Genetic inhibition of IL-12β suppresses systolic overload-induced cardiac oxidative stress, inflammation, and heart failure development

Figures

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  Fig. 1. Cardiac IL-12β expression was significantly increased after TAC. (A) Western blots and quantified data of LV IL-12β expression in WT sham and WT TAC mice. (B) Representative LV IL-12β staining images and quantified data of IL-12β+ area. (C) Representative images of IL-12β and CD45 co-immunostaining of WT mice LV tissue under sham and TAC conditions. The green stain in panel B is due to cardiac autofluorescence recorded at FITC channel. ∗p < 0.05; n = 5 per group.

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  Fig. 2. IL-12β KO significantly attenuated TAC-induced cardiac dysfunction, an increase in LV weight, LA weight, lung weight, and RV weight in male and female mice. (A) Representative M-mode echocardiographic images of WT and IL-12β KO male mice: pre-TAC, 2 weeks, 4 weeks, and 6 weeks after TAC, and Quantified data of echocardiographic measurements of LVEF, LVFS, LVESD, and LVEDD of both sexes. (B) Survival curves of WT TAC and IL-12β KO TAC mice of both sexes (log-rank test). (C–F) The ratio of LV weight, left atrial (LA) weight, lung weight, RV weight to tibial length (TL) of the indicated groups. (G) Representative LV WGA staining images and quantified data of cardiomyocyte cross-sectional areas. ∗p < 0.05; #p < 0.05 compared to WT sham; †p < 0.05 compared to WT TAC; $p < 0.05 compared to IL-12β KO sham; n = 7 to 22 per group for panels A-F and n = 4-5 per group for panel G; LVEF, LV ejection fraction; LVFS, LV fractional shortening; LVESD, LV end-systolic diameter; LVEDD, LV end-diastolic diameter.

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  Fig. 3. IL-12β KO attenuated TAC-induced alterations in the gene expression related to fibrosis and inflammation (A) Principal component analysis of WT and IL-12β KO mice, under sham or TAC conditions. (B) Venn diagram showing differentially expressed genes (DEGs) of WT and IL-12β KO mice under sham or TAC conditions, as well as the shared and uniquely changed genes. (C) DEGs cluster heatmap in WT mice compared to IL-12β KO mice, under sham or TAC conditions. (D) Volcano plots showing upregulated and downregulated genes among the groups. (E) Gene ontology (GO) biological processes enrichment bubble chart in WT TAC mice compared to IL-12β KO TAC. (F) GO cellular components enrichment histogram shows the enriched cellular components between KO and WT mice after TAC. n = 2 per group for sham and n = 3 per group for TAC.

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  Fig. 4. IL-12β KO suppressed LV genetic pathways associated with extracellular matrix remodeling, inflammation, and LV fibrosis in mice after TAC. (A) The top 20 upregulated and downregulated pathways between KO and WT mice after TAC, identified by GSEA with ReactomeGSA. (B) Representative GSEA plots of collagen formation and extracellular matrix organization. (C, D) Representative images and quantified data of LV interstitial and perivascular fibrosis performed by Sirius Red/Fast Green staining in male and female mice. #p < 0.05 compared to WT sham; †p < 0.05 compared to WT TAC; $p < 0.05 compared to IL-12β KO sham; n = 3 per group for panels A-B and n = 4-5 per group for panel D.

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  Fig. 5. IL-12β KO significantly attenuated TAC-induced LV inflammation in mice. (A) The top 20 upregulated and downregulated pathways between KO and WT mice after TAC were identified by GSEA with KEGG pathway analysis. (B) Heatmap shows the top LV immune and/or inflammation-related genes in KO and WT mice after TAC. (C, D) Representative LV CD45+ immuno-staining images and quantified data of LV CD45+ of the indicated groups. (E) Percentage distribution of major immune cell subsets within LV CD45+ cells of the indicated groups determined by flow cytometry. The green stain in panel C is due to cardiac autofluorescence recorded at FITC channel. #p < 0.05 compared to WT sham; †p < 0.05 compared to WT TAC; $p < 0.05 compared to IL-12β KO sham; n = 2-3 per group for panels A-B and n = 4-5 per group for panels D-E.

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  Fig. 6. IL-12β KO attenuated TAC-induced pulmonary inflammation, fibrosis, and vessel remodeling in male and female mice. (A, B) Representative images and quantified data of infiltrated CD45+ leukocytes in the lung performed by immuno-histological staining. (C, D) Representative images and quantified data of lung fibrosis performed by Sirius Red/Fast Green staining. (E, F) Representative images and quantified data of lung vessel remodeling performed by immuno-histological staining. #p < 0.05 compared to WT sham; †p < 0.05 compared to WT TAC; $p < 0.05 compared to IL-12β KO sham; n = 4-5 per group.

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  Fig. 7. IL-12β KO significantly attenuated TAC-induced early-phase LV dysfunction, cardiomyocyte hypertrophy, and inflammation. (A) Representative M-mode echocardiographic images and quantified data of echocardiographic measurements of LVEF, LVFS, LVESD, and LVEDD of the indicated groups. (B) The ratios of cardiac and pulmonary tissues to tibial length (TL) of the indicated groups. (C, D) Representative images and quantified data of LV cardiomyocyte size and LV CD45+ cells of the indicated groups. (E) Percentage of major immune cell subsets within LV CD45+ cells determined by flow cytometry. (F) Representative flow cytometry plots and quantified data of CD44+CD3+, CD44+CD4+, and CD44+CD8+ T cells within the corresponding T cells. The green stain in panel D is due to cardiac autofluorescence recorded at FITC channel. ∗p < 0.05; n = 5-10 per group.

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  Fig. 8. IL-12β KO significantly attenuated "stem cell-like" central memory T cell activation and their IFNγ production in the drainage lymph nodes. (A, B) Percentages of major immune cell subsets within CD45+ cells in the drainage lymph nodes determined by flow cytometry. (C) Percentages of IFNγ+CD8+ and TNFα+CD8+ within CD8+ T cells. (D, E) Percentages of CXCR3+CD4+ and CXCR3+CD8+ T cells within CD4+ and CD8+ T cells, respectively. (F) Percentage of effective memory (CD44+CD62L−), naïve (CD44−CD62L+), and central memory (CD44+CD62L+) T cells of CD8+ T cells. ∗p < 0.05; TEM, Effective Memory T Cells; Tnaïve, Naïve T Cells; TCM, Central Memory T Cells; n = 4-5 per group.

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  Fig. 9. IL-12β KO and anti-IL-12β antibody treatment significantly attenuated TAC-induced LV oxidative stress. (A) Representative dihydroethidium (DHE) staining images and quantified data of DHE intensity of the indicated groups. (B) Representative western blots and quantification of LV 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) of the indicated groups. (C-E) Representative DHE, 3-NT, and 4-HNE staining images, and quantified data of relative DHE, 3-NT, and 4-HNE intensity of the indicated groups. ∗p < 0.05; n = 5 to 6 per group.