Kynurenic Acid/GPR35 Signaling Protects the Infarcted Heart by Suppressing Macrophage mtDNA-Triggered cGAS-STING Activation

Figures

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  Figure 1. KynA attenuates cardiac dysfunction after MI. (A) Experimental design. Sham-operated mice received vehicle alone for 7 days, while MI mice were administered intraperitoneal KynA or vehicle for 7 days post-MI. Echocardiography and Masson’s trichrome staining were performed at day 28. (B) Representative M-mode echocardiogram images acquired at 28 days post-MI. (C) Echocardiographic analysis of LVEF, LVFS, LVEDD, and LVESD was performed 28 days post-MI. n = 5–11 per group. (D) HW/BW and LW/HW ratios were determined 28 days post-MI. n = 5–11 per group. (E) Representative images (Left) and quantitative analysis (Right) of infarct scar size using Masson’s trichrome staining. Scale bar: 500 μM. n = 9–11 per group. (F) Representative images (Left) and quantitative analysis (Right) of cardiac fibrosis, as assessed by Masson’s trichrome staining, 28 days post-MI. Scale bar: 100 μm. n = 9–11 per group. Statistical comparisons: one-way ANOVA was performed in (C,D); 2-tailed unpaired t-test was performed in (E,F). * p < 0.05, ** p < 0.01, *** p < 0.001. KynA, Kynurenic Acid; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDD, left ventricular…

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  Figure 2. KynA attenuates proinflammatory macrophage population recruitment in the heart following MI. (A–E) Mice received daily vehicle or KynA treatment after MI. The animals were sacrificed on day 5 after MI and heart tissues were harvested for flow cytometric analysis. n = 6 per group. (A) The gating strategy of macrophages in the hearts. (B) Proportions of macrophages in the hearts of MI mice treated with vehicle or KynA. (C) Proportions of CD86+ (Left) and CD86− (Right) macrophages in the hearts of MI mice treated with vehicle or KynA. (D) Quantification of cardiac infiltrating macrophages post-MI. (E) Quantification of cardiac infiltrating subsets of CD86+ (Left) and CD86− (Right) macrophages post-MI. (F) Immunofluorescence co-staining for CD68 (yellow) and iNOS (green), scale bar = 50 μm, n = 5 per group. (G) Il1b, Il6 and Tnf mRNA expression levels in the hearts of mice treated with vehicle or KynA. n = 5–6 per group. (H) Experimental design. After MI, mice received PKH26-labelled BMDMs. The mice were then randomised to receive vehicle or KynA treatment for 5 days. (I) Representative flow cytometry plots (Left) and proportions (Right) of PHK26+ macrophages in the heart. n = 5–6…

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  Figure 3. The depletion of endogenous macrophages diminishes the protective effects of KynA. (A) Mice received intraperitoneal administration of clodronate liposomes or control liposomes. After 3 days, flow cytometry was applied to confirm systemic macrophage depletion. Representative flow cytometry plots (Left) and percentages (Right) of F4/80+ macrophages in the spleens of mice. n = 4–5 per group. (B) Experimental design. Mice received intraperitoneal administration of clodronate liposomes on day -1 before MI and days 2 and 5 after MI to deplete macrophages; control liposomes were used as the control. Mice received intraperitoneal injections of KynA or vehicle for 8 consecutive days after MI. Echocardiography and Masson’s trichrome staining were performed at day 28. (C) Representative echocardiogram images in M-mode (Top) and echocardiographic assessment of LVEF, LVFS LVEDD, and LVESD (Bottom) at 28 days after MI. n = 6–7 per group. (D) HW/BW and LW/HW ratios determined at 28 days after MI. n = 6–7 per group. (E) Representative images (Left) and quantitative analysis (Right) of infarct scar size using Masson’s trichrome staining. Scale bar: 1 mm. n = 6–7 per group. (F) Representative…

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  Figure 4. KynA attenuates the proinflammatory macrophage phenotype. BMDMs were collected after being treated with vehicle, LPS/IFNγ, or 10 mM KynA in combination with LPS/IFNγ for 12 h. (A) The mRNA expression of Nos2 across the experimental groups. n = 3–4 per group. (B) Representative flow cytometry plots (Left) and percentages (Right) of CD86+ macrophages among the indicated groups. n = 3 per group. (C) Representative flow cytometry histograms (Top) and MFI (Bottom) of ROS levels across the experimental groups. n = 3 per group. (D) Representative immunofluorescence images of DHE staining. Scale bar: 50 μm. n = 3 per group. (E) Representative flow cytometry histograms (Top) and MFI (Bottom) of mtROS levels across the experimental groups. n = 3 per group. (F) Representative immunofluorescence images of MitoSox staining. Scale bar: 50 μm. n = 3 per group. (G) Representative flow cytometry histograms (Top) and (Bottom) of MMP levels across the experimental groups. n = 3 per group. (H) Representative immunofluorescence images of Rho-123 staining. Scale bar: 50 μm. n = 3 per group. (I) Il1b, Il6 and Tnf mRNA expression levels among the indicated groups. n = 3–4 per group. (J) ELISA…

