Employing disease-associated genetic variants to study the influence of TLR5 and NLRP6 on gut immunity in humans

Abstract

The Nod-like receptor 6 (NLRP6) is a central innate immune receptor, which has drawn attention in mice models, particularly for its relevance in maintaining gut homeostasis. However, mechanistic insights on the molecular level and data regarding the role of NLRP6 in humans are limited. NLRP6 has been described to recruit the adaptor protein ASC and thereby activate NF-κB. Published data from our group indicates that a polymorphism coding for Leucine instead of Methionine at position 163 (M163L) within NLRP6 is associated with significantly higher risk for colorectal cancer (CRC) in humans. Another described exchange from Phenylalanine to Tyrosine (F361Y) is associated with SNP M163L. However, a functional impact of these SNPs on the molecular level and their contribution towards the development of CRC have not been investigated to date. I therefore studied these genetic variants in molecular model systems to gain an insight into possible functional changes and thereby glean general insights into how NLRP6 may participate in gut innate immunity. My in vitro studies showed that NLRP6 SNPs M163L and F361Y together enhance the interaction of NLRP6 with ASC, and F361Y induces higher NF-κB activation. This fits with the observation that CRC is induced in SNP carriers. Whole blood stimulation with respective TLR ligands and assessment of cytokine expression did not show a role of these SNPs in modulating different TLR signaling. Previously, Klimosch et al. reported functional SNPs of the flagellin receptor, TLR5, to be associated with CRC survival. Thus, we included these in the stool sample analysis on the effect of functional TLR5 and NLRP6 SNPs on gut immune parameters. By stimulation of, HEK cells stably expressing TLR5 with stool content (which contains shed intestinal flagellin) from different SNP carriers, we observed that TLR5 activation correlated with TLR5 SNP carriage: Stool samples from carriers of hyperactive TLR5 alleles revealed significantly reduced TLR5 activation, while increased TLR5 activation was observed for stool from loss-of-function carriers. For calprotectin levels, in stool samples from homozygous carriers for a hypofunctional TLR5 allele significantly reduced levels were observed. There was also a significant correlation with secretory IgA, a key regulator of intestinal homeostasis. Collectively, my results indicate reduced correlation between immune parameters, inflammatory stool characteristics and SNP carriage, and thus a direct relevance for TLR5 and NLRP6 variants for gut homeostasis and CRC. Metagenomic sequencing of the same samples is thus a plausible next step and currently conducted by our collaboration partners. Complementary analysis on immune and microbial parameters in stool samples of CRC patients and comparison with our results could thus further advance our knowledge in changes upon CRC disease development and find possible targets for individual genotype specific treatments or prevention strategies.