The nucleotide-binding domain (NBD) leucine rich repeat (LRR) containing proteins, NLRs, are intracellular sensors of PAMPs and DAMPs. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of pro-caspase-1 to caspase-1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoetic cells and has not been studied for inflammasome function. RNAi-mediated knockdown of NLRC5 nearly eliminated caspase-1, IL-1β and IL-18 processing in response to bacterial infection, PAMPs and DAMPs. This was confirmed in primary human monocytic cells. NLRC5 together with procaspase-1, pro-IL-1β and the inflammasome adaptor, ASC, reconstituted inflammasome activity which showed cooperativity with NLPR3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in an NBD-dependent but LRR-inhibitory fashion. These results invoke a model where NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.

The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat containing (NLR) family, in the development of allergic airway disease is currently controversial. Here, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathology, mucus production and airway hyperreactivity between wild type and Nlrp3-/- mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we also did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite (HDM) antigen exposure. While we did not observe significant phenotypic differences in any of the models tested, we did observe a significant reduction of IL-13 and IL-33 in Nlrp3-/- mice compared to wild type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the levels of the NLRP3 inflammasome associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However this difference did not greatly alter the clinical outcome of the disease. This suggests that the role of NLRP3 in allergic asthma has to be re-evaluated.

The cell type-specific expression of a gene is dependent on developmentally regulated modifications in chromatin structure that allow accessibility of basal and inducible transcription factors. In this study, we demonstrate that a cis-acting element in the second intron of the murine IL-4 gene has a dual function in regulating transcription in mast cells as well as chromatin accessibility of the IL-4 gene locus through its influence on the methylation state of the gene. Previous studies have shown that mast cell-restricted transcription factors GATA-1/2 and PU.1 associate with the intron element and regulate its activity. In this study, we use DNase I footprinting and mutational analyses to identify two additional sites that contribute to the element’s ability to enhance transcription. One of these sites associates preferentially with STAT5a and STAT5b. We also demonstrate that deletion of the element or mutation of the GATA binding site in the context of a stably integrated IL-4 genomic construct prevents maintenance of a demethylated locus in IL-4-producing mast cells. These data indicate that, analogous to Ig and TCR intron regulatory elements, the intron enhancer has an essential role in maintaining developmentally regulated demethylation at the IL-4 gene locus. In addition, they indicate that members of the GATA family of transcription factors likely play an important role in these processes.

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