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  Figure 5. KynA inhibits proinflammatory macrophages via GPR35. BMDMs were transfected with either control siRNA or GPR35 siRNA. Subsequently, the cells were treated with either LPS/IFNγ alone or 10 mM KynA administered in combination with LPS/IFNγ for a duration of 12 h. (A) The mRNA expression of Nos2 across the experimental groups. n = 3 per group. (B) Representative flow cytometry plots (Left) and percentages (Right) of CD86+ macrophages among the indicated groups. n = 3 per group. (C) Representative flow cytometry histograms (Top) and MFI (Bottom) of ROS levels across the experimental groups. n = 3 per group. (D) Representative immunofluorescence images of DHE staining. Scale bar: 50 μm. n = 3 per group. (E) Representative flow cytometry histograms (Top) and MFI (Bottom) of mtROS levels across the experimental groups. n = 3 per group. (F) Representative immunofluorescence images of MitoSox staining. Scale bar: 50 μm. n = 3 per group. (G) Representative flow cytometry histograms (Top) and MFI (Bottom) of MMP levels across the experimental groups. n = 3 per group. (H) Representative immunofluorescence images of Rho-123 staining. Scale bar: 50 μm. n = 3 per group. (I) Il1b, Il6 and Tnf…

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  Figure 6. KynA-mediated inhibition of mtDNA leakage results in downregulation of the cGAS/STING pathway. (A,B) BMDMs were treated with either LPS/IFNγ alone or with 10 mM KynA in combination with LPS/IFNγ for 12 h. The BMDMs were harvested for RNA sequencing (RNA-seq) analysis after treatment. n = 4 per group. (A) Enrichment analysis of downregulated genes in the LPS/IFNγ + KynA treated group versus the LPS/IFNγ group. (B) Heatmap of selected enriched pathways based on downregulated genes in the LPS/IFNγ + KynA-treated group compared to the LPS/IFNγ group. (C) The mRNA expression of Cgas and Sting1 among the indicated groups. n = 3–4 per group. (D) Representative Western blotting images of the indicated protein expression levels across experimental groups. n = 3 per group. (E) Quantification of the indicated protein expression levels across experimental groups. n = 3 per group. (F) Quantitative analysis of mtDNA content, including Cox2, Atp6, and D-loop regions, across the experimental groups. n = 3 per group. Statistical comparisons: 2-tailed unpaired t-test was performed in (E); one-way ANOVA was performed in (C,F). * p < 0.05, ** p < 0.01, *** p < 0.001. GO, Gene Ontology; KEGG, Kyoto…

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  Figure 7. KynA suppresses the cGAS/STING pathway through GPR35-mediated inhibition of mtDNA leakage in macrophages. (A) The mRNA expression of Cgas and Sting1 was analyzed across the experimental groups. n = 3 per group. (B) Quantitative assessment of mtDNA content, including Cox2, Atp6, and D-loop regions, among the experimental groups. (C) Representative immunoblotting images of the indicated protein across the experimental groups. n = 4 per group. (D) Quantification analysis of the indicated protein across the experimental groups. n = 4 per group. (E) Schematic illustration of the experimental design for mtDNA stimulation in BMDMs. The mtDNA was extracted and transfected into BMDMs with 5 μg/mL after treated with either LPS/IFNγ alone or with KynA in combination with LPS/IFNγ. (F) Representative Western blotting images of the indicated protein across the experimental groups. n = 4 per group. (G) Quantification analysis of the indicated protein across the experimental groups. n = 4 per group. (H) ELISA quantification of IL-1β, IL-6, and TNF-α secretion in cell culture supernatants among indicated groups. n = 3 per group. Statistical comparisons: two-way ANOVA was performed in…

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  Figure 8. KynA/GPR35-mediated VDAC1 oligomerization controls cytosolic mtDNA release in macrophages. (A) Representative immunoblot images (Left) and corresponding quantitative analysis (Right) of VDAC1 expression across the experimental groups. n = 3 per group. (B) Representative immunoblot images (Left) and corresponding quantitative analysis (Right) of VDAC1 expression across the experimental groups. n = 4 per group. Statistical comparisons: 2-tailed unpaired t-test was performed in (A); two-way ANOVA was performed in (B). ** p < 0.01, *** p < 0.001. VDAC1, Voltage-dependent anion channel 1; Di, Dimers; Muti, multimers.