Gut microbial Nordihydroguaiaretic acid suppresses macrophage pyroptosis to regulate epithelial homeostasis and inflammation

Abstract

Background: Aging is associated with increased severity of inflammatory bowel disease (IBD). Gut senescence and altered environmental factors contribute to changes in the intestinal metabolome, particularly in frail older individuals. However, the role of age-associated dysbiosis, characterized by a decline in beneficial gut microbiota and their metabolites, in exacerbating IBD remains unclear.

Methods: To investigate the impact of aging-associated dysbiosis on colitis development, we employed fecal microbiota transplantation (FMT) in wild-type and IL-10-deficient mice. Aged mice were treated with gut microbiota from either young or aged mice and then subjected to dextran sulfate sodium (DSS) to induce experimental colitis. 16S rDNA sequencing and metabolomics were used to analyze microbial and metabolite profiles. Single-cell RNA sequencing (scRNA-seq) was performed to characterize lamina propria CD45⁺ immune cell composition.

Results: Aged mice receiving microbiota from young mice exhibited less severe colitis than those receiving microbiota from aged mice, as evidenced by reduced disease activity, weight loss, and colonic shortening. Besides, aged mice displayed a significant decrease in the Lactobacillus population, accompanied by a reduction in Nordihydroguaiaretic acid (NDGA) levels. Decreased fecal NDGA levels were also observed in both IBD patients and elderly individuals. Administration of NDGA alleviated experimental colitis by downregulating the GSDMD/NR4A1/NLRP3 axis-mediated macrophage pyroptosis. Deletion of GSDMD in macrophages significantly diminished the protective effect of NDGA on colitis.

Conclusions: Our findings demonstrate that aging is associated with dysbiosis and reduced NDGA production, which increases susceptibility to intestinal inflammation. Gut microbial NDGA exhibits potential anti-inflammatory activity in colitis, suggesting a promising therapeutic target for aged-related IBD.

Keywords: Aging; IBD; NDGA; gut microbiota; macrophage pyroptosis.

Figure 1.

Gut microbiota remodeling alleviates experimental colitis in aged mice. (a) Experimental design of fecal bacterial transplantation; (b) The overall survival curves of DSS-treated mice; (c) bloody stools of DSS-treated mice; (d) body weight changes were daily monitored after DSS administration; (e) DAI were scored in DSS-treated mice; (f–g) mice were euthanized on day 7, and colon lengths were measured; (h-i) representative images of the histological-examined colon sections; (j-k) representative immunofluorescence images of tight junction proteins (occludin, ZO-1) in the DSS-treated colonic tissues and its quantitative analysis.

Figure 2.

Aging alters the gut microbiota community and leads to reduced fecal NDGA. (a–b) Box plots of alpha-diversity indices (Shannon and Chao); (c) principle coordination analysis (PCoA) between group Olderly(O) and group young(Y) microbiota colonies based on OTU abundance;(d) relative gut microbiota composition at the phylum level;(e) volcano plot showing the differential metabolites of group O and Y; (f) Principal component analysis (PCA) showing the correlation between the differential metabolites and the gut microbiota composition at the phylum level; (g) differential metabolites of group O and Y (TOP 10 differential metabolites); (h-i) fecal and serum NDGA levels were detected by targeted metabolomics in mice; (j-k) fecal NDGA levels were detected by targeted metabolomics in human; (l) correlation analysis between fecal NDGA levels and the clinical severity of IBD; (m) correlation heatmaps illustrate the relationship between differential strains and metabolites; (n-o) sector and Sankey plots demonstrate the association between differential strains and metabolites. (for 16s-seq and untargeted metabolomics, n = 10. For targeted metabolomics in mice, n = 5-8. For targeted metabolomics in human, young n = 20, aged n = 18, healthy control n = 38, UC n = 25 and CD n = 25).

Figure 3.

