Pycard and BC017158 Candidate Genes of Irm1 Locus Modulate Inflammasome Activation for IL-1ß Production.

We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1ß, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1ß production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between "High" and "Low" responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-ß production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1ß response and the formation of ASC specks in stimulated cells. IL-1ß and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.

Novel Pycard gene polymorphism impairs Nlpr3 inflammasome-induced IL-1β production in mice selected for low inflammatory response

A SNP based linkage study in mouse lines phenotypically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR), mapped a major locus, Inflammatory response modulator 1 (Irm1) at 128Mb (3.5Mb interval) in chr 7 controlling AIR, measured by leukocyte and IL-6 levels in exudates and IL-1β production by circulating leukocytes after Nlpr3 inflammasome activation. Sequencing of this region in mice with extreme high or low IL-1β levels revealed 14 SNPs between the two groups, narrowing the locus interval to 420Kb. Candidate genes at Irm1 include Pycard with an undescribed exon 3 (C/T) mutation leading to E19K substitution at the pyrin domain, and Itgam, Rgs10 and BC017158 with intronic SNPs. In Pycard, the C allele was fixed in high responder AIRmax mice whereas the C and T alleles frequencies were 39% and 61%, respectively in AIRmin. We then investigated the effect of this novel Pycard SNP in inflammation phenotypes.Methods AIRmin mice bearing the 3 genotypes at Pycard: CC, CT, TT were produced by genotype-assisted mating. Inflammatory response was measured in AIRmax and in the 3 AIRmin sublines by the number of infiltrating cells and IL-6 concentration in the 24h exudate induced by sc Biogel P-100 bead injection and ex vivo IL-1β production by circulating leukocytes after E coli LPS (1 ug) and ATP (5mM) activation.Results IL-1β levels were similar in AIRmaxCC (4.5-±0.4 ng/ml) and AIRminCC (3.4±2.4 ng/ml) whereas AIRminCT produced 0.3±0.5 and AIRminTT <0.05 ng/ml IL-1β. Leukocyte influx and IL-6 levels in inflammatory exudates were not affected.Conclusion The E19K substitution in Pycard causes a negative effect in inflammasome activation for IL-1β production, without interfering in other inflammation phenotypes.