The number of Th17 cells in the spleen and cardiac tissue is decreased by Ad-IL-17AR:Fc.

<p>(<i>A</i>) Representative flow cytometry images of Th17 (CD4⁺ IL-17⁺) cells from each group gated on CD4⁺ T cells. Numbers in the upper right quadrants and lower right quadrants indicate the separate percentages of Th17 cells and CD4⁺ T cells, respectively. PE = phycoerythrin; FITC = fluorescein isothiocyanate. (<i>B</i>) The percentage of Th17 (CD4⁺ IL-17⁺) cells in each group was analyzed using CellQuest software. (<i>C</i>) The levels of IL-6 and TNF-α in the supernatants of heart homogenates detected using ELISA. The data represent the mean ± SEM for 6 mice per group. **<i>P</i><0.01 vs control group, *<i>p</i><0.05 vs control group.</p

In Vivo Delivery of Adenoviral Vector Containing Interleukin-17 Receptor A Reduces Cardiac Remodeling and Improves Myocardial Function in Viral Myocarditis Leading to Dilated Cardiomyopathy

<div><p>Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.</p></div

IL-17-stimulated MMPs/TIMPs protein expression, collagen deposition and proliferation in CFs.

<p>(<i>A</i>) Representative western blot for ADAMTS-1 protein levels in CFs treated with different doses (0, 10, and 50 ng/ml) of IL-17 for 24 h, and at 0, 24 and 48 h. (<i>B</i>) IL-17-mediated expression of ADAMTS-1 and MMP-2/TIMP-1 in the CFs treated with IL-17 (10 ng/ml) for 24 h, using TGF-β (10 ng/ml) as a control. (<i>C</i>) RT-PCR in CFs showing the IL-17-induced expression of COI1α2 and COI3α1 mRNAs. (<i>D</i>) The proliferation of CFs following the administration of different doses of IL-17 using cell counting kit-8 (CCK-8). (<i>E</i>) Western blot showing the expression of DDR2 at the different concentrations of IL-17. (<i>F</i>) Immunofluorescence in the cultured CFs showing the expression of the DDR2 protein in the 10 ng/ml IL-17, 5 ng/ml IL-17 and control groups (magnification ×200). The values in the IL-17-treated CFs were normalized to the values in the control cells (n = 5 per group). The data represent the mean ± SEM. *<i>P</i><0.05 vs control group, **<i>P</i><0.01 vs control group.</p

Ad-IL-17AR:Fc administration reduces IL-17A production in the blood and the heart.

<p>(<i>A</i>) Serum IL-6 concentrations at post-infection 7 days, 2mons and 3mons in PBS-, Ad-IL-17AR:Fc- and Ad:null-treated mice (n = 5) were measured using ELISA, and the data were analyzed by a 2-factor ANOVA. ^##<i>p</i><0.01, ^#<i>p</i><0.05 vs 7 Days group, **<i>p</i><0.01, *<i>p</i><0.05 vs control group, (<i>B</i>) Serum TNF-α concentrations at post-infection 7 days, 2mons and 3mons in PBS-, AdIL-17R:Fc- and Ad:null-treated mice (n = 5) were measured using ELISA, and the data were analyzed by a 2-factor ANOVA. ^#<i>p</i><0.05 vs 7 Days group, *<i>p</i><0.05 vs control group. The data represent the mean ± SEM for 6 mice per group. (<i>C</i>)The IL-17A protein extracted from the left ventricle of the mice in the three groups at post-infection day 90, was subjected to western blotting and was probed with the indicated antibodies (n = 3). (D)The IL-17A protein level is expressed as a ratio of the GAPDH level in the same sample; the data are expressed as the mean± SEM. *<i>P</i><0.05 vs the control group. (<i>E</i>) The IL-17RA protein extracted from the left ventricle of the mice in the three groups at post-infection day 90, was subjected to western blotting and was probed with the indicated antibodies (n = 3). (<i>F</i>) The IL-17RA protein level is expressed as a ratio of the GAPDH level in the same sample; the data are expressed as the mean± SEM.</p

The levels of IL-6 and TNF-α in the supernatants of cardiac fibroblast cultures treated with IL-17.

<p>The IL-17 (10 ng/ml)-treated group produced higher levels of IL-6 and TNF-α cytokines than the control- and IL-17 (5 ng/ml)-treated groups. Moreover, IL-6 in the IL-17 (5 ng/ml)-treated group was highly expressed in the supernatants of the heart homogenates (ELISA). The values in the IL-17-treated CFs were normalized to the values in the control cells (n = 5 per group). The data represent the mean ± SEM. **<i>P</i><0.01 vs control group, ^##<i>P</i><0.01 vs control group.</p

Degradation of CFs related to the MMPs/TIMPs expression following neutralization of IL-17.

<p>(<i>A</i>) Representative western blots showing the expression of the cardiac ADAMTS-1 protein in each group. (<i>B</i>) Representative western blots showing the expression of the cardiac TIMP-1 protein in each group. (<i>C</i>) Representative western blots showing the expression of the cardiac MMP-2 protein in each group. The expression of the cardiac ADAMTS-1 and MMP-2/TIMP-1 proteins in each group is expressed as a ratio of the GAPDH expression for the same sample. On day 90, 3 mice per group were analyzed. *<i>P</i><0.05 vs control group.</p