Peripheral arterial disease (PAD) is a worldwide disease caused by atherosclerosis. It is a circulatory condition where narrowed blood vessels reduce blood flow to the peripheral such as legs. Although current gold standard treatment for advanced PAD patients is still based on surgical revascularization, there is no effective therapy for many patients that are not suitable for surgery. In addition, better recovery from surgical revascularization largely relies on angiogenesis in the adjacent ischemic tissue. Thus, novel pro-angiogenic therapies to improve post-ischemic neovascularization are urgently desired. However, current poor understanding of the roles of anti-inflammatory cytokines in angiogenesis prevents the development of these new therapies. We and others have reported that IL-35 is a newly identified inducible immunosuppressive heterodimeric cytokine in the IL-12 family. IL-35 is composed of p35 (IL-12A) and EBI3, and its receptors are comprised of homodimers or heterodimer of IL-12Rb2 and gp130 (IL-6ST). We have shown that IL-35 inhibits endothelial cell (EC) activation induced by lipopolysaccharide (LPS) or atherogenic lysophosphatidylcholine (LPC). At least partially through these new EC-dependent mechanisms, IL-35 inhibits inflammation in autoimmune diseases, infectious diseases, atherosclerosis, and tumors. Recent studies have indicated the role of IL-35 in angiogenesis in rheumatoid arthritis and different tumors. However, whether and how IL-35 regulates post-ischemic angiogenesis in peripheral artery disease are unrevealed. In our study, we used hindlimb ischemia (HLI) and Matrigel plug assay as in vivo angiogenesis models and wound healing assay as in vitro angiogenesis model to study the role and underlying mechanisms of IL-35-mediated angiogenesis. We made the following findings: 1) muscle in human and mouse has high angiogenic potential in physiological conditions; 2) angiogenic cytokines and chemokines including anti-inflammatory cytokines are predominantly regulated by inflammatory transcription factors; 3) IL-35 signaling is induced in ischemic muscle; 4) IL-12Rb2, but not IL-6ST, is the key receptor component of IL-35 signaling in ischemic muscle and hypoxic human microvascular endothelial cells (HMVECs); 5) hyperlipidemia (atherogenic factor) impairs angiogenesis in vivo and in vitro, which partially acts through the induction of IL-35; 6) IL-12Rb2 deficiency improves HLI-induced angiogenesis in both WT or apolipoprotein E (ApoE) -/- mice (an atherosclerosis model); 7) IL-35 injection inhibits HLI-induced angiogenesis in WT mice but not that in the IL-12Rb2 deficient mice; 8) IL-35 injection enlarges the avascular area in gastrocnemius muscle after HLI; 9) IL-35 obstructs fibroblast growth factor-2 (FGF2)-induced angiogenesis in Matrigel plug assay in vivo; 10) CD45-CD31+ ECs from the IL-35-injected ischemic muscle at day 14 of HLI have an abnormal extracellular matrix organization, activated integrin pathways (cell-matrix adhesions), disrupted vascular endothelial (VE)-cadherin-plakoglobin complex (cell-cell adhesions), and increased infiltration and migration of bone marrow-derived leukocytes; 11) IL-35 inhibits HMVEC migration in wound healing assay in vitro presumably through upregulation of anti-angiogenic proteins including pigment epithelium-derived factor (PEDF), serpin family B member 5 (SERPINB5, Maspin), and thrombospondin (THBS)-1. These results suggest that anti-inflammatory cytokine IL-35, signaling through the key receptor subunit IL-12Rb2, inhibits HLI-induced angiogenesis and delays tissue repair by dysregulating cell-cell and cell-matrix adhesions, which leads to the impaired vascular adhesion junction and maturation of blood vessels. / Biomedical Sciences

Type 1 diabetes (T1D) is etiologically considered as an autoimmune disease, where insulin-producing β-cells are damaged by autoimmune attacks. Regulatory T (Treg) cells are immune homeostasis cells. In the present thesis I aimed to investigate the role of Treg cells and other immune cells in the early development of T1D. In order to do that, we first determined which immune cells that are altered at an early stage of the T1D development. We found that dendritic cells and plasmacytoid dendritic cells induce the initial immune response. Next, we investigated the role of Treg cells in multiple low dose streptozotocin (MLDSTZ) induced T1D and in NOD mice. We found that the numbers of Treg cells were increased in both MLDSTZ and NOD mice when the MLDSTZ mice were hyperglycemic. However, the increased Treg cells showed a decreased production of anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) and an increased expression of pro-inflammatory cytokines (IFN-γ and IL-17a). These results revealed that Treg cells switch their phenotype under T1D conditions. IL-35 administration effectively prevented the development of, and reversed established MLDSTZ induced T1D. Treg cells from IL-35 treated mice showed an increased expression of the Eos transcription factor, accompanied by an increased expression of IL-35 and a decreased expression of IFN-γ and IL-17a. These data indicate that IL-35 administration counteracted the early development of T1D by maintaining the phenotype of the Treg cells. Furthermore, IL-35 administration reversed established T1D in the NOD mouse model by maintaining the phenotype of Treg cells, seemingly by inducing the expression of Eos. Moreover, the circulating level of IL-35 was significantly lowered in both new onset and long-standing T1D patients compared to healthy controls. In addition, patients with T1D with remaining C-peptide had significantly higher levels of IL-35 than patients lacking C-peptide, suggesting that IL-35 might prevent the loss of β-cell mass. In line with this hypothesis, we found that LADA patients had a higher proportion of IL-35+ tolerogenic antigen presenting cells than T1D patients. Subsequently, we determined the proportions of IL-35+ Treg cells and IL-17a+ Treg cells in T1D patients with diabetic nephropathy (DN), which were age, sex and BMI matched with healthy controls and T1D patients. The proportion of IL-35+ Treg cells was decreased in DN and T1D patients, but IL-17a+ Treg cells were more abundant than in healthy controls. Furthermore, we found that the number of Foxp3+ Treg cells was increased in the kidneys of MLDSTZ mice. However, infiltration of mononuclear cells was seen in kidneys of these mice. In addition, kidney tissues of IL-35 treated MLDSTZ mice did not show any mononuclear cell infiltration. These results demonstrate that IL-35 may be used to prevent mononuclear cell infiltration in kidney diseases. Our findings indicate that the numbers of Foxp3+ Treg cells are increased in T1D, but that these Treg cells fail to counteract the ongoing immune assault in islets and kidneys of hyperglycemic mice. This could be explained by a phenotypic shift of the Treg cells under hyperglycemic conditions. IL-35 administration reversed established T1D in two different animal models of T1D and prevented mononuclear cell infiltration in the kidneys by maintaining the phenotype of Treg cells.