West Nile virus triggers intestinal dysmotility via T cell–mediated enteric nervous system injury
Hana Janova, … , Thaddeus S. Stappenbeck, Michael S. Diamond
Hana Janova, … , Thaddeus S. Stappenbeck, Michael S. Diamond
Published August 29, 2024
Citation Information: J Clin Invest. 2024;134(21):e181421. https://doi.org/10.1172/JCI181421.
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Research Article Gastroenterology Infectious disease

West Nile virus triggers intestinal dysmotility via T cell–mediated enteric nervous system injury

Abstract

Intestinal dysmotility syndromes have been epidemiologically associated with several antecedent bacterial and viral infections. To model this phenotype, we previously infected mice with the neurotropic flavivirus West Nile virus (WNV) and demonstrated intestinal transit defects. Here, we found that within 1 week of WNV infection, enteric neurons and glia became damaged, resulting in sustained reductions of neuronal cells and their networks of connecting fibers. Using cell-depleting antibodies, adoptive transfer experiments, and mice lacking specific immune cells or immune functions, we show that infiltrating WNV-specific CD4+ and CD8+ T cells damaged the enteric nervous system (ENS) and glia, which led to intestinal dysmotility; these T cells used multiple and redundant effector molecules including perforin and Fas ligand. In comparison, WNV-triggered ENS injury and intestinal dysmotility appeared to not require infiltrating monocytes, and damage may have been limited by resident muscularis macrophages. Overall, our experiments support a model in which antigen-specific T cell subsets and their effector molecules responding to WNV infection direct immune pathology against enteric neurons and supporting glia that results in intestinal dysmotility.

Authors

Hana Janova, Fang R. Zhao, Pritesh Desai, Matthias Mack, Larissa B. Thackray, Thaddeus S. Stappenbeck, Michael S. Diamond

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Figure 3

WNV infection promotes infiltration of monocytes into the intestine.

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WNV infection promotes infiltration of monocytes into the intestine. (A–...
(A–E) Whole-mount preparations of the muscularis externa were isolated from the middle and distal regions of the small intestine from WNV-infected heterozygous Ccr2-GFP mice at (A and B) 6 or (C) 15 dpi and stained for (A) neuron (HuC/D) and macrophage (Iba1) markers, (B) WNV antigen and macrophage markers, or (C–E) macrophage markers. Yellow arrowheads indicate monocytes (CCR2 GFP+Iba1– cells). Scale bars: 100 μm. (A–C) Images were obtained from the myenteric plexus of the middle region of the small intestine from at least 2 experiments. (D) Monocytes (Ccr2 GFP+Iba1–) in the myenteric plexus are shown as the numbers of cells per mm2. (E) The fraction of Ccr2 GFP+ area (representing monocytes and/or monocyte-derived macrophages) in the myenteric plexus of WNV- or sham-infected mice. (F and G) Muscularis externa of the middle and distal small intestines from sham- or WNV-infected mice harvested at 15, 28, or 65 dpi were stained for Iba1+ macrophages. Macrophages in (F) the myenteric plexus and (G) the circular muscle layer are shown as the number of Iba1+ cells per mm2. Images of Iba1 staining in sham- or WNV-infected mice at 65 dpi. Scale bars: 100 μm. (H–J) GI transit was measured after oral gavage of carmine red dye (H) in sham- or WNV-infected mice (at 7 dpi) after treatment with anti-CCR2 or isotype mAbs (I) in WNV-infected Ccr2+/– and Ccr2–/– mice, and (J) in sham- or WNV-infected mice after treatment with anti-CSF1R or an isotype control mAb. (K–M). Whole-mount preparations of the muscularis externa were isolated from the middle region of small intestine of WNV-infected mice treated with anti-CSF1R or isotype mAbs and stained for (K) nNOS+ and calretinin+ neurons, (L) 5-HT+ neurons, or (M) S100β+ glia. Scale bars: 100 μm. The fraction of the area that stained positive for calretinin, nNOS, 5-HT, or S100β; values were normalized to those for sham-infected mice treated with an isotype control mAb. Data were pooled from (D and E) 2; (F and G) 2 (15 dpi); 3 (28 dpi) and 4 (65 dpi); (H) 3; (I) 1; (J) 3; and (K–M) 3 experiments. The numbers of mice per group were as follows: (D and E) n = 4–7; (F) n = 9–13; (G) n = 10–12; (H) n = 5–20; (I) n = 7–10; (J) n = 7–16; (K) n = 6–13; (L) n = 5–10; and (M) n = 7–13. Column heights in D–G and J–L indicate mean values, and lines in H–J indicate median values. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by (D, F and G) 2-tailed Mann-Whitney U test and (L) Kruskal-Wallis ANOVA with Dunn’s post test.