Matthew Butcher

Blockade of Interleukin-17A Results in Reduced Atherosclerosis in Apolipoprotein E–Deficient Mice

Background— T cells play an important role during the immune response that accompanies atherosclerosis. To date, the role for interleukin (IL)-17A in atherogenesis is not well defined. Here, we tested the hypothesis that atherosclerosis-prone conditions induce the differentiation of IL-17A–producing T cells, which in turn promote atherosclerosis. Methods and Results— IL-17A was found to be elevated in the plasma and tissues of apolipoprotein E–deficient (Apoe−/−) mice. IL-17A–expressing T cells were significantly increased in the aortas, spleen, and lamina propria of aged Apoe−/− mice compared with age-matched C57BL/6 mice. IL-17A+ T cells resided in both adventitia and aortas of aged Apoe−/− mice fed a chow diet. Elevated levels of IL-17A+ T cells were also detected in the aortas of 21-week-old Apoe−/− mice fed a Western diet for 15 weeks. IL-17A+ T cells were characterized as predominantly CD4+ T helper 17 (Th17) cells and &ggr;&dgr;+ T cells. Blockade of IL-17A in Apoe−/− mice by use of adenovirus-produced IL-17 receptor A reduced plaque burden in Apoe−/− mice fed a Western diet for 15 weeks. In addition, the treatment diminished circulating IL-6 and granulocyte colony-stimulating factor levels and limited CXCL1 expression and macrophage content within the aortas. Conversely, IL-17A treatment of whole aorta isolated from Apoe−/− mice promoted aortic CXCL1 expression and monocyte adhesion in an ex vivo adhesion assay. Conclusions— These results demonstrate that atherosclerosis-prone conditions induce the differentiation of IL-17A–producing T cells. IL-17A plays a proatherogenic inflammatory role during atherogenesis by promoting monocyte/macrophage recruitment into the aortic wall.

The IL-17A/IL-17RA Axis Plays a Proatherogenic Role via the Regulation of Aortic Myeloid Cell Recruitment

Rationale: Atherosclerosis is a disease of large- and medium-sized arteries that is characterized by chronic vascular inflammation. While the role of Th1, Th2, and T-regulatory subsets in atherogenesis is established, the involvement of IL-17A-producing cells remains unclear. Objective: To investigate the role of the IL-17A/IL-17RA axis in atherosclerosis. Methods and Results: We bred apolipoprotein-E-deficient (Apoe−/−) mice with IL-17A-deficient and IL-17 receptor A-deficient mice to generate Il17a−/−Apoe−/− and Il17ra−/−Apoe−/− mice. Western diet fed Il17a−/−Apoe−/− and Il17ra−/−Apoe−/− mice had smaller atherosclerotic plaques in the aortic arch and aortic roots, but showed little difference in plaque burden in the thoracoabdominal aorta in comparison with Apoe−/− controls. Flow cytometric analysis of Il17a−/−Apoe−/− and Il17ra−/−Apoe−/− aortas revealed that deficiency of IL-17A/IL-17RA preferentially reduced aortic arch, but not thoracoabdominal aortic T cell, neutrophil, and macrophage content in comparison with Apoe−/− aortic segments. In contrast to ubiquitous IL-17RA expression throughout the aorta, IL-17A was preferentially expressed within the aortic arch of WD-fed Apoe−/− mice. Deficiency of IL-17A or IL-17RA reduced aortic arch, but not thoracoabdominal aortic TNF&agr; and CXCL2 expression. Aortic vascular IL-17RA supports monocyte adherence to explanted aortas in ex vivo adhesion assays. Short-term homing experiments revealed that the recruitment of adoptively transferred monocytes and neutrophils to the aortas of Il17ra−/−Apoe−/− mice is impaired in comparison with Apoe−/− recipients. Conclusions: The IL-17A/IL-17RA axis increases aortic arch inflammation during atherogenesis through the induction of aortic chemokines, and the acceleration of neutrophil and monocyte recruitment to this site.

Phenotypic and functional heterogeneity of macrophages and dendritic cell subsets in the healthy and atherosclerosis-prone aorta.

Atherosclerosis continues to be the leading cause of cardiovascular disease. Development of atherosclerosis depends on chronic inflammation in the aorta and multiple immune cells are involved in this process. Importantly, resident macrophages and dendritic cells (DCs) are present within the healthy aorta, but the functions of these cells remain poorly characterized. Local inflammation within the aortic wall promotes the recruitment of monocytes and DC precursors to the aorta and micro-environmental factors direct the differentiation of these emigrated cells into multiple subsets of macrophages and DCs. Recent data suggest that several populations of macrophages and DCs can co-exist within the aorta. Although the functions of M1, M2, Mox, and M4 macrophages are well characterized in vitro, there is a limited set of data on the role of these populations in atherogenesis in vivo. Recent studies on the origin and the potential role of aortic DCs provide novel insights into the biology of aortic DC subsets and prospective mechanisms of the immune response in atherosclerosis. This review integrates the results of experiments analyzing heterogeneity of DCs and macrophage subsets in healthy and diseased vessels and briefly discusses the known and potential functions of these cells in atherogenesis.

