rasmussen's encephalitis is characterized by relatively lower production of IFN-β and activated cytotoxic T cell upon herpes viruses infection.
Journal of neuroinflammation
BACKGROUND:The etiology of Rasmussen's encephalitis (RE), a rare chronic neurological disorder characterized by CD8+ T cell infiltration and unihemispheric brain atrophy, is still unknown. Various human herpes viruses (HHVs) have been detected in RE brain, but their contribution to RE pathogenesis is unclear. METHODS:HHVs infection and relevant immune response were compared among brain tissues from RE, temporal lobe epilepsy (TLE) and traumatic brain injury (TBI) patients. Viral antigen or genome, CD8+ T cells, microglia and innate immunity molecules were analyzed by immunohistochemical staining, DNA dot blot assay or immunofluorescence double staining. Cytokines were measured by multiplex flow cytometry. Cell apoptosis was visualized by TUNEL staining. Viral infection, immune response and the severity of unihemispheric atrophy were subjected to correlation analysis. RESULTS:Antigens of various HHVs were prevalent in RE and TLE brains, and the cumulative viral score of HHVs positively correlated with the unihemispheric atrophy in RE patients. CD8+ T cells infiltration were observed in both RE and TLE brains and showed co-localization with HHV antigens, but their activation, as revealed by Granzyme B (GZMB) release and apoptosis, was found only in RE. In comparison to TLE, RE brain tissues contained higher level of inflammatory cytokines, but the interferon-β level, which was negatively correlated with cumulative viral score, was relatively lower. In line with this, the DNA sensor STING and IFI16, rather than other innate immunity signaling molecules, were insufficiently activated in RE. CONCLUSIONS:Compared with TBI, both RE and TLE had prevalently HHV infection and immune response in brain tissues. However, in comparison to TLE, RE showed insufficient activation of antiviral innate immunity but overactivation of cytotoxic T cells. Our results show the relatively lower level of antiviral innate immunity and overactivation of cytotoxic T cells in RE cases upon HHV infection, the overactivated T cells might be a compensate to the innate immunity but the causative evidence is lack in our study and need more investigation in the future.
10.1186/s12974-022-02379-0
Evaluation of cerebrospinal fluid CXCL13 concentrations and lymphocyte subsets in tick-borne encephalitis.
Toczylowski Kacper,Grygorczuk Sambor,Osada Joanna,Wojtkowska Malgorzata,Bojkiewicz Ewa,Wozinska-Klepadlo Marta,Potocka Paulina,Sulik Artur
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
OBJECTIVES:Recent studies suggest that the clinical presentation of tick-borne encephalitis (TBE) is determined by the host immune responses to the tick-borne encephalitis virus (TBEV). The aim of the study was to characterize immune responses in TBE to give a better insight into the immunopathogenesis of this disease. METHODS:Anti-TBEV antibody levels, cerebrospinal fluid (CSF) and blood lymphoid populations, and concentrations of CXCL13 (a potent B-cell and T-cell chemoattractant), were analyzed in 35 patients with TBE (20 adults and 15 children). RESULTS:When compared with the blood, the CSF lymphoid population was significantly enriched in CD4+ T-cells and relatively depleted in natural killer (NK) cells and B lymphocytes. In comparison with TBE meningitis, patients suffering from TBE meningoencephalitis (n = 11, 31%) had a 3.5-fold higher median CSF CXCL13 concentration, 1.8-fold higher CSF/serum ratio of anti-TBEV IgG antibodies, and 1.8-fold higher median CSF cell count. CSF CXCL13 levels did not change significantly in children with TBE meningitis receiving supportive treatment, but decreased in children with TBE meningoencephalitis who received intravenous steroids. CONCLUSIONS:CD4+ cells are abundant in the CSF of patients with TBE. CXCL13 may be involved in the neuropathology of TBE by attracting different subsets of lymphocytes to the CSF.
10.1016/j.ijid.2020.01.023
Baseline Inflammatory Status Reveals Dichotomic Immune Mechanisms Involved In Primary-Progressive Multiple Sclerosis Pathology.
