Dominant Effects of the Diet on the Microbiome and the Local and Systemic Immune Response in Mice

Outside the nutrition community the effects of diet on immune-mediated diseases and experimental outcomes have not been appreciated. Investigators that study immune-mediated diseases and/or the microbiome have overlooked the potential of diet to impact disease phenotype. We aimed to determine the effects of diet on the bacterial microbiota and immune-mediated diseases. Three different laboratory diets were fed to wild-type mice for 2 weeks and resulted in three distinct susceptibilities to dextran sodium sulfate (DSS)-induced colitis. Examination of the fecal microbiota demonstrated a diet-mediated effect on the bacteria found there. Broad-spectrum antibiotics disturbed the gut microbiome and partially eliminated the diet-mediated changes in DSS susceptibility. Dietary changes 2 days after DSS treatment were protective and suggested that the diet-mediated effect occurred quickly. There were no diet-mediated effects on DSS susceptibility in germ-free mice. In addition, the diet-mediated effects were evident in a gastrointestinal infection model (Citrobacter rodentium) and in experimental autoimmune encephalomyelitis. Taken together, our study demonstrates a dominant effect of diet on immune-mediated diseases that act rapidly by changing the microbiota. These findings highlight the potential of using dietary manipulation to control the microbiome and prevent/treat immune-mediated disease.     

Roles of platelet and endothelial cell COX-1 in hypercholesterolemia-induced microvascular dysfunction

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.     

Flaxseed oil and inflammation-associated bone abnormalities in interleukin-10 knockout mice

Interleukin-10-/- (IL-10) knockout (KO) mice develop an intestinal inflammation that closely mimics human inflammatory bowel disease (IBD) which is accompanied by inflammation-associated bone abnormalities and elevated serum proinflammatory cytokines. The objective of this study was to use the IL-10 KO mouse model to determine whether flaxseed oil (FO) diet, rich in alpha-linolenic acid (ALA), attenuates intestinal inflammation and inflammation-associated bone abnormalities, compared to a corn oil (CO) control diet. Male wild-type (WT) or IL-10 KO mice were fed a 10% CO or 10% FO diet from weaning (postnatal day 28) for 9 weeks. At necropsy, serum, intestine, femurs and lumbar vertebrae were collected and analyzed. IL-10 KO mice fed CO had lower femur bone mineral content (BMC; P<.001), bone mineral density (BMD; P<.001), peak load (P=.033) and lumbar vertebrae BMD (P=.02) compared to WT mice fed either diet. Flaxseed oil had a modest, favorable effect on IL-10 KO mice as femur BMC, BMD and peak load were similar to WT mice fed CO or FO. In addition, lumbar vertebra BMD was similar among IL-10 KO mice fed FO and WT mice fed CO or FO. The fact that FO attenuated serum tumor necrosis factor-alpha (TNF-alpha) among IL-10 KO mice suggests that the positive effects of FO on femur BMC, BMD, peak load and vertebral BMD in IL-10 KO mice may have been partly mediated by changes in serum TNF-alpha. In conclusion, these findings suggest that a dietary level of ALA attainable from a 10% flaxseed oil diet results in modest improvements in some bone outcomes but does not attenuate intestinal inflammation that is characteristic of IL-10 KO mice.     

Intestinal fatty acid‐binding protein mediates atherosclerotic progress through increasing intestinal inflammation and permeability

Atherosclerosis is one of leading phenotypes of cardiovascular diseases, featured with increased vascular intima‐media thickness (IMT) and unstable plaques. The interaction between gastrointestinal system and cardiovascular homeostasis is emerging as a hot topic. Therefore, the present study aimed to explore the role of an intestinal protein, intestinal fatty acid‐binding protein (I‐FABP/FABP2) in the atherosclerotic progress. In western diet–fed ApoE^?/? mice, FABP2 was highly expressed in intestine. Silence of intestinal Fabp2 attenuated western diet–induced atherosclerotic phenotypes, including decreasing toxic lipid accumulation, vascular fibrosis and inflammatory response. Mechanistically, intestinal Fabp2 knockdown improved intestinal permeability through increasing the expression of tight junction proteins. Meanwhile, intestinal Fabp2 knockdown mice exhibited down‐regulation of intestinal inflammation in western diet–fed ApoE^?/? mice. In clinical patients, the circulating level of FABP2 was obviously increased in patients with cardiovascular disease and positively correlated with the value of carotid intima‐media thickness, total cholesterol and triglyceride. In conclusion, FABP2‐induced intestinal permeability could address a potential role of gastrointestinal system in the development of atherosclerosis, and targeting on intestinal FABP2 might provide a therapeutic approach to protect against atherosclerosis.     

Sex differences in inflammation and inflammatory pain in cyclooxygenase-deficient mice

There are two cyclooxygenase (COX) genes encoding characterized enzymes, COX-1 and COX-2. Nonsteroidal anti-inflammatory drugs are commonly used as analgesics in inflammatory arthritis, and these often inhibit both cyclooxygenases. Recently, inhibitors of COX-2 have been used in the treatment of inflammatory arthritis, as this isoform is thought to be critical in inflammation and pain. The objective of this study was to determine the effect of COX-1 or COX-2 gene disruption on the development of chronic Freund's adjuvant-induced arthritis and inflammatory pain in male and female mice. The effect of COX-1 or COX-2 gene disruption on inflammatory hyperalgesia, allodynia, inflammatory edema, and arthritic joint destruction was studied. COX-2 knockout mice (COX-2-/-) showed reduced edema and joint destruction in female, but not male, animals. In addition, neither male nor female COX-2-/- mice developed thermal hyperalgesia or mechanical allodynia, either ipsilateral or contralateral to the inflammation. COX-1 gene disruption also reduced inflammatory edema and joint destruction in female, but not male mice, although females of both COX-/- lines did show some bony destruction. There was no difference in ipsilateral allodynia between COX-1 knockout and wild-type animals, but female COX-1-/- mice showed reduced contralateral allodynia compared with male COX-1-/- or wild-type mice. These data show that the gene products of both COX genes contribute to pain and local inflammation in inflammatory arthritis. There are sex differences in some of these effects, and this suggests that the effects of COX inhibitors may be sex dependent.     

Oral administration of food antigen induces T cell mediated intestinal inflammation: a model using TCR-transgenic mice

To investigate the mechanisms inducing food-sensitive intestinal inflammation, we focused on the OVA23-3 mouse, a transgenic mouse strain expressing a T cell receptor that recognizes ovalbumin (OVA). Mice administered an egg-white (EW) diet containing OVA showed a trend of loose feces and significant weight loss. Histology of the jejunum showed severe inflammation with villous atrophy. Thus, we studied the role of T cells and intestinal microflora in the development of the inflammation. Severe villous disruption was observed in sections of the jejunum from OVA23-3 mice and RAG-2 gene-deficient OVA23-3 mice fed with EW-diet. Further, a larger number of T cells was found in the lamina propria of the jejunum of EW-diet fed OVA23-3 mice, RAG-2 gene-deficient mice and germfree OVA23-3 mice compared with those of control-diet fed mice. However, severe inflammation was not detected in the jejunum of germfree OVA23-3 mice. CD4+ T cells from the MLN of EW-diet fed OVA23-3 mice showed a Th2 cytokine secretion profile. These observations have thus clarified that antigen-specific Th2 cells play important roles in the development of intestinal inflammation. Although the presence of indigenous bacteria was not essential for the inflammation, T cells could mediate a more severe inflammatory response in their presence.     

Cyclooxygenase-1 overexpression decreases Basal airway responsiveