Interleukin-13 protects against experimental autoimmune myocarditis by regulating macrophage differentiation

Abstract

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

Figure 1

IL-13 deficiency enhances chronic postviral autoimmune myocarditis. A: IL-13-deficient mice (filled bar) develop severe CVB3-induced myocarditis at day 35 after infection (P = 0.001), compared to WT BALB/c mice (open bar). B: Post-CB3 infection survival of IL-13 KO mice (thick solid line) was significantly diminished, compared to WT BALB/c control mice (thin dashed line) (P < 0.0001). Sixty percent of IL-13 KO mice died by day 21 after infection compared to 100% survival in WT BALB/c mice. C: IL-13 KO mice (filled bar) had increased MyHCα_614–629-reactive serum total IgG antibodies compared to WT BALB/c control mice (open bar) at day 35 after infection. IL-13 KO mice had severely affected cardiac function on day 21 of EAM: LVEDD (D), LVESD (E), %FS (F), %EF (G). Viral replication in the heart at days: 7 (H), 11 (I), and 14 (J) of CVB3-induced myocarditis. Diamonds represent individual animals, and are representative of three experiments with 6 to 12 mice per group. Bars represent the means of each group, ±SD. Statistics are by two-tailed Student’s t-test.

Figure 2

IL-13-deficient mice develop severe myocarditogenic peptide-induced myocarditis. A: Representative EAM in an IL-13 KO mouse. B: Representative EAM in a WT BALB/c mouse. C: The differences in the EAM severity in IL-13 KO mice (filled diamonds) were highly significant, compared to WT BALB/c mice (open diamonds) at day 21 after infection. Data represent individual animals, and are representative of five repetitions with 8 to 14 mice per group. Statistics are by Mann-Whitney rank sum U-test. D: Total cardiac-infiltrating CD45⁺ leukocytes, as calculated by flow cytometry and manual enumeration. E: IL-13 KO mice (filled bars, n = 12) had increased MyHCα_614–629-reactive serum antibodies compared to WT BALB/c control mice (open bars, n = 11) at day 21 after infection. Statistics are by two-tailed Student’s t-test. F: Blocking IL-13 with αIL-13 mAb increased severity of EAM, compared to isotype-treated control. Original magnifications: ×5 (A, B; left) and ×64 (A, B; right).

Figure 3

IL-13 KO mice had increased levels of proinflammatory cytokines, histamine, and fibrosis in the heart. A: Levels of IFN-γ, IL-1β, IL-4, IL-18, TGF-β1, and histamine from heart homogenates of IL-13 KO mice (filled bars, n = 12) were significantly higher than in WT BALB/c control mice (open bars, n = 11) at day 21 after infection of EAM as determined by ELISA. Data represent the means of each group, plus SD, and are normalized to wet heart weight. Statistics are by two-tailed Student’s t-test. B: IL-13 KO (filled diamonds) mice had significantly increased caspase-1 activity in splenocytes on day 21 of EAM, compared to WT mice (open diamonds). C: IL-13 KO mice (filled diamonds) develop increased fibrosis on day 30 of EAM compared to WT BALB/c mice (open diamonds). Fibrosis was assessed as the area of the heart section with collagen deposition, which stains bright blue with Masson’s Trichrome stain. Representative hearts with fibrotic changes in an IL-13 KO mouse (D) and WT BALB/c mouse (E) are shown.

Figure 4

IL-13 KO mice with EAM developed DCM with impaired cardiac function. A: Representative M-mode echocardiography of an IL-13 KO mouse at day 50 after infection. B: Representative M-mode echocardiography of a WT BALB/c control mouse. The hearts of IL-13 KO mice (filled bar, n = 10) had significantly increased LVEDD (C), LVESD (D), and significantly decreased %FS (E) and %EF (F), compared to WT BALB/c control mice (open bar, n = 10) at day 50 after infection of EAM.