Frontiers in immunology
Objective:To ascertain the role of inflammation in the response to ocrelizumab in primary-progressive multiple sclerosis (PPMS). Methods:Multicenter prospective study including 69 patients with PPMS who initiated ocrelizumab treatment, classified according to baseline presence [Gd+, n=16] or absence [Gd-, n=53] of gadolinium-enhancing lesions in brain MRI. Ten Gd+ (62.5%) and 41 Gd- patients (77.4%) showed non-evidence of disease activity (NEDA) defined as no disability progression or new MRI lesions after 1 year of treatment. Blood immune cell subsets were characterized by flow cytometry, serum immunoglobulins by nephelometry, and serum neurofilament light-chains (sNfL) by SIMOA. Statistical analyses were corrected with the Bonferroni formula. Results:More than 60% of patients reached NEDA after a year of treatment, regardless of their baseline characteristics. In Gd+ patients, it associated with a low repopulation rate of inflammatory B cells accompanied by a reduction of sNfL values 6 months after their first ocrelizumab dose. Patients in Gd- group also had low B cell numbers and sNfL values 6 months after initiating treatment, independent of their treatment response. In these patients, NEDA status was associated with a tolerogenic remodeling of the T and innate immune cell compartments, and with a clear increase of serum IgA levels. Conclusion:Baseline inflammation influences which immunological pathways predominate in patients with PPMS. Inflammatory B cells played a pivotal role in the Gd+ group and inflammatory T and innate immune cells in Gd- patients. B cell depletion can modulate both mechanisms.
10.3389/fimmu.2022.842354
Intrathecal B-cell accumulation and axonal damage distinguish MRI-based benign from aggressive onset in MS.
Engel Sinah,Friedrich Michaela,Muthuraman Muthuraman,Steffen Falk,Poplawski Alicia,Groppa Sergiu,Bittner Stefan,Zipp Frauke,Luessi Felix
Neurology(R) neuroimmunology & neuroinflammation
OBJECTIVE:We explored the incremental value of adding multiple disease activity biomarkers in CSF and serum for distinguishing MRI-based benign from aggressive MS in early disease course. METHODS:Ninety-three patients diagnosed with clinically isolated syndrome (CIS) or early MS were divided into 3 nonoverlapping severity groups defined by objective MRI criteria. Ninety-seven patients with noninflammatory neurologic disorders and 48 patients with other inflammatory neurologic diseases served as controls. Leukocyte subsets in the CSF were analyzed by flow cytometry. CSF neurofilament light chain (NfL) and chitinase-3-like protein 1 (CHI3L1) levels were measured by ELISA. Serum NfL levels were examined using single molecule array technology. RESULTS:CSF CD20+/CD14+ ratios and NfL levels in CSF and serum were significantly different between high and low MRI severity groups, whereas no difference was found for CSF CHI3L1 levels. NfL levels in CSF and serum highly correlated. Receiver operating characteristic analysis demonstrated that the cumulative sums combining CSF CD20+/CD14+ ratios and NfL levels in serum or CSF considerably improved diagnostic accuracy. A composite score built from these 2 cumulative sums best distinguished MRI severity. These findings were validated by support vector machine analysis, which confirmed that the accuracy of the cumulative sums and composite score outperforms single biomarkers. CONCLUSION:Patients with extreme manifestations of CIS or early MS defined by strict MRI parameters can be best distinguished by combining markers of intrathecal B-cell accumulation and axonal damage. This could stratify individual treatment decisions toward a more personalized immunotherapy.
10.1212/NXI.0000000000000595
Monocytes inhibit NK activity TGF-β in patients with obstructive sleep apnoea.
Hernández-Jiménez Enrique,Cubillos-Zapata Carolina,Toledano Victor,Pérez de Diego Rebeca,Fernández-Navarro Isabel,Casitas Raquel,Carpio Carlos,Casas-Martín Jose,Valentín Jaime,Varela-Serrano Anibal,Avendaño-Ortiz Jose,Alvarez Enrique,Aguirre Luis A,Pérez-Martínez Antonio,De Miguel Maria P,Belda-Iniesta Cristobal,García-Río Francisco,López-Collazo Eduardo
The European respiratory journal
Obstructive sleep apnoea (OSA) is associated with cancer incidence and mortality. The contribution of the immune system appears to be crucial; however, the potential role of monocytes and natural killer (NK) cells remains unclear.Quantitative reverse transcriptase PCR, flow cytometry and assays were used to analyse the phenotype and immune response activity in 92 patients with OSA (60 recently diagnosed untreated patients and 32 patients after 6 months of treatment with continuous positive airway pressure (CPAP)) and 29 healthy volunteers (HV).We determined that monocytes in patients with OSA exhibit an immunosuppressive phenotype, including surface expression of glycoprotein-A repetitions predominant protein (GARP) and transforming growth factor-β (TGF-β), in contrast to those from the HV and CPAP groups. High levels of TGF-β were detected in OSA sera. TGF-β release by GARP monocytes impaired NK cytotoxicity and maturation. This altered phenotype correlated with the hypoxic severity clinical score (CT90). Reoxygenation eventually restored the altered phenotypes and cytotoxicity.This study demonstrates that GARP monocytes from untreated patients with OSA have an NK-suppressing role through their release of TGF-β. Our findings show that monocyte plasticity immunomodulates NK activity in this pathology, suggesting a potential role in cancer incidence.
