Inhibition of NK1.1 signaling attenuates pressure overload-induced heart failure, and consequent pulmonary inflammation and remodeling

Figures

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  Figure 1. Heart failure (HF) is associated with increased pulmonary IFN-γ+ NK1.1+ cells. (A, B) Flow cytometry analysis of the lung tissue shows an increased percentage of IFN-γ+ cells in the CD45+ subset in the HF mice. n=9-11. (C–E) In the IFN-γ+ subset, T cells (CD3+) and natural killer (NK1.1+) cells are the major cell populations (n=9-11). (F, G) Changes of percentages of IFN-γ+ CD4 T cells, CD8 T cells, NK1.1+NK cells, F4/80 macrophage, γδ T cells, and other immune cells after HF (n=4-5). (H, I) the alterations of IFN-γ producing capacity in the CD4+ T cells, CD8+ T cells, NK1.1+NK cells, and γδ T cells, as indicated by the histogram and geometric mean fluorescent intensity (n=4-5). ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 2. Depletion of NK1.1+ cells attenuated transverse aortic constriction (TAC)-induced left ventricle (LV) dysfunction in mice. (A) Schematic of experimental design for depletion of NK1.1+ cells with or without TAC-induced HF. (B) Representative images of echocardiography in the indicated groups. (C) Summarized echocardiography measurements of LV ejection fraction (LV EF), fractional shortening (LV FS). n=5-13. (D) Echocardiographic measurements of LV end-systolic diameter, and end-diastolic diameter. n=5-13. (E, F) Representative images of wheat germ agglutinin (WGA) staining and quantification of cardiomyocyte size in the indicated groups, showing that TAC-induced cardiomyocyte hypertrophy was not affected by depletion of NK1.1+ cells. (G-I) Tissue weight (LV, RV, and lung) to tibial length ratio. n=5-26. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CSA, cross-sectional area.

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  Figure 3. Depletion of NK1.1+ cells attenuated TAC-induced LV leukocyte infiltration and fibrosis in mice. (A, B) Representative images of Sirius Red staining and quantified LV interstitial fibrosis in the indicated groups. (C, D) Representative images of immunostaining of CD45 or MAC-2 and quantification of LV infiltration of CD45+ leukocytes in the indicated groups. (E, F) Representative images of immunostaining of LV MAC-2+ macrophage and quantification of the infiltration of CD45+ leukocytes or MAC-2+ macrophages. n=4-5. *p<0.05, **p<0.01, ***p<0.001.

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  Figure 4. Depletion of NK1.1+ cells attenuated TAC-induced RV inflammation and hypertrophy in mice. (A, B) Representative images of WGA staining and quantification of cardiomyocyte size in the indicated groups, showing that TAC-induced RV hypertrophy was not attenuated by depletion of NK1.1+ cells. (C, D) Representative images of Sirius Red staining and quantification of interstitial fibrosis in the indicated groups in the RV. (E, F) Representative images of immunostaining of CD45 and quantification of the infiltration of CD45+ leukocytes in the RV of the indicated groups. (G, H) Representative images of immunostaining of MAC-2 and quantification of the infiltration of MAC-2+ macrophages in the RV of the indicated groups. n=4-5. *p<0.05, **p<0.01, ****p<0.0001. CSA, cross-sectional area.

