Khatuna Gabunia

The Anti-Inflammatory Cytokine Interleukin 19 Is Expressed By and Angiogenic for Human Endothelial Cells

Objective—To characterize the expression and function of interleukin (IL) 19, a recently described T-helper 2 anti-inflammatory IL, on endothelial cell (EC) pathophysiological features. Methods and Results—The expression and effects of anti-inflammatory ILs on EC activation and development of angiogenesis are uncharacterized. We demonstrate by immunohistochemistry and immunoblot that IL-19 is expressed in inflamed, but not normal, human coronary endothelium and can be induced in cultured human ECs by serum and basic fibroblast growth factor. IL-19 is mitogenic and chemotactic, and it promotes EC spreading. IL-19 activates the signaling proteins STAT3, p44/42, and Rac1. In functional ex vivo studies, IL-19 promotes cordlike structure formation of cultured ECs and enhances microvessel sprouting in the mouse aortic ring assay. IL-19 induces tube formation in gelatinous protein (Matrigel) plugs in vivo. Conclusion—To our knowledge, these data are the first to report expression of the anti-inflammatory agent, IL-19, in ECs; and the first to indicate that IL-19 is mitogenic and chemotactic for ECs and can induce the angiogenic potential of ECs.

Attenuation of Experimental Atherosclerosis by Interleukin-19

Objective—Interleukin-19 (IL-19) is a putative Th2, anti-inflammatory interleukin. Its expression and potential role in atherogenesis are unknown. IL-19 is not detected in normal artery and is expressed to a greater degree in plaque from symptomatic versus asymptomatic patients, suggesting a compensatory counter-regulatory function. We tested whether IL-19 could reduce atherosclerosis in susceptible mice and identified plausible mechanisms. Approach and Results—LDLR−/− mice fed an atherogenic diet and injected with either 1.0 or 10.0 ng/g per day recombinant mouse IL-19 had significantly less plaque area in the aortic arch compared with controls (P<0.0001). Weight gain, cholesterol, and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19–treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, interferon-&ggr;, interleukin-1&bgr;, and interleukin-12&bgr; and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19–treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated that IL-19–treated mice have significantly less macrophage infiltrate compared with controls (P<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells, vascular smooth muscle cells, and bone marrow–derived macrophages with IL-19 resulted in a significant decrease in chemokine mRNA and mRNA stability protein human antigen R. Conclusions—These data suggest that IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.

Interleukin-19 (IL-19) Induces Heme Oxygenase-1 (HO-1) Expression and Decreases Reactive Oxygen Species in Human Vascular Smooth Muscle Cells

Background: IL-19 is a Th2 interleukin recently recognized to have anti-inflammatory activity in vascular cells. Results: IL-19 induced HO-1 expression, decreased apoptosis, and ROS abundance in vascular cells in an HO-1-dependent fashion. Conclusion: IL-19 represents a link for two previously unassociated protective processes; Th2 cytokine mediated anti-inflammation and ROS reduction. Significance: IL-19 may represent a potential therapeutic for vascular diseases. Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease.

IL-19 Reduces Ligation-Mediated Neointimal Hyperplasia by Reducing Vascular Smooth Muscle Cell Activation

We tested the hypothesis that IL-19, a putative member of the type 2 helper T-cell family of anti-inflammatory interleukins, can attenuate intimal hyperplasia and modulate the vascular smooth muscle cell (VSMC) response to injury. Ligated carotid artery of IL-19 knockout (KO) mice demonstrated a significantly higher neointima/intima ratio compared with wild-type (WT) mice (P = 0.04). More important, the increased neointima/intima ratio in the KO could be reversed by injection of 10 ng/g per day recombinant IL-19 into the KO mouse (P = 0.04). VSMCs explanted from IL-19 KO mice proliferated significantly more rapidly than WT. This could be inhibited by addition of IL-19 to KO VSMCs (P = 0.04 and P < 0.01). IL-19 KO VSMCs migrated more rapidly compared with WT (P < 0.01). Interestingly, there was no type 1 helper T-cell polarization in the KO mouse, but there was significantly greater leukocyte infiltrate in the ligated artery in these mice compared with WT. IL-19 KO VSMCs expressed significantly greater levels of inflammatory mRNA, including IL-1β, tumor necrosis factor α, and monocyte chemoattractant protein-1 in response to tumor necrosis factor α stimulation (P < 0.01 for all). KO VSMCs expressed greater adhesion molecule expression and adherence to monocytes. Together, these data indicate that IL-19 is a previously unrecognized counterregulatory factor for VSMCs, and its expression is an important protective mechanism in regulation of vascular restenosis.

Interleukin-19 increases angiogenesis in ischemic hind limbs by direct effects on both endothelial cells and macrophage polarization

Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. There is a gap in our knowledge concerning the role of anti-inflammatory interleukins in angiogenesis, macrophage polarization, and endothelial cell activation. Interleukin-19 is a unique anti-inflammatory Th2 cytokine that promotes angiogenic effects in cultured endothelial cells (EC); the purpose of this study was to characterize a role for IL-19 in restoration of blood flow in hind-limb ischemia, and define potential mechanisms. Hind limb ischemia was induced by femoral artery ligation, and perfusion quantitated using Laser Doppler Perfusion Imaging (LDPI). Wild type mice which received i.p. injections of rIL-19 (10ng/g/day) showed significantly increased levels of perfusion compared to PBS controls. LDPI values were significantly decreased in IL-19(-/-) mice when compared to wild type mice. IL-19(-/-) mice injected with rIL-19 had significantly increased LDPI compared with PBS control mice. Significantly increased capillary density was quantitated in rIL-19 treated mice, and significantly less capillary density in IL-19(-/-) mice. Multiple cell types participate in IL-19 induced angiogenesis. IL-19 treatment of human microvascular EC induced expression of angiogenic cytokines. M2 macrophage marker and VEGF-A expression were significantly increased in macrophage and the spleen from rIL-19 injected mice, and M1 marker expression was significantly increased in the spleen from IL-19(-/-) compared with controls. Plasma VEGF-A levels are higher in rIL-19 injected mice. IL-19 decreased the expression of anti-angiogenic IL-12 in the spleen and macrophage. This study is the first to implicate IL-19 as a novel pro-angiogenic interleukin and suggests therapeutic potential for this cytokine.

Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC

The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

Interleukin-19 induces angiogenesis in the absence of hypoxia by direct and indirect immune mechanisms

Neovascularization and inflammation are independent biological processes but are linked in response to injury. The role of inflammation-dampening cytokines in the regulation of angiogenesis remains to be clarified. The purpose of this work was to test the hypothesis that IL-19 can induce angiogenesis in the absence of tissue hypoxia and to identify potential mechanisms. Using the aortic ring model of angiogenesis, we found significantly reduced sprouting capacity in aortic rings from IL-19(-/-) compared with wild-type mice. Using an in vivo assay, we found that IL-19(-/-) mice respond to vascular endothelial growth factor (VEGF) significantly less than wild-type mice and demonstrate decreased capillary formation in Matrigel plugs. IL-19 signals through the IL-20 receptor complex, and IL-19 induces IL-20 receptor subunit expression in aortic rings and cultured human vascular smooth muscle cells, but not endothelial cells, in a peroxisome proliferator-activated receptor-γ-dependent mechanism. IL-19 activates STAT3, and IL-19 angiogenic activity in aortic rings is STAT3-dependent. Using a quantitative RT-PCR screening assay, we determined that IL-19 has direct proangiogenic effects on aortic rings by inducing angiogenic gene expression. M2 macrophages participate in angiogenesis, and IL-19 has indirect angiogenic effects, as IL-19-stimulated bone marrow-derived macrophages secrete proangiogenic factors that induce greater sprouting of aortic rings than unstimulated controls. Using a quantitative RT-PCR screen, we determined that IL-19 induces expression of angiogenic cytokines in bone marrow-derived macrophages. Together, these data suggest that IL-19 can promote angiogenesis in the absence of hypoxia by at least two distinct mechanisms: 1) direct effects on vascular cells and 2) indirect effects by stimulation of macrophages.

Heterologous protein incites abnormal plasma cell accumulation and autoimmunity in MRL-MpJ mice.

Although it is evident that there is complex interplay among genetic and environmental factors contributing to systemic autoimmunity, the events inciting autoreactivity are incompletely understood. Previously we demonstrated that MRL-MpJ mice posses a genetic background susceptible to autoimmunity development under conditions of altered inhibitory signaling. To gain better understanding of the influence of exogenous factors on autoreactivity in susceptible individuals, young MRL-MpJ mice were challenged with a single injection of heterologous protein and evaluated for evidence of autoimmunity. We found that MRL-MpJ mice developed high titer serum reactivity to DNA within 1 week of protein administration reaching maximal levels within 1 month. Importantly, the level of autoimmunity was sustained for an extended period of time (6 months). This was accompanied by a substantial increase in germinal center B cell and plasma cell numbers. In contrast, control mice showed no change in autoreactivity or lymphocyte homeostasis. Autoimmunity was dependent on marginal zone B cells as their depletion reduced serum auto-reactivity after challenge, thus suggesting immune stimulation with heterologous proteins can precipitate loss of B cell tolerance and autoimmunity in genetically prone individuals. This model may provide an important tool to further investigate the mechanisms whereby environmental stimuli trigger autoimmune reactivity in susceptible hosts.

Genetic Deletion of IL-19 (Interleukin-19) Exacerbates Atherogenesis in Il19−/−×Ldlr−/− Double Knockout Mice by Dysregulation of mRNA Stability Protein HuR (Human Antigen R)

Objective— To test the hypothesis that loss of IL-19 (interleukin-19) exacerbates atherosclerosis. Approach and Results— Il19−/− mice were crossed into Ldlr−/− (low-density lipoprotein receptor knock out) mice. Double knockout (dKO) mice had increased plaque burden in aortic arch and root compared with Ldlr−/− controls after 14 weeks of high-fat diet (HFD). dKO mice injected with 10 ng/g per day rmIL-19 had significantly less plaque compared with controls. qRT-PCR and Western blot analysis revealed dKO mice had increased systemic and intraplaque polarization of T cells and macrophages to proinflammatory Th1 and M1 phenotypes, and also significantly increased TNF (tumor necrosis factor)-&agr; expression in spleen and aortic arch compared with Ldlr−/− controls. Bone marrow transplantation suggests that immune cells participate in IL-19 protection. Bone marrow-derived macrophages and vascular smooth muscle cells isolated from dKO mice had a significantly greater expression of inflammatory cytokine mRNA and protein compared with controls. Spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stability protein HuR (human antigen R). Bone marrow-derived macrophage and vascular smooth muscle cell isolated from dKO mice also had greater HuR abundance. HuR stabilizes proinflammatory transcripts by binding AU-rich elements in the 3′ untranslated region. Cytokine and HuR mRNA stability were increased in dKO bone marrow-derived macrophage and vascular smooth muscle cell, which was rescued by addition of IL-19 to these cells. IL-19–induced expression of miR133a, which targets and reduced HuR abundance; miR133a levels were lower in dKO mice compared with controls. Conclusions— These data indicate that IL-19 is an atheroprotective cytokine which decreases the abundance of HuR, leading to reduced inflammatory mRNA stability.