Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice

Abstract Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle is necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Intestinal parasitic infection alters bone marrow derived dendritic cell inflammatory cytokine production in response to bacterial endotoxin in a diet-dependent manner.

Giardia lamblia is a common intestinal parasitic infection that although often acutely asymptomatic, is associated with debilitating chronic intestinal and extra-intestinal sequelae. In previously healthy adults, a primary sporadic Giardia infection can lead to gut dysfunction and fatigue. These symptoms correlate with markers of inflammation that persist well after the infection is cleared. In contrast, in endemic settings, first exposure occurs in children who are frequently malnourished and also co-infected with other enteropathogens. In these children, Giardia rarely causes symptoms and associates with several decreased markers of inflammation. Mechanisms underlying these disparate and potentially enduring outcomes following Giardia infection are not presently well understood. A body of work suggests that the outcome of experimental Giardia infection is influenced by the nutritional status of the host. Here, we explore the consequences of experimental Giardia infection under conditions of protein sufficiency or deficiency on cytokine responses of ex vivo bone marrow derived dendritic cells (BMDCs) to endotoxin stimulation. We show that BMDCs from Giardia- challenged mice on a protein sufficient diet produce more IL-23 when compared to uninfected controls whereas BMDCs from Giardia challenged mice fed a protein deficient diet do not. Further, in vivo co-infection with Giardia attenuates robust IL-23 responses in endotoxin-stimulated BMDCs from protein deficient mice harboring enteroaggregative Escherichia coli. These results suggest that intestinal Giardia infection may have extra-intestinal effects on BMDC inflammatory cytokine production in a diet dependent manner, and that Giardia may influence the severity of the innate immune response to other enteropathogens. This work supports recent findings that intestinal microbial exposure may have lasting influences on systemic inflammatory responses, and may provide better understanding of potential mechanisms of post-infectious sequelae and clinical variation during Giardia and enteropathogen co-infection.

5-Fluorouracil Induces Enteric Neuron Death and Glial Activation During Intestinal Mucositis via a S100B-RAGE-NFκB-Dependent Pathway.

5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system (ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury.

Disentangling Microbial Mediators of Malnutrition: Modeling Environmental Enteric Dysfunction.

Environmental enteric dysfunction (EED) (also referred to as environmental enteropathy) is a subclinical chronic intestinal disorder that is an emerging contributor to early childhood malnutrition. EED is common in resource-limited settings, and is postulated to consist of small intestinal injury, dysfunctional nutrient absorption, and chronic inflammation that results in impaired early child growth attainment. Although there is emerging interest in the hypothetical potential for chemical toxins in the environmental exposome to contribute to EED, the propensity of published data, and hence the focus of this review, implicates a critical role of environmental microbes. Early childhood malnutrition and EED are most prevalent in resource-limited settings where food is limited, and inadequate access to clean water and sanitation results in frequent gastrointestinal pathogen exposures. Even as overt diarrhea rates in these settings decline, silent enteric infections and faltering growth persist. Furthermore, beyond restricted physical growth, EED and/or enteric pathogens also associate with impaired oral vaccine responses, impaired cognitive development, and may even accelerate metabolic syndrome and its cardiovascular consequences. As these potentially costly long-term consequences of early childhood enteric infections increasingly are appreciated, novel therapeutic strategies that reverse damage resulting from nutritional deficiencies and microbial insults in the developing small intestine are needed. Given the inherent limitations in investigating how specific intestinal pathogens directly injure the small intestine in children, animal models provide an affordable and controlled opportunity to elucidate causal sequelae of specific enteric infections, to differentiate consequences of defined nutrient deprivation alone from co-incident enteropathogen insults, and to correlate the resulting gut pathologies with their functional impact during vulnerable early life windows.

Increased Urinary Trimethylamine N-Oxide Following Cryptosporidium Infection and Protein Malnutrition Independent of Microbiome Effects.

Cryptosporidium infections have been associated with growth stunting, even in the absence of diarrhea. Having previously detailed the effects of protein deficiency on both microbiome and metabolome in this model, we now describe the specific gut microbial and biochemical effects of Cryptosporidium infection. Protein-deficient mice were infected with Cryptosporidium parvum oocysts for 6-13 days and compared with uninfected controls. Following infection, there was an increase in the urinary excretion of choline- and amino-acid-derived metabolites. Conversely, infection reduced the excretion of the microbial-host cometabolite (3-hydroxyphenyl)propionate-sulfate and disrupted metabolites involved in the tricarboxylic acid (TCA) cycle. Correlation analysis of microbial and biochemical profiles resulted in associations between various microbiota members and TCA cycle metabolites, as well as some microbial-specific degradation products. However, no correlation was observed between the majority of the infection-associated metabolites and the fecal bacteria, suggesting that these biochemical perturbations are independent of concurrent changes in the relative abundance of members of the microbiota. We conclude that cryptosporidial infection in protein-deficient mice can mimic some metabolic changes seen in malnourished children and may help elucidate our understanding of long-term metabolic consequences of early childhood enteric infections.

Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype.

BACKGROUND: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota. OBJECTIVE: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls. DESIGN: With the use of a 1H nuclear magnetic resonance spectroscopy-based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model. RESULTS: We showed considerable shifts within the intestinal microbiota 14-24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both well-nourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed. CONCLUSIONS: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction.

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.

Intestinal cell kinase is a novel participant in intestinal cell signaling responses to protein malnutrition.

