Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis
Charlotte E. Egan, … , John A. Ozolek, David J. Hackam
Charlotte E. Egan, … , John A. Ozolek, David J. Hackam
Published December 21, 2015
Citation Information: J Clin Invest. 2016;126(2):495-508. https://doi.org/10.1172/JCI83356.
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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 1

Development of NEC is associated with induction of Th17 cells and reduction in Tregs in the newborn intestine of mice and humans.

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Development of NEC is associated with induction of Th17 cells and reduct...
(A–H) Representative confocal micrographs from sections of the terminal ileum in mouse (A–D) and human infant (E–H) with or without NEC, immunostained for CD3 or CD4 as indicated. Characterization of the immune cell infiltrate in the lamina propria in mouse (I) and human (J) is shown. Scale bars: 10 μm. (K–M) qRT-PCR expression of the lamina propria CD4+ T cells in mice with and without NEC for the indicated lymphocyte transcription factors in murine CD4+ T cells. Ctrl, control. (N–R) Flow cytometric analysis of Foxp3+ (N and O) or Rorγt+ (P and Q) cells in the lamina propria of mice with and without NEC; quantification is shown in R. (S–W) Flow cytometric analysis of GFP+CD4+ cells in the small intestine of mice without (S and V) and with (T and W) NEC that expressed GFP either on the IL-17 promoter (S–U) or the Rorγt promoter (V–X); quantification is shown in U and X. (Y and Z) IL-17A ELISA in the intestinal lysates of mouse (Y) and human (Z). Human data are representative of 6 individuals for flow cytometry, 4 individuals for immunohistochemistry, and 6 for cytokine analysis. Mouse data are representative of 3 independent experiments with 5 mice per group in all cases. Error bars indicate mean ± SD. **P ≤ 0.01; ***P ≤ 0.001, Student’s t test when comparisons of 2 groups are made, by ANOVA for multiple comparisons.