Inflammasome Activation Contributes to Interleukin-23 Production in Response to Clostridium difficile

C. difficile toxins and PAMPs induce IL-23 expression in human cells. MoDCs were generated from monocytes isolated from healthy human peripheral blood samples and were treated for 24 h with purified toxins A and B (2 ng/ml) or with filter-sterilized culture supernatants from C. difficile strain r20291 or an isogenic toxin mutant, R20291 AB-. RNA was isolated and assayed by quantitative reverse transcription-PCR for IL-23 gene expression. Student’s t test was used to determine statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data shown are representative of three independent experiments.

IL-1β secretion by BMDCs in response to C. difficile toxins is enhanced by a priming signal. (A) BMDCs were treated for 24 h with purified toxins A and B (2 ng/ml), LPS (100 ng/ml), or filter-sterilized culture supernatants from C. difficile strain R20291 or an isogenic toxin mutant, R20291 AB-. BMDC medium was assayed for IL-1β via an ELISA. (B) BMDCs were incubated with serum amyloid A (500 ng/ml) with or without toxins A and B (2 ng/ml). (C) Western blotting results for supernatants (Sup) and whole-cell lysate (Lys) from treated BMDCs. Blots were probed with antibodies directed against IL-1β p17 (supernatant) and β-actin (lysate). Lane 1 contains recombinant IL-1β as a positive control. Student’s t test was used to determine statsitical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data shown were combined from at least 3 experiments.

TLR4 and MyD88 signaling contribute to IL-23 production in response to C. difficile. BMDCs from C57BL6/J or MyD88^−/− mice (A) or C57BL6/J, TLR2^−/−, TLR4^−/−, or TLR5^−/− mice (B) were treated for 24 h with either LPS or filter-sterilized culture supernatant from C. difficile strain r20291 or the nontoxigenic strain R20291 AB-, in the presence or absence of purified toxins A and B (2 ng/ml). IL-23 protein levels were quantified with an ELISA; data in panel B are relative to 100% of C57BL6 levels under each condition. Student’s t test was used to determine statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. #, result below the detection level. Data shown were combined from 3 independent experiments.

IL-1β signaling contributes to IL-23 production. BMDCs were treated for 24 h with filter-sterilized culture supernatant from C. difficile strain R20291 or R20291 AB-. (A) Recombinant murine IL-1β (rIL-1β; 50 ng/ml), IL-1β neutralizing antibody (aIL-1β; 1 µg/ml), IL-1 receptor antagonist (IL-1RA; 500 ng/ml), or IgG isotype control antibody (1 µg/ml) was added, as indicated. (B) BMDCs from C57BL6 (wild-type [WT]) or IL-1R^−/− mice were treated for 24 h with filtrates from C. difficile strain R20291 or R20291 AB-, in the presence or absence of purified toxins A and B. Data are shown relative to 100% of the C57BL6 results under each condition. Data were combined from at least 3 independent experiments. Student’s t test was used to determine statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Inflammasome inhibition reduces IL-23 secretion. BMDCs were treated with filter-sterilized culture supernatant from strain R20291 in the presence of 40 mM KCl, 40 mM NaCl (A and B) or 20 µM YVAD-FMK or 25 µg/ml glybenclamide (C and D) for 24 h. IL-1β (A and C) and IL-23 (B and D) in the cell supernatant were measured in an ELISA. Student’s t test was used to determine statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were combined from at least 3 independent experiments.