Interleukin‐22 Directly Activates Myocardial STAT3 (Signal Transducer and Activator of Transcription‐3) Signaling Pathway and Prevents Myocardial Ischemia Reperfusion Injury

Figures

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  Figure 1. IL‐22 activates the STAT3 signaling pathway in heart tissue. A, Total cell lysates were prepared from mouse hearts at the indicated times after injection of IL‐22 or PBS. Blots were probed using antibodies against phosphorylated STAT3 (P‐STAT3), STAT3, and GAPDH. Graphs represent quantitative differences in the expression between the ratio of P‐STAT3 to STAT3 (n=3 per group). *P<0.05 vs pre‐injection, † P<0.01 vs pre‐injection (Dunn's test). B, Total cell lysates were prepared from mouse hearts at 30 minutes after injection of IL‐10 or IL‐22. Blots were probed using antibodies against P‐STAT3, STAT3, and GAPDH. Graphs represent quantitative differences in the expression ratio of P‐STAT3 to STAT3 (n=3 per group). *P<0.05 vs PBS injection (Dunn's test). AU indicates arbitrary units; IL, interleukin; Pre, pre‐injection; P‐STAT3, phosphorylated STAT3; STAT3, signal transducer and activator of transcription‐3.

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  Figure 2. IL‐22 activates the myocardial STAT3 both in vivo and in vitro. A, Double immunofluorescence staining of P‐STAT3 (green) and α‐actinin (red) in mouse heart tissue at 30 minutes after IL‐22 or PBS injection (n=3 for each group). Scale bar=100 μm. Representative photographs of hearts from each group are shown. Values indicate the percentage of P‐STAT3–positive cells in the hearts. † P<0.01 vs PBS injection (Wilcoxon rank‐sum test). B, IL‐22 enhances STAT3 activation in mouse ventricular cardiomyocytes. After overnight serum depletion, myocytes were stimulated with PBS or 10 ng/mL IL‐22 for 15 minutes, and total cell lysates were prepared. Blots were probed using antibodies against P‐STAT3, STAT3, and GAPDH. Graphs represent quantitative differences in the expression between the ratio of P‐STAT3 to STAT3 (n=3 per group). † P<0.01 vs PBS injection (Wilcoxon rank‐sum test). AU indicates arbitrary units; IL, interleukin; P‐STAT3, phosphorylated STAT3; STAT3, signal transducer and activator of transcription‐3.

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  Figure 3. IL‐22 receptor expression in the heart tissue of intact mice. A, From peripheral leukocytes and hearts of intact mice, mRNA was prepared and subjected to real‐time polymerase chain reaction analyses for the IL‐22 receptors, including IL‐22R1 and IL‐10R2, and IL‐22BP. Presented values are normalized to GAPDH expression (n=3 per group). *P<0.05 vs leukocytes (Wilcoxon rank‐sum test). B, Total cell lysates were prepared from the livers and hearts of intact mice. Blots were probed using antibodies against IL‐22R1 and GAPDH (n=3 per group). C, Double immunofluorescence staining of IL‐22R1 and α‐actinin in heart tissue from intact mice. Frozen sections were stained with antibodies against IL‐22R1 (green) and anti‐α‐actinin (red). Isotype IgG was used as a negative control for anti‐IL‐22R1 antibody. Scale bar=100 μm. D, IL‐22R1 expression in mouse ventricular cardiomyocytes. After overnight serum depletion, myocytes were stimulated with PBS or 10 ng/mL IL‐22 for 15 minutes, and total cell lysates were prepared. Blots were probed using antibodies against IL‐22R1 and GAPDH (n=3 per group). DAPI indicates 4′,6‐diamidino‐2‐phenylindole; HepG2, hepatoma G2; IgG, immunoglobulin G; IL,…

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  Figure 4. Expression of IL‐22 and its receptors in heart tissue after ischemia reperfusion. A, From mouse hearts at the indicated times after ischemia reperfusion, mRNA was prepared and subjected to real‐time polymerase chain reaction analyses for IL‐22R1, IL‐10R2, IL‐22, and IL‐22BP. Presented values are normalized to GAPDH and expressed as the fold‐change from pre‐ischemia values (n=4–5 per group). *P<0.05 vs pre‐ischemia. † P<0.01 vs pre‐ischemia (Dunn's test). B, Total cell lysates were prepared from mouse hearts of at the indicated times after ischemia reperfusion. Blots were probed using antibodies against IL‐22R1 and GAPDH. Graphs represent quantitative differences in the expression between the ratio of IL‐22R1 to GAPDH (n=4–5 per group). *P<0.05 vs pre‐ischemia (Dunn's test). C, Serum IL‐22 levels at the indicated times after ischemia reperfusion, determined by Bio‐Plex (n=3). *P<0.05 vs pre‐ischemia (Dunn's test). IL indicates interleukin; IL‐10R2, IL‐10 receptor 2; IL‐22BP, IL‐22 binding protein; IL‐22R1, IL‐22 receptor subunit 1; pre, pre‐ischemia.

