Summary There is a significant recruitment of leucocytes into aortas during atherogenesis. L-selectin regulates leucocyte migration into secondary lymphoid and peripheral tissues and was proposed to play a role in leucocyte homing into aortas. Here, we determine the role of L-selectin in atherosclerosis. L-selectin-deficient Apoe−/− (Sell−/−Apoe−/−) mice had a 74% increase in plaque burden compared to Apoe−/− mice fed a chow diet for 50 weeks. Elevated atherosclerosis was accompanied by increased aortic leucocyte content, but a 50% reduction in aortic B cells despite elevated B cell counts in the blood. Follicular B cells represented 65%, whereas B1a and regulatory B cells (Breg) comprised 5% of aortic B cells. B1a and Breg cell subsets were reduced in Sell−/−Apoe−/− aortas with accompanied 2-fold decrease in aortic T15 antibody and 1.2-fold decrease of IL-10 levels. L-selectin was required for B1 cell homing to the atherosclerotic aorta, as demonstrated by a 1.5-fold decrease in the migration of Sell−/−Apoe−/− versus Apoe−/− cells. Notably, we found a 1.6-fold increase in CD68hi macrophages in Sell−/−Apoe−/− compared to Apoe−/− aortas, despite comparable blood monocyte numbers and L-selectin-dependent aortic homing. L-selectin had no effect on neutrophil migration into aorta, but led to elevated blood neutrophil numbers, suggesting a potential involvement of neutrophils in atherogenesis of Sell−/−Apoe−/− mice. Thus, L-selectin deficiency increases peripheral blood neutrophil and lymphocyte numbers, decreases aortic B1a and Breg populations, T15 antibody and IL-10 levels, and increases aortic macrophage content of Sell−/−Apoe−/− mice. Altogether, these data provide evidence for an overall athero-protective role of L-selectin.

Adipose tissue (AT) inflammation is an emerging factor contributing to cardiovascular disease. STAT4 is a transcription factor expressed in adipocytes and in immune cells and contributes to AT inflammation and insulin resistance in obesity. The objective of this study was to determine the effect of STAT4 deficiency on visceral and peri-aortic AT inflammation in a model of atherosclerosis without obesity. Stat4-/-Apoe-/- mice and Apoe-/- controls were kept either on chow or western diet for 12 weeks. Visceral and peri-aortic AT were collected and analyzed for immune composition by flow cytometry and for cytokine/chemokine expression by real-time PCR. Stat4-/-Apoe-/- and Apoe-/- mice had similar body weight, plasma glucose and lipids. Western diet significantly increased macrophage, CD4+, CD8+ and NK cells in peri-aortic and visceral fat in Apoe-/- mice. In contrast, in Stat4-/-Apoe-/- mice, western diet failed to increase the percentage of immune cells infiltrating the AT. Also, IL12p40, TNFα, CCL5, CXCL10 and CX3CL1 were significantly reduced in the peri-aortic fat in Stat4-/-Apoe-/- mice. Importantly, Stat4-/-Apoe-/- mice on western diet had significantly reduced plaque burden vs. Apoe-/- controls. In conclusion, STAT4 deletion reduces inflammation in peri-vascular and visceral AT and this may contribute via direct or indirect effects to reduced atheroma formation.

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, and dendritic cells 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 macrophages and dendritic cells, focusing particularly on the dynamic regulation of these subsets in the arterial wall and novel, emerging functions of these cells during atherogenesis.

The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis.

The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. While the pro-inflammatory role of signal transducer and activator of transcription 4 (STAT4) in atherosclerosis and diet-induced insulin resistance (IR) was recently established, an impact of STAT4 on atherogenesis in conditions of IR is not known. Here we generated Stat4−/−Ldlr−/− mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC fed Stat4−/−Ldlr−/− mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr−/− controls. Interestingly, we detected a reduction in T follicular helper (Tfh) and plasma B cells, but a sharp elevation in CD8+ Tregs in spleens and aortas of Stat4−/−Ldlr−/− versus Ldlr−/− mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. Stat4-deficient CD8+ Tregs suppressed Tfh and germinal center B cell development upon immunization with KLH indicating an important role for STAT4 in CD8+ Treg functions in vivo. Furthermore, adoptive transfer of Stat4−/−Ldlr−/− CD8+ Tregs vs Ldlr−/− CD8+ Tregs resulted in a significant reduction of plaque burden, suppression of Tfh and germinal center B cells in DDC fed Ldlr−/− recipients. STAT4 expression in macrophages also affected the Tfh/CD8+Treg axis, as conditioned media from Stat4−/−Ldlr−/− MΦs supported CD8+ Treg but not Tfh cell differentiation in TGFβ-dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr−/− mice via STAT4-dependent macrophage as well as cell-intrinsic suppression of CD8+ Treg generation and functions and maintenance of Tfh cell generation and accompanied humoral immune response.