Nutritional deficiency and stress can severely impair intestinal architecture, integrity and host immune defense, leading to increased susceptibility to infection and cancer. Although the intestine has an inherent capability to adapt to environmental stress, the molecular mechanisms by which the intestine senses and responds to malnutrition are not completely understood. We hereby report that intestinal cell kinase (ICK), a highly conserved serine/threonine protein kinase, is a novel component of the adaptive cell signaling responses to protein malnutrition in murine small intestine. Using an experimental mouse model, we demonstrated that intestinal ICK protein level was markedly and transiently elevated upon protein deprivation, concomitant with activation of prominent pro-proliferation and pro-survival pathways of Wnt/ß-catenin, mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and protein kinase B (PKB/Akt) as well as increased expression of intestinal stem cell markers. Using the human ileocecal epithelial cell line HCT-8 as an in vitro model, we further demonstrated that serum starvation was able to induce up-regulation of ICK protein in intestinal epithelial cells in a reversible manner, and that serum albumin partially contributed to this effect. Knockdown of ICK expression in HCT-8 cells significantly impaired cell proliferation and down-regulated active ß-catenin signal. Furthermore, reduced ICK expression in HCT-8 cells induced apoptosis through a caspase-dependent mechanism. Taken together, our findings suggest that increased ICK expression/activity in response to protein deprivation likely provides a novel protective mechanism to limit apoptosis and support compensatory mucosal growth under nutritional stress.

The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host.

Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics-growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells-were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism's ability to form biofilm (P<0.001), adhere to IEC-6 epithelial cells (P<0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P<0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (> 3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P<0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (10 (10) CFU/mouse) significantly abrogated growth shortfalls (by>90%; P<0.01); furthermore, stool shedding was reduced (days 9-11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium's effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

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. CONCLUSIONS: ABCG1 deficiency in aortic endothelial cells activates endothelial IL-6-IL-6 receptor-STAT3 signaling, thereby increasing monocyte-endothelial interactions and vascular inflammation.

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 kappaB, as well as production of interleukin-12p40, tumor necrosis factor alpha, 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.

The G protein-coupled receptor G2A: involvement in hepatic lipid metabolism and gallstone formation in mice.

UNLABELLED: The G2A receptor is a member of the ovarian cancer G protein-coupled receptor 1 family of stress-inducible G protein-coupled receptors. In this study, we examined the hepatobiliary effects of loss of function of G2A in mice fed either a chow or lithogenic diet. G2A-deficient (G2A(-/-)) mice fed chow had a 25% reduction in biliary phosphatidylcholine content, reduced hepatic gene expression of the phosphatidylcholine transporter adenosine triphosphate-binding cassette B4, and an 8-fold increase in expression of the nuclear receptor liver X receptor (LXR). Despite the increased expression of LXR, transcription of several LXR target genes was reduced. G2A(-/-) mice fed a lithogenic diet had rapid gallstone formation, an increased cholesterol saturation index, a 2.5-fold increase in farnesoid X receptor expression, a 5-fold increase in LXR expression, and a 90% reduction in cholesterol 7alpha-hydroxylase expression in comparison with wild-type mice. There were no changes in gallbladder volume. CONCLUSION: These data demonstrate that the G2A receptor is important for hepatobiliary bile salt, cholesterol, and phospholipid homeostasis and for the pathogenesis of cholesterol gallstone formation.

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 kappaB 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.

Sphingosine-1-phosphate prevents tumor necrosis factor-{alpha}-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)-alpha in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-alpha-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-alpha 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-alpha. 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-alpha-mediated monocyte-EC interactions in vivo.

Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice.

Atherosclerosis is a major complication of diabetes. Up to 16 weeks of age, the db/db mouse is insulin-resistant and hyperglycemic and is a good model of Type 2 diabetes. After approximately 16 weeks of age, the mice develop pancreatic beta cell failure that can progress to a Type 1 diabetes phenotype. We have previously shown that glucose increases production of endothelial 12/15 lipoxygenase (12/15LO) products in vitro. In young 10-week-old Type 2 diabetic db/db mice, we found significant elevations in levels of urinary 12/15LO products, 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 13S-hydroxyoctadecaenoic acid (13S-HODE) in vivo compared with C57BLKS/J mice. Using isolated primary aortic endothelial cells (ECs) from db/db mice and WEHI78/24 mouse monocyte cells in static adhesion assays, we found increased WEHI monocyte adhesion to db/db ECs (14 +/- 2 monocytes/field for db/db ECs versus 4 +/- 1 monocytes/field for C57BLKS/J ECs, p < 0.002). Thus, ECs from db/db mice appear to be "pre-activated" to bind monocytes. Analysis of db/db ECs revealed a 2-fold elevation in 12/15LO protein compared with C57BLKS/J EC. To determine that 12/15LO products were responsible for the increased monocyte adhesion observed with db/db ECs, we inhibited expression of murine 12/15LO using either an adenovirus expressing a ribozyme to 12/15LO (AdRZ) or with the 12/15LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 microm cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate significantly reduced monocyte adhesion to db/db endothelium (p < 0.009). Thus, inhibition of the murine 12/15LO in db/db mice significantly reduced monocyte/endothelial interactions. We also found that adhesion of monocytes to diabetic db/db ECs was mediated by interactions of alpha4beta1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of beta2 integrins with endothelial intercellular adhesion molecule 1. In summary, regulation of the 12/15LO pathway is important for mediating early vascular changes in diabetes. Modulation of the 12/15LO pathway in the vessel wall may provide therapeutic benefit for early vascular inflammatory events in diabetes.