Anthony C. Bruce

L-Selectin, α4β1, and α4β7 Integrins Participate in CD4+ T Cell Recruitment to Chronically Inflamed Small Intestine

CD4+ T cells are essential for development and perpetuation of Crohn’s disease, a chronic immune-mediated condition that affects primarily the small intestine. Using novel models of Crohn’s disease-like ileitis (i.e., SAMP1/YitFc and CD4+ T cell transfer models), we have begun to understand the adhesive pathways that mediate lymphocyte trafficking to the chronically inflamed small bowel. Expansion of the CD4/β7+ population and increased mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression were observed within the intestinal lamina propria with disease progression. However, Ab blockade of the β7 integrin, the α4β7 heterodimer, MAdCAM-1, or L-selectin did not attenuate inflammation. Blockade of two pathways (L-selectin and MAdCAM-1 or α4 integrins) was required to improve ileitis. Further analyses showed that 55 ± 7% of the mesenteric lymph node α4β7+CD4 expressed L-selectin. These L-selectin+ T cells were the main producers of TNF-α and the predominant ileitis-inducing subpopulation. Mechanistically, combined blockade of L-selectin and MAdCAM-1 depleted the intestinal lamina propria of CD4+ T cells that aberrantly coexpressed α4β7 and α4β1 integrins, markedly decreasing local production of TNF-α and IFN-γ. Thus, pathogenic CD4+ T cells not only use the physiologic α4β7/MAdCAM-1 pathway, but alternatively engage α4β1 and L-selectin to recirculate to the chronically inflamed small intestine.

CXCR6 Promotes Atherosclerosis by Supporting T-Cell Homing, Interferon-γ Production, and Macrophage Accumulation in the Aortic Wall

Background— T lymphocytes are thought to be important in atherosclerosis, but very little is known about the mechanisms of lymphocyte recruitment into atherosclerosis-prone aortas. In this study we tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of CD4+ T helper 1 cells and natural killer T cells, is involved in lymphocyte homing into the aortic wall and modulates the development and progression of atherosclerosis. Methods and Results— To investigate the role of CXCR6 in the development and progression of atherosclerosis, we bred CXCR6-deficient (CXCR6GFP/GFP) mice with apolipoprotein E–deficient (ApoE−/−) mice. We found that CXCR6GFP/GFP/ApoE−/− mice fed a Western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE−/− controls. Flow cytometry analysis of the aortas from CXCR6GFP/GFP/ApoE−/− mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas resulted in significantly diminished production of interferon-&ggr; and reduction of CD11b+/CD68+ macrophages in the aorta. Conclusions— These data provide evidence for a proatherosclerotic role of CXCR6. Absence of CXCR6 alters the recruitment of CXCR6+ leukocytes and modulates the local immune response within the aortic wall.

IL-17A Inhibits the Expansion of IL-17A-Producing T Cells in Mice through “Short-Loop” Inhibition via IL-17 Receptor

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4−CD8−αβlow, CD4+CD8−αβ+ (Th17), and γδ+ T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra−/−) mice and Il17ra−/−Itgb2−/− mice that lack both IL-17R and all four β2 integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra−/− and Il17ra−/−Itgb2−/− mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.

Leukocyte phosphoinositide‐3 kinase γ is required for chemokine‐induced, sustained adhesion under flow in vivo

During inflammation, leukocytes roll along the wall of postcapillary venules scanning the surface for immobilized CXCL1, a chemokine that triggers firm adhesion by activating CXCR2 on the neutrophil. PI‐3K are signaling molecules important in cellular processes, ranging from cellular differentiation to leukocyte migration. PI‐3Kγ can be activated directly by the βγ dimer of heterotrimeric G proteins coupled to CXCR2. Here, we used in vivo and ex vivo intravital microscopy models to test the role of PI‐3Kγ in leukocyte arrest. PI‐3Kγ null mice showed an 80% decrease in CXCL1‐induced leukocyte adhesion in venules of the exteriorized mouse cremaster muscle. In wild‐type mice, rolling leukocytes showed rapid and sustained adhesion, but in PI‐3Kγ−/− mice, adhesion was not triggered at all or was transient, suggesting that absence of PI‐3Kγ interferes with integrin bond strengthening. Wild‐type mice reconstituted with PI‐3Kγ null bone marrow showed a 50% decrease in CXCL1‐induced leukocyte adhesion. In a blood‐perfused micro‐flow chamber, leukocytes from PI‐3Kγ−/− mice showed a defect in adhesion on a P‐selectin/ICAM‐1/CXCL1 substrate, indicating that leukocyte PI‐3Kγ was required for adhesion. The adhesion defect in PI‐3Kγ−/− mice was as severe as that in mice lacking LFA‐1, the major integrin responsible for neutrophil adhesion. We conclude that the γ isoform of PI‐3K must be functional in leukocytes to allow efficient adhesion from rolling in response to chemokine stimulation.

Endothelial Cell PECAM-1 Promotes Atherosclerotic Lesions in Areas of Disturbed Flow in ApoE-Deficient Mice

Objective—Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. Methods and Results—We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-&kgr;B were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-&kgr;B activity and VCAM-1 expression under conditions of atheroprone flow. Conclusion—These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-&kgr;B–mediated gene expression.

