Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) Experimental scheme of scRNA-seq analysis of epithelial cells. Infant mice (P5) (n=3/group) were infected with V. cholerae and sacrificed 20 hours post-infection (hpi). The proximal and distal SI were collected, and epithelial cells were isolated. Single-cell RNA libraries were prepared from sorted Epcam+ cells. (b) UMAP of the distal small intestine epithelial single-cell RNA-seq data from uninfected and V. cholerae infected P5 infant mice (n=3 for each condition, cells were pooled); EEC, enteroendocrine cells. The arrow indicates cluster 5 enterocytes in infected animals in the distal SI. ( c) Top-hit pathways from the Single Cell Pathway Analysis (Qval > 8.75 in any cluster). Dot plot represents enrichment in color and Qval in circle size. Stem, stem cells, EC, enterocytes, Gob, Goblet cells, EEC, enteroendocrine cells. ( d) Volcano plot showing q-value (Qval) and pathway enrichment from Single Cell Pathway Analysis. Pathway enrichment indicates a mean pathway change comparing uninfected and V. cholerae -infected conditions. Colors represent the following pathways: IFN (red), TNFα and Inflammatory (yellow), Mitosis (green), Translation and Ribosome (light blue), Golgi, ER, and mTOR (pink), and Metabolism (dark blue). ( e) Percentage of cells in each cluster vs the total cell number in the distal SI. ( f) GO pathway analysis of cluster 5 representative genes. (g) Fgb and Sult6b2 gene detection by single molecule RNA FISH co-stained with DAPI (blue) in the distal SI tissue at 20 hpi. White arrowheads in the higher magnification image indicate the expression of Fgb (green) and Sult6b2 (magenta) mRNA in epithelial cells. Dotted line indicates the epithelial cell membrane. Signal intensity was adjusted to visualize single puncta in the high magnification image. Color-separated images are shown in Extended Data. Fig.3b. ( h) Representative immunostaining of Reg3β (red), V. cholerae (magenta), and DAPI (blue) in the distal SI from an uninfected animal (left) or V. cholerae -infected animal (right). Scale bar: 100 μm. White arrows show the colocalization of Reg3β and V. cholerae on the epithelium. ( i) Deconvoluted z-stack image of V. cholerae (green) and Reg3β (red) from a puncta of Reg3β/ V. cholerae colocalization image. Overlay image was taken by confocal microscope using x100 lens and z-stack with 0.3 micron gaps per layer. (j) Pearson’s correlation coefficient between Reg3β and V. cholerae co-localization using the images of ( h ) from infected animals. 55 images from 4 animals were analyzed and plotted in a violin plot. ( is partially created in https://BioRender.com .)

Article Snippet: Reg3β knockout (C57BL/6NCrl-Reg3bem1(IMPC)Mbp/Mmucd, RRID:MMRRC_067360-UCD) was obtained from the Mutant Mouse Resource and Research Center (MMRRC) at University of California at Davis, an NIH-funded strain repository, and was donated to the MMRRC by Kent Lloyd, D.V.M., University of California, Davis.

Techniques: Infection, Isolation, RNA Sequencing, Staining, Expressing, Membrane, Immunostaining, Microscopy

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) UMAP of epithelial single-cell RNA-seq data of the distal small intestine in P5 infant mice in control and IL22Fc-treated animals. ( b) Comparison of cluster abundance between IL22Fc-treated and untreated control animals. ( c) Dot plot representing control vs. IL22Fc-treated gene expression changes of cluster 5 cell marker genes (Fig. S3A) in epithelial cell subsets. Color represents average log 2 fold change of gene expression compared to untreated control. Dot size represents negative log 10 -scaled adjusted p-value. ( d) V. cholerae growth curve measured by OD600 after incubation with native or heat-inactivated Reg3β (left). Percent V. cholerae survival after incubation with indicated concentration of Reg3β (right). Data represent mean ± SD for experimental replicates; native Reg3β (n = 4/condition), heat-inactivated Reg3β (n = 2/condition). ( e-g) CFU (e), founding population (Ns) (f), and CFU/Ns (g) of V. cholerae in the proximal and distal SI at 16 hpi. Infant mice were orally administered 2 doses of vehicle (PBS, n = 4) or Reg3β recombinant protein (33 μg/dose, n = 5) at −0.5 and +1 hpi. Bars represent the geometric mean for biological replicates. One-way ANOVA. ( h-j) CFU (g), Ns (h), and CFU/Ns (i) of V. cholerae in the proximal and distal SI of P5 Reg3β-/- mice and heterozygous littermate at 16 hpi. Bars represent the geometric mean for biological replicates. Open circles indicate no CFU detected. One-way ANOVA. Sample groups were compared among the same tissue. P-values are shown.

Article Snippet: Reg3β knockout (C57BL/6NCrl-Reg3bem1(IMPC)Mbp/Mmucd, RRID:MMRRC_067360-UCD) was obtained from the Mutant Mouse Resource and Research Center (MMRRC) at University of California at Davis, an NIH-funded strain repository, and was donated to the MMRRC by Kent Lloyd, D.V.M., University of California, Davis.

Techniques: RNA Sequencing, Control, Comparison, Gene Expression, Marker, Incubation, Concentration Assay, Recombinant