Wild-type (WT) (B6.WT, black, n=13 for (A–E), and both co-housed B6.WT and B6.Nlrc4+/–Casp11+/–, black, n=9 for (F–O)), B6.Nlrc4–/–, and B6.Nlrc4–/–Casp11–/– mice were treated orally with 25 mg streptomycin sulfate in water and orally challenged the next day with 107 colony forming units (CFUs) of WT Shigella flexneri. In (A–E), B6.Nlrc4–/– mice received 200 μg of either TNFα neutralizing antibody (pink, n=13) or isotype control antibody (light blue, n=14) by intraperitoneal injection daily from 1 day before infection through sacrifice at 2 days post-infection. In (F–O), B6.Nlrc4–/–Casp11–/– mice received 200 μg of either TNFα neutralizing antibody (teal, n=12) or isotype control antibody (maroon, n=13) by intraperitoneal injection daily from 1 day before infection through sacrifice at 2 days post-infection. (A, G) Mouse weights from 0 through 2 days post-infection. Each symbol represents the mean for all mice of the indicated group. (B, H) Shigella CFUs per million cells from the combined intestinal epithelial cell (IEC) enriched fraction of gentamicin-treated cecum and colon tissue. (C, D, L–N) CXCL1, IL-1β, and TNFα levels measured by ELISA from homogenized cecum and colon tissue of infected mice. (E, O) Blood scores from feces collected at 2 days post-infection. 1=occult blood, 2=macroscopic blood. (F) Representative images of the cecum and colon from B6.Nlrc4–/–Casp11–/– mice receiving either isotype control or TNFα neutralizing antibody. (I, J) Quantification of cecum and colon lengths normalized to mouse weight prior to infection; cecum or colon length (cm)/mouse weight (g). (K) The ratio of fecal pellet weight when wet (fresh) divided by the fecal pellet weight after overnight drying. Pellets were collected at day 2 post-infection. (B–E, H–O) Each symbol represents one mouse. Data collected from three independent experiments (A–E) and two independent experiments (F–O). Mean ± SD is shown in (A, C, D, G, I–N). Geometric mean ± SD is shown in (B, H). Statistical significance was calculated by Mann-Whitney test in (A (day 2), B–D, G (day 2), H–M), by one-way ANOVA with Tukey’s multiple comparison test in (N), and by Fisher’s exact test in (E, O) where data were stratified by presence (score = 1 or 2) or absence (score = 0) of blood. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant (p>0.05).