Figure 5

A: IL-4-deficient mice develop mild MyHCα_614–629-induced myocarditis (filled diamonds), comparable to WT BALB/c mice (open diamonds). Data represent individual animals, and three repetitions with 8 to 10 mice per group. B: There were no differences in levels of intracardiac IL-1β, IL-13, IL-18, or IFN-γ between IL-4 KO (filled bars, n = 8) and WT BALB/c control mice (open bars, n = 8) at day 21 after infection of EAM as determined by ELISA. Statistics are by two-tailed Student’s t-test. Data are normalized to wet heart weight. MyHCα_614–629-reactive serum antibodies by subclass: total IgG (C), IgG1 (D), IgG2a (E), IgG2b (F). IL-4 KO (filled bars, n = 8) had higher levels of IgG2a and IgG2b MyHCα_614–629-reactive serum antibodies than WT BALB/c control mice (open bars, n = 8) at day 21. G: Similar to IL-4 KO mice, we did not observe any differences in EAM severity after treatment with anti-IL-4 mAb, compared to isotype-treated controls. Severity statistics are by Mann-Whitney U-test.

Figure 6

A: IL-13/IL-4 DKO mice (filled diamonds) develop myocarditis resembling severe IL-13 KO EAM, which was significantly greater than myocarditis severity in WT BALB/c control mice (open diamonds) at day 21 after infection. Data represent individual animals. Individual experiments were conducted five times with 8 to 14 mice per group. B: Intracardiac IL-18 was significantly increased in IL-13/IL-4 DKO mice compared to WT BALB/c mice at day 21 after infection of EAM as determined by ELISA. Data are normalized to wet heart weight. C: Total IgG MyHCα_614–629-reactive serum antibodies and its subclasses IgG2a and IgG2b were significantly increased in IL-13/IL-4 DKO (filled bars, n = 8), compared to WT BALB/c control mice (open bars, n = 8) at day 21 after infection. IgG1 was significantly decreased in IL-13/IL-4 DKO mice. D: CD4⁺CD25⁺Foxp3⁺ T cells were decreased in IL-13/IL-4 DKO spleens (filled diamonds), compared to WT BALB/c control mice (open diamonds) at day 21 of EAM. Statistics are by two-tailed Student’s t-test.

Figure 7

IL-13 KO mice display greater spleen T-cell activation. A: Activated CD4⁺ (left) and CD8α⁺ (right) T cells were determined by surface CD62L^lo expression. B: Activated CD4⁺ (left) and CD8α⁺ (right) T cells were determined by surface CD28 expression. Data represent percentage of CD4⁺ or CD8α⁺-gated cells in individual IL-13 KO (filled diamonds) or WT (open diamonds) animals. Horizontal bars indicate the mean of each group ± the SD. Data are representative of two independent experiments. C: Seventy-two-hour proliferation of spleen cells from IL-13 KO mice (filled bar) is greater than from WT BALB/c mice (open bar) in response to 10 μg/ml of MyHCα_614–629 at day 21 after infection, as determined by [³H]Me-thymidine uptake. Data indicate the mean δ cpm (stimulated cpm − unstimulated cpm; values for individual animals are the mean of triplicate wells) for each group (n = 3); plus the SD. D: CD4⁺CD25⁺Foxp3⁺ Treg cells as a proportion of spleen lymphoid cells, in IL-13 KO (filled diamonds) or WT (open diamonds) animals at day 21 after infection. E: Intracardiac CD4⁺ T cells as a proportion of total CD45⁺ leukocytes (left) and calculated absolute numbers (right) in IL-13 KO (filled diamonds) or WT (open diamonds) animals at day 21 after infection. Horizontal bars indicate the mean of each group ± the SD. Statistics are by two-tailed Student’s t-test.

Figure 8

Monocyte and macrophage phenotypes in the hearts of IL-13 KO mice at day 21 of EAM. A: Representative gating of intracardiac monocyte and macrophage populations. Infiltrating CD45⁺ leukocytes are subsetted by bivariate analysis of F4/80 and CD11b expression, revealing distinct populations: UL, CD11b^hiF4/80⁻ monocytes; UR, CD11b^hiF4/80^int macrophages; LR, F4/80^hiCD11b^int macrophages. CD204 and CD206 expression are then determined, based on gates set from total CD45⁺ cells. B: CD204⁺CD206⁺ double-positive expression on CD11b^hiF4/80⁻ monocytes, as a proportion of CD45⁺ cells. C: CD204⁺ or CD206⁺ expression, as a proportion of total F4/80⁺ macrophages. CD204⁻CD206⁻ double-negative CD11b^hiF4/80⁻ monocytes (D) and F4/80⁺ macrophages (E), as a proportion of total CD45⁺ cells. Individual IL-13 KO (filled) and WT (open) animals are shown. Horizontal bars indicate the mean of each group ± the SD. Statistics are by two-tailed Student’s t-test.