Bacterial flagellin is a dominant, stable innate immune activator in the gastrointestinal contents of mice and rats

Abstract

Intestinal contents comprise the largest repository of immunogenic ligands of microbial origin. We undertook this study to assess the predominant microbe-associated molecular patterns (MAMPs) present therein and the receptors) that mediate the innate immune responses to them. Here, we demonstrated that intestinal contents from conventional, but not germ-free, mice and rats triggered robust innate immune responses in vitro and in vivo. Such immune responses were abrogated in the absence of either myeloid differentiation factor 88 (MyD88) or Toll-like receptor (TLR) 5, but not TLR4, suggesting that the stimuli was flagellin (i.e., protein subunit of flagella that drives bacterial motility). Accordingly, pre-treating intestinal extracts with proteinase, thereby degrading flagellin, was sufficient to block their ability to activate innate immune responses. Taken together, this work serves to underscore flagellin as a major, heat-stable and bioactive MAMP in the intestinal content that confers this milieu strong potential to trigger innate immune responses.

Keywords: Gut microbiota; IL-10; LPS; MyD88; cytokines; toll-like Receptor-4; toll-like Receptor-5.

Figure 1.

In vivo administration of intestinal extracts from conventional but not germ-free mice induced robust pro-inflammatory cytokine and chemokine responses. Eight-week-old female C57BL/6 mice (n = 5) were administered FE (2.0 mg equivalent of dry feces weight; i.P.) from either conventional (Conv) mice or germ-free (GF) mice. Control mice were given sterile PBS. (a) Body weight (%) at 0 and 24 h post-treatment. After 2 h post-treatment, mice were bled and hemolysis-free sera were analyzed for (b) IL-6, (c) KC, (d) serum amyloid a (SAA) and (e) Lipocalin-2 (Lcn2). Results were expressed as mean ± SEM. Statistical significance calculated using two-tailed Student’s t test for (a) and one-way ANOVA with Tukey’s post-hoc test for (b-e). **p < 0.01, ***p < 0.001.

Figure 2.

Intestinal extracts from conventional mice failed to induce pro-inflammatory cytokine response in Myd88 deficient mice. Eight-week-old male MyD88-deficient (Myd88KO) mice (n = 5) and their WT littermates (n = 3) were administered FE (2.0 mg equivalent of dry feces weight; i.P.) from conventional mice. Control (Con) mice were given sterile PBS. After 2 h, mice were bled, and hemolysis-free sera were analyzed for (a) IL-6 and (b) KC. Results were expressed as mean ± SEM. Statistical significance calculated using two-way ANOVA with Tukey’s post-hoc test. ***p < 0.01.

Figure 3.

Intestinal extracts-induced pro-inflammatory response is TLR5-dependent. Eight-week-old male WT, TLR4-deficient (Tlr4KO) or TLR5-deficient (Tlr5KO) mice (n = 3–5) were administered FE (2.0 mg equivalent of dry feces weight; i.P.) from conventional mice. Control (Con) mice were given sterile PBS. After 2 h, mice were bled, and hemolysis-free sera were analyzed for (a&b) IL-6 and KC in WT and Tlr4KO mice and (c&d) IL-6 and KC in WT and Tlr5KO mice. Results were expressed as mean ± SEM. Statistical significance calculated using two-way ANOVA with Tukey’s post-hoc test. ***p < 0.001.

Figure 4.

The immunogenicity of fecal extract was attributable to flagellin, which can be dampened by proteinase treatment or heightened by heating and depolymerization. (a) Flagellin immunoblot showing the absence and presence of flagellin in germ-free (GF) and conventional (Conv) mouse feces, respectively. (b&c) FE from Conv mice were digested with or without Proteinase K (1 mg/ml) overnight at 37ºC. Samples were boiled for 10 min to inactivate Proteinase K prior to administering to eight-week-old male mice (n = 6) at a dose of 200 µg protein equivalent (i.P.). After 2 h, mice were bled and hemolysis-free sera were analyzed for (b) IL-6 and (c) KC. (d&e) in a separate experiment, eight-week-old female mice (n = 3–5) were challenged with FE (2.0 mg equivalent of dry feces weight; i.P.) that were either semi-processed (SP: without boiling or sonication) or fully processed (FP: with boiling and sonication); see Methods section for details. After 2 h, mice were bled and hemolysis-free sera were analyzed for (d) IL-6 and (e) KC. Results were expressed as mean ± SEM. Statistical significance calculated using two-tailed Student’s t test for (b-c) and one-way ANOVA with Tukey’s post-hoc test for (d-e). **p < 0.01, ***p < 0.001.