10.1183/13993003.02456-2016
Myeloid Dendritic Cells Are Major Producers of IFN-β in Dermatomyositis and May Contribute to Hydroxychloroquine Refractoriness.
The Journal of investigative dermatology
Dermatomyositis pathogenesis remains incompletely understood; however, recent work suggests a predominant IFN-1 response. We explored dermatomyositis pathogenesis by quantifying the inflammatory cells in the skin, comparing myeloid with plasmacytoid dendritic cell release of IFN-β, and assessing myeloid dendritic cell (mDC) contribution to hydroxychloroquine refractoriness. Immunohistochemistry was performed to assess cell-type expression in lesional skin biopsies from 12 patients with moderate-to-severe cutaneous dermatomyositis. Immunofluorescence, laser-capture microdissection, and flow cytometry were used to assess mDC release of IFN-β in lesional skin biopsies and blood of patients with dermatomyositis. Immunohistochemistry was utilized to determine whether myeloid or plasmacytoid dendritic cells were increased in hydroxychloroquine nonresponders. CD4+, CD11c+, and CD69+ cells were more populous in lesional skin of patients with dermatomyositis. mDCs colocalized with IFN-β by immunofluorescence and laser-capture microdissection revealed increased IFN-β mRNA expression by mDCs in lesional skin of patients with dermatomyositis. In blood, both mDCs and plasmacytoid dendritic cells were major producers of IFN-β in patients with dermatomyositis, whereas plasmacytoid dendritic cells predominately released IFN-β in healthy controls (P < 0.01). mDCs were significantly increased in the skin of hydroxychloroquine nonresponders compared with that in the skin of responders (P < 0.05). mDCs cells appear to play an important role in dermatomyositis pathogenesis and IFN-β production.
10.1016/j.jid.2020.12.032
Soluble glucocorticoid-induced tumor necrosis factor receptor regulates Helios expression in myasthenia gravis.
Li Yi,Yang Shumei,Li Zhibin,Meng Huanyu,Jin Wanling,Yang Huan,Yin Weifan
Journal of translational medicine
BACKGROUND:Helios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios. METHOD:Multicolor flow cytometry was performed to analyze Helios Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry. RESULT:Both Helios Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios Tregs possessed greater immunosuppressive capacity compared to Helios Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL. CONCLUSION:This work demonstrates abnormal changes in Helios Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.
10.1186/s12967-019-1916-1
Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.
Lawson Brian R,Gonzalez-Quintial Rosana,Eleftheriadis Theodoros,Farrar Michael A,Miller Stephen D,Sauer Karsten,McGavern Dorian B,Kono Dwight H,Baccala Roberto,Theofilopoulos Argyrios N
Clinical immunology (Orlando, Fla.)
IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.
10.1016/j.clim.2015.08.007
IL-27 shapes the immune properties of human astrocytes and their impact on encountered human T lymphocytes.