IL-23 Is Required for Neutrophil Homeostasis in Normal and Neutrophilic Mice

IL-23 is secreted by macrophages and dendritic cells in response to microbial products and inflammatory cytokines. IL-23 is a heterodimer composed of the unique IL-23p19 subunit linked to the common p40 subunit that it shares with IL-12. IL-23 is implicated in autoimmune diseases, where it supports the expansion of IL-17A-producing CD4+ Th17 cells. IL-23 also regulates granulopoiesis in a neutrostat regulatory feedback loop through IL-17A-producing neutrophil regulatory (Tn) cells, most of which express γδ TCR. This homeostatic system is disrupted in mice lacking adhesion molecules like β2-integrins (Itgb2−/−) which have defective neutrophil trafficking and neutrophilia. To test the role of IL-23 in the homeostatic regulation of circulating neutrophil numbers, we measured blood neutrophil numbers in p40-deficient (IL12b−/−) mice and found them reduced compared with wild-type mice. IL12b−/−Itgb2−/− mice, lacking β2-integrins, IL-12, and IL-23 showed significantly blunted neutrophilia compared with Itgb2−/− mice. Treatment of both IL12b−/− and IL12b−/−Itgb2−/− mice with IL-23, but not IL-12, restored circulating neutrophil counts. Serum levels of IL-17A were readily detectable in Itgb2−/− mice, but not in IL12b−/−Itgb2−/− mice, suggesting that IL-17A production is reduced when IL-23 is absent. Similarly, tissue mRNA expression of IL-17A was reduced in IL12b−/−Itgb2−/−mice compared with Itgb2−/− controls. The total number of CD3+ IL-17A-producing Tn cells were significantly reduced in the spleen and lamina propria of IL12b−/−Itgb2−/− mice, with the largest reduction found in γδ+ T cells. Our results suggest a prominent role of IL-23 in the regulation of granulopoiesis and the prevalence of IL-17A-producing Tn cells.

ABCG1 Deficiency in Mice Promotes Endothelial Activation and Monocyte–Endothelial Interactions

Objective—Activated endothelium and increased monocyte–endothelial interactions in the vessel wall are key early events in atherogenesis. ATP binding cassette (ABC) transporters play important roles in regulating sterol homeostasis in many cell types. Endothelial cells (EC) have a high capacity to efflux sterols and express the ABC transporter, ABCG1. Here, we define the role of ABCG1 in the regulation of lipid homeostasis and inflammation in aortic EC. Methods and Results—Using EC isolated from ABCG1-deficient mice (ABCG1 KO), we observed reduced cholesterol efflux to high-density lipoprotein compared to C57BL/6 (B6) EC. However, total cholesteryl ester levels were not changed in ABCG1 KO EC. Secretions of KC, MCP-1, and IL-6 by ABCG1 KO EC were significantly increased, and surface expressions of intercellular adhesion molecule-1 and E-selectin were increased several-fold on ABCG1 KO EC. Concomitant with these findings, we observed a 4-fold increase in monocyte adhesion to the intact aortic endothelium of ABCG1 KO mice ex vivo and to isolated aortic EC from these mice in vitro. In a gain-of-function study in vitro, restoration of ABCG1 expression in ABCG1 KO EC reduced monocyte–endothelial interactions. Utilizing pharmacological inhibitors for STAT3 and the IL-6 receptor, we found that blockade of STAT3 and IL-6 receptor signaling in ABCG1 KO EC completely abrogated monocyte adhesion to ABCG1 KO endothelium. Conclusion—ABCG1 deficiency in aortic endothelial cells activates endothelial IL-6–IL-6 receptor–STAT3 signaling, thereby increasing monocyte–endothelial interactions and vascular inflammation.

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.

Monocytes Are Recruited From Venules During Arteriogenesis in the Murine Spinotrapezius Ligation Model

Objective— Chronic arterial occlusion results in arteriogenesis of collateral blood vessels. This process has been shown to be dependent on the recruitment of growth-promoting macrophages to remodeling collaterals. However, the potential role of venules in monocyte recruitment during microvascular arteriogenesis is not well demonstrated. First, we aim to document that arteriogenesis occurs in the mouse spinotrapezius ligation model. Then, we investigate the temporal and spatial distribution, as well as proliferation, of monocytes/macrophages recruited to collateral arterioles in response to elevated fluid shear stress. Approach and Results— Laser speckle flowmetry confirmed a postligation increase in blood velocity within collateral arterioles but not within venules. After 72 hours post ligation, collateral arteriole diameters were increased, proliferating cells were identified in vessel walls of shear-activated collaterals, and perivascular CD206+ macrophages demonstrated proliferation. A 5-ethynyl-2′-deoxyuridine assay identified proliferation. CD68+CD206+ cells around collaterals were increased 96%, whereas CX3CR1(+/GFP) cells were increased 126% in ligated versus sham groups after 72 hours. CX3CR1(+/GFP) cells were predominately venule associated at 6 hours after ligation; and CX3CR1(+/GFP hi) cells shifted from venule to arteriole associated between 6 and 72 hours after surgery exclusively in ligated muscle. We report accumulation and extravasation of adhered CX3CR1(+/GFP) cells in and from venules, but not from arterioles, after ligation. Conclusions— Our results demonstrate that arteriogenesis occurs in the murine spinotrapezius ligation model and implicate postcapillary venules as the site of tissue entry for circulating monocytes. Local proliferation of macrophages is also documented. These data open up questions about the role of arteriole–venule communication during monocyte recruitment.

Adipose-Derived Stem Cells From Diabetic Mice Show Impaired Vascular Stabilization in a Murine Model of Diabetic Retinopathy

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose‐derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose‐derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high‐throughput enzyme‐linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.