Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
The rheumatoid arthritis (RA) risk locus CCR6 rs3093024 SNP is associated with increased risk of RA in a sex-specific pattern in Asian populations. Specifically, the variant allele was associated with increased RA disease risk in ACPA-positive women. This SNP is in tight linkage disequilibrium with rs3093023, which has been associated with both RA risk and increased serum IL17 levels. We previously reported that these two SNPs altered estrogen receptor binding to estrogen response elements and resulted in the induction of CCR6 by estradiol (E2) in a SNP-dependent fashion. Specifically, CCR6 expression could be induced by E2 only in cells carrying the variant genotype. Strikingly, IL17A and IL17RA responded in a parallel fashion. The present study was designed to study mechanisms underlying the CCR6 SNP effect on the estrogen-dependent regulation of immune mediators in relation to the pathogenesis of RA—a disease for which 2/3 of patients are women.
“Human Variation Panel” lymphoblastoid cell lines (LCLs) consisting of LCLs from 300 subjects were used to obtain genome-wide mRNA expression and genome-wide SNP data, and to perform functional genomic studies including site-directed mutagenesis, luciferase reporter assays, qPCR, NF-κB reporter assays and co-immunoprecipitation.
A reporter construct with variant genotypes for the CCR6 SNPs increased luciferase activity in response to E2 treatment as compared to wild-type (p<0.001), providing direct evidence for the functional role of these SNPs in the regulation of CCR6 transcription. Use of the LCL model system allowed us to determine global mRNA expression profiling for functional study. We then performed pathway analysis using genes correlated with CCR6 expression with p≤1E-08 and found that those genes were enriched in immunological regulation pathways. Next, we validated genes significantly correlated with CCR6 expression, with a focus on immunological genes, including cytokines, chemokines and toll-like receptors, all of which have been implicated in the pathogenesis of RA. CCR6 knock down using LCLs with known CCR6 SNP genotypes resulted in significant changes (p<0.05) in the expression of CCL20, IL7R, IL7, IL17RA, IL17A, IL6R, IL6, TLR2, TLR4 and NF-κB p65. Additionally, significant induction for IL17RA, TLR2, TLR4 and NF-κB p65 in response to E2 treatment was observed only in variant genotype cells (p<0.05). In parallel, NF-κB transcriptional activity responded in a CCR6 SNP and E2-dependent fashion. Finally, CCR6 could physically interact with NF-kB p65 as determined by co-immunoprecipitation. These results suggest that NF-κB signaling might be associated with CCR6 SNP and estrogen-dependent effects.
CCR6 SNPs are functionally relevant to the pathogenesis of RA. CCR6 is estrogen inducible in a SNP-dependent fashion, resulting in downstream effects modulating the expression of proinflammatory cytokines and NF-κB transcriptional activity. Our results provide a mechanistic explanation for the results of previous genome-wide association studies that have linked these SNPs in the CCR6 gene to RA risk and the differential effects of these SNPs on E2 treatment.
To cite this abstract in AMA style:Ho MF, Bongartz T, Ingle JN, Wang L, Weinshilboum RM. Rheumatoid Arthritis Risk Polymorphisms in CCR6, SNP and Estrogen-Dependent Response to Immune Mediator Gene Expression, and NF-κb Transcriptional Activity: Crosstalk between the Immune and Endocrine Systems [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/rheumatoid-arthritis-risk-polymorphisms-in-ccr6-snp-and-estrogen-dependent-response-to-immune-mediator-gene-expression-and-nf-%ce%bab-transcriptional-activity-crosstalk-between-the-immune-and-endoc/. Accessed October 17, 2021.
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