Pharmacological inhibition of IL12β is effective in treating pressure overload-induced cardiac inflammation and heart failure

Abstract

Background and objective: Emerging evidence indicates that inflammation regulates cardiac remodeling and heart failure (HF). IL12β is a subunit for proinflammatory cytokines IL12 and IL23. However, the effect of IL12β inhibition on HF development and the underlying mechanism is not understood.

Methods: We determined the effect of pharmacological inhibition of IL12β using IL12β blocking antibody on transverse aortic constriction (TAC)-induced left ventricular (LV) inflammation and HF development.

Results: IL12β blocking antibody significantly attenuated TAC-induced LV immune cell infiltration, hypertrophy, fibrosis, dysfunction, and the consequent pulmonary inflammation and remodeling. More specifically, we found that IL12β blocking antibody significantly attenuated TAC-induced LV and pulmonary infiltration of neutrophils, macrophages, CD11c⁺ dendritic cells, CD8⁺ T cells, and CD4⁺ T cells. Moreover, IL12β blocking antibody significantly suppressed the production of pro-inflammatory cytokine pro-IL1β and IFNγ by macrophages and IFNγ by CD8⁺ T cells and/or CD4⁺ T cells.

Conclusions: These findings indicate that pharmacological inhibition of IL12β effectively protected the heart from systolic overload-induced inflammation, remodeling, and dysfunction by reducing the proinflammatory signaling from both innate and adaptive immune responses.

Keywords: IL12β; T cells; heart failure; inflammation; lung remodeling; macrophages.

Figure 1

Pharmacological inhibition of IL12β attenuated TAC-induced cardiac dysfunction, LV hypertrophy, increases in lung weight, and RV hypertrophy in mice. (A) Schematic diagram of the experimental design. (B) Representative M-mode echocardiographic images of the indicated groups. (C–H) Quantified data of echocardiographic measurements of LV ejection fraction (LVEF), LV fractional shortening (LVFS), LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV end-systolic volume (LVESV), and LV end-diastolic volume (LVEDV), respectively. (I) The ratio of LV weight, left atrial (LA) weight, lung weight, RV weight, right atrial (RA), and total heart weight to tibial length (TL) of the indicated groups. (J, K) Representative wheat germ agglutinin (WGA) staining images and quantified data of LV cardiomyocyte cross-sectional area of the indicated groups. (L, M) Representative images and quantification of β-MHC expression in LV tissues of the indicated groups. *p<0.05; ^#p<0.05 IgG-treated TAC mice compared with the control; ^†p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; ^$p<0.05 anti-IL12β-treated TAC mice compared with the control; n = 5–7 mice per group.

Figure 2

Pharmacological inhibition of IL12β attenuated TAC-induced LV inflammation. (A, B) Representative images and quantified data of LV CD45⁺ leukocytes performed by immuno-histological staining. (C) Flow cytometry plot for the identification of CD45⁺ leukocytes in the LV. (D) Quantified data of the number of CD45⁺ leukocytes per LV. (E) The percentage of immune cell subsets within CD45⁺ leukocytes. (F) Quantified data of number of different immune cell subsets per LV. *p<0.05; ^#p<0.05 IgG-treated TAC mice compared with the control; ^† p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; Neutro, Neutrophils; Mφ, Macrophage; Mono, Monocytes; DCs, Dendritic Cells; NK, Natural Killer Cells; n = 3–5 mice per group.

Figure 3

Pharmacological inhibition of IL12β attenuated TAC-induced T cell accumulation in the LV and LV fibrosis. (A) Flow cytometry plots for the identification of CD3⁺ T cells in the LV. (B) Quantified data of the number of CD3⁺ T cells per LV. (C) The percentage of immune cell subsets within CD3⁺ T cells. (D) The number of different immune cell subsets per LV. (E–H) Representative LV interstitial and perivascular fibrosis and quantified data of fibrosis of the indicated groups, respectively. *p<0.05; ^#p<0.05 IgG-treated TAC mice compared with the control; ^† p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; n = 3–5 mice per group.

Figure 4

Pharmacological inhibition of IL12β attenuated pulmonary dysfunction and remodeling in wild-type mice. (A–G) Quantified data of resistance of respiratory system (Rrs), elastance of respiratory system (Ers), tissue damping (G), tissue elastance (H), inspiratory capacity (IC), compliance of respiratory system (Crs), and quasi-static compliance (Cst) of the indicated groups, respectively. (H, I) Representative images and quantified data of lung fibrosis performed by Sirius Red/Fast Green staining. (J, K) Representative images and quantified data of lung vessel remodeling performed by immuno-histological staining. *p<0.05; n = 5–7 mice per group.

Figure 5

Pharmacological inhibition of IL12β attenuated TAC-induced pulmonary inflammation. (A, B) Representative images and quantified data of infiltrated CD45⁺ leukocytes in the lung performed by immuno-histological staining. (C) The percentage of immune cell subsets within CD45⁺ leukocytes. (D) The percentage of different macrophage subsets within F4/80 macrophages. (E) The percentage of immune cell subsets within CD3⁺ T cells. *p<0.05; ^#p<0.05 IgG-treated TAC mice compared with the control; ^† p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; ^$p<0.05 anti-IL12β-treated TAC mice compared with the control; Mφ, Macrophage; DCs, Dendritic Cells; NK, Natural Killer Cells; AMφ, Alveolar Mφ; IMφ, Interstitial Mφ; MdMφ, Monocyte-derived Mφ; NKT, Natural Killer T Cells; n = 4–5 mice per group.