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  Figure 5. IL‐22 expression in the heart, peripheral leukocytes, spleen, and thymus after ischemia reperfusion. Real‐time polymerase chain reaction analyses for IL‐22 was performed using mRNA from each tissue, pre‐ischemia, or at 3 hours after ischemia reperfusion. The presented values are normalized to GAPDH (n=3 per group). *P<0.05 vs pre‐ischemia. † P<0.01 vs pre‐ischemia (Wilcoxon rank‐sum test). IL indicates interleukin; IRI, ischemia reperfusion injury; pre, pre‐ischemia.

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  Figure 6. IL‐22 prevents myocardial injury after ischemia reperfusion. A, Representative images of Evans blue and triphenyltetrazolium chloride staining in PBS‐treated or IL‐22‐treated mice at 24 hours postreperfusion (n=6–7 per group). The infarct size of the LV is expressed as a percentage of the AAR for each group. Graphs show quantification of AAR/LV and infarct area/AAR. *P<0.05 vs PBS‐treated mice (Wilcoxon rank‐sum test). B, Echocardiography was performed in PBS‐treated or IL‐22‐treated mice at 24 hour postreperfusion (n=4–7 per group). *P<0.05 vs PBS‐treated mice (Wilcoxon rank‐sum test). AAR indicates area at risk; EF, ejection fraction; FS, fractional shortening; IL, interleukin; LV, left ventricle; LVESD, left ventricular end systolic dimension; MI, myocardial infarction.

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  Figure 7. IL‐22 inhibits myocardial apoptosis after ischemic reperfusion injury. A, TUNEL assays of heart tissue from PBS‐treated or IL‐22‐treated mice at 3 hours postreperfusion (n=5–6 per group). Representative images of the ischemic areas of hearts from each group. Graph shows the percentage of TUNEL‐positive cells. *P<0.05 vs PBS‐treated mice (Wilcoxon rank‐sum test). B, Total cell lysates were prepared from the left ventricle of PBS‐treated or IL‐22‐treated mice at 30 minutes postreperfusion. Blots were probed using antibodies against P‐STAT3, STAT3, and GAPDH. Graphs represent quantitative differences in the expression between the ratio of P‐STAT3 to STAT3 (n=3 per group). *P<0.05 vs PBS‐treated mice (Wilcoxon rank‐sum test). AU indicates arbitrary units; IRI, ischemia reperfusion injury; IL, interleukin; P‐STAT3, phosphorylated STAT3; STAT3, signal transducer and activator of transcription‐3; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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  Figure 8. Expression of apoptosis‐related molecules in hearts of IL‐22‐treated mice. A, Total cell lysates were prepared from hearts of intact mice at 3 or 6 hours after injection of IL‐22 or PBS. Blots were probed using antibodies against BNIP3L, Bok, caspase 6, CIDEA, Diablo, Nod1, cleaved‐caspase 3, Bcl‐xL, Mcl‐1, Bax, Bcl‐2, P‐p53, p53, and GAPDH. Graphs represent quantitative differences in the expression ratio of P‐p53 to p53 (n=3 per group). *P<0.05 vs PBS injection (Wilcoxon rank‐sum test). B, Total cell lysates were prepared from the left ventricle of PBS‐treated or IL‐22‐treated mice at 3 hours after ischemia reperfusion. Blots were probed using antibodies against P‐p53, p53, and GAPDH. Graphs represent quantitative differences in the expression ratio of P‐p53 to p53 (n=3 per group). *P<0.05 vs PBS injection (Wilcoxon rank‐sum test). AU indicates arbitrary units; Bax, Bcl‐2‐associated X protein; Bcl‐2, B‐cell CLL/lymphoma 2; Bcl‐xL, B‐cell lymphoma‐extra large; BNIP3L, BCL2/adenovirus E1B interacting protein 3‐like; Bok, BCL2‐related ovarian killer protein; CIDEA, Cell death‐inducing DNA fragmentation factor, α subunit‐like effector A; IL, interleukin; Nod1, nucleotide‐binding…