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  Figure 5. Depletion of NK1.1+ cells attenuated pulmonary leukocyte infiltration, fibrosis, and vascular remodeling. (A-F) Representative images of immunostaining of pulmonary CD45, MAC-2, or NK1.1 and quantified data in the indicated groups. (G, H) Representative images of Sirius Red staining and quantified interstitial fibrosis in the indicated groups in the lung. (I, J) Representative images of co-staining CD31 and α-smooth muscle actin (α-SMA) in the indicated groups, showing pulmonary vessel muscularization and remodeling. n=4-6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 6. Anti-NK1.1 antibodies effectively depleted NK1.1+ cells and cytokine-producing NK1.1+NK and NK1.1+NKT cells. (A, B) Representative plots of flow cytometry and quantification of NK1.1+ cell populations in the indicated groups. (C-E) Representative plots of flow cytometry and quantification of the percentage of NK1.1+NK and NK1.1+NKT cells in the CD45+ cell subset. n=4-6. (F-K) Quantification of the percentage of IFN-γ+, TNF-α+, granzyme A+ (GZMA), CD69+, NKG2A+, or perforin+ (PRF) NK1.1+NK cells in the CD45+ cell subset. n=4-6. **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 7. Effect of depletion of NK1.1+ cells on pulmonary IFN-γ+ CD4+ and CD8+ T cell subsets. (A, B) Representative images of flow cytometry plots of and relative percentage of IFN-γ+ cells in the CD4+ subset. (C, D) Representative images of flow cytometry plots of and relative percentage of IFN-γ+ cells in the CD8+ subset. (E, F) Relative percentage of TNF-α+ and PD1+ cells in the CD4+ subset. n=4-6. (G, H) Relative percentage of TNF-α+ and PD1+ cells in the CD8+ subset. n=4-6. *p<0.05, **p<0.01, ****p<0.0001.

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  Figure 8. Effect of depletion of NK1.1+ cells on pulmonary CD4+ and CD8+ T cell activation. (A-D) Representative images of flow cytometry plots of pulmonary CD4+ T cells and the relative percentage of naïve (CD44-CD62L+CD4+), effector memory (CD44+CD62L-CD4+), and central memory (CD44+CD62L+CD4+) subsets. (E–H) Representative images of flow cytometry plots of and the percentage of pulmonary naïve (CD44-CD62L+CD8+), effector memory (CD44+CD62L-CD8+), and central memory cells (CD44+CD62L+CD8+) in CD8+ T cells. n=4-6. *p<0.05, **p<0.01.

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  Figure 9. Effect of depletion of NK1.1+ cells on pulmonary macrophage and dendritic cell infiltration. (A, B) Representative images of flow cytometry plots of F4/80+ macrophages in the CD45+ population and relative percentages. n=4-6. (C, D) Representative images of flow cytometry plots of and relative percentage of F4/80+CD11c+ alveolar macrophages (AMφ) cells in the CD45+ subset. (E, F) Representative images of flow cytometry plots of and relative percentage of Ly6Clow/CD11b+ interstitial Mφ (IMφ) in the CD45+ subset. (G, H) Relative percentage of CD11c+ dendritic cells in the CD45+ subset. n=4-6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 10. Effect of depletion of NK1.1+ cells on pulmonary macrophage and dendritic cell activation. (A, B) Representative images of flow cytometry plots and relative percentage of MHC-II+ cells in the total F4/80+ Mφ subset. n=4-6. (C, D) Representative images of flow cytometry plots and relative percentage of MHC-II+ cells in the AMφ subset. n=4-6. (E, F) Representative images of flow cytometry plots of and relative percentage of MHC-II+ cells in the MdMφ population. (G, H) Representative images of flow cytometry plots of and relative percentage of MHC-II+ cells in the IMφ population. (I, J) Relative percentage of MHC-II+ cells in the dendritic cell subset. n=4-6. (K-N) Geometric mean of fluorescent intensity of MHC-II in the AMφ, MDMφ, IMφ, and DC population. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GEO mean, geometric mean.

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  Figure 11. Schematic showing NK and NKT-derived IFN-γ was significantly increased in pulmonary tissues after HF. Depletion of NK/NKT cells using anti-NK1.1 blocking antibodies significantly attenuated systolic overload-induced left ventricular inflammation, fibrosis, and dysfunction, as well as pulmonary leukocyte infiltration, fibrosis, vessel remodeling, and right ventricular hypertrophy. In summary, NK and NKT cells regulate systolic overload-induced HF development and progression by modulating cardiopulmonary inflammatory response.