The findings on PDWGF-induced pro-IL-1�� and selleck screening library IL-1�� production in mouse BMDC were further confirmed in a human system, as anti-TLR4 mAb substantially reduced PDWGF-induced IL-1�� release by 70% in celiac PBMC, while anti-TLR2 mAb revealed no effect on PDWGF-induced IL-1�� release (Fig. 6E). Very recently, Junker et. al. [3] reported that nongluten wheat amylase inhibitors (AI) that copurify with ��-gliadins are present in gliadin digest and can stimulate IL-8 cytokine production via TLR4 pathway. Moreover, the highly disulfide-linked secondary structure of AI is necessary to activate TLR4. Thus, we tested whether wheat AI are able to stimulate IL-1�� secretion in our system. We found that AI stimulated celiac PBMC to robust secretion of IL-1��, comparable to those induced by PDWGF (Fig. 7).
Next, we evaluated whether reduction and alkylation of AI as well as PDWGF will affect the capacity to induce IL-1�� secretion. We found that reduction and alkylation of AI as well as PDWGF completely abolished IL-1�� secretion from celiac PBMC (Fig. 7). Figure 7 Reduction and alkylation (R/A) of PDWGF led to abrogated IL-1�� production. Discussion We have shown here, in line with previous study [7], that PDWGF is capable of inducing robust IL-1�� production by monocytes and PBMC in celiac patients. Moreover, we show for the first time that PDWGF induces significantly increased amounts of IL-1�� from monocytes and PBMC in celiac patients, and slightly elevated amounts of IL-18. Next, we investigated the molecular mechanisms underlying the PDWGF-induced production of IL-1�� in celiac PBMC.
In this study we clearly document that PDWGF-induced IL-1�� production by celiac PBMC is caspase-1 dependent. Interestingly, we observed that active caspase-1 was already present in unstimulated PBMC and PDWGF was able to markedly increase caspase-1 activation and the processing of pro-IL-1�� in celiac PBMC, in contrast to those of healthy donors. Our data correlate with prior findings that celiac patients display the active form of caspase-1 and mature IL-18 protein in small bowel mucosa [31]. Moreover, we have confirmed that PDWGF-induced IL-1�� release was dependent on NLRP3 and ASC, as shown by the stimulation of NLRP3?/? and ASC?/? BMDC. The role of the NLRP molecule in the predisposition to, or progression of CD remains unclear.
Our data propose that the GG genotype of the SNP rs10754558 NALP3 gene could play a protective role in celiac disease. Interestingly, a similar trend has been seen in the first study of Pontillo et al. [32], where Anacetrapib the G allele was protective against the development of CD (non-significantly), but this effect has not been confirmed in their following study [33]. However, similarly with our observation, neither study has found any NALP1 allele/genotype association with autoimmune disease.