Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis
Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam
Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam
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Research Article Gastroenterology

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.

Authors

Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam

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Figure 3

TLR4-dependent CCL25 release recruits pathogenic CD4+ T cells to the gut, leading to the development of NEC.

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TLR4-dependent CCL25 release recruits pathogenic CD4+ T cells to the gut...
(A–C) Flow cytometric analysis of CD4+ T cells (A), qRT-PCR for Foxp3 (B), and RORγt (C) in either WT mice, mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC), or mice overexpressing TLR4 in the intestinal epithelium (TLR4IEC–over). (D–G) Representative confocal micrographs of the terminal ileum from mouse (D and E) or human (F and G) without and with NEC stained for CCL25 (red), E-cadherin (green), and nuclei (blue). Scale bars: 50 μm. (H–J) qRT-PCR for CCL25 in the indicated strain in the presence or absence of NEC as indicated (H); expression of CCL25 in the supernatant by ELISA obtained from cultured enteroids from adult or newborn WT or TLR4ΔIEC mice that had been exposed to either saline or LPS (I); qRT-PCR showing expression of CCL25 in the newborn human intestine with or without NEC as indicated (J). (K–M) Representative H&E sections of the terminal ileum of C57BL/6 mice that were either control (K), subjected to NEC after administration of lentivirus with scrambled shRNA (L), or subjected to NEC after administration of shCCL25 lentivirus (M). Scale bars: 50 μm. Shown are the expression of iNOS in the terminal ileum (N), NEC severity score (O), percentage of CD4+ cells in the lamina propria (P), and expression of CCL25 by qRT-PCR in the indicated groups (Q). Mouse data are representative of at least 4 independent experiments with at least 4 mice per group for the in vitro data and 6 mice per group for in vivo analysis. Error bars indicate mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, by ANOVA for multiple comparisons.