Human Fetal TNF-α-Cytokine-Producing CD4 Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.
Schreurs Renée R C E,Baumdick Martin E,Sagebiel Adrian F,Kaufmann Max,Mokry Michal,Klarenbeek Paul L,Schaltenberg Nicola,Steinert Fenja L,van Rijn Jorik M,Drewniak Agata,The Sarah-May M L,Bakx Roel,Derikx Joep P M,de Vries Niek,Corpeleijn Willemijn E,Pals Steven T,Gagliani Nicola,Friese Manuel A,Middendorp Sabine,Nieuwenhuis Edward E S,Reinshagen Konrad,Geijtenbeek Teunis B H,van Goudoever Johannes B,Bunders Madeleine J
Immunity
Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)CD4CD69 T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4 T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4 T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4 Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4 T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.
10.1016/j.immuni.2018.12.010
Toll-like receptor-4 inhibits enterocyte proliferation via impaired beta-catenin signaling in necrotizing enterocolitis.
Sodhi Chhinder P,Shi Xia-Hua,Richardson Ward M,Grant Zachary S,Shapiro Richard A,Prindle Thomas,Branca Maria,Russo Anthony,Gribar Steven C,Ma Congrong,Hackam David J
Gastroenterology
BACKGROUND & AIMS:Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-catenin signaling. METHODS:Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4(-/-) mice after induction of NEC or endotoxemia. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. RESULTS:TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. CONCLUSION:We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.
10.1053/j.gastro.2009.09.045
Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.
Gastroenterology
BACKGROUND & AIMS:Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS:Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS:TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS:TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.
10.1053/j.gastro.2012.05.053
Necrotizing enterocolitis induces T lymphocyte-mediated injury in the developing mammalian brain.
Zhou Qinjie,Niño Diego F,Yamaguchi Yukihiro,Wang Sanxia,Fulton William B,Jia Hongpeng,Lu Peng,Prindle Thomas,Pamies David,Morris Meaghan,Chen Liam L,Sodhi Chhinder P,Hackam David J
Science translational medicine
Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4 T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into recipient mice lacking CD4 T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4 T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4 T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4 T lymphocytes into naïve recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ-releasing CD4 T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.
10.1126/scitranslmed.aay6621
Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis.
Egan Charlotte E,Sodhi Chhinder P,Good Misty,Lin Joyce,Jia Hongpeng,Yamaguchi Yukihiro,Lu Peng,Ma Congrong,Branca Maria F,Weyandt Samantha,Fulton William B,Niño Diego F,Prindle Thomas,Ozolek John A,Hackam David J
The Journal of clinical investigation
The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.
10.1172/JCI83356
Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities.
Cho Steven X,Rudloff Ina,Lao Jason C,Pang Merrin A,Goldberg Rimma,Bui Christine B,McLean Catriona A,Stock Magdalena,Klassert Tilman E,Slevogt Hortense,Mangan Niamh E,Cheng Wei,Fischer Doris,Gfroerer Stefan,Sandhu Manjeet K,Ngo Devi,Bujotzek Alexander,Lariviere Laurent,Schumacher Felix,Tiefenthaler Georg,Beker Friederike,Collins Clare,Kamlin C Omar F,König Kai,Malhotra Atul,Tan Kenneth,Theda Christiane,Veldman Alex,Ellisdon Andrew M,Whisstock James C,Berger Philip J,Nold-Petry Claudia A,Nold Marcel F
Nature communications
Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/T17 polarization. In murine NEC, pro-inflammatory type 3 NKp46RORγtTbet innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46RORγt ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.
10.1038/s41467-020-19400-w