Journal of neuroinflammation
BACKGROUND:Interleukin-27 (IL-27) can trigger both pro- and anti-inflammatory responses. This cytokine is elevated in the central nervous system (CNS) of multiple sclerosis (MS) patients, but how it influences neuroinflammatory processes remains unclear. As astrocytes express the receptor for IL-27, we sought to determine how these glial cells respond to this cytokine and whether such exposure alters their interactions with infiltrating activated T lymphocytes. To determine whether inflammation shapes the impact of IL-27, we compared the effects of this cytokine in non-inflamed and inflamed conditions induced by an IL-1β exposure. MAIN BODY:Transcriptomic analysis of IL-27-exposed human astrocytes showed an upregulation of multiple immune genes. Human astrocytes increased the secretion of chemokines (CXCL9, CXCL10, and CXCL11) and the surface expression of proteins (PD-L1, HLA-E, and ICAM-1) following IL-27 exposure. To assess whether exposure of astrocytes to IL-27 influences the profile of activated T lymphocytes infiltrating the CNS, we used an astrocyte/T lymphocyte co-culture model. Activated human CD4 or CD8 T lymphocytes were co-cultured with astrocytes that have been either untreated or pre-exposed to IL‑27 or IL-1β. After 24 h, we analyzed T lymphocytes by flow cytometry for transcription factors and immune molecules. The contact with IL-27-exposed astrocytes increased the percentages of T-bet, Eomes, CD95, IL-18Rα, ICAM-1, and PD-L1 expressing CD4 and CD8 T lymphocytes and reduced the proportion of CXCR3-positive CD8 T lymphocytes. Human CD8 T lymphocytes co-cultured with human IL-27-treated astrocytes exhibited higher motility than when in contact with untreated astrocytes. These results suggested a preponderance of kinapse-like over synapse-like interactions between CD8 T lymphocytes and IL-27-treated astrocytes. Finally, CD8 T lymphocytes from MS patients showed higher motility in contact with IL-27-exposed astrocytes compared to healthy donors' cells. CONCLUSION:Our results establish that IL-27 alters the immune functions of human astrocytes and shapes the profile and motility of encountered T lymphocytes, especially CD8 T lymphocytes from MS patients.
10.1186/s12974-022-02572-1
Ofatumumab Modulates Inflammatory T Cell Responses and Migratory Potential in Patients With Multiple Sclerosis.
Neurology(R) neuroimmunology & neuroinflammation
BACKGROUND AND OBJECTIVES:The anti-CD20 antibody ofatumumab is an efficacious therapy for multiple sclerosis (MS) through depletion of B cells. The purpose of this study was to examine the derivative effects of B cell depletion on the peripheral immune system and a direct treatment effect on T cells expressing CD20. METHODS:Frequency and absolute numbers of peripheral leukocytes of treatment-naive patients with relapsing-remitting MS (RRMS) and patients treated with ofatumumab for a mean of 482 days were assessed in this observational study by flow cytometry. In addition, effector function and CNS migration of T cells using a human in vitro blood-brain barrier (BBB) assay were analyzed. RESULTS:This study showed that ofatumumab treatment of patients with RRMS increased the control of effector T cells and decreased T cell autoreactivity. It also showed that ofatumumab reduced the level of peripheral CD20 T cells and that the observed decrease in CNS-migratory capacity of T cells was caused by the depletion of CD20 T cells. Finally, our study pointed out a bias in the measurement of CD20 cells due to a steric hindrance between the treatment antibody and the flow cytometry antibody. DISCUSSION:The substantial ofatumumab-induced alteration in the T cell compartment including a severely decreased CNS-migratory capacity of T cells could partly be attributed to the depletion of CD20 T cells. Therefore, we propose that depletion of CD20 T cells contributes to the positive treatment effect of ofatumumab and suggests that ofatumumab therapy should be considered a B cell and CD20 T cell depletion therapy. CLASSIFICATION OF EVIDENCE:This study provides Class IV evidence that compared with treatment-naive patients, ofatumumab treatment of patients with RRMS decreases peripheral CD20 T cells, increases effector T cell control, and decreases T cell autoreactivity.
10.1212/NXI.0000000000200004
Circulating epithelial tumor cell analysis in CSF in patients with leptomeningeal metastases.