Figure 6

Anti-IL12β antibody treatment attenuated TAC-induced pulmonary F4/80⁺ macrophage accumulation and activation. (A) Flow cytometry plots for F4/80⁺ macrophages. (B) Quantified data of the percentage of F4/80⁺ cells within CD45⁺ cells. (C) Flow cytometry plots for the detection of MHCII expression in F4/80⁺ cells. (D, E) Quantified data of the percentage of MHCII^highF4/80⁺ cells within F4/80⁺ and CD45⁺ cells, respectively. (F) Quantified data of mean fluorescent intensity of MHCII in F4/80⁺ cells. (G) Representative histograms of MHCII expression in F4/80⁺ cells of the indicated groups. *p<0.05; n = 4–5 mice per group.

Figure 7

Anti-IL12β antibody treatment attenuated TAC-induced alveolar macrophage accumulation and activation of alveolar and interstitial macrophages. (A) Flow cytometry plots of lung alveolar macrophages (AMφ). (B) Quantified data of the percentage of AMφ, Ly6C^low interstitial macrophages (IMφ), and monocyte-derived Ly6C^high interstitial macrophages (MdMφ) within CD45⁺ cells. (C) Flow cytometry plots for the identification of IMφ and MdMφ. (D) Quantified data of the percentage of MHCII^highAMφ, MHCII^highIMφ, and MHCII^highMdMφ within CD45⁺ leukocytes. (E) Flow cytometry plots for the detection of MHCII expression in AMφ. (F) Quantified data of mean fluorescent intensity of MHCII in AMφ, IMφ, and MdMφ. (G-I) Quantified data of MHCII^highAMφ, MHCII^highIMφ, and MHCII^highMdMφ within AMφ, IMφ, and MdMφ, respectively. (J) Representative histograms of MHCII expression in AMφ, IMφ, and MdMφ of the indicated groups. *p<0.05; ^#p<0.05 IgG-treated TAC mice compared with the control; ^†p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; ^$p<0.05 anti-IL12β-treated TAC mice compared with the control; MHCII^high (MHCII⁺); n = 4–5 mice per group.

Figure 8

Anti-IL12β antibody treatment attenuated TAC-induced activation of pulmonary dendritic cells (CD11c^highF4/80^-). (A) Flow cytometry plots of lung CD11c^highF4/80^- cells. (B) Quantified data of the percentage of CD11c^highF4/80^- cells within CD45⁺ cells. (C) Flow cytometry plots for the detection of MHCII expression in CD11c^highF4/80^- cells. (D, E) Quantified data of the percentage of MHCII^highCD11c^highF4/80^- cells within CD11c^highF4/80^- cells and CD45⁺ cells, respectively. (F) Quantified data of mean fluorescent intensity of MHCII in CD11c^highF4/80^- cells. (G) Representative histograms of MHCII expression in CD11c^highF4/80^- cells of the indicated groups. *p<0.05; n = 4–5 mice per group.

Figure 9

Anti-IL12β antibody treatment attenuated TAC-induced pulmonary T cell activation. (A) Flow cytometry plots for the detection of the activation status of CD4⁺ T cells. (B) Quantified data of the percentage of CD44⁺CD62L^-CD4⁺ effector memory (T_EM) cells, CD44^-CD62L⁺CD4⁺ naïve cells (T_naïve), and CD44⁺CD62L⁺CD4⁺ central memory (T_CM) cells within CD4⁺ T cells. (C) Flow cytometry plots for the detection of the activation status of CD8⁺ T cells. (D) Quantified data of the percentage of CD44⁺CD62L^-CD8⁺ T_EM cells, CD44^-CD62L⁺CD8⁺ T_naïve cells, and CD44⁺CD62L⁺CD8⁺ T_CM cells within CD8⁺ T cells. (E) Quantified data of mean fluorescent intensity of CD44 in CD4⁺ T and CD8⁺ T cells. (F) Representative histograms of CD44 expression in CD4⁺ T and CD8⁺ T cells of the indicated groups. ^#p<0.05 IgG-treated TAC mice compared with the control; ^† p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; ^$p<0.05 anti-IL12β-treated TAC mice compared with the control; n = 4–5 mice per group.

Figure 10

Anti-IL12β antibody treatment attenuated TAC-induced pro-inflammatory cytokine production by pulmonary T cells and macrophages. (A) Flow cytometry plots for the detection of IFNγ production by CD4⁺ T cells. (B) Quantified data of IFNγ⁺ and IL17⁺ cells within CD4⁺ cells. (C) Flow cytometry plots for the detection of IFNγ production by CD8⁺ T cells. (D) Quantified data of IFNγ⁺ and IL17⁺ cells within CD8⁺ cells. (E) Flow cytometry plots for the detection of pro-IL1β production by F4/80⁺ macrophages. (F) Quantified data of IFNγ⁺, pro-IL1β⁺, and IL10⁺ cells within F4/80⁺ macrophages. ^#p<0.05 IgG-treated TAC mice compared with the control; ^†p<0.05 anti-IL12β-treated TAC mice compared with IgG-treated TAC mice; ^$p<0.05 anti-IL12β-treated TAC mice compared with the control; n = 4–5 per group.

Figure 11

Schematic diagram showing effect of anti-IL12β antibody treatment on TAC-induced heart failure development and progression. Neutralizing IL12β using anti-IL12β antibody attenuates TAC-induced LV inflammation, hypertrophy, dysfunction, and consequent pulmonary inflammation, remodeling, and right ventricular hypertrophy. DAMPs, Damage-associated molecular patterns; APC, Antigen presenting cell.