Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis

Figure 1.. IL-3 exacerbates cardiac inflammation in EAM. (A) Schematic diagram of the experimental design. EAM was induced by injecting 100 µg cardiac αMHC emulsified in CFA on days 0 and 7. Mice were killed before or 10, 21, or 45 d after the first immunization. (B) Representative H&E staining of the hearts from WT and Il3−/− mice at peak of inflammation (day 21). Bars, 100 µm. (C) H&E-stained sections as in B were scored for inflammation by percentage of myocardium infiltrated with mononuclear cells (n = 6–7 per group of two independent experiments). (D) Serum cardiac troponin-I (cTnI) levels in WT and Il3−/− mice were measured by ELISA on day 21 (n = 6–7 per group of two independent experiments). (E) Representative flow dot plots of WT and Il3−/− heart tissue cell suspensions to assess inflammatory cells on day 21. (F and G) Flow cytometry–based quantification of indicated leukocyte subsets in the hearts of WT and Il3−/− mice before and 10 and 21 d after the first immunization (n = 5–15 per group of at least two independent experiments). (H) Representative Azan staining of the hearts from WT and Il3−/− mice on day 45 and quantification of cardiac fibrosis (n = 7 per group of two…

Figure 2.. T cell–derived IL-3 is essential to cardiac inflammation in myocarditis. (A) Il3 mRNA levels in the heart (HT), BM, spleen (Sp), draining LN, thymus (TH), and lung (LG) before and 8, 14, and 21 d after the first immunization (n = 6–9 per group representing two independent experiments). nd, not detected. (B) Representative flow dot plots of heart tissue cell suspensions to identify IL-3+ cells on day 21. (C) Further flow cytometric characterization of IL-3–producing CD4+ T cells by costaining for IFN-γ, IL-17A, and IL-4 in the inflamed heart. (D) T cells were isolated by draining LNs of either WT or Il3−/− immunized mice on day 14 and culturing with WT BMDCs in the presence or absence of the indicated peptide (10 µg/ml) for 72 h. Culture supernatants were collected, and IL-3 levels were measured by ELISA. MOG, myelin oligodendrocyte glycoprotein. (E) Schematic diagram of T cell adoptive transfer–induced EAM. (F) Quantification of total leukocyte numbers in the hearts of recipient Scid mice (n = 6–7 per group of two independent experiments). (G and H) WT mice were lethally irradiated and reconstituted with a mixture of BM cells obtained from Rag1−/− and either WT or Il3−/− mice at…

Figure 3.. IL-3 is dispensable for T cell sensitization. (A) In vivo T cell proliferation in the draining LNs was measured by BrdU incorporation before and 10 and 21 d after the first immunization (n = 4–8 per group of two independent experiments). BrdU was injected intraperitoneally 2 h before the sacrifice. (B) In vitro T cell proliferation was assessed by a cell tracer dye, Cell Trace Violet. CD4+ T cells obtained from LNs of immunized WT or Il3−/− mice were stained with Cell Trace Violet and cultured at indicated conditions for 72 h (n = 4–8 per group of three independent experiments). (C) Enumeration of CD4+ T cell numbers in the draining LNs before and 10 d after the first immunization (n = 4–8 per group of two independent experiments). (D) Representative flow dot plots to identify DC subsets in the draining LNs. (E) Quantification of migratory cDCs, resident cDCs, and moDCs in WT and Il3−/− draining LNs on days 0 and 10 (n = 4 per group of two independent experiments). (F) Production of IFN-γ and IL-17A by WT and Il3−/− CD4+ T cells in the draining LNs on day 21. (G) Percentage of IFN-γ+ or IL-17A+ CD4+ T cells in the draining LNs on day 21 (n = 4–8 per group of two independent…

