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) Violin plot of traditional marker gene expression in stem cells, secretory progenitor cells, and goblet cells. Lgr5 and Olfm4 : stem cell marker, Mki67 ; proliferating cell marker, Atoh1 and Spdef ; secretory progenitor cell marker, Muc2 : Goblet cell marker, Lyz1 : Paneth cell marker. ( b) Heatmap of goblet and Paneth cell marker expression in adult (Haber et al: GSE92332( 16 )) and IL22Fc-treated P5 infant mice. ( c) Representative RNAScope images of Atoh1 (green) and Spdef (magenta) expression in the epithelial cells in the crypts of the distal small intestine of vehicle- and IL22Fc-treated infant mice. ( d, e) The number of Atoh1 + (d), or Spdef + (e) cells in the SI epithelium. Vehicle: n = 122 images from 4 animals, IL22Fc: n = 141 images from 4 animals. Unpaired t-test. ( f) Representative images of immunohistochemistry of E-Cadherin (red), Muc2 (green), and DAPI (blue) in the distal small intestine of vehicle- and IL22Fc-treated animals. White arrowheads point to the Muc2+ cells in the crypts. ( g) PAS staining of the distal small intestine of vehicle and IL22Fc treated animals. Arrowheads indicate PAS+ cells in the crypt. Vehicle: n = 63 images from 3 animals, IL22Fc: n = 65 images from 3 animals. ( h) Enumeration of PAS+ cells in the crypts. Unpaired t-test with Welch’s correction. ( i ) Western blot image of SI luminal wash detected with anti-Muc2 antibody. n = 5/group. (i) Appearance of luminal washes collected from control and IL22Fc-treated infant mice (left). Turbidity of luminal washes were measured as OD600 in a spectrophotometer (right). (j) Immunoprecipitation-western blot for Muc2 in luminal washes (right blot) and the signal intensity analyzed by densitometry (right bar graph). Signal intensity was normalized by the average intensity in control samples. n =5/group. ( k-m) CFU (k), FP (l), and replication (CFU/FP) (m) of V. cholerae in the small intestine of P5 infant Muc2 −/− vs littermate wild type or heterozygous controls (Muc2 +/+, +/− ) animals treated with (+) or without (−) IL22Fc. n = 6-28 animals/group. Bars represent the geometric mean for biological replicates. One-way ANOVA. Sample groups were compared among the same tissue. P-values are indicated in individual plots. Open circles indicate no CFU detected.

Article Snippet: Sections were incubated with antigen retrieval buffer (citrate buffer, pH 6.0) and microwaved for 10 min. Rehydrated tissue was blocked in blocking buffer for 30 min, stained with rabbit anti-Muc2 antibody (1:100, Proteintech) in blocking buffer overnight, washed in PBS, and stained with anti-rabbit IgG Alexa Flour 488 (1:200, ThermoFisher) at room temperature for 30 min.

Techniques: Marker, Gene Expression, Expressing, RNAscope, Immunohistochemistry, Staining, Western Blot, Control, Spectrophotometry, Immunoprecipitation