Fig 1: Schematic illustration of F. nucleatum-induced macrophage inflammatory activation and colitis exacerbation through the succinic acid–SUCNR1–NF-κB axis. F. nucleatum colonization increases succinic acid levels in the intestine and circulation. Succinic acid engages its cognate receptor, SUCNR1, on intestinal macrophages and activates downstream NF-κB signaling, thereby promoting pro-inflammatory macrophage activation characterized by increased expression of CD86 and iNOS and enhanced production of inflammatory cytokines, including IL-6, TNF-a, and IL-1ß. These macrophage-associated inflammatory responses are accompanied by epithelial barrier disruption, reduced goblet cell abundance and mucus barrier integrity, increased epithelial apoptosis, and aggravated mucosal and systemic inflammation. By contrast, the fumarate reductase-deficient (frdA-KO) F. nucleatum strain, which exhibits impaired succinic acid production, shows a reduced capacity to induce macrophage inflammatory activation or aggravate colitis. Exogenous succinic acid restores these effects, whereas SUCNR1 knockdown or NF-κB inhibition attenuates succinic acid-induced macrophage inflammatory activation. Together, these findings support a model in which F. nucleatum aggravates colitis through a macrophage-centered succinic acid–SUCNR1–NF-κB signaling axis.
Fig 2: F. nucleatum-derived succinic acid promotes pro-inflammatory macrophage activation through SUCNR1. (A) Scatter plot showing differential metabolites between the culture supernatant of F. nucleatum (CSF, n = 4) and bacterial culture medium control (BHC, n = 3). Metabolites enriched in CSF are shown in red, and metabolites decreased relative to BHC are shown in green. (B) Venn diagram showing the overlap between CSF-enriched metabolites and significantly upregulated fecal metabolites in an independent quantitative IBD metabolomics cohort reported by Ning et al. (C) Quantification of succinic acid concentrations in CSF and BHC. (D, E) Succinic acid concentrations in colonic tissues (D) and serum (E) from mice after oral administration of F. nucleatum or PBS control (n = 6 per group). (F) Representative immunofluorescence images of SUCNR1 (red), F4/80 (green), and nuclei (DAPI, blue) in mouse colon tissues. Scale bars, 50 µm. (G) Western blot analysis of SUCNR1 expression in BMDMs. (H) Representative immunofluorescence images of SUCNR1 (red) and nuclei (DAPI, blue) in BMDMs. Scale bars, 20 µm. (I-J) RT-qPCR analysis of NOS2 and TNF-a mRNA expression in BMDMs. (K) Representative flow cytometry plots showing CD86 and CD206 expression in BMDMs. (L) Quantification of CD86+CD206− BMDMs. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 3: Succinic acid restores the impaired pro-inflammatory macrophage activation induced by the frdA-KO F. nucleatum strain. (A) Experimental design. C57BL/6J mice (n = 5 per group) were pretreated with antibiotics for 3 days, followed by oral gavage of wild-type F. nucleatum (Fn-WT), frdA-KO F. nucleatum, or PBS beginning on Day 0. Colitis was induced by administering 2.5% (w/v) DSS in drinking water from Day 14 to Day 22. Succinic acid supplementation (3%, w/v) was initiated 3 days before DSS exposure and continued throughout DSS treatment. (B) Representative immunofluorescence staining of F4/80 (green), iNOS (red), and nuclei (DAPI, blue) in colon tissues. (C, D) Representative flow cytometry plots and quantification of CD86+CD206− macrophages in colonic lamina propria. (E) Representative histogram showing CD86+CD206− macrophages in mouse intestinal tissues under different treatments. (F, G) Representative flow cytometry plots and quantification of CD86+CD206− BMDMs treated with LPS, Fn-WT + LPS, frdA-KO F. nucleatum + LPS, or frdA-KO F. nucleatum + succinic acid + LPS. (H) Representative immunofluorescence staining of iNOS (green) and nuclei (DAPI, blue) in BMDMs. (I) Western blot analysis of iNOS, Arg-1, and SUCNR1 expression in BMDMs. ß-tubulin was used as the loading control. (J–N) RT-qPCR analysis of NOS2, IL-1ß, TNF-a, IL-6, and CD206 mRNA expression in BMDMs. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.
Fig 4: F. nucleatum promotes a pro-inflammatory macrophage phenotype during intestinal inflammation. (A) Representative flow cytometry plots showing CD86 and CD206 expression in colonic lamina propria macrophages from PBS-, F. nucleatum-, DSS-, and F. nucleatum + DSS-treated mice. (B) Quantification of CD86+CD206- macrophages in colonic lamina propria.(C) Quantification of Ly6C+MHCII- macrophages in colonic lamina propria. (D, E) Representative histogram and quantification of TNF-a expression in intestinal macrophages. (F, G) Representative histogram and quantification of iNOS expression in intestinal macrophages. (H, I) Representative histogram and quantification of Arg1 expression in intestinal macrophages. (J) Representative immunofluorescence staining of F4/80 and iNOS in colonic tissues from the indicated groups.(K) Quantification of CD86+CD206- BMDMs under LPS-induced inflammatory conditions. (L) Representative flow cytometry plots showing CD86 and CD206 expression in BMDMs treated with PBS, F. nucleatum, LPS, or F. nucleatum + LPS.(M) Western blot analysis of iNOS and Arg1 expression in BMDMs from the indicated groups. ß-tubulin was used as the loading control. (N) Representative immunofluorescence staining of iNOS in BMDMs. (O) Representative immunofluorescence staining of CD206 in BMDMs. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001; ns, not significant.
Fig 5: Low-molecular-weight metabolites from F. nucleatum culture supernatant promote pro-inflammatory macrophage activation. (A) Representative flow cytometry plots showing CD86 and CD206 expression in BMDMs treated with LPS, F. nucleatum + LPS, HCSF + LPS, or LCSF + LPS. (B) Representative histogram showing the CD86+CD206− macrophage population in the indicated groups. (C) Quantification of CD86+CD206− BMDMs. (D) Western blot analysis of iNOS and Arg-1 expression in BMDMs from the indicated groups. ß-actin was used as the loading control. (E) Quantification of iNOS and Arg-1 protein expression normalized to ß-actin. (F) Representative immunofluorescence staining of iNOS in BMDMs. Nuclei were counterstained with DAPI. (G–H) RT-qPCR analysis of pro-inflammatory mediators, including NOS2 and TNF-a, in BMDMs. (I–J) RT-qPCR analysis of IL-10 and CD206 expression in BMDMs. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.
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