ANGPTL2 promotes immune checkpoint inhibitor-related murine autoimmune myocarditis

Fig. 1. ICI administration exacerbates EAM. a Schematic illustrating experimental design of the EAM model. b Representative images of H&E-stained heart tissues from indicated groups at day 10 of the EAM model. Scale bar, 100 μm. Naive mice were not subjected to the EAM model or treated with ICIs. c The area showing signs of inflammatory infiltration was scored from H&E-stained sections of ventricle shown in (b). Data are means ± SD; n = 4 per group. ****P < 0.0001; ***P < 0.001; *P < 0.05, one-way ANOVA test followed by Tukey’s multiple comparison test. d Immunofluorescent staining for CD45 (red) in heart tissues in naive mice or on day 10 of the EAM or EAM + ICI model. Nuclei are counterstained with DAPI (blue). Scale bar, 100 μm. e, f Percentage of indicated leukocyte subsets infiltrating the heart among total CD45+ cells at day 10 of the EAM or EAM + ICI model, based on flow cytometric analysis. Data are means ± SD; n = 4 per group. ns, not significant (P > 0.05); ***P < 0.001; **P < 0.01; *P < 0.05, two-way ANOVA test followed by Sidak’s multiple comparison test. g Absolute number of indicated leukocyte subsets per milligram heart tissue on day 10 of indicated groups. Data are means…

Fig. 2. Phenotypic characterization of cardiac fibroblasts in the EAM. a Schematic showing isolation of CD45-CD31-PDGFRα+ cardiac fibroblasts from naive, EAM only, and EAM + ICI mice. b qRT-PCR analysis of CD45-CD31-PDGFRα+ cardiac fibroblasts for transcripts of markers indicated at the top in indicated groups. Naive levels were set to 1. Data are means ± SD; n = 3 per group. ns, not significant (P > 0.05); ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA test followed by Tukey’s multiple comparison test.

Fig. 3. ANGPTL2 in heart is expressed predominantly by cardiac fibroblasts. a mRNA levels of indicated genes in WT whole heart tissues, BMDMs, in vitro-stimulated T cells, or CD45-CD31-PDGFRα+ cardiac fibroblasts (CFs). Values are normalized to those of 18s RNA. b Representative images of ANGPTL2 immunostaining in heart tissues from indicated groups at day 10. Scale bar, 100 μm.

Fig. 4. Angptl2-deficient mice show attenuated ICI-related EAM. a Schematic illustrating experimental design of the EAM + ICI model. b Representative images of H&E-stained heart tissues from WT and Angptl2-/- mice at day 10 of the EAM + ICI model. Scale bar, 100 μm. c Areas of inflammatory infiltration were scored from H&E-stained sections of ventricle shown in (b). Data are means ± SD; n = 4 for WT group, n = 7 for Angptl2-/- group. ***P < 0.001, unpaired t test. d Immunofluorescent staining for CD45 (red) in heart tissues from indicated genotypes on day 10 of the EAM + ICI model. Nuclei are counterstained with DAPI (blue). Scale bar, 100 μm. e Absolute number of indicated leukocyte subsets per milligram heart tissue from WT and Angptl2-/- mice on day 10 of the EAM + ICI model. Data are means ± SD; n = 6 for WT group, n = 9 for Angptl2-/- group. ns, not significant (P > 0.05); ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05, unpaired t test or Mann–Whitney test. f Representative M-mode echocardiograms from indicated groups at day 30 of the EAM + ICI model. Arrows indicate distance between systolic contraction (LVESD) and diastolic relaxation (LVEDD). FS (%) (g) and Heart weight/Body…

Fig. 5. Chemokine expression is downregulated in Angptl2-/- myCFs. a Myofibroblastic cardiac fibroblasts (myCFs) were isolated from heart and cultured. b Pdgfra and Acta2 transcript levels in CD45-CD31-PDGFRα+ CFs or myCFs. CF levels were set to 1. Data are means ± SD; n = 3 per group. **P < 0.01; *P < 0.05, unpaired t test with Welch’s correction. c Representative immunoblotting for ANGPTL2 in WT and Angptl2-/- myCFs. HSC70 served as a loading control. d Pdgfra and Acta2 transcript levels in WT or Angptl2-/- myCFs. WT myCF levels were set to 1. Data are means ± SD; n = 3 per group. ns, not significant (P > 0.05), unpaired t test. e qRT-PCR analysis of transcripts of indicated genes in WT or Angptl2-/- myCFs. WT myCF levels were set to 1. Data are means ± SD; n = 4 per group. ****P < 0.0001; ***P < 0.001; *P < 0.05, unpaired t test or Mann–Whitney test. f Schematic showing design of T cell migration assay. g Quantification of results from T cell migration assay of indicated groups based on flow cytometric analysis. WT myCF levels were set to 1. Data are means ± SD; n = 4 per group. ****P < 0.0001, unpaired t test.

Fig. 6. ANGPTL2 promotes chemokine expression in myCFs via NF-кB signaling. a qRT-PCR analysis of transcripts of indicated genes in Angptl2-/- myCFs treated with or without rANGPTL2. Levels in ANGPTL2-untreated Angptl2-/- myCF were set to 1. Data are means ± SD; n = 3 per group. ns, not significant (P > 0.05); ****P < 0.0001; **P < 0.01, unpaired t test or unpaired t test with Welch’s correction. b Quantification of T cell migration assay of indicated groups based on flow cytometric analysis. Levels in the presence of ANGPTL2-untreated Angptl2-/- myCF were set to 1. Data are means ± SD; n = 4 per group. ****P < 0.0001, unpaired t test. c Representative histograms of cell surface expression of integrin α5, β1, or PIR-B in WT myCFs. d qRT-PCR analysis of transcripts of indicated genes in rANGPTL2-stimulated Angptl2-/- myCFs pretreated with the NF-κB inhibitor BAY11-7085 (BAY) or vehicle (DMSO). Expression levels in Angptl2-/- myCFs not treated with either rANGPTL2 or BAY were set to 1. Data are means ± SD; n = 4 per group. ****P < 0.0001; ***P < 0.001, one-way ANOVA test followed by Tukey’s multiple comparison test.

Fig. 7. Model proposing regulation of ICI-related autoimmune myocarditis by ANGPTL2 derived from cardiac fibroblasts. In heart tissues, cardiac myofibroblasts express abundant ANGPTL2, when then promotes chemokine expression in myofibroblasts via integrin α5β1 signaling, enhancing recruitment of T cells and contributing to autoimmune inflammation.