van Bussel Mark T J,Pluim Dick,Milojkovic Kerklaan Bojana,Bol Mijke,Sikorska Karolina,Linders Dorothé T C,van den Broek Daan,Beijnen Jos H,Schellens Jan H M,Brandsma Dieta
Neurology
OBJECTIVE:The primary objective was to determine the sensitivity and specificity of epithelial cell adhesion molecule (EpCAM) immunoflow cytometry circulating tumor cells (CTC) analysis in CSF in patients with suspected leptomeningeal metastases (LM). The secondary objective was to explore the distribution of driver mutations in the primary tumor, plasma, cell free CSF (cfCSF), and isolated CTC from CSF in non-small cell lung cancer (NSCLC). METHODS:We tested the performance of the CTC assay vs CSF cytology in a prospective study in 81 patients with a clinical suspicion of LM but a nonconfirmatory MRI. In an NSCLC subcohort, we analyzed circulating tumor (ct)DNA of the selected driver mutations by digital droplet PCR (ddPCR). RESULTS:The sensitivity of the CTC assay was 94% (95% confidence interval [CI] 80-99) and the specificity was 100% (95% CI 91-100) at the optimal cutoff of 0.9 CTC/mL. The sensitivity of cytology was 76% (95% CI 58-89). Twelve of the 23 patients with NSCLC had mutated epidermal growth factor receptor (). All 5 tested patients with LM demonstrated the primary driver mutation in cfCSF. The driver mutation could also be detected in CTC isolated from CSF. CONCLUSION:CTC in CSF are detected with a high sensitivity for the diagnosis of LM. ddPCR can determine mutations in both cfCSF and isolated CTC from CSF of patients with -mutated NSCLC and LM. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that EpCAM-based immunoflow cytometry analysis of CSF accurately identifies patients with LM.
10.1212/WNL.0000000000008751
Identification of a novel role for matrix metalloproteinase-3 in the modulation of B cell responses in multiple sclerosis.
Frontiers in immunology
There has been a growing interest in the presence and role of B cell aggregates within the central nervous system of multiple sclerosis patients. However, very little is known about the expression profile of molecules associated with these aggregates and how they might be influencing aggregate development or persistence in the brain. The current study focuses on the effect of matrix metalloproteinase-3, which is associated with B cell aggregates in autopsied multiple sclerosis brain tissue, on B cells. Autopsied brain sections from multiple sclerosis cases and controls were screened for the presence of CD20 B cell aggregates and expression of matrix metalloproteinase-3. Using flow cytometry, enzyme-linked immunosorbent assay and gene array as methods, studies were conducted using peripheral blood of healthy volunteers to demonstrate the effect of matrix metalloproteinase-3 on B cells. Autopsied brain sections from multiple sclerosis patients containing aggregates of B cells expressed a significantly higher amount of matrix metalloproteinase-3 compared to controls. experiments demonstrated that matrix metalloproteinase-3 dampened the overall activation status of B cells by downregulating CD69, CD80 and CD86. Furthermore, matrix metalloproteinase-3-treated B cells produced significantly lower amounts of interleukin-6. Gene array data confirmed that matrix metalloproteinase-3 altered the proliferation and survival profiles of B cells. Taken together, out data indicate a role for B cell modulatory properties of matrix metalloproteinase-3.
10.3389/fimmu.2022.1025377
Dysfunction of endothelial progenitor cells is associated with the type I IFN pathway in patients with polymyositis and dermatomyositis.
Ekholm Louise,Kahlenberg J Michelle,Barbasso Helmers Sevim,Tjärnlund Anna,Yalavarthi Srilakshmi,Zhao Wenpu,Seto Nickie,Betteridge Zoe,Lundberg Ingrid E,Kaplan Mariana J
Rheumatology (Oxford, England)
OBJECTIVE:Alterations in phenotype and function of endothelial progenitor cells (EPCs) have been associated with poor vascular outcomes and impaired vascular repair in various conditions. Our hypothesis was that patients with PM and DM have dysregulation of EPCs driven by type I IFN and IL-18 similar to other autoimmune diseases. METHODS:Quantification of circulating EPCs was performed by flow cytometry in patients with PM/DM and matched healthy controls. The ability of EPCs to differentiate into mature endothelial cells was investigated by light and fluorescence microscopy quantification in the presence or absence of PM/DM or control serum, neutralizing antibodies to type I IFN receptor or IL-18. Serum type I IFN activity was quantified by induction of type I IFN-inducible genes in HeLa cells. Circulating IL-18 concentrations were assessed by ELISA. RESULTS:Circulating EPCs were significantly lower in PM/DM patients compared with controls. PM/DM EPCs displayed a decreased capacity to differentiate into mature endothelial cells and PM/DM serum significantly inhibited differentiation of control EPCs. This effect was reversed in the majority of samples with neutralizing antibodies to IL-18 or to type I IFN receptor or by a combination of these antibodies. Patients with associated impairments in EPC function had higher type I IFN serum activity. CONCLUSION:PM/DM is associated with dysregulation of EPC phenotype and function that may be attributed, at least in part, to aberrant IL-18 and type I IFN pathways. The implication of these vasculopathic findings for disease prognosis and complications remains to be determined.
