Adeno-associated Virus Vector-mediated Interleukin-10 Induction Prevents Vascular Inflammation in a Murine Model of Kawasaki Disease

Figures

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  Figure 1. The AAV-mediated induction of IL-10 improves CAWS-induced cardiac dysfunction (A) Mice were injected intramuscularly with AAV-GFP (control) or AAV-IL-10. The plasma IL-10 levels were assessed at 2 and 4 weeks after the injection of AAV (n = 11–13). ND indicates not detected. (B–F) Mice were treated intraperitoneally with CAWS or vehicle at 2 weeks after the AAV injection. The survival of CAWS-treated AAV-GFP and AAV-IL-10 mice was analyzed using Kaplan-Meier method (n = 10–12) (B). Echocardiography was performed at 28 and 56 days after CAWS or vehicle treatment. Two-dimensional M-mode echocardiograms at 56 days are shown (C). The cardiac function %FS, (D) and dimension LVEDD, (E), and the heart weight/body weight ratio (F) were assessed at 28 and 56 days after CAWS or vehicle treatment (%FS, n = 7–17 [day 28]; n = 4–7 [day 56], LVEDD, n = 7–17 [day 28]; n = 3–6 [day 56], the heart weight/body weight ratio (n = 10–14 [day 28]; n = 5–9 [day 56]). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.

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  Figure 2. The AAV-mediated induction of IL-10 reduces CAWS-induced vascular inflammation and fibrosis Mice were treated intraperitoneally with CAWS or vehicle, 2 weeks after the injection of AAV-GFP (control) or AAV-IL-10. Heart sections were obtained at day 28 after CAWS or vehicle treatment and stained with HE, EVG, MT, and SR. (A,B) Representative images of the aortic root and coronary artery are shown. (C) The fibrotic area of the aortic root and coronary artery was quantified (n = 3–4). Data are expressed as the mean ± SEM. *p < 0.05.

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  Figure 3. The AAV-mediated induction of IL-10 reduces CAWS-induced inflammatory cell infiltration Mice were treated intraperitoneally with CAWS or vehicle, 2 weeks after the injection of AAV-GFP (control) or AAV-IL-10. (A) Heart sections were obtained at day 28 after CAWS or vehicle treatment and subjected to immunohistochemical staining for CD45, Mac2, Gr-1, and CD3. (B) The Mac2+ and Gr-1+ areas were quantified (n = 4–6). Data are expressed as the mean ± SEM. **p < 0.01.

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  Figure 4. The AAV-mediated induction of IL-10 reduces CAWS-induced inflammatory and fibrotic responses Mice were treated intraperitoneally with CAWS or vehicle, 2 weeks after the injection of AAV-GFP (control) or AAV-IL-10. The heart samples were obtained at day 28 after CAWS or vehicle treatment. The mRNA levels of Tnfa, Il6, Il1b, Il17a, Ccl2, Ccr2 (A), Mmp2, Mmp9, Timp1, Col1a1, Col3a1, and Tgfb1 (B) were assessed by a real-time RT-PCR analysis (n = 4–6). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.

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  Figure 5. CD11b+Dectin-2+ cells are involved in the inhibitory effect of AAV-IL-10 Mice were treated intraperitoneally with CAWS or vehicle, 2 weeks after the injection of AAV-GFP (control) or AAV-IL-10. Heart samples were obtained at day 28 after CAWS or vehicle treatment. (A) The sections were subjected to immunohistochemical staining for Dectin-2. (B) The mRNA levels of Clec4n were assessed by a real-time RT-PCR analysis (n = 4–5). (C) The population of CD11b+Dectin-2+CD45+ cells was assessed by flow cytometry (n = 7–8). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.

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  Figure 6. IL-10 inhibits the CAWS-induced production of inflammatory cytokines in BMDCs (A–D) Primary murine peritoneal macrophages (A), BMMs (B), BMDCs (C), and splenocytes (D) were treated with CAWS (10 μg/mL) and LPS (100 ng/mL) for 24 h (peritoneal macrophages) or 48 h (BMMs, BMDCs, and splenocytes). The TNF-α and IL-6 protein levels in the supernatants were assessed (n = 3). (E) BMDCs were pretreated with or without IL-10 (40 ng/mL) for 1 h, and then treated with CAWS (1–10 μg/mL) for 48 h. The TNF-α and IL-6 protein levels in the supernatants were assessed (n = 3). (F) The mRNA levels of Clec4n, Clec7a, and Clec4e in BMCs, BMDCs, and BMMs were assessed by a real-time RT-PCR analysis (n = 4–6). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.

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  Figure 7. GM-CSF enhances the CAWS-induced inflammatory cytokine production in BMMs BMMs were pretreated with or without IL-10 (40 ng/mL) for 1 h, and then treated with CAWS (10 μg/mL) and/or GM-CSF (1 ng/mL) for 48 h. (A) The TNF-α and IL-6 protein levels in the supernatants were assessed (n = 3). (B) The mRNA levels of Clec4n were assessed by a real-time RT-PCR analysis (n = 3). Data are expressed as the mean ± SEM. *p < 0.05. (C) BMMs were treated with or without GM-CSF (1 ng/mL) for 48 h. The cells were pretreated with or without IL-10 (40 ng/mL) for 1 h, and then treated CAWS (10 μg/mL) for the indicated periods. Cell lysates were prepared and analyzed by Western blotting with antibodies against Syk, p-Syk, ERK1/2, p-ERK1/2, and β-actin.

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  Figure 8. IL-10 inhibits the expression of TNF-α and IL-6, but not GM-CSF and Dectin-2, in the early phase of CAWS-induced vasculitis (A) Mice were treated intraperitoneally with a single administration of CAWS, and heart samples were obtained at 6 and 24 h after the administration. The mRNA levels of Tnfa, Il6, Csf2, Clec4n were assessed by a real-time RT-PCR analysis (n = 4). (B) Mice were treated intraperitoneally with a single administration of CAWS, 2 weeks after the injection of AAV-GFP (control) or AAV-IL-10. Heart samples were obtained at 24 h after the administration. The mRNA levels of Tnfa, Il6, Csf2, Clec4n were assessed by a real-time RT-PCR analysis (n = 8–10). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.