Interleukin-23 receptor expressing γδ T cells locally promote early atherosclerotic lesion formation and plaque necrosis in mice

Aims Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and gamma delta T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and gamma delta T cells in atherosclerosis. Methods and results IL-23R(+) cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr(-/-)Il23r(gfp/+)), and mostly identified as gamma delta T cells that express IL-17 and GM-CSF. scRNA-seq confirmed gamma delta T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr(-)(/)(-)Il23r(gfp/gfp) mice deficient in IL-23R showed a loss of IL-23R(+) cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr(-/-) controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr(-/-)Tcr delta(-/-) mice lacking all gamma delta T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr(-)(/-) mice. In both HFD-fed Ldlr(-/-)Il23r(gfp/gfp) and Ldlr(-/-)Tcr delta(-/-) mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. Conclusions IL-23R(+) gamma delta T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development

Extracellular matrix protein-1 as a mediator of inflammation-induced fibrosis after myocardial infarction

Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+) regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4(+) T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-)Pd1(-/-)) displayed striking increases in systemic CD4(+) and CD8(+) T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-)Pd1(-/-) mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/-) mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis

Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice

<div><p>Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs) bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3) is required for the development of classical CD8α⁺ and CD103⁺ DCs. By crossing mice deficient in <i>Batf3</i> with atherosclerosis-prone low density lipoprotein receptor (<i>Ldlr</i>^<i>-/-</i><i>)</i>-deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α⁺ and CD103⁺ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.</p></div

T cell activation is impaired in the spleen of <i>Batf3</i>^<i>-/-</i> mice after 8 weeks of HFD.

<p>Flow cytometric analyses of spleen cells obtained from atherosclerotic <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed a HFD for 8 weeks. (a) Frequencies of activated CD44^highCD62L^low CD4⁺ and (b) CD8⁺ T cells (representative dot plots are shown; values indicate gated events among CD4⁺ T cells). (c) Frequencies of FoxP3⁺CD25⁺CD4⁺ Tregs, (d) IFNγ⁺CD4⁺ T cells (representative dot plots are shown, values indicate gated events among CD4⁺ T cells), (e) IL-17a⁺CD4⁺ T cells and (f) IFNγ⁺CD8⁺ T cells. Data ara presented as mean ± SEM; *p<0.5;,**p<0.01; ns, non significant.</p

Immune responses are mildy alter in the spleen of <i>Batf3</i>-deficient mice after 8 weeks of HFD.

<p>(a-d) Flow cytometric analyses of Ly6G⁺ neutrophils (a), Ly6C^high and Ly6C^low monocytes (b), and of CD3⁺ T cells (c) among CD45⁺ leukocytes, and of frequencies of CD4⁺ and CD8⁺ T cells among CD3⁺ T cells (d) in spleens from atherosclerotic <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed a HFD for 8 weeks. Data ara presented as mean ± SEM; **p<0.01; ***p<0.001; ns, non significant.</p

Lipid levels of <i>Ldlr</i>^<i>-/-</i> and <i>Ldlr</i>^<i>-/-</i><i>Batf3</i>^<i>-/-</i> mice fed a high fat diet for 8 weeks.

<p>Lipid levels of <i>Ldlr</i>^<i>-/-</i> and <i>Ldlr</i>^<i>-/-</i><i>Batf3</i>^<i>-/-</i> mice fed a high fat diet for 8 weeks.</p

Deficiency in <i>Batf3</i> does not alter atherosclerotic lesion development.

<p>(a,b) Single cell suspensions from aortae and splenocytes were obtained from <i>Ldlr</i>^-/- and <i>Ldlr</i>^<i>-/-</i><i>Batf3</i>^<i>-/-</i> mice fed a HFD for 8 weeks and analyzed by flow cytometry. CD11c⁺ MHCII⁺ DCs were gated and further discriminated by expression of CD11b and CD103 in the aorta (a) or CD8α in the spleen (b). (c-d) Quantification of plaque area in Oil-Red-O stained aortae and in Aldehyde Fuchsin-stained aortic roots in atherosclerotic <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed a HFD for 8 weeks; representative images of the aorta and aortic root sections are shown. Data ara presented as mean ± SEM; ns, non significant.</p

Plaque composition is not altered by <i>Batf3</i> deficiency.

<p>Quantification of the area positive for Mac-2 (a) representative images of immunofluorescence staining are shown; scale bars: 50μm; cell nuclei were counterstained with DAPI (blue), α-smooth muscle actin (b), Sirius-red (c), and of the necrotic core (d) in the aortic root from <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed with 8 weeks of HFD. Data ara presented as mean ± SEM; ns, non significant.</p