10.1093/rheumatology/kew288
Immunoadsorption versus double-dose methylprednisolone in refractory multiple sclerosis relapses.
Journal of neuroinflammation
OBJECTIVE:Intravenous methylprednisolone is the standard treatment for a multiple sclerosis relapse; however, this fails to improve symptoms in up to one quarter of patients. Immunoadsorption is an accepted treatment for refractory relapses, but prospective comparator-controlled studies are missing. METHODS:In this observational study, patients with steroid-refractory acute multiple sclerosis relapses receiving either six courses of tryptophan-immunoadsorption or double-dose methylprednisolone therapy were analysed. Outcomes were evaluated at discharge and three months later. Immune profiling of blood lymphocytes and proteomic analysis were performed by multi-parameter flow cytometry and Olink analysis, respectively (NCT04450030). RESULTS:42 patients were enrolled (methylprednisolone: 26 patients; immunoadsorption: 16 patients). For determination of the primary outcome, treatment response was stratified according to relative function system score changes ("full/best" vs. "average" vs. "worse/none"). Upon discharge, the adjusted odds ratio for any treatment response ("full/best" + "average" vs. "worse/none") was 10.697 favouring immunoadsorption (p = 0.005 compared to methylprednisolone). At follow-up, the adjusted odds ratio for the best treatment response ("full/best" vs. "average" + "worse/none") was 103.236 favouring IA patients (p = 0.001 compared to methylprednisolone). Similar results were observed regarding evoked potentials and quality of life outcomes, as well as serum neurofilament light-chain levels. Flow cytometry revealed a profound reduction of B cell subsets following immunoadsorption, which was closely correlated to clinical outcomes, whereas methylprednisolone had a minimal effect on B cell populations. Immunoadsorption treatment skewed the blood cytokine network, reduced levels of B cell-related cytokines and reduced immunoglobulin levels as well as levels of certain coagulation factors. INTERPRETATION:Immunoadsorption demonstrated favourable outcomes compared to double-dose methylprednisolone. Outcome differences were significant at discharge and follow-up. Further analyses identified modulation of B cell function as a potential mechanism of action for immunoadsorption, as reduction of B cell subsets correlated with clinical improvement.
10.1186/s12974-022-02583-y
TLR expression in peripheral monocyte subsets of patients with idiopathic inflammatory myopathies: association with clinical and immunological features.
Torres-Ruiz Jiram,Carrillo-Vazquez Daniel Alberto,Padilla-Ortiz Diana Marcela,Vazquez-Rodriguez Ricardo,Nuñez-Alvarez Carlos,Juarez-Vega Guillermo,Gomez-Martin Diana
Journal of translational medicine
BACKGROUND:Monocytes and toll-like receptors (TLR) have been found in the inflammatory infiltrate of muscle biopsies in patients with idiopathic inflammatory myopathies (IIM), suggesting an important role of these cells in the pathogenesis of myositis. The monocyte subsets, their TLR expression in peripheral blood and their relationship with the clinical characteristics of patients with IIM has not been addressed. METHODS:We recruited 45 patients with IIM diagnosis and 15 age and sex-adjusted healthy controls. We assessed the disease activity and damage, performed a nailfold capillaroscopy and registered the cardio-pulmonary parameters from the medical charts. Monocyte subsets, their expression of TLR2 and TLR4 and the serum Th1/Th2/Th17 cytokines levels were evaluated by flow cytometry. We expressed quantitative variables as medians and interquartile ranges (IQR) or minimum and maximum (min-max). Differences between groups were assessed with Mann-Whitney U and the Kruskal-Wallis tests. Correlation between quantitative variables was assessed with Spearman Rho. RESULTS:Twenty-nine patients were women (64.4%) and 32 (71.1%) had dermatomyositis. In comparison to healthy controls, patients with active IIM had a higher percentage of intermediate monocytes and lower amounts of classical monocytes. Patients with IIM had a higher expression of TLR4 in all their monocyte subsets, regardless of disease activity and prednisone treatment. Serum IL-6 correlated with the TLR2 expression in every monocyte subset and the expression of TLR2 in intermediate monocytes was higher among patients with dysphagia. Subjects with nailfold capillaroscopy abnormalities had a higher amount of TLR2+ classical and non-classical monocytes and those with interstitial lung disease (ILD) had a higher percentage of TLR4+ non-classical monocytes. The classical and intermediate monocytes from patients with anti Mi2 antibodies had a higher expression of TLR4. The percentage of intermediate monocytes and the expression of TLR4 in all monocyte subsets showed a good diagnostic capacity in patients with IIM. CONCLUSION:Patients with IIM have a differential pool of monocyte subsets with an enhanced expression of TLR2 and TLR4, which correlates with disease activity and distinctive clinical features including dysphagia, ILD, vasculopathy, and pro-inflammatory cytokines. These immunological features might be useful as a potential diagnostic tool as well as novel disease activity biomarkers in IIM.
