As shown in Fig. 5A, the stimulation of WT BMDC with PDWGF led to increased IL-1�� secretion that was markedly elevated when exogenous ATP was added. In contrast, PDWGF and ATP-stimulated BMDC �C deficient in NLRP3 or ASC proteins �C secreted very low levels of IL-1�� compared to the Ivacaftor WT BMDC. On the other hand, NLRP3 and ASC molecules were dispensable for the PDWGF-induced production of TNF-�� (data not shown), indicating an unimpaired cytokine production capacity of BMDC from these mouse strains. Additionally, in contrast to the induction of IL-1��, flow cytometric analysis of maturation markers (CD40, CD80, CD86) on BMDC revealed that PDWGF and LPS (as positive control) induced a similar increased maturation of BMDC, and that this effect was not dependent on the inflammasome component NLRP3 (Fig.

5B). Moreover, we found that, similar to human PBMC, treatment of mouse BMDC with caspase-1 inhibitor Z-YVAD-fmk resulted in a marked decrease of IL-1�� production from PDWGF and ATP-treated cells (Fig. 5C). These data suggest that PDWGF digest induces IL-1�� production through the NLRP3 inflammasome complex, and that caspase-1 is necessary for IL-1�� secretion in BMDC. Figure 5 PDWGF stimulates BMDC to IL-1�� production through NLRP3 and ASC. PDWGF Stimulates BMDC to IL-1�� Production through TLR, MyD88 and TRIF Our data showing the involvement of MAPKs JNK, ERK, and p38, and of the NF-��B signaling pathway in PDWGF-induced IL-1�� production from celiac PBMC (Fig. 4), led us to study upstream TLR signaling. First, we assessed the role of the MyD88 adaptor molecules, as well as TRIF, in the induction of IL-1�� by PDWGF.

BMDC from C57BL10 WT, MyD88, and TRIF KO mice were stimulated with PDWGF (100 ��g/ml) and ATP (2 mM), as described above. We found that the secretion of IL-1�� induced by PDWGF alone or in combination with ATP, was significantly reduced in MyD88 and TRIF-deficient BMDC (Fig. 6A). Consistently, the induction of pro-IL-1�� in response to PDWGF was abrogated in MyD88 KO BMDC and markedly reduced in TRIF KO BMDC (Fig. 6B). In contrast, the induction and secretion of IL-1�� was not reduced in BMDC deficient in IL-1R (Fig. 6B). These results suggest that TLR signaling is essential for pro-IL-1�� induction in response to PDWGF digest. Figure 6 TLR signaling is required for pro-IL-1�� synthesis in response to PDWGF.

By analyzing PDWGF-induced IL-1�� secretion in TLR2, TLR4, and TLR2/4 KO mice, we found that the secretion of IL-1��, induced by PDWGF, alone or in combination with ATP, was significantly reduced in TLR4 KO BMDC and abrogated in BMDC from mice deficient for both TLR2 and TLR4 Batimastat (Fig. 6C). Consistently, the induction of pro-IL-1�� in response to PDWGF was significantly reduced in BMDC deficient in TLR4, but not TLR2 (Fig. 6D).