Effect of interleukin-17A on anti-ANT antibodies as well as cytokines in viral myocarditis mice

Objective

To observe the effect of interleukin-17A (IL-17A) on serum anti-mitochondrial endosomal ADP/ATP carrier autoantibody (anti-ANT antibody) levels and cytokines in viral myocarditis (VMC) mice.

Methods

Male wild-type (WT) and IL-17A knockout (IL-17A^−/−) BALB/c mice were intraperitoneally injected with coxsackievirus B3 (CVB3) to establish a VMC model (VMC-WT and VMC-IL-17A^−/−), while WT BALB/c mice were injected with PBS intraperitoneally to establish a normal control group (WT group). After 14 days, myocardial tissues were taken to calculate heart mass, and paraffin sections were prepared and stained with HE staining to observe the pathological changes and calculate the pathological score of myocardial tissues. Flow cytometry was used to detect the level of CD4⁺ T lymphocytes in peripheral blood, ELISA was used to determine the level of anti-ANT antibody, IL-17 and IL-23 in serum, and Western blotting was used to detect the expression level of IL-17 and IL-23 proteins in myocardial tissue.

Results

VMC mice were successfully constructed. Mice in the WT group showed no abnormal activity; Mice in the VMC-WT group gradually showed behaviors such as huddling, shrugging, trembling, and poor response from the third day of injection; Mice in the VMC-IL-17A^−/− group showed the above symptoms to a lesser extent. In the VMC-WT group, the HM and pathological score were 5.62 ± 0.27 g/kg and 3.12 ± 0.45 score. The HM and pathologic score in the VMC-IL-17A^−/− group were lower than those in the VMC-WT group. The pathological examination of WT mice showed no inflammatory cell infiltration and patchy necrosis. The rats in VMC-WT group showed extensive inflammatory cell infiltration and large sheet necrosis of cardiomyocytes under microscope. Compared with the VMC-WT group, the VMC-IL-17A^−/− group showed focal myocardial necrosis and significantly reduced inflammatory cell infiltration 14 days after CVB3 infection. At the same time, the percentage of CD4⁺T was remarkably lower in the VMC-WT mice compared with the WT mice. The percentage of CD4⁺T was higher in the VMC-IL-17A^−/− group (27.54 ± 3.62) than in the VMC-WT group (16.97 ± 2.18). In addition, anti-ANT antibody (1.48 ± 0.31 μg/L), IL-17 (33.47 ± 4.26 pg/mL) and IL-23 (32.42 ± 4.31 pg/mL) levels were significantly lower in serum of VMC-IL-17A^−/− mice than in the VMC-WT mice. At the protein level, the expression of IL-17 and IL-23 showed consistent results.

Conclusion

Serum anti-ANT antibody, IL-17, and IL-23 levels were significantly elevated in VMC mice. Although the ELISA results suggest IL-17A is involved in the production of anti-ANT antibody in VMC mice, the possibility of cross-reactivity cannot be completely ruled out; therefore, the results should be interpreted with caution. The knockdown of IL-17A gene was associated with inhibition of myocardial inflammation, elevation of peripheral blood CD4+ T lymphocytes, and attenuation of myocardial injury in VMC mice.