Doses of PDWGF correspond to the expected concentrations Romidepsin of gliadin in the small intestine after a gluten-containing meal ,  and were used earlier in in vitro studies , . Subsequently, the secretion of IL-1��, IL-1�� and IL-18 in culture supernatants was evaluated. Unstimulated monocytes and PBMC from healthy donors, as well as from celiac patients, spontaneously secreted negligible levels of IL-1�� (Fig. 1A). Cells from healthy donors slightly increased IL-1�� production upon PDWGF stimulation, while patient? monocytes or PBMC strongly secreted (~2-3-fold higher) IL-1��, even already when stimulated with 50 ��g/ml. In contrast to robust IL-1�� production, we found measurable but far lower levels of PDWGF-induced IL-18 in cells from healthy donors and from celiac patients (Fig.
1B). Next, we tested the production of IL-1��. Celiac PBMC and monocytes secreted significantly higher levels of IL-1��, in a dose-dependent manner after PDWGF treatment, when compared to the cells from healthy donors (Fig. 1C). Figure 1 PDWGF digest induces IL-1��, IL-18, and IL-1�� release in monocytes and PBMC from CD patients. To exclude that the observed effect of PDWGF is due to LPS contamination, PDWGF was preincubated with PmB for neutralization of endotoxin. LPS preincubated with PmB was used as a control. As PmB was shown to directly trigger IL-1�� secretion by activating the NLRP3 inflammasome , we tested the effect of PmB on PDWGF-induced TNF-�� and IL-6 secretion. Preincubation with PmB completely abrogated LPS-induced TNF-�� and IL-6 production, but had no significant effect on PDWGF-induced TNF-�� and IL-6 production by celiac PBMC (Fig.
1D). PDWGF Induces IL-1�� Production in a Caspase-1 Dependent Manner To determine whether caspase-1 is required for PDWGF-induced IL-1�� production, we used Z-YVAD-fmk, a specific caspase-1 inhibitor. PBMC were pretreated with Z-YVAD-fmk (10 ��M) for 30 min and subsequently stimulated with PDWGF for 24 h. As shown in Fig. 2A, Z-YVAD-fmk reduced PDWGF-induced IL-1�� production by 70��10% in PBMC from CD patients, but had no significant effect on PDWGF-induced IL-1�� and TNF-�� production. Figure 2 The role of caspase-1 in PDWGF-treated PBMC. We further confirmed that PDWGF-induced IL-1�� production is caspase-1 dependent by western blot analysis. As shown in Fig.
2B, celiac PBMC treated with PDWGF for 24 h up regulated the expression of intracellular IL-1��, followed by the secretion and accumulation of mature IL-1�� into supernatants. Caspase-1 inhibitor Z-YVAD-fmk strongly reduced the amount of the processed form of IL-1�� in cell lysates, as well as supernatants, but had no effect on pro-IL-1�� expression. Entinostat In contrast, PDWGF stimulation of PBMC from healthy donors led to weak pro-IL-1�� production with no detectable mature 17 kDa IL-1�� form in cell lysates, nor cell culture supernatants.