Contribution of flagellin pattern recognition to intestinal inflammation during Salmonella enterica serotype typhimurium infection

Abstract

Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3alpha and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.

FIG. 1.

In vitro characterization of serotype Typhimurium flagellar mutants. The genotypes of the serotype Typhimurium strains IR715 (wild-type), SW473 (fliC fljB), SW215 (flgK), SPN315 (flgK fliC fljB), SW399 (invA), and SW358 (flgK fliC fljB invA) are indicated at the bottom of each panel. Strains chosen for in vivo comparison (flgK mutant and flgK fliC fljB mutant) are highlighted by a box (A to C) or black bars (D). (A) Staining for flagella using crystal violet. Bar, 5 μm. (B) Motility of serotype Typhimurium mutants on plates containing 0.3% agar. (C) Determination of flagellin amounts exported by serotype Typhimurium strains regardless of assembly into filaments. Sheared surface-associated structures, as well as bacterial culture supernatant, were precipitated with trichloroacetic acid and analyzed by Western blotting with rabbit anti-Hi serum (αHi). The molecular mass and position of standard protein is indicated on the left. (D) Invasion of T84 epithelial cells by serotype Typhimurium. Cells were infected for 1 h, washed with PBS, and then treated with gentamicin for 90 min before bacteria were counted. The data are shown as geometric means (bars) from three independent experiments ± the standard error. ns, not statistically significant.

FIG. 2.

MIP3A (A) and IL-8 (B) transcription in T84 cells is induced by bacterial flagellin. Bacterial culture supernatants were prepared by shearing off surface-associated structures, removing bacterial cells by centrifugation, and filter sterilization. T84 cells were stimulated for 1 h with 0.005 ml of sterile bacterial culture supernatant or 2 mg of purified serotype Typhimurium flagellin/liter. RNA was extracted and relative gene expression was determined by real-time qRT-PCR. The genotypes of the serotype Typhimurium strains IR715 (wild-type), SW473 (fliC fljB), SW215 (flgK), and SPN315 (flgK fliC fljB) are indicated at the bottom of each panel. Strains chosen for in vivo comparison (flgK mutant and flgK fliC fljB mutant) are highlighted by black bars. The data are expressed as fold increases of gene expression in infected cells over uninfected cells. The data are shown as geometric means (bars) from three independent experiments ± the standard error. ns, not statistically significant.

FIG. 3.

Flagellin-dependent IL-1β secretion by BMDM. BMDM from wild-type (A) or caspase-1-deficient (B) mice were treated with 100 ng of LPS/ml for 4 h and infected with serotype Typhimurium strains IR715 (wild-type), SW473 (fliC fljB), SW399 (invA), SW215 (flgK), or SPN315 (flgK fliC fljB) at an MOI of 5 for 18 h. The concentration of IL-1β in the culture supernatant was determined by ELISA. The data are shown as geometric means (bars) from three (A) or four (B) independent experiments ± the standard error. Strains chosen for in vivo comparison (flgK mutant and flgK fliC fljB mutant) are highlighted by black bars. ns, not statistically significant.

FIG. 4.

Changes in mRNA levels in the murine intestinal mucosa 12 h after serotype Typhimurium infection. (A to C) Relative expression of Il17A (A), Il6 (B), an Mip3a (C) in the middle cecum. Mip3a mRNA levels in the cecal patch containing distal cecum (D) and in ileal Peyer's patches (E). Streptomycin pretreated C57BL/6 wild-type and caspase-1 mice were infected with 10⁹ CFU of IR715 (wild-type), SW215 (flgK), or SPN315 (flgK fliC fljB). Fold changes were standardized to the mean expression level of the respective transcript in uninfected wild-type mice. The genotype of the mice infected with each bacterial strain is indicated below each panel. The data are shown as geometric means (bars) ± the standard error. Strains that differ only in their capacity to induce flagellin pattern recognition (flgK mutant and flgK fliC fljB mutant) are highlighted by black or white bars. ns, not statistically significant.

FIG. 5.

Changes in relative expression of the caspase-1-dependent transcripts Ifng (A), Mip2 (B), and Kc (C) in the middle cecum of streptomycin-pretreated mice induced by IR715 (wild-type), SW215 (flgK), or SPN315 (flgK fliC fljB). Relative gene expression was determined by real-time qRT-PCR, and fold changes were standardized to the mean expression level of the respective transcripts in uninfected wild-type mice. The genotype of the mice infected with each bacterial strain is indicated below each panel. The data are shown as geometric means (bars) ± the standard error. Strains that differ only in their capacity to induce flagellin pattern recognition (flgK mutant and flgK fliC fljB mutant) are highlighted by black or white bars. ns, not statistically significant.

FIG. 6.

Contribution of motility-mediated invasion and pattern recognition to fluid accumulation in bovine ligated ileal loops. Loops were infected with IR715 (wild type), SW215 (flgK), or SPN315 (flgK fliC fljB) for 2 h. The genotype of each bacterial strain is indicated below each panel. (A) Recovery of serotype Typhimurium from Peyer's patch-associated mucosa. Punches (6 mm) were washed three times with PBS and treated for 1 h with 0.1 mg of gentamicin/ml before determination of bacterial numbers. The data are shown as geometric means (bars) from four animals ± the standard error. (B) Fluid accumulation elicited by serotype Typhimurium. The data are shown as geometric means (bars) from three animals ± the standard error. Strains that differ only in their capacity to induce flagellin pattern recognition (flgK mutant and flgK fliC fljB mutant) are highlighted by black bars. ns, not statistically significant.

FIG. 7.

Changes in the relative expression of MIP3A (A), IL-8 (B), GRO (C), IL17A (D), and IFNG (E) in the mucosa of bovine ligated ileal loops 2 h after serotype Typhimurium infection. Loops were infected with IR715 (wild type), SW215 (flgK), or SPN315 (flgK fliC fljB). The genotype of each bacterial strain is indicated below each panel. The data are shown as geometric means (bars) from four animals ± the standard error. Strains that differ only in their capacity to induce flagellin pattern recognition (flgK mutant and flgK fliC fljB mutant) are highlighted by black bars. ns, not statistically significant.