Fig. 2: Loss of NLRP6 results in intestinal dysbiosis and barrier impairment correlating with steatohepatitis activity and tumor burden.

a Cecal microbiota composition of 13-week-old mice (WT (n = 8), Nlrp6^−/− (n = 9), NEMO^∆hepa (n = 13), NEMO^∆hepa/Nlrp6^−/− (n = 11)) was analyzed using 16S rRNA gene amplicon sequencing. Bar chart based on permutational multivariate analysis of variance (ADONIS) presenting the percentage of variance of the gut microbiota explained by the factors “Genotype” (R² = 0.057, **p < 0.006), “Cage” (R² = 0.073, **p < 0.004), “Nemo” (R² = 0.079, **p < 0.002),” Line”. Genotype - all included genotypes; Cage – individual cages; Nemo – WT & Nlrp6^−/− mice vs. NEMO^∆hepa & NEMO^∆hepa/Nlrp6^−/−; Line – WT & NEMO^∆hepa vs. Nlrp6^−/− & NEMO^∆hepa/Nlrp6^−/− (R² = 0.16, ***p < 0.001). b Analysis for differential abundance of microbiota via DESeq analysis of 13 weeks old NEMO^∆hepa (n = 13) and NEMO^∆hepa/Nlrp6^−/− (n = 11) mice. c Linear discriminant analysis (LDA) of effect size (LEfSe) between NEMO^∆hepa (n = 13) and NEMO^∆hepa/Nlrp6^−/− (n = 11). d Representative pictures of IF stainings of ZO-1 in ileum and colon of NEMO^∆hepa, NEMO^∆hepa/Nlrp6^−/− and respective controls (WT, Nlrp6^−/−). Nuclei were counterstained with DAPI, representative of 2 independent experiments; Scale bar: 100 µm. e, f Immunoblot analysis of ileum and colon protein extracts from 52-week-old mice for Occludin and β-actin as loading control of indicated genotypes, representative of 2 experiments. g RT-qPCR analysis of inflammatory mRNA expression (IL-1β, Il-18, TNFα, Mcp1, Ccl5) in the ileum of NEMO^∆hepa (n = 12) and NEMO^∆hepa/Nlrp6^−/− (n = 12) mice, unpaired t-test (IL-1β: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI 0.0235–2.421, p = 0.046; Il-18: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI 0.0293–0.7369, p = 0.035; TNFα: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI −0.210 to 0.595, p = n.s.; Mcp1: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI 0.097–1.932, p = 0.032; Ccl5: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI 1.023–4.706, p = 0.0038. h In vivo intestinal permeability assessed by oral gavage of FITC-dextran. Rel. induction of FITC-dextran permeability in NEMO^∆hepa (n = 6) and NEMO^∆hepa/Nlrp6^−/− mice (n = 4), unpaired t-test: NEMO^∆hepa vs. NEMO^∆hepa/Nlrp6^−/−, 95% CI 0.101–0.615, p = 0.012), pooled data from 2 independent experiments. i Strong correlation (Spearman) of intestinal barrier function evidenced by FITC-dextran permeability with tumor number in NEMO^∆hepa (blue dots) and NEMO^∆hepa/Nlrp6^−/− (red dots) mice (8 pairs), p = 0.0012 (two-sided), r = 0.96. All Data are presented as the mean ± standard error of the mean (SEM; experiments are considered significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Individual data points represent biological replicates unless otherwise stated. Source data are provided as a Source data file.