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 6

TLR4-dependent proinflammatory environment of the premature intestinal mucosa predisposes to the development of NEC through the reversible STAT3-dependent induction of Th17 cells.

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TLR4-dependent proinflammatory environment of the premature intestinal m...
(A–J) qRT-PCR showing the expression of the indicated cytokines in human intestine from fetal bowel, active NEC, or healed NEC as indicated (A–D) or from mice without or with NEC from the indicated strain (F–I). (E and J) SDS-PAGE showing the expression of pSTAT3 in intestinal lysates from human (E) or mouse (J) as indicated; blots were stripped and reprobed for STAT3. (K–R) Representative H&E sections (K–N) and confocal micrographs stained for 3-nitrotyrosine (O–R) obtained from terminal ileum of control mice (K and O) or mice induced to develop NEC in the absence (L and P) or presence of the STAT3 inhibitor WP1066 (100 mg/kg/d oral, M and Q) or ATRA (50 μg oral daily, N and R). Scale bars: 50 μm. (S–W) Panels pertain to the groups of mice in K–R. Shown are the NEC severity score (S), qRT-PCR showing the expression of iNOS (T), flow cytometric analysis showing the percentage of positive CD4+ cells that costained for Foxp3+ or RORγt (U and V), and qRT-PCR showing the expression of IL-17 in the terminal ileum of mice as indicated (W). Data are representative of 2 experiments with 10 mice per group. Error bars indicate mean ± SD. ***P ≤ 0.001. ANOVA was used for multiple comparisons.