Current views on the functions of interleukin-17A-producing cells in atherosclerosis

Multiple components of the immune response are involved in the initiation, progression and persistence of atherosclerosis. Interleukin (IL)-17A is produced by a broad variety of leukocytes and plays an important role in host defense. IL-17A is also involved in the pathology of several autoimmune diseases mainly via the regulation of chemokine expression and leukocyte migration to the site of inflammation. There is an increasing body of evidence indicating an association between elevated levels of IL-17A and cardiovascular diseases. Interestingly, this IL-17A-dependent response occurs in parallel with the Th1-dominant immune response during atherogenesis. To date, the precise role of IL-17A+ cells in atherosclerosis is controversial. Several studies have suggested a pro-atherogenic role of IL-17A via the regulation of aortic macrophage numbers, Th1-related cytokines and aortic chemokine expression. However, two studies recently described anti-inflammatory effects of IL-17A on mouse plaque burden via possible regulation of aortic VCAM-1 expression and T cell content. Furthermore, an initial study using IL-17A-deficient mice demonstrated that IL-17A affects the immune composition and inflammatory phenotype of the aortic wall; however, no effects were observed on atherosclerosis. Further studies are necessary to fully address the role of IL-17A and other IL-17 family members in atherosclerosis.

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes.

Aims/hypothesisChronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets.MethodsHuman islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS.ResultsType 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45+ leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c+ cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets.Conclusions/interpretationElevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.

Interaction between cytokines and inflammatory cells in islet dysfunction, insulin resistance and vascular disease

Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in sustained inflammation in diabetes and atherosclerosis. 12‐ and 15‐lipoxygenase (LO), such as 12/15‐LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12‐ and 15‐LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue‐specific knockout of 12/15‐LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12‐ and 15‐LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12‐ and 15‐LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)‐12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐1 (NOX‐1) in islets and IL‐17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.

Accelerated Atherosclerosis in Apoe−/− Mice Heterozygous for the Insulin Receptor and the Insulin Receptor Substrate-1

Objective—Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. Methods and Results—To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr+/−Irs1+/−Apoe−/−) mice. Insr+/−Irs1+/−Apoe−/− mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr+/+Irs1+/+Apoe−/− mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr+/−Irs1+/−Apoe−/− and Insr+/+Irs1+/+Apoe−/− mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr+/−Irs1+/−Apoe−/− compared to Insr+/+Irs1+/+Apoe−/− mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr+/−Irs1+/− to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr+/−Irs1+/−Apoe−/− mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr+/−Irs1+/−Apoe−/− mice. Conclusion—These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr+/−Irs1+/−Apoe−/− mice by promoting vascular dysfunction and inflammation.

Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ+ Th1/Tregs.

RATIONALE Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. OBJECTIVE Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. METHODS AND RESULTS We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. CONCLUSIONS Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.

Smooth Muscle Cell–Derived Interleukin-17C Plays an Atherogenic Role via the Recruitment of Proinflammatory Interleukin-17A + T Cells to the Aorta

Objective—Atherosclerosis is characterized by frequent communication between infiltrating leukocytes and vascular cells, through chemokine and cytokine networks. Interleukin-17C (IL-17C) is detectable within atherosclerotic lesions; however, the potential involvement of this cytokine has not been examined. Thus, we sought to investigate the role of IL-17C in atherosclerosis. Approach and Results—The expression of IL-17 cytokines was profiled within aortas of apolipoprotein E double knockout (Apoe−/−) mice, and Il17c expression was elevated. Flow cytometry experiments revealed a major population of aortic IL-17C–producing smooth muscle cells. Next, we generated Il17c−/−Apoe−/− mice and demonstrated that atherosclerotic lesion and collagen content was diminished within Western diet–fed Il17c−/−Apoe−/− aortas and aortic roots in comparison to Apoe−/− controls. Smooth muscle cells and fibroblasts were mainly responsible for the reduced Col1A1 expression in the aorta of Il17c−/−Apoe−/− mice. Importantly, IL-17C–treated Apoe−/− aortas showed upregulated Col1A1 expression ex vivo. Il17c−/−Apoe−/− mice displayed a proportional reduction in aortic macrophages, neutrophils, T cells, T helper 1 cells, and T regulatory cells, without corresponding changes in the peripheral immune composition. Examination of aortic IL-17A+ T-cell receptor &ggr;&dgr; T cells and Th17 cells demonstrated a stark reduction in the percentage and number of these subsets within Il17c−/−Apoe−/− versus Apoe−/− mice. Explanted 12-week Western diet–fed Apoe−/− aortas treated with IL-17C resulted in the induction of multiple vascular chemokines and cytokines. Th17 cells demonstrated attenuated migration toward supernatants from cultures of Il17c−/−Apoe−/− smooth muscle cells, and short-term homing experiments revealed diminished recruitment of Th17 cells to the aorta of Il17c−/−Apoe−/− recipients. Conclusions—Smooth muscle cell–derived IL-17C plays a proatherogenic role by supporting the recruitment of Th17 cells to atherosclerotic lesions.

The dynamic lives of macrophage and dendritic cell subsets in atherosclerosis.

Atherosclerosis, the major pathological process through which arterial plaques are formed, is a dynamic chronic inflammatory disease of large‐ and medium‐sized arteries in which the vasculature, lipid metabolism, and the immune system all play integral roles. Both the innate and adaptive immune systems are involved in the development and progression of atherosclerosis but myeloid cells represent the major component of the burgeoning atherosclerotic plaque. Various myeloid cells, including monocytes, macrophages (MΦs), and dendritic cells (DCs) can be found within the healthy and atherosclerotic arterial wall, where they can contribute to or regulate inflammation. However, the precise behaviors and functions of these cells in situ are still active areas of investigation that continue to yield exciting and surprising new data. Here, we review recent progress in understanding of the complex biology of MΦs and DCs, focusing particularly on the dynamic regulation of these subsets in the arterial wall and novel, emerging functions of these cells during atherogenesis.