David T. Bolick

Sphingosine-1-Phosphate Prevents Tumor Necrosis Factor-α-Mediated Monocyte Adhesion to Aortic Endothelium in Mice

Objective—Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte–EC interactions in vivo. Methods and Results—We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-&agr; in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-&agr;–injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-&agr; plus SEW2871-treated mice had few monocytes bound (P<0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte–EC interactions in response to TNF-&agr;. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun. Conclusions—Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-&agr;–mediated monocyte–EC interactions in vivo.

Sphingosine-1 Phosphate Prevents Monocyte/Endothelial Interactions in Type 1 Diabetic NOD Mice Through Activation of the S1P1 Receptor

Monocyte recruitment and adhesion to vascular endothelium are key early events in atherosclerosis. We examined the role of sphingosine-1-phosphate (S1P) on modulating monocyte/endothelial interactions in the NOD/LtJ (NOD) mouse model of type 1 diabetes. Aortas from nondiabetic and diabetic NOD mice were incubated in the absence or presence of 100 nmol/L S1P. Fluorescently labeled monocytes were incubated with the aortas. Aortas from NOD diabetic mice bound 7-fold more monocytes than nondiabetic littermates (10±1 monocytes bound/field for nondiabetic mice vs 74±12 monocytes bound/field for diabetic mice, P<0.0001). Incubation of diabetic aortas with 100 nmol/L S1P reduced monocyte adhesion to endothelium by 90%. We found expression of S1P1, S1P2, and S1P3 receptors on NOD aortic endothelial cells. The S1P1 receptor-specific agonist SEW2871 inhibited monocyte adhesion to diabetic aortas. Studies in diabetic S1P3-deficient mice revealed that the S1P3 receptor did not play a pivotal role in this process. S1P reduced endothelial VCAM-1 induction in type 1 diabetic NOD mice, most likely through inhibition of nuclear factor &kgr;B translocation to the nucleus. Thus, S1P activation of the S1P1 receptor functions in an antiinflammatory manner in type 1 diabetic vascular endothelium to prevent monocyte/endothelial interactions. S1P may play an important role in the prevention of vascular complications of type 1 diabetes.

12/15-Lipoxygenase Regulates Intercellular Adhesion Molecule-1 Expression and Monocyte Adhesion to Endothelium Through Activation of RhoA and Nuclear Factor-κB

Background—12/15-lipoxygenase (12/15-LO) activity leads to the production of the proinflammatory eicosanoids 12-S-hydroxyeicosatetraenoic acid (12SHETE) and 13-S-hydroxyoctadecadienoic acid. We have previously shown a 3.5-fold increase in endothelial intercellular adhesion molecule (ICAM)-1 expression in mice overexpressing the 12/15-LO gene. We examined whether 12/15-LO activity regulated endothelial ICAM-1 expression. Methods and Results—Freshly isolated aortic endothelial cells (EC) from 12/15-LO transgenic mice had significantly greater nuclear factor-&kgr;B (NF-&kgr;B) activation and ICAM mRNA expression compared with C57BL/6J control. 12/15-LO transgenic EC showed elevated RhoA activity, and inhibition of RhoA using either C3 toxin or the Rho-kinase inhibitor Y-27632 blocked NF-&kgr;B activation, ICAM-1 induction, and monocyte adhesion. Furthermore, we show that 12SHETE activates protein kinase C&agr;, which forms a complex with active RhoA and is required for NF-&kgr;B–dependent ICAM expression in response to 12SHETE. Conclusions—The 12/15-LO pathway stimulates ICAM-1 expression through the RhoA/protein kinase C&agr;-dependent activation of NF-&kgr;B. These findings identify a major signaling pathway in EC through which 12/15-LO contributes to vascular inflammation and atherosclerosis.

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.

Biomarkers of environmental enteropathy, inflammation, stunting, and impaired growth in children in Northeast Brazil

Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6–26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment. Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin. We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.