NDGA dose-dependently ameliorates DSS-induced colonic inflammation. (a) Experimental design of NDGA treatment in DSS-induced colitis; (b) the overall survival curves of DSS-treated mice; (c) body weight changes were daily monitored after DSS administration; (d) DAI was measured in DSS-treated mice; (e) bloody stools of DSS-treated mice; (f–g) mice were euthanized on day 7, and colon lengths were measured; (h–i) representative images of the histological-examined colon sections and the pathological scores were quantified; (j) fecal NDGA levels were detected by targeted metabolomics in mice.

Figure 4.

NDGA decreases the accumulation of colonic inflammatory macrophage subsets. (a) Column plots of different cell subsets in three samples; (b) tSNE mapping of cellular subpopulations (labeled datasets are inflammatory macrophage subpopulations); (c) tSNE distribution of the Lyz2 and IL-1β-positive cells; (d) KEGG pathway enrichment analysis of different macrophage subsets; (e) genes bubble maps of indicated macrophage subsets; (f) heatmap of overall differential gene expression of indicated immune cell clusters.

Figure 5.

NDGA decreases the accumulation of colonic inflammatory macrophage in the DSS-induced colitis model. (a) qRT-PCR detected pro-inflammatory factor levels in mouse colon tissues; (b) Immunofluorescence detection of F4/80 and IL-1β-positive cells in mouse colon tissues; (c–e) Counts of F4/80-positive, IL-1β-positive, and F4/80 and IL-1β-positive cells.

Figure 6.

NDGA inhibits LPS/ATP-induced macrophage pyroptosis in BMDMs. (a) qRT-PCR detected pro-inflammatory factor levels in BMDMs treated with NDGA at the indicated dosage; (b) qRT-PCR detected pro-inflammatory factor levels in BMDMs treated with NDGA (5μM) at the indicated time; (c–d) PI staining of BMDMs and the quantitative analysis of positive cells; (e) levels of LDH released from the supernatants of BMDMs.

Figure 7.

NDGA modulates the NR4A1-NLRP3 axis in pyroptotic macrophages. (a) Histogram of the number of differential genes between the two groups of BMDMs (n = 4); (b) volcano plot of differential gene expression; (c) principle coordination analysis between the two groups of BMDMs; (d–e) KEGG and GO signaling pathway enrichment analyses of the differential genes in BMDMs; (f) GSEA analyses demonstrating the pathways inhibited by NDGA;(g) heatmap showing the differential gene expression in two groups of BMDMs;(h) PPI analyses of the differential genes in BMDMs; (i–j) immunofluorescent co-localization assay of NLRP3 and NR4A1 in BMDMs and their quantitative analysis. (k–l) immunoblot detection of GSDMD, NR4A1, NLRP3 and Caspase1 protein levels in BMDMs and their quantitative analysis; (m) the caspase-1 activity in BMDMs detected using a specific detection kit; (n) Co-IP analysis of the protein interaction between NR4A1 and NLRP3 in BMDMs.

Figure 8.

NDGA protects against DSS-induced colitis in a GSDMD-dependent manner. (a) Experimental design of NDGA treatment in GSDMD^fl/flLyz2-Cre mice; (b) The overall survival curves of DSS-treated mice; (c) Bloody stools of DSS-treated mice; (d) Body weight changes were daily monitored after DSS administration;(e) DAI was measured in DSS-treated mice; (f–g) mice were euthanized on day 7, and colon lengths were measured. (h) representative images of the histological-examined colon sections and the pathological scores were quantified. (i) representative images of PAS staining in the DSS-treated colon and its quantitative analysis. (j–k) representative immunofluorescence images of tight junction proteins (occludin, ZO-1) in the DSS-treated colonic tissues and its quantitative analysis.

Figure 9.

Gut microbial NDGA suppresses inflammatory macrophage activation to regulate epithelial homeostasis and inflammation.This study demonstrates that age-associated dysbiosis leads to reduced NDGA production, resulting in the activation of inflammatory macrophages during colitis development. Conversely, NDGA intervention ameliorates intestinal inflammation and improves barrier function by inhibiting GSDMD/NR4A1/NLRP3-mediated pyroptotic cell death and cytokine release in macrophages.