Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

Abstract

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.

Commensal Propionibacterium strain UF1 mitigates intestinal inflammation via Th17 cell regulation

“…With this notion in mind, our studies demonstrate that HBM directs the fate of the bacterial composition of preterm infants and promotes the propagation of beneficial bacteria, including P.UF1, within preterm infants' gut microbiota; this critical trend was lacking in preterm infants fed formula. Importantly, this deficiency in the levels of beneficial bacteria undoubtedly contributes to uncontrolled inflammation that may potentially result in serious proinflammatory diseases such as NEC, wherein functional gut homeostasis is significantly impaired (14). This was evidenced by the fact that transfaunation of GF mice with FF fecal microbiota augmented proinflammation; however, addition of P. UF1 to this transfaunation mitigated the inflammatory responses and increased protective Th17 cells and Tregs.…”

“…However, further mechanistic insights are required to better understand the immune regulation orchestrated by HBM, the beneficial bacteria within, and critical induced metabolites, all of which may control T cell responses (e.g., Th17 cells) intricately involved in mucosal protection and the pathogenesis of proinflammatory diseases (13). This includes NEC, in which the recruitment of inflammatory cells is robustly increased, while functional Tregs are significantly decreased (14). Thus, if severe induced inflammation leads to NEC, its redirection by novel therapeutic approaches involving beneficial commensals, including Propionibacterium (P.) University of Florida 1 (UF1) (P. UF1), may enhance proinflammatory NEC regression.…”

Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18

“…Intriguingly, gut IL-17A mRNA was increased in experimental NEC in baboons (28), and recently IL-17A protein was shown to impair enterocyte tight junctions, increase enterocyte apoptosis, and reduce enterocyte proliferation leading to NEC in mice (29), highlighting the potential role of the IL-18/IL-17A axis in other neonatal inflammatory diseases. The deleterious role of excessive IL-17A, produced by many different cellular sources including a subset of γδT cells that can produce IL-17A rapidly via an IL-1R1-dependent pathway (30,31), is well established in the pathogenesis of severe inflammatory and ischemic-injury models, including sepsis (23,32,33).…”

“…Recently, IL-22 mRNA was shown to be modestly elevated (2-to 2.5-fold) above a fetal baseline in the intestine of humans with NEC and also elevated 1-to 1.25-fold above levels in control mice in neonatal mice with NEC (29). Because IL-18 plays a strongly deleterious role in the setting of sepsis, further studies are needed to determine the factors, including IL-22, contributing toward IL-18 production in neonates.…”

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

“…Human fetal tissue from less than 24-week gestation was obtained from the University of Pittsburgh Health Sciences Tissue Bank through an honest broker system after approval from the University of Pittsburgh Institutional Review Board and in accordance with the University of Pittsburgh anatomical tissue procurement guidelines. Approximately 100 isolated crypts were plated in each well of a 48-well plate onto a thin layer of Matrigel (Corning) and were grown in crypt culture media comprised of Advanced DMEM/F12 (Invitrogen) with 20% (vol/vol) HyClone ES Screened FBS (Fisher), 1% Penicillin/Streptomycin (Invitrogen), 1% L-glutamine, Gentamycin, 0.2% Amphotericin B, 1% N-acetylcysteine (100 mM; Sigma), 1% N-2 supplement (100×; Invitrogen), 2% (vol/vol) B27 supplement (50×; Invitrogen), Gibco Hepes (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 0.05 mM; Invitrogen), and ROCK Inhibitor Y-27632 (1 mM, 100×; Sigma) and supplemented with the following growth factors for the remainder of the respective experiments, with media changes occurring every 48 hours: 100 ng/mL WNT3a (Fisher), 500 ng/mL R-spondin (R&D), 100 ng/mL Noggin (Peprotech), and 50 ng/mL EGF (Fisher) (15,16). For image-based applications, enteroids were plated onto Matrigel in eight-well chamber slides (Nuc LabTek-II).…”