Fig. 1: Cathepsins B plays a major role in the regulation of NAIP/NLRC4 dependent-responses to cytosolic flagellin.

A Starch-elicited peritoneal macrophages (PMs) isolated from C57BL/6j wild-type (WT) mice were treated with LPS (500 ng/ml, 3 h) and stimulated with ultrapure flagellin extracted from Salmonella typhimurium in its free form (Fli) (1 μg/ml, 6 h), with empty DOTAP vesicles or with flagellin inserted into DOTAP (FliDot) (1 μg/ml, 6 h). IL-1β secretion was assessed in the culture supernatant by ELISA. The bars represent the average of four independent experiments performed in technical triplicates ± SD, **p < 0.01 (One-way ANOVA) when compared to cells treated with LPS alone or with LPS + DOTAP. B The secretion of the active forms of IL-1β and caspase-1 were detected by western blot (WB) of the cell culture supernatant. Pro-caspase-1, pro-IL-1β and β-actin were detected by WB in cell lysates (lys). Representative data from two independent experiments. C, D Bone marrow-derived macrophages (BMDM) from WT, Naip1-7^−/− and Nlrc4^−/− mice were treated with LPS and stimulated with empty DOTAP or FliDot. C IL-1β secretion was evaluated in the culture supernatant by ELISA. The numbers represent the mean ± SD of experimental triplicates. ****p < 0.0001 (Two-way ANOVA) when compared to WT cells. Data representative of three independent experiments. D Cytotoxicity was assessed by LDH release in the culture supernatant. The numbers represent the mean ± SD of experimental triplicates. ****p < 0.0001 (Two-way ANOVA) when compared to WT cells. Data representative of three independent experiments. E WT BMDMs were pretreated with the cathepsin B inhibitor Ca-074Me (25 μM) and primed with LPS. Next, the cells were stimulated with FliDot (1 μg/ml, 6 h). IL-1β release was evaluated in the culture supernatants by HTRF. The bars represent the average of three independent experiments performed in technical triplicates ± SD. **p < 0.01; ****p < 0.0001 (Two-way ANOVA) when compared to untreated cells. F PMA-differentiated (200 ng/mL, 24 h) THP-1 monocytic cells were transfected with ultrapure flagellin from S. typhimurium using Lipofectamine 3000 (1 μg/ml, 4 h) pretreated or not with cathepsin inhibitor Ca-074Me (25 μM). IL-1β secretion was assessed in the culture supernatant by ELISA. The bars represent quintuplicate of two independent experiments ± SD, *p < 0.05, ***p < 0.001 (One-way ANOVA) when compared to untreated cells. G WT BMDMs were pretreated with Ca-074Me or with the cathepsin L inhibitor CAA 0225, primed with LPS and stimulated with silica (250 μg/ml, 6 h) or with FliDot (1 μg/ml, 6 h). The bars represent the average of two independent experiments performed in technical triplicates ± SD. *p < 0.05 **p < 0.01; ****p < 0.0001 (Two-way ANOVA) when compared to untreated cells. H PMA-differentiated (200 ng/mL, 24 h) shRNA-scramble and shRNA-CTSB THP-1 cells were transfected with ultrapure flagellin from S. typhimurium using Lipofectamine 3000 (1 μg/ml, 4 h). IL-1β secretion was assessed in the culture supernatant by ELISA. The numbers represent the mean ± SD of experimental triplicates **p < 0.01, (Two-way ANOVA) when compared to untreated cells. Representative data from two independent experiments.