IL-9 Inhibits Viral Replication in Coxsackievirus B3-Induced Myocarditis

Abstract

Myocardial injuries in viral myocarditis (VMC) are caused by viral infection and related autoimmune disorders. Recent studies suggest that IL-9 mediated both antimicrobial immune and autoimmune responses in addition to allergic diseases. However, the role of IL-9 in viral infection and VMC remains controversial and uncertain. In this study, we infected Balb/c mice with Coxsackievirus B3 (CVB3), and found that IL-9 was enriched in the blood and hearts of VMC mice on days 5 and 7 after virus infection. Most of IL-9 was secreted by CD8⁺ T cells on day 5 and CD4⁺ T cells on day 7 in the myocardium. Further, IL-9 knockout exacerbated cardiac damage following CVB3 infection, along with a sharp increase in viral replication and IL-17a expression, as well as a decrease in TGF-β. In contrast, the repletion of IL-9 in Balb/c mice with CVB infection induced the opposite effect. Studies in vitro further revealed that IL-9 directly inhibited viral replication in cardiomyocytes by reducing coxsackie and adenovirus receptor expression, which might be associated with upregulation of TGF-β autocrine effect in these cells. However, IL-9 had no direct effect on apoptosis in cardiomyocytes. Our data indicated that IL-9 played a protective role in disease progression by inhibiting CVB3 replication in the early stages of VMC.

Keywords: IL-9; TGF-β; coxsackie and adenovirus receptor; coxsackievirus B3; viral myocarditis.

Figure 1

IL-9 expressions were increased in myocardium of VMC mice. (A) The results of immunohistochemistry (magnification 400×) in the heart tissue showed that IL-9 distributed in the lesions with inflammation on days 5 and 7. (B) CD45⁺ cells were gated. And the isotype control of IL-9 was showed. (C) The representative pictures for IL-9-secreting leukocyte (CD45⁺IL-9⁺) levels in different groups. (D) The results of statistical analysis for IL-9-secreting leukocyte levels by flow cytometry in different groups. (E) The CD45⁺IL-9⁺ cells were gated and further analyzed for CD11b, Gr-1, CD49b, CD4, and CD8 expressions to detect the cellular source of IL-9. **P < 0.01 vs. control group; ^##P < 0.01 vs. IL-9KO group. Values are means ± SEM. Ten mice were euthanized in each group separately on days 5 and 7.

Figure 1

IL-9 expressions were increased in myocardium of VMC mice. (A) The results of immunohistochemistry (magnification 400×) in the heart tissue showed that IL-9 distributed in the lesions with inflammation on days 5 and 7. (B) CD45⁺ cells were gated. And the isotype control of IL-9 was showed. (C) The representative pictures for IL-9-secreting leukocyte (CD45⁺IL-9⁺) levels in different groups. (D) The results of statistical analysis for IL-9-secreting leukocyte levels by flow cytometry in different groups. (E) The CD45⁺IL-9⁺ cells were gated and further analyzed for CD11b, Gr-1, CD49b, CD4, and CD8 expressions to detect the cellular source of IL-9. **P < 0.01 vs. control group; ^##P < 0.01 vs. IL-9KO group. Values are means ± SEM. Ten mice were euthanized in each group separately on days 5 and 7.

Figure 2

IL-9 attenuated the severity of VMC mice. (A) The representative pictures of histopathology (magnification 200×) in heart tissue. (B) The ratios of HM/BW in different groups. (C) The pathological scores in different groups. (D) The levels of serum cTnI in different groups. *P < 0.05 vs. control group; **P < 0.01 vs. control group; ^#P < 0.05 vs. WT group; ^##P < 0.01 vs. WT group. Values are means ± SEM. Ten mice were euthanized in each group separately on days 5 and 7. HM/BW, the ratios of heart weight to body weight; cTNT, cardiac troponin I.

Figure 3

IL-9 inhibited cardiac viral replication in VMC mice. (A) The levels of cardiac CVB3 titers were showed on days 5 and 7. Data represent mean values of CVB3 PFU per gram of the hearts. (B) The mRNA levels of cardiac CAR expression. (C) The protein levels of cardiac CAR expression. *P < 0.05 vs. control group; **P < 0.01 vs. control group; ^#P < 0.05 vs. WT group; ^##P < 0.01 vs. WT group. Values are means ± SEM. Ten mice were euthanized in each group separately on days 5 and 7.

Figure 4

IL-9 regulated serum IL-17a and TGF-β expressions in VMC mice. The levels of serum IL-9, IL-17a, IL-10, TGF-β, TNF-α, IFN-γ, IFN-α, and IFN-β in Control, WT, IL-9KO, and rIL-9 groups. *P < 0.05 vs. control group; **P < 0.01 vs. control group; ^#P < 0.05 vs. WT group; ^##P < 0.01 vs. WT group. Values are means ± SEM. Ten mice were euthanized in each group separately on days 5 and 7.

Figure 5

IL-9 directly suppressed CVB3 replication in cardiomyocyte in vitro. (A) Representative pictures of plaque assay of CVB3 in different groups. (B) The results of statistical analysis for CVB3 titers in Control, CVB3, and IL-9 groups. (C) The changes of IL-9R and CAR on myocardial cells in different groups. **P < 0.01 vs. control group; ^#P < 0.05 vs. CVB3 group; ^##P < 0.01 vs. CVB3 group. Values are means ± SEM. Each experiment was independently performed three times.

Figure 6

IL-9 facilitated TGF-β autocrine effect from cardiomyocyte in vitro. (A) The levels of autocrine TGF-β, TNF-α, IFN-α, and IFN-β from myocardial cells. (B) The changes of signal molecule phosphorylated ERK1/2 and RhoA were showed in different groups. (C) The levels of CVB3 titer, CAR mRNA, and CAR protein of myocardial cells treated with IL-9 or IL-9 + TGF-β mAb. *P < 0.05 vs. control group; **P < 0.01 vs. control group; ^#P < 0.05 vs. CVB3 group; ^##P < 0.01 vs. CVB3 group; $P < 0.05 vs. IL-9 group. Values are means ± SEM. Each experiment was independently performed three times.

Figure 7

IL-9 did not directly influence cardiomyocyte apoptosis in vitro. (A) The representative pictures for TUNEL-positive myocardial cells. α-SMA: red. DAPI: blue. TUNEL: green. (B) The statistical analysis for the number of TUNEL-positive cardiomyocytes. (C) The ratio of Bax/Bcl-2 mRNA was analyzed. *P < 0.05 vs. control group; **P < 0.01 vs. control group. Values are means ± SEM. Each experiment was independently performed three times.