10.1186/s12967-020-02290-3
Portrait of blood-derived extracellular vesicles in patients with Parkinson's disease.
Lamontagne-Proulx Jérôme,St-Amour Isabelle,Labib Richard,Pilon Jérémie,Denis Hélèna L,Cloutier Nathalie,Roux-Dalvai Florence,Vincent Antony T,Mason Sarah L,Williams-Gray Caroline,Duchez Anne-Claire,Droit Arnaud,Lacroix Steve,Dupré Nicolas,Langlois Mélanie,Chouinard Sylvain,Panisset Michel,Barker Roger A,Boilard Eric,Cicchetti Francesca
Neurobiology of disease
The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) - for comparative purposes in individuals with another chronic neurodegenerative condition - and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.
10.1016/j.nbd.2018.11.002
Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.
Liu Yongji,Shi Ling,Liu Yuan,Li Peng,Jiang Guoping,Gao Xiaoning,Zhang Yongbin,Jiang Chuanwu,Zhu Weiping,Han Hongxing,Ju Fang
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
BACKGROUND:Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. MATERIALS AND METHODS:Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. RESULTS:Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. CONCLUSION:Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM.
10.1016/j.biopha.2018.02.006
Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy.
Lieberman Nicole A P,DeGolier Kole,Kovar Heather M,Davis Amira,Hoglund Virginia,Stevens Jeffrey,Winter Conrad,Deutsch Gail,Furlan Scott N,Vitanza Nicholas A,Leary Sarah E S,Crane Courtney A
Neuro-oncology
Background:Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Methods:Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. Results:DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor β1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. Conclusions:DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.
10.1093/neuonc/noy145
Physalin F, a seco-steroid from Physalis angulata L., has immunosuppressive activity in peripheral blood mononuclear cells from patients with HTLV1-associated myelopathy.
Pinto Lorena A,Meira Cássio S,Villarreal Cristiane F,Vannier-Santos Marcos A,de Souza Claudia V C,Ribeiro Ivone M,Tomassini Therezinha C B,Galvão-Castro Bernardo,Soares Milena B P,Grassi Maria F R
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Human T-lymphotropic virus type 1 (HTLV-1) induces a strong activation of the immune system, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Physalin F is a secosteroid with potent anti-inflammatory and immunomodulatory activities. The present study aimed to investigate the effects of physalin F on peripheral blood mononuclear cells (PBMC) of HAM/TSP subjects. A concentration-dependent inhibition of spontaneous proliferation of PBMC from HAM/TSP subjects was observed in the presence of physalin F, as evaluated by (3)H-thymidine uptake. The IC50 for physalin F was 0.97 ± 0.11 μM. Flow cytometry analysis using Cytometric Bead Array (CBA) showed that physalin F (10 μM) significantly reduced the levels of IL-2, IL-6, IL-10, TNF-α and IFN-γ, but not IL-17A, in supernatants of PBMC cultures. Next, apoptosis induction was addressed by using flow cytometry to evaluate annexin V expression. Treatment with physalin F (10 μM) increased the apoptotic population of PBMC in HAM/TSP subjects. Transmission electron microscopy analysis of PBMC showed that physalin F induced ultrastructural changes, such as pyknotic nuclei, damaged mitochondria, enhanced autophagic vacuole formation, and the presence of myelin-like figures. In conclusion, physalin F induces apoptosis of PBMC, decreasing the spontaneous proliferation and cytokine production caused by HTLV-1 infection.
10.1016/j.biopha.2016.01.041