Coding variants in NOD-like receptors: An association study on risk and survival of colorectal cancer

Nod-like receptors (NLRs) are important innate pattern recognition receptors and regulators of inflammation or play a role during development. We systematically analysed 41 non-synonymous single nucleotide polymorphisms (SNPs) in 21 NLR genes in a Czech discovery cohort of sporadic colorectal cancer (CRC) (1237 cases, 787 controls) for their association with CRC risk and survival. Five SNPs were found to be associated with CRC risk and eight with survival at 5% significance level. In a replication analysis using data of two large genome-wide association studies (GWASs) from Germany (DACHS: 1798 cases and 1810 controls) and Scotland (2210 cases and 9350 controls) the associations found in the Czech discovery set were not confirmed. However, expression analysis in human gut-related tissues and immune cells revealed that the NLRs associated with CRC risk or survival in the discovery set were expressed in primary human colon or rectum cells, CRC tissue and/or cell lines, providing preliminary evidence for a potential involvement of NLRs in general in CRC development and/or progression. Most interesting was the finding that the enigmatic development-related NLRP5 (also known as MATER) was not expressed in normal colon tissue but in colon cancer tissue and cell lines. Future studies may show whether regulatory variants instead of coding variants might affect the expression of NLRs and contribute to CRC risk and survival

Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression

Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42

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

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.Dissertation ist gesperrt bis 22.03.2023

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease

Expression of selected CRC-associated NLRs in immune cells, primary tissue samples or CRC cell lines.

<p>mRNA expression of NLRP2 (A,B), NLRP5 (C), NLRP13 (D), NLRC5 (E) and NLRP3 (F) was determined relative to the housekeeper TBP by performing triplicate (means +SD show) qPCR using TaqMan gene-specific primers and probes on the indicated samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199350#sec002" target="_blank">Methods</a>). In the case of (E) HCT cells were treated with 1000 U/ml IFNγ or 50 ng/ml S. typhimurium Flagellin for 3 or 6 hours as indicated. TBP-relative ΔCt values were normalized to a reference sample (labelled R, ΔΔCt method). N denotes samples in which no expression was detectable above Ct within 40 cycles.</p

CRC risk: genotype distribution of SNPs analyzed in the Czech case-control population for SNPs with p ≤ 0.05.

<p>Amino acid changes are given as <> with the amino acid position indicated. Data adjusted for age at diagnosis and sex. Nominal significance at p≤0.05; significance level corrected for multiple testing (39 genotyped SNPs) at p≤0.001.</p