Recombinant IL-37 Exerts an Anti-inflammatory Effect on Human Aortic Valve Interstitial Cells through Extracellular and Intracellular Actions

Figures

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  Figure 1. Recombinant IL-37 suppresses soluble ECM proteins-induced adhesion molecules and pro-inflammatory cytokines in AVICs. (A and B) Normal human AVICs were treated with recombinant IL-37 (1 ng/mL) for 1 hour or left untreated, followed by stimulation with recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL) for 48 hours. Representative immunoblots (upper) and densitometric data (lower) show that IL-37 abolished the effect of matrilin-2 and biglycan on the production of ICAM-1 and VCAM-1. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. #P<0.05 vs. matrilin-2 alone or biglycan alone. (C) Cytokine production of AVICs after stimulated with recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL) for 48 hours. IL-37 suppressed CCL2 and IL-6 production in AVIC exposed to soluble ECM proteins. Values are mean ± SEM, n = 3 donors. *P<0.05 vs. control. #P<0.05 vs. matrilin-2 alone or biglycan alone. (D and E) AVICs from wild type mice and IL-37tg mice were treated with recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL) for 48 hours or left untreated. Representative immunoblots (upper) and densitometric data (lower) show that expression of IL-37 in murine AVICs abolished…

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  Figure 2. IL-37 inhibits ECM protein-induced PKR and NF-κB activation. (A and B) Normal human AVICs were treated with recombinant IL-37 (1 ng/mL) for 1 hour or left untreated. The cells were then stimulated with recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL) for different times. Representative immunoblots (upper) and densitometric data (lower) show that IL-37 markedly reduced the phosphorylation of PKR and NF-κB p65. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. #P<0.05 vs. cells treated with matrilin-2 alone or biglycan alone at the same time point.

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  Figure 3. Neutralization of IL-18Rα attenuates the anti-inflammatory effect of IL-37 on human AVICs. Human AVICs were treated with IL-18Rα neutralizing antibodies (5 µg/mL) prior to recombinant IL-37. ICAM-1 levels were examined 48 hours after stimulation with recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL). Representative immunoblots (upper) and densitometric data (lower) show that neutralization of IL-18Rα attenuated the effect of IL-37 in suppressing ICAM-1 levels. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. #P<0.05 vs. cells treated with matrilin-2 alone or biglycan alone.

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  Figure 4. Recombinant IL-37 internalized by human AVICs in an IL-18Rα-independent fashion. (A) Human AVICs were left untreated or treated with recombinant IL-37 (1 ng/mL) for various times. After treatment, cells were washed with either PBS or PBS containing detergent. Representative immunoblots (upper) and densitometric data (lower) show that treatment with recombinant IL-37 increased cellular IL-37 levels. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. (B) Human AVICs were treated with tagged IL-37 protein. Representative images of immunofluorescence staining show tagged IL-37 (red) is localized in the perinuclear region of human AVICs. Alexa Fluor 488 conjugate of wheat germ agglutinin (WGA) was applied to label plasma membrane (green). DAPI (40 ,6-diamidino-2-phenylindole) was used to stain nuclei (blue). Original magnification, 40x objective. (C) Representative immunofluorescence staining for tagged IL-37 (red), WGA (green), and DAPI (blue) on human AVICs. Original magnification, 63x objective. Neutralization of IL-18Rα does not alter the internalization of recombinant IL-37 into human AVICs.

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  Figure 5. Internalization of recombinant IL-37 into human AVICs is associated with early endosome and blocking the endocytosis pathway reduced the internalization and anti-inflammatory potency of recombinant IL-37. (A) Human AVICs were treated with tagged IL-37 protein. Representative images of immunofluorescence staining show tagged IL-37 (red), EEA1 (green), and DAPI (blue). Original magnification, 63x objective. IL-37 is partially co-localized with EEA1 (early endosome). (B) Human AVICs were treated with specific inhibitors of endocytic pathways (Nystatin (10 µM), EIPA (20 µM) and Chlorpromazine (10 µM)) prior to recombinant IL-37 (1 ng/mL). Representative immunoblots (upper) and densitometric data (lower) show that Nystatin and EIPA reduce cellular levels of IL-37 in human AVICs treated with recombinant IL-37. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. #P<0.05 vs. cells treated with matrilin-2 alone or biglycan alone. (C and D) Human AVICs were treated with specific inhibitors of endocytic pathways prior to recombinant IL-37 (1 ng/mL) and matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL). Representative immunoblots (upper) and densitometric data (lower) show that inhibition…

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  Figure 6. Nuclear translocation appears to be involved in the mechanism underlying the anti-inflammatory effect of intracellular IL-37. (A and B) Human AVICs were overexpressed with plasmid EGFP-IL-37 and EGFP-IL-37D20A and treated with matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL) for 48 hours. While expression of EGFP-IL-37 abolished NF-κB, and ICAM-1; expression EGFP-IL-37D20A did not. Values are mean ± SEM, n = 4 donors. *P<0.05 vs. control. #P<0.05 vs. cells treated with matrilin-2 alone or biglycan alone. (C-E) AVICs from wild type, IL-37tg and IL-37D20Atg mice were treated with or without recombinant matrilin-2 (2 µg/mL) or biglycan (0.2 µg/mL). While expression of IL-37 in murine AVICs markedly reduced the phosphorylation of NF-κB p65 and inflammatory response, expression of mutant IL-37 (D20A that has no nucleus-targeting sequence) did not. Values are mean ± SEM, n = 4 mice per group. *P<0.05 vs. control. #P<0.05 vs. cells treated with matrilin-2 alone or biglycan alone.