Fig 4. Caspase-1 and Caspase-3 inhibition reduces shedding of infected epithelial cells in the lumen of PMN-HIOs and differentially affect bacterial burden and bacterial association with the epithelium.

A. Representative fluorescence microscopy images of TUNEL staining of HIO and PMN-HIO histology sections. HIOs were microinjected with STM and either cultured alone or co-cultured with PMNs in the presence of inhibitors for Caspase-1 (z-YVAD), Caspase-3 (z-DEVD), or DMSO control. B. Quantitation of the percent of lumen filled with TUNEL-positive cells of STM-infected HIOs or PMN-HIOs with indicated treatments. C. Quantitation of the percent of infected cells per HIO based on 3 fields per view per HIO. D. Quantitation of number of bacteria per infected cell in PMN-HIOs based on 3 fields per view per HIO. E. Quantitation of epithelium associated bacteria. Number of bacteria within 50μm of the apical epithelial surface were counted and normalized per 100μm distance. F. Fluorescent microscopy images of STM-infected PMN-HIO histology sections. Samples were stained for Salmonella (green), E-cadherin (red), and DAPI (blue). Unless otherwise stated, graphs show the mean +/-SD of n≥ 10 HIOs represented by dots from at least two independent experiments. Outliers were removed using the ROUT method with Q = 0.1%. Significance was determined by one-way ANOVA with post-Tukey’s test for multiple comparisons where *p<0.05, **p<0.01.