AAV9-mediated functional screening for cardioprotective cytokines in Coxsackievirus-B3-induced myocarditis

Figures

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  Figure 1. FunSel identifies IL9 as a cardioprotective agent in the sub-acute phase during VM. (A) Graphical representation of the events included in the Functional Selection (FunSel) technique to screen potential cardioprotective targets under the selective pressure of viral myocarditis (VM). NGS Next-generation sequencing. B Results of competitive selection of the most efficiently expressed cytokines (46 of the initial 60) in Pool 60. Each dot represents the relative enrichment of each cytokine vector detected by barcode sequencing, 2 weeks after Coxsackievirus B3 (CVB3)-infection (1.00 = no enrichment, dotted line; > 1.00 = positive selection; < 1.00 = negative selection) from pools of 3 animals per group. From this first set of results, 10 enriched cytokines (in yellow) and 5 depleted cytokines (in red) were selected to obtain Pool 15, with which a second FunSel cycle was performed. (C) Same as in (B) after overexpression of pool 15, 2 weeks after CVB3-infection from biological pools of 3 animals per group. In yellow, cytokines enriched in VM, chosen to study further (IL9, IL3, IL15, IL13, and IL4); in red, cytokines most consistently depleted in both rounds of VM (IL18, IL17b, and…

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  Figure 2. Synergic overexpression of the top listed cytokines (Pool 5) decreases cardiac necrosis in VM. (A,B) Cardiac necrosis and (C) average lesion size detected by hematoxylin and eosin detected by hematoxylin and eosin (H&E) staining 14 days after CVB3 infection in Pool 5-overexpressing hearts and controls. Scale bar = 500 µm. Significance assessed by Mann–Whitney U test with *p ≤ 0.05; ***p ≤ 0.001. (D) Relative Cvb3 viral genome expression levels at 14 days post-infection in Pool 5-overexpressing hearts and controls. Significance assessed by Mann–Whitney U test with ***p ≤ 0.001. All values are expressed as mean ± SEM.

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  Figure 3. Overexpression of IL9 plus IL3, IL4, IL13, IL15 (Pool 5) decreases T lymphocyte response in the acute phase. (A,B) CD45 staining (yellow patches, leukocytes) at 14 days post-infection in Pool 5-overexpressing hearts and controls. Scale bar = 500 µm. Significance assessed by two-way ANOVA followed by Tukey’s test with ***p ≤ 0.001. (C,D) CD3+ staining (T lymphocytes) 14 days after CVB3 infection in Pool 5-overexpressing hearts and controls. Scale bar = 100 µm. Significance assessed by two-way ANOVA followed by Tukey’s test with *p ≤ 0.05; **p ≤ 0.01. All values are expressed as mean ± SEM.

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  Figure 4. Overexpression of Pool 5 alters cardiac T cytotoxic and T helper lymphocyte balance in the acute phase. (A,B) Flow cytometry gating strategy and hierarchy to detect T lymphocyte subpopulation at 14 days post-infection in Pool 5-overexpressing hearts and controls. (C) T cytotoxic and (D) T helper lymphocytes detected by flow cytometry in Pool 5-overexpressing hearts and controls at 14 days post-infection. Significance assessed by a Kruskal–Wallis test (C) and a two-way ANOVA (D), followed by Tukey’s test with *p ≤ 0.05; ***p ≤ 0.001. (E) T helper 2 lymphocytes (Th2), (F) Th17, (G) Th22, (H) Th1, and (I) Th9 detected by flow cytometry in infected Pool 5-overexpressing hearts and infected controls 14 days after CVB3 infection. Significance assessed by Kruskal–Wallis test with *p ≤ 0.05 for panels (E), (F), and (G) or two-way ANOVA followed by Tukey’s test for panels (H) and (I). All values are expressed as mean ± SEM.

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  Figure 5. Overexpression of Pool 5 improves cardiac function in the chronic phase during VM. (A) End-diastolic volume (EDV) and (B) ejection fraction (EF) measured by echocardiography in infected Pool 5-overexpressing C3H mice and infected controls 35 days after CVB3 infection. Significance assessed by two-way ANOVA followed by Tukey’s test with *p ≤ 0.05, ***p ≤ 0.001. (C,D) Fibrosis detected by Sirius Red staining in infected Pool 5-overexpressing hearts and controls 35 days after CVB3 infection. Scale bar = 500 µm. Significance assessed by two-way ANOVA followed by Tukey’s test with *p ≤ 0.05; ***p ≤ 0.001. All values are expressed as mean ± SEM.

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  Figure 6. IL9 overexpression improves VM outcome, decreasing T lymphocyte response in the acute phase. (A,B) Cardiac necrosis detected by hematoxylin and eosin (H&E) staining 14 days after CVB3 infection in IL9-overexpressing hearts and controls. Scale bar = 500 µm. Significance assessed by Mann–Whitney U test with **p ≤ 0.01. (C,D) CD45 staining (leukocytes) in infected IL9-overexpressing and infected control hearts at 14 days post-infection. Scale bar = 500 µm. Significance assessed by two-way ANOVA followed by Tukey’s test with ***p ≤ 0.001. (E,F) CD3 staining (T lymphocytes) in infected IL9-overexpressing and infected control hearts 14 days after CVB3 infection. Scale bar = 100 µm. Significance assessed by two-way ANOVA followed by Tukey’s test with ***p ≤ 0.001. All values are expressed as mean ± SEM.

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  Figure 7. IL9 overexpression alters T helper subpopulations, enhancing Th2 and reducing Th17/22 presence in the acute phase. (A) T cytotoxic and (B) T helper lymphocytes detected by flow cytometry in IL9-overexpressing hearts and controls at 14 days post-infection. Significance assessed by two-way ANOVA, followed by Tukey’s test. (C) T helper 2 lymphocyte (Th2), (D) Th17, (E) Th22, (F) Th1, and (G) Th9 cardiac subpopulations detected by flow cytometry in CVB3-infected IL9-overexpressing hearts and infected controls at 14 days post-infection. Significance assessed by two-way ANOVA followed by Tukey’s test or by Kruskal–Wallis test (F), with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. All values are expressed as mean ± SEM.

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  Figure 8. IL9 overexpression is not sufficient to improve cardiac function in the chronic phase during VM. (A) End-diastolic volume (EDV) and (B) ejection fraction (EF) measured by echocardiography in infected IL9-overexpressing C3H mice and infected controls 35 days after CVB3 infection. Significance assessed by two-way ANOVA, followed by Tukey’s test. (C,D) Fibrosis detected by Sirius Red staining in infected IL9-overexpressing hearts and controls 35 days after CVB3 infection. Scale bar = 500 µm. Significance assessed by two-way ANOVA, followed by Tukey’s test with *p ≤ 0.05. All values are expressed as mean ± SEM.