Expression of IL-23/Th17 pathway in a murine model of Coxsackie virus B3-induced viral myocarditis

Abstract

Background: The IL-23/Th17 pathway is implicated in the pathogenesis of a number of chronic inflammatory and autoimmune diseases. Whether it regulates the viral myocarditis (VMC) is unknown.

Results: To examine the pathogenesis role of IL-23/Th17 axis in VMC, we used male BALB/c mice to induced VMC by Coxsackie virus B3 (CVB3) peritoneal injection. IL-23, IL-17, and signal transducer and activator of transcription 3 (STAT3) mRNA in the myocardium of VMC mice were assessed by semi-quantitative RT-PCR. IL-23 and IL-17 protein from blood serum were evaluated by ELISA. Phosphorylated-STAT3 (p-STAT3) protein expression in the myocardium was evaluated by immunohistochemical staining. Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets. Isolated CD4+ T cells from VMC mice were cultured with recombinant IL-23(rIL-23) in vitro. In addition, a STAT3-specific inhibitor (S3I-201) was used to test whether regulation of STAT3 could be partly responsible for Th17 diminution. Results showed that expression of IL-23, IL-17, STAT3 mRNA and protein increased in VMC mice. When purified CD4+ T cells derived from VMC mice were cultured in vitro with rIL-23, the frequency of Th17 cells was dramatically increased, accompanied by significantly enhanced production of IL-17 in the supernatants of cultured CD4+ T cells. S3I-201 significantly restrained Th17 cell proliferation.

Conclusions: The IL-23/Th17 pathway axis is strongly expressed in murine VMC, identifying a novel pathway of potential significance in viral myocarditis.

Figure 1

Histopathologic changes of heart tissue in VMC mice. A. Representative image of inflammation in VMC heart tissue (H&E, original magnification × 400). B The pathological scores at different times, *P < 0.05, versus control group, ^ΔP < 0.05, versus week 0, 1, 3, 4, and 6 VMC mice, ^※P < 0.05, versus week 4 and 6 VMC mice. The number of mice in each time point of VMC group was 8, 8, 5, 6, 6 and 5 respectively. The number of mice in each time points of control was 5.

Figure 2

IL-23, IL-17 mRNA and protein expression in the myocardium of mice. A. Strong expression of IL-23, IL-17 occurred from the first week to the sixth week after CVB3 infection inVMC mice. M: Maker; 1: week 0; 2: week 1; 3: week 2; 4: week 3; 5: week 4; 6: week 6. B. Densitometric quantitation of IL-23. IL-23 mRNA expression was elevated in VMC mice from the first week to the sixth week. ^ΔP < 0.01, versus control group, ^ΔP < 0.05, versus week 0, 1, and 2 VMC mice. C. Densitometric quantitation of IL-17. IL-17 mRNA expression was elevated in VMC mice from the first week, and the arbitrary units were higher than those of controls. ^ΔP < 0.01 versus controls, ^ΔP < 0.05, versus week 0 VMC mice. D. Representative of IL-17 immunohistochemistry images in heart tissue of VMC mice (Dark brown granules, original magnification × 400). E. Morphometric quantitation of IL-17 protein expression. ^ΔP < 0.01, versus control group, ^ΔP < 0.05, versus week 0, 1, 2 and 6 VMC mice. F. IL-23 protein levels were steadily increased in the VMC mice, ^ΔP < 0.05, versus control group, ^ΔP < 0.05, versus week 0, and 6 VMC mice. G, IL-17 protein levels were steadily increased in the VMC mice from 1 week after i.p, and reached statistical difference comparing with those of controls, ^ΔP < 0.05. The highest level of IL-17 in VMC occurred on 4th week, ^ΔP < 0.05, versus week 0, 1 VMC mice. ^※P < 0.05, versus week 0, 1 VMC mice.

Figure 3

Th17 frequencies in VMC mice. A. Representative pictures of CD4⁺IL-17⁺ Th17 cells in VMC mice. Numbers in the upper right quadrants indicate the mean percentages of CD4⁺ Th17 cells in VMC mice. B. The results of the Th17 cells statistical analysis. ^ΔP < 0.01, versus control group. ^ΔP < 0.05, versus week 0, 1, 2, 3, and 6 VMC mice.

Figure 4

STAT3 mRNA and p-STAT3 protein expression in the myocardium of VMC mice. A. Strong expression of STAT3 occurred from 1st week to 6th week after CVB3 infection. M: Maker; 1: week 0; 2: week 1; 3: week 2; 4: week 3; 5: week 4; 6: week 6. B. Densitometric quantitation of STAT3. STAT3 mRNA expression was elevated in VMC mice from 1st week to 6th week, ^ΔP < 0.01, versus control group. ^ΔP < 0.05, versus week 0, 1 and 2. C. Representative of p-STAT3 immunohistochemistry images in heart tissue of VMC mice (Dark brown stain in cytoplasm, original magnification × 400). D. Morphometric quantitation of p-STAT3 protein expression in heart. ^ΔP < 0.05, versus control group, ^ΔP < 0.05, versus week 0, 1, 2 VMC mice.

Figure 5

Th17/IL-17 production stimulated by rIL-23. A. Representative pictures of CD4⁺IL-17⁺ Th17 cells stimulated by rIL-23. The numbers in the upper right quadrants represents the mean percentage of Th17 in response to 5 d of culture with or without IL-23. B. The results of Th17 cells statistical analysis. *P < 0.05. C. Strong expression of IL-17 and STAT3 after rIL-23 stimulation. D. Densitometric quantitation of IL-17 and STAT3 mRNA in the cultured cells after rIL-23 stimulation. *P < 0.05. E. IL-17 protein levels in the culture supernatant were significantly increased after rIL-23 stimulation *P < 0.01.

Figure 6

Intracellular IL-17 production inhibited by S3I-201. A. Representative pictures of CD4⁺IL-17⁺ Th17 cells inhibited by S3I-201. The numbers in upper right quadrants represent the mean percentage of Th17 cells in response to S3I-201. B. The results of Th17 cells statistical analysis, *P < 0.05. C. Decreased expression of IL-17 and STAT3 after S3I-201 inhibition. D. Densitometric quantitation of IL-17 and STAT3 mRNA in the cultured cells after S3I-201 inhibition. *P < 0.05. E. IL-17 protein levels in the culture supernatant were significantly decreased after S3I-201 inhibition, *P < 0.01.