Journal: Cell reports
Article Title: MyD88 signaling in myeloid cells induces gastrointestinal tract injury and systemic inflammation after West Nile virus infection
doi: 10.1016/j.celrep.2025.116852
Figure Lengend Snippet: WT C57BL/6J mice were treated with anti-IFNAR1 mAb plus isotype control or anti-CCR2 mAb (25 μg/mouse) and subcutaneously inoculated with 10 2 FFU of WNV 1 day later. Two additional doses of isotype control or anti-CCR2 mAb were administered at 1 and 3 dpi. At 5 dpi, mice were administered fluorescently conjugated dextrans by oral gavage. (A-B and E-F) Confocal microscopy images of duodenal sections at 5 dpi. (A) WNV antigen (red), monocytes (CCR2, green), EpCAM (white), and nuclei (Hoechst 33258, blue). Scale bars, 100 μm (upper panel) or 50 μm (lower panel). (B) Quantification of CCR2-positive cells in the GI tract, expressed as percentage of total Hoechst 33258-positive cells per field. (C and D) Serum levels of 10 (C) and 250 (D) kDa dextran. (E and F) Quantification of WNV antigen-positive cells in the GI tract, expressed as percentage of total Hoechst 33258-positive cells per field (E) or as percentage of total EpCAM-positive epithelial cells per field (F). (G and H) Hematoxylin and eosin staining of liver sections from mice at 5 dpi. Scale bars, 100 μm (G). (H) Quantitation of liver injury. (I–K) Confocal microscopy images of cerebral cortex sections at 5 dpi showing Iba1 (white) and nuclei (Hoechst 33258, blue). Scale bars, 100 μm (upper panel) or 50 μm (lower panel) (I). (J) Iba1-positive area was quantified from at least 15 microglia/macrophage cells per field and normalized to the number of microglia/macrophage nuclei counted. (K) Numbers of Iba1-positive microglia/macrophages per field. Each data point is derived from at least three independent fields per mouse brain (J–K). (L–O) Cytokine levels of TNF (L and M) and IL-6 (N and O) in serum (L and N) and brain homogenates (M and O). (P–S) WNV RNA levels in the serum (P), spleen (Q), liver (R), and brain (S) at 5 dpi. Bars indicate median values from two experiments; dotted lines show LODs; n = 10 mice (A–F and I–S); n = 6 mice (G–H). Statistical analysis: Mann-Whitney test (ns: non-significant, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).
Article Snippet: Sections were incubated overnight at 4°C with rat anti-WNV hyperimmune serum (1:750 dilution), rabbit anti-EpCAM polyclonal serum (1:2000, Abcam) and goat anti-Iba1 polyclonal serum (1:500, ThermoFisher) or rabbit anti-CCR2 polyclonal serum (1:500, Novus Biologicals) diluted in 1X TBS containing 1% BSA, 3% normal donkey serum, and 0.1% Triton X-100.
Techniques: Control, Confocal Microscopy, Staining, Quantitation Assay, Derivative Assay, MANN-WHITNEY
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