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 2

NEC development requires an influx of lymphocytes into the lamina propria of the newborn mouse and human intestine.

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NEC development requires an influx of lymphocytes into the lamina propri...
(A–F) Representative H&E-stained histomicrographs of the terminal ileum from WT (A and B), Rag1–/– (C and D), or Rag1–/– mice that had been repopulated with naive donor CD4+ T cells (E and F) and were either control (A, C, and E) or induced to develop NEC (B, D, and F). Scale bars: 50 μm. (G and H) NEC severity score (G) and expression of IL-1β in the terminal ileum (H) for the indicated groups. (I–K, M–O) Representative confocal micrographs for DAPI (blue), E-cadherin (green), and 3-nitrotyrosine (red) (I–K) and H&E-stained sections (M–O) of the terminal ileum in mice 48 hours after the injection of CD4+ T cells elicited from mice with NEC into naive Rag1–/– hosts. Scale bars: 50 μm. (L) qRT-PCR showing the expression of IL-1β in the terminal ileum of Rag1–/– mice that had been injected either with no cells or with either CD4– or CD4+ cells from the terminal ileum of mice with NEC. (P–S) Flow cytometry showing the distribution of CD4+ T cells from mice pertaining to experiments shown in E and F or I–O above. Mouse data are representative of 3 independent experiments with 5 or more mice per group, as shown. Error bars indicate mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, by ANOVA for multiple comparisons.