Fig 2: Neutrophils are activated (showing histone citrullination) in the circulation and myocardium of mice with collagen-induced arthritis (CIA).(A) Representative immunofluorescence images of isolated neutrophils from blood of mice with CIA or healthy control mice. Neutrophils from mice with CIA are pre-activated (H3Cit+; pink) and show a propensity for spontaneous NET formation. Representative microscopic picture of isolated neutrophils are shown (Scale bar 50 μm). Healthy control (left) and CIA (right). * = activated H3Cit+ neutrophil. (n = 6). (B) Graph illustrating, as a function of time, the neutrophil-to-lymphocyte ratio a marker of hyperinflammatory response, measured in peripheral blood in DBA/1 J mice with CIA and a healthy control, respectively (n = 6). (C) Gating and quantification of infiltrating Ly6G+/CD45+ and CD45+ cells using flow cytometry in healthy control and CIA myocardial tissue (n = 14). (D) Quantitative comparison of tissue levels of IL-1β an inflammatory cytokine-mediating fibrosis measured using ELISA (n = 9). (E) Representative LV sections of CIA mice and healthy control mice stained for DAPI+ (blue), Ly6G+ (red), and H3Cit+ (green) cells (scale bar: 100 μm) and a representative image of an activated neutrophil in the right panel with co-staining of Ly6G+ (red) and H3Cit+ (green) (scale bar 10 μm). Comparative analysis of the total number of Ly6G+ (red) and H3cit+ (green) cells in LV heart sections of CIA mice and healthy controls (n = 8). Data are mean ± SEM. (C, E) *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U test (C, E) t test.

Fig 3: Inhibition of PAD4 dampens thromboinflammation by decreased neutrophil infiltration, neutrophil H3Cit expression and reduced endothelial activation.(A) Representative immunostainings of LV sections and quantification of double-positive (Ly6G+ [red] and H3cit+ [green]) cells indicative of activated neutrophils in the myocardium of mice with collagen-induced arthritis (CIA) treated with the vehicle and mice with CIA treated with the PAD4 inhibitor, respectively (scale bar: 50 μm) (n = 7–8). (B) Quantification of infiltrating CD45+ and LY6G+ cells in the myocardial tissue of mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor, respectively, assessed by flow cytometry (n = 8). (C) Representative LV sections stained with DAPI+ (blue) and VWF+ (green). Quantification of VWF deposition in the myocardium of mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor (scale bar: 200 μm). (D) Myocardial tissue levels of IL-1β in mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired T test. (B) Wilcoxon matched pairs signed rank test.

Fig 4: scRNA-seq of CD45+ TILs from CMT167WT or CMT167AhR-KO five-week tumors reveals differences in TIL composition.A) CMT167WT or CMT167AhR-KO tumors excised five weeks after transplantation as in Fig. 7 were digested and sorted by flow cytometry for CD45+ cells. RNA from single cells was then sequenced. Greater than 2000 CD3high T cells were recovered from each sample. Sixteen unique CD3 Seurat clusters (#0–15) were identified using the Immunological Genome Project (ImmGen) reference compendium (43) and the singleR annotation method. B) Clusters were overlayed in green and purple to designate CD4 and CD8 cells respectively. C,D) Violin plots identify distinct CD4 (C) and CD8 (D) T cell populations. E) Clusters were overlayed burnt orange or teal to designate cells from CMT167WT or CMT167AhR-KO tumors, respectively. The orange polygon indicates the relative transcriptomic resemblance of clusters 2, 5, 6, 8 from CMT167WT tumors and the teal polygon indicates the relative transcriptomic similarity of clusters 13 and 14 from CMTAhR-KO tumors. F) Proportion of cells originating from CMT167WT (orange) and CMT167AhR-KO (teal) tumors within each Seurat cluster. The exact percentage of T cells from CMT167WT tumors is presented at the top.

Fig 5: CMT167AhR-KO tumors have a higher density of infiltrating CD4+ and CD8+ T cells than CMT167WT tumors.106 CMT167WT or CMT167AhR-KO cells were injected subcutaneously into syngeneic C57BL/6 mice. Tumors, if present, were excised between two and five weeks after cell injection. Approximately half of each five-week tumor was fixed and sectioned for immunofluorescent studies and the remaining tumor half digested to recover infiltrating leukocytes. A) Representative immunofluorescent images from a total of five CMT167WT and four CMT167AhR-KO five-week tumors (three sections/tumor) stained with DAPI (blue), AhR-specific antibody (green), and CD45-specific antibody (red) are shown. B) Mean density (number of cells/tumor mm2) + SE of CD45+ cells from five CMT167WT and four CMT167AhR-KO five-week tumors. C-E) Tumor infiltrating cells were recovered, counted, and stained for CD45, CD4, CD8, PD-1, and CD44 and analyzed by flow cytometry. Each dot represents the number of cells/mm3 (i.e. cell density) from one tumor. Data were obtained from two to 10 mice per group per week depending on tumors available to excise. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Multiple comparisons t tests). F). The percent of all CD4+PD-1+ (left) or CD8+PD-1+ (right) T cells that are also CD44high. Data from two independent experiments, six mice/condition/experiment, are presented as the percent positive + SE. **p<0.01, p<0.001 (Student’s t-test, equal variance).