Fig 2: Cardiomyopcyte‐derived IL‐18 by β‐AR activation contributed to Cx43 up‐regulation in fibroblasts in a paracrine fashion. Quantification of IL‐18 expression in cardiomyocytes by Western blotting analysis (A) (n = 6 mice/group), or concentrations by ELISA (B) in myocardium tissue lysate (n = 6 mice/group) and (C) in plasma (n = 6 mice/group) in 5‐month‐old NTG and β2‐TG mice. D, Immunofluorescence staining of WGA (green), IL‐18 (yellow), α‐SMA (red) and DAPI (blue) in LV myocardium from 5‐month‐old NTG and β2‐TG mice. Scale bar: 20 µm. Concentration of IL‐18 (E) in myocardium tissue lysate and (F) in plasma from control and 7‐day after ISO treatment measured by ELISA (n = 5‐7 mice/group). Concentration of IL‐18 (G) in primary cardiomyocytes in culture or (H) culture media harvested from cardiomyocytes with ISO treatment (1 μmol/L, 48 h) by ELISA (n = 4‐6 independent isolation/2 hearts). I, Western blotting images and quantification of band intensity for Cx43 expression in adult mouse cardiac fibroblasts treated with IL‐18 (10 ng/mL) for 48 h (n = 5 independent isolation/5 hearts). J, Representative images of Cx43 IHC staining in LV sections and (K) quantificational analysis for Cx43 positive area and the ratio of lateral to ID localized Cx43 area in LV sections of mice treated daily with IL‐18 nAb or IgG for 7 d commencing from ISO infusion (n = 7 mice/group). Scale bar: 50 µm. Arrows indicate lateralized Cx43 and arrowheads for ID Cx43 localization. Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs NTG or control. Statistical significance was determined by two‐tailed unpaired Student's t test

Fig 3: Schematics depicting gap junction remodelling in cardiomyocytes and fibroblasts induced by cardiac β‐AR activation. In both ISO stimulation and β2‐TG mouse models, β‐AR activation in cardiomyocytes suppressed Cx43 expression and shifted Cx43 localization from ID to the lateral side of cardiomyocytes. Meanwhile, Cx43 expression in fibroblasts, when tested in vivo and in vitro, were up‐regulated via direct activation of β2‐AR/cAMP/PKA signalling cascade as well as stimulation by IL‐18 released from cardiomyocytes upon β‐AR activation (curved arrow). These changes would increase the probability of intercellular coupling via gap junctions

Fig 4: Loss of NR2F6 reduces the splenic cDC1 and macrophage compartment.A Representative dot-plots of splenic DC (CD11c+MHC-II+) populations (out of CD45+Lin-BB20-Ly6C-F4/80-) in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. B Representative dot-plots of splenic XCR1+cDC1 (XCR1+CD11b-) populations derived from CD45+Lin-BB20-Ly6C-F4/80- CD11c+MHC-II+ cells in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. C Quantification of the frequencies of parent and of CD45+ splenic XCR1+cDC1 cells in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. D Quantification of IL-15Rα MFI in splenic DC populations in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. E Representative dot-plots of splenic Ly6C-B220-, monocyte (Ly6C+B220-), plasmacytoid DC (Ly6C+B220+), and B cell (Ly6C-B220+) populations in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. F Representative dot-plots of splenic macrophage (CD11bmidF4/80+) populations derived from Ly6C-B220- in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. G Quantification of frequency of parent and of CD45+ total splenic macrophages (CD45+Lin-B220-Ly6C-CD11bmidF4/80+) in wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. H Quantification of IL-15Rα expression (MFI) in splenic macrophage populations of wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. I Schematic overview of the experimental setup, splenic NK cells of wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice were isolated and expanded in vitro for 7 days with 50 ng/ml IL-15. NK cells were left unstimulated (M) or were stimulated for 5 hours with IL-12 + IL-18, α-NKp46, or co-cultured with B16-F10 tumor cells, IFNγ and TNFα cytokine levels were measured. J Frequency of IFNγ producing NK cells, K MFI of IFNγ expressing NK cells and L frequency of TNFα producing NK cells were quantified for wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) NK cells. A–H Representative data are shown as pooled experiments of two independent experiments n = 8. J–L The representative data shown are from one independent experiment out of two replicative experiments, with n = 4 per group and experiment. Each dot represents the data of an individual mouse. Results are shown as mean ± SD. The normality of data was evaluated by the Shapiro–Wilk test. An asterisk indicates statistically significant differences between genotypes calculated using Student’s t-test, or Mann–Whitney U test. A p value < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 5: NKp46 expression within Nr2f6-deficient NK cells in the blood is highly enhanced.A Representative dot plots, quantification of total cell numbers and NKp46 expression (MFI) of blood-derived NK cells (CD3-CD19-NK1.1+NKp46+) from wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. B Representative dot plots and quantification of percent of DNAM-1+ blood NK cells (CD3-CD19-NK1.1+NKp46+) from wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. C NKp46 expression (MFI) of isolated splenic NK cells from wild-type (Nr2f6+/+) or Nr2f6-deficient (Nr2f6−/−) mice. NK cells were cultured in vitro for 7 days in the presence of IL-15 and subsequently left unstimulated (M) or stimulated for 5 hours with IL-12 + IL-18, IL-15, or co-cultured with B16-F10 tumor cells. D Prediction of putative NR2F6 (NR2F COUP-TF) binding sites of the mouse Ncr1 promoter based on position weight matrix from the TRANSFAC database [35]. E NR2F6 binding to the Ncr1 promoter at -912bp was investigated by ChIP. Nr2f6+/+ or Nr2f6−/− sorted splenic NK cells were used with anti-NR2F6 or IgG2b control precipitation, Ncr1 promoter was quantified by qPCR, data are presented as relative to input. A, B Representative data is shown as pooled experiments of at least three independent experiments n = 11. C One of two independent experiments is shown with n = 4 per genotype and experiment. E Representative data is shown as pooled experiments of three independent experiments with n = 1 per genotype and experiment. Each dot represents the data of an individual mouse. Results are shown as mean ± SD. The normality of data was evaluated by the Shapiro-Wilk test. An asterisk indicates statistically significant differences between genotypes calculated using Student’s t-test. A p value < 0.05 was considered statistically significant. **p < 0.01***p < 0.001; ****p < 0.0001.