Figure 5. TLR2 mediates CDT recognition and is required for eosinophil suppression.

(A) BMDCs were generated from TLR2^−/−, TLR4^−/−, and TLR5^−/− mice before treatment with 200 ng/mL CDTa and 200 ng/mL CDTb (CDT) and 2 ng/mL Toxin A and 2 ng/mL Toxin B (TcdA/B) for 24 hours. IL-1β secretion was assessed by ELISA. (B) BMDCs were treated with 200 ng/mL CDTa and 200 ng/mL CDTb (CDT) and 2 ng/mL Toxin A and 2 ng/mL Toxin B (TcdA/B) for 24 hours in the presence of a TLR2 neutralizing antibody or an isotype control. IL-1β was assessed by ELISA. Data shown combined from three independent experiments of 3 replicates each (A–B). (C) 8 week old TLR2 knockout mice or C57BL/6J mice were infected with R20291 or R20291 CdtB- and monitored for survival (data combined from two independent experiments, n=14). (D) Mice were sacrificed on day 3 of infection and colonic eosinophils (CD45⁺ CD11b⁺ SiglecF⁺ SSC^hi) were measured by flow cytometry following tissue processing and staining (data combined from two independent experiments, n=8). (E) C57BL/6 mice received 4×10⁵ TLR2^−/− or B6 bone marrow-derived eosinophils (TLR2^−/− Eo or B6 Eo) via IP injection one day prior and for 3 subsequent days following infection with R20291 or R20291 CdtB-. Mice were monitored daily for survival (data combined from two independent experiments, n=13). (F) BM Eos were incubated for 8 hours with 200 ng/mL CDTa and 200 ng/mL CDTb in the presence or absence of anti-TLR2 neutralizing antibody (aTLR2) or anti-CDT neutralizing nanobody (aCDT). Eosinophils were stained with Live dead or Annexin V and cell death was assessed by flow cytometry, data shown are representative of 3 independent experiments assayed in duplicate. * = p value < 0.05, ** = p value < 0.01, *** = p value < 0.001 by Mann-Whitney test (A), Welch’s unequal variance t-test (B, D, F) or Kaplan-Meier Analysis (C, E). NS = not significant. Error bars shown represent S.E.M.