Figure 4.. Monocyte-derived APCs promote local T cell proliferation in the inflamed heart. (A) CD4+ T cell proliferation was measured by BrdU incorporation in the inflamed hearts (HT) and blood of WT and Il3−/− mice 21 d after the first immunization. BrdU was injected 2 h before the sacrifice. (B) Percentage of BrdU+ cells in cardiac and blood (BL) CD4+ T cells of WT and Il3−/− mice at the indicated time points (n = 4–8 per group of two independent experiments). (C) Representative flow cytometric dot plots of activated caspase-3 expression in WT and Il3−/− CD4+ T cells in the heart 21 d after the first immunization. (D) Percentage of activated caspase-3+ cells shown in C (n = 4 per group of two independent experiments). (E) Quantification of IFN-γ+, IL-17A+, and GM-CSF+ CD4+ T cells in the hearts on days 0 and 21 (n = 4–8 per group of two independent experiments). (F) 5 × 104 of autoreactive T cells and 104 of each of the sorted cardiac populations were cultured at indicated conditions for 3 d. The percentage of proliferating T cells was evaluated by Cell Trace Violet dye and normalized to T cells cultured with moDCs in the presence of αMHC peptide (n = 4–7 per group of three independent…

Figure 5.. IL-3 attracts monocytes by inducing cardiac macrophage chemokine production. (A) Flow cytometric analysis for IL-3 receptor α subunit (IL-3Rα: CD123)–positive cells in the inflamed heart on day 21. Representative flow dot plots for CD123 as well as the β subunit of the receptor (CD131) are shown. (B) Comparison of Ccl2, Ccl7, and Ccl12 gene expression in different leukocyte subsets sorted from WT myocarditis hearts on day 21. Values were normalized to that of Ly-6Chigh monocytes (n = 4 per each population of two independent experiments). (C) Depiction of a lentiviral vector containing the sgRNA targeting IL-3Rα from a U6 promoter (U6) and Cas9 from a short EF1a promoter (EFS) with eGFP from a picornavirus-derived 2A autocleavage site (P2A). (D) Schematic diagram for experimental design. Lentiviral particles were transfected into BM lineage-negative (Lin−) cells, which were subsequently transferred into lethally irradiated WT mice, and myocarditis was induced 6 wk later. Il3rα+/+ (GFP−) and Il3rα−/− (GFP+) cardiac macrophages were sorted at the peak of inflammation. (E) Representative flow dot plots to identify GFP+ cells in blood after gating on Ly-6Chigh monocytes. (F)…

Figure 6.. Anti-IL-3 therapy ameliorates acute inflammation and fibrosis in myocarditis. After injecting activated BMDCs pulsed with αMHC into WT mice on days 0, 2, and 4, the mice were randomly assigned to either anti–IL-3 neutralizing antibody–treated or control IgG–treated groups. The antibodies were intraperitoneally injected once a day from day 4, and the hearts were harvested 10 and 28 d after the first BMDC injection. (A) Flow cytometric quantification of indicated cells in the hearts of both groups on day 10 are shown (n = 9 per group of three independent experiments). (B) Cardiac fibrosis was assessed and quantified on day 28 by Azan staining (n = 10–11 per group of three independent experiments). Representative images of Azan-stained sections from both groups are depicted. Bars, 100 µm. (C and D) Heart weight (HW) to body weight (BW) ratios (C) and LVEDD, LVESD, LVEF, and LV mass at 28 d after the first BMDC injection in control IgG–treated and anti-IL-3–treated mice (D; n = 10–11 per group of three independent experiments). *, P < 0.05. For statistical analysis, a two-tailed Mann-Whitney U test or unpaired t test was applied to compare two groups. Results are shown as mean ± SEM.

Figure 7.. Proposed model. We propose that IL-3’s function in autoimmune disease is as follows: During sensitization, DCs present self-antigen to T cells, and this process is IL-3 independent (1). Self-reactive T cells circulate and travel to their destination tissue, where they are reactivated by tissue APCs (2). T cell–derived IL-3 acts on macrophages to produce CCL2, CCL7, and CCL12 (3). Recruited CCR2+ Ly-6Chigh monocytes differentiate into moDCs and MHCII+ macrophages that preferentially proliferate and activate T cells, leading to enhanced T cell proliferation and production of T cell–derived cytokines, including IL-3 (4). Monocytes also give rise to macrophages that further produce monocyte-attracting chemokines in response to IL-3 (5). This IL-3–dependent T cell–macrophage–moDC tricellular amplification loop is dismantled by anti-IL-3 treatment that mitigates tissue inflammation and damage (6).