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. 2020 Oct 19;9:e59022. doi: 10.7554/eLife.59022

Figure 4. Shigella-infected 129.Nlrc4–/– mice exhibit hallmarks of severe human shigellosis.

(A–H) 129.Nlrc4+/– and 129.Nlrc4–/– littermates were infected as described for Figure 2. Endpoint harvests were performed at 48 hr post-infection (p.i.). (A) Representative images of H&E stained cecum and colon tissue from infected mice. Scale bar, 200 μm. (B) Blinded quantification of histology score (cumulative) for tissues in (A). Edema, hyperplasia, inflammatory infiltrate, and epithelial cell death were scored from 0 to 4. The final score is the sum of individual scores from each category. (C) Quantification of cecum and colon lengths. Values were normalized to mouse weight prior to infection; cecum length (cm) / mouse weight (g). (D) Quantification of feces weights before and after dehydration at 2 days p.i. A larger ratio indicates diarrhea. (E) Mouse weights at 0 through 2 days p.i. Each symbol represents the mean for all mice of the indicated condition. Statistics refer to both WT Shigella-infected 129.Nlrc4+/– and 129.Nlrc4–/– mice and WT versus BS103 Shigella-infected 129.Nlrc4–/– mice at both 1 and 2 days p.i. All other comparisons were non-significant. (F) MPO levels measured by ELISA from feces of 129.Nlrc4+/– and 129.Nlrc4–/– mice collected -1 through 2 days p.i. (G) CFU determination from gentamicin-treated whole tissue homogenates from the cecum or colon (H) CFU determination from the IEC enriched fraction of gentamicin-treated cecum and colon tissue (combined). (I,J) Fecal blood scores from feces at two days p.i. 1 = occult blood, 2 = macroscopic blood. (I and J) show scores from two representative experiments. (K) Representative images of the cecum and colon dissected from 129.Nlrc4+/– and 129.Nlrc4–/– mice. Note the cecum tissue thickening (size reduction), macroscopic edema, and loose stool (absence of arrows), and vascular lesions and bleeding. (B–D,F–J) Each symbol represents one mouse. Filled symbols, WT Shigella; open symbols, BS103. Data are representative of three independent experiments. Mean ± SD is shown in (B, C, D, E, F). Geometric mean ± SD is shown in (G and H). Mann-Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant (p > 0.05).

Figure 4.

Figure 4—figure supplement 1. Construction and functional characterization of Nlrc4 knockout mice on the 129S1/SvImJ genetic background.

Figure 4—figure supplement 1.

(A) The targeted wild-type Nlrc4 sequence (chromosome 17, NC_000083.6, exon 5) aligned to the Nlrc4 guide RNA. The protospacer adjacent motif (PAM) is indicated. Below is a schematic of the Sanger sequencing verified product of CRISPR/Cas9-editing (129 Nlrc4 del), which results in a 10 base pair deletion and an in-frame early TGA stop codon in exon 5 of Nlrc4. (B) Quantification of cell death in 129 WT or Nlrc4–/– bone-marrow-derived macrophages (BMMs) treated with 10 µg/mL PA alone or PA + 10 µg/mL LFn-FlaA (LFn fused to Legionella pneumophila flagellin, ‘FlaTox’). Cell death was measured 30 min post-infection by propidium iodide uptake and reported as percent death relative to 100% killing by treatment with Triton X-100. (C) WT or 129.Nlrc4–/– mice were injected intravenously with 0.2 µg/g body weight PA + 0.1 µg/g body weight LFn-FlaA and body temperature was monitored for the indicated times (minutes) post-treatment. The initial temperature decrease in all mice is due to isoflurane treatment.