G2A Deficiency in Mice Promotes Macrophage Activation and Atherosclerosis

G2A is a stress-inducible G protein–coupled receptor that is expressed on several cell types within atherosclerotic lesions. We demonstrated previously that G2A deficiency in mice increased aortic monocyte recruitment and increased monocyte:endothelial interactions. To investigate the impact of G2A deficiency in macrophages, we isolated peritoneal macrophages from G2A+/+ApoE−/− and G2A−/−ApoE−/− mice. G2A−/−ApoE−/− macrophages had significantly lower apoptosis than control macrophages. The prosurvival genes BCL-2, BCL-xL, and cFLIP were increased in G2A−/−ApoE−/− macrophages. Macrophages from G2A−/−ApoE−/− mice also had increased proinflammatory status that was indicative of a M1 macrophage phenotype. This was indicated by significantly increased nuclear translocation of nuclear factor &kgr;B, as well as production of interleukin-12p40, tumor necrosis factor &agr;, and interleukin-6, and reduced expression of arginase-I. Moreover, G2A−/−ApoE−/− macrophages had reduced ability to engulf apoptotic cells in vitro. We examined atherosclerosis in mice fed a Western diet for 10 weeks and found that G2A deficiency increased lesion size in the aortic root by 50%. Plasma lipid levels were not changed in G2A−/−ApoE−/− mice. However, we found that absence of G2A increased the number of aortic macrophages and attenuated apoptosis in this cell type. Moreover, bone marrow transplantation studies indicated that deficiency of G2A in marrow-derived cells significantly contributed to atherosclerosis development. In the absence of G2A, increased macrophage activation and decreased apoptosis is associated with accumulation of macrophages in the aorta and increased atherosclerosis.

12/15 Lipoxygenase Mediates Monocyte Adhesion to Aortic Endothelium in Apolipoprotein E–Deficient Mice Through Activation of RhoA and NF-κB

Objectives—12/15 lipoxygenase (12/15LO) has been implicated as a mediator of inflammation and atherosclerosis. In the current study, we identified mechanisms through which 12/15LO mediates monocyte:endothelial interactions in vivo in apolipoprotein E-deficient mice (apoEKO), a well-characterized mouse model of atherosclerosis. Methods and Results—In apoEKO mice that are also deficient in 12/15LO (doubleKO), monocyte adhesion to aorta in vivo was reduced by 95% in doubleKO mice compared with apoEKO mice. Inhibition of 12/15LO in apoEKO mice in vivo using CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) prevented monocyte adhesion to aortic endothelium in apoEKO mice. Aortic endothelium of apoEKO mice had significant activation of rhoA compared with doubleKO aortic endothelium. Further, apoEKO aorta displayed significant activation of NF-&kgr;B. DoubleKO aorta displayed little nuclear localization of NF-&kgr;B. Finally, we found significant upregulation of intercellular adhesion molecule-1 (ICAM-1) on apoEKO aortic endothelium compared with doubleKO endothelium. Inhibition of rhoA and PKC&agr; significantly reduced NF-&kgr;B activation, ICAM-1 induction, and monocyte adhesion to aorta. Conclusions—We conclude that 12/15LO products activate endothelial rhoA and PKC&agr;. Activation of rhoA and PKC&agr; cause activation and translocation of NF-&kgr;B to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.

Absence of the G Protein-Coupled Receptor G2A In Mice Promotes Monocyte/Endothelial Interactions in Aorta

The G protein–coupled receptor G2A is highly expressed on macrophages and lymphocytes and has been localized to atherosclerotic plaques. We examined the role of G2A in modulating monocyte/endothelial interactions in the vessel wall. We measured adhesion of WEHI 78/24 monocytes to aortas of C57BL/6 (B6) and G2A-deficient (G2A−/−) mice using an ex vivo adhesion assay. G2A−/− mice had 10-fold elevations in adhesion of monocytes to aortas. Injection of GFP-expressing wild-type macrophages into B6 and G2A−/− mice in vivo showed increased macrophage accumulation in the aortic wall of G2A−/− mice. We isolated aortic endothelial cells (ECs) from B6 and G2A−/− mice and found a 2-fold increase in intercellular adhesion molecule-1 and E-selectin surface expression on G2A−/− ECs using flow cytometry. Using ELISA, we found a 3-fold increase in interleukin-6 and monocyte chemoattractant protein-1 production by G2A−/− ECs compared with B6 ECs. We found a dramatic increase in nuclear localization of the p65 subunit of nuclear factor &kgr;B in G2A−/− ECs. Transfection of G2A into G2A−/− ECs to restore normal expression levels reduced p65 nuclear localization to 35%. Restoration of G2A expression in G2A−/− ECs significantly reduced intercellular adhesion molecule-1 and endothelial selectin surface expression and reduced monocyte chemoattractant protein-1 and interleukin-6 production. Restoring G2A to G2A−/− ECs reduced monocyte adhesion by 80% compared with G2A−/− ECs in a flow chamber assay. Absence of G2A in endothelium results in proinflammatory signaling and increased monocyte/endothelial interactions in the aortic wall. Thus, endothelial G2A expression may aid in prevention of vascular inflammation and atherosclerosis.

Sphingosine-1-Phosphate Reduces CD4+ T-Cell Activation in Type 1 Diabetes Through Regulation of Hypoxia-Inducible Factor Short Isoform I.1 and CD69

OBJECTIVES—Non-obese diabetic (NOD) mice develop spontaneous type 1 diabetes. We have shown that sphingosine-1-phosphate (S1P) reduces activation of NOD diabetic endothelium via the S1P1 receptor. In the current study, we tested the hypothesis that S1P could inhibit CD4+ T-cell activation, further reducing inflammatory events associated with diabetes. RESEARCH DESIGN AND METHODS—CD4+ T-cells were isolated from diabetic and nondiabetic NOD mouse splenocytes and treated in the absence or presence of S1P or the S1P1 receptor-specific agonist, SEW2871. Lymphocyte activation was examined using flow cytometry, cytokine bead assays, and a lymphocyte:endothelial adhesion assay. RESULTS—Diabetic T-cells secreted twofold more γ-interferon (IFN-γ) and interleukin-17 than nondiabetic lymphocytes. Pretreatment with either S1P or SEW2871 significantly reduced cytokine secretion by ∼50%. Flow cytometry analysis showed increased expression of CD69, a marker of lymphocyte activation, on diabetic T-cells. Both S1P and SEW2871 prevented upregulation of CD69 on CD4+ cells. Quantitative RT-PCR showed that lymphocytes from diabetic NOD mice had 2.5-fold lower hypoxia-inducible factor (HIF)-1α short isoform I.1 (HIF1αI.1) mRNA levels than control. HIF1αI.1 is a negative regulator of lymphocyte activation. S1P significantly increased HIF1α I.1 mRNA levels in both control and diabetic groups. IFN-γ production and surface CD69 expression was significantly increased in lymphocytes of HIF1αI.1-deficient mice. S1P did not reduce either CD69 or IFN-γ expression in lymphocytes from HIF1αI.1-deficient mice. CONCLUSIONS—S1P acts through the S1P1 receptor and HIF1α I.1 to negatively regulate T-cell activation, providing a potential therapeutic target for prevention of diabetes and its vascular complications.

Zinc deficiency alters host response and pathogen virulence in a mouse model of enteroaggregative escherichia coli-induced diarrhea

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrheal disease globally. In the current study, we investigated the impact of zinc deficiency on the host and pathogenesis of EAEC. Several outcomes of EAEC infection were investigated including weight loss, EAEC shedding and tissue burden, leukocyte recruitment, intestinal cytokine expression, and virulence expression of the pathogen in vivo. Mice fed a protein source defined zinc deficient diet (dZD) had an 80% reduction of serum zinc and a 50% reduction of zinc in luminal contents of the bowel compared to mice fed a protein source defined control diet (dC). When challenged with EAEC, dZD mice had significantly greater weight loss, stool shedding, mucus production, and, most notably, diarrhea compared to dC mice. Zinc deficient mice had reduced infiltration of leukocytes into the ileum in response to infection suggesting an impaired immune response. Interestingly, expression of several EAEC virulence factors were increased in luminal contents of dZD mice. These data show a dual effect of dietary zinc in benefitting the host while impairing virulence of the pathogen. The study demonstrates the critical importance of zinc and may help elucidate the benefits of zinc supplementation in cases of childhood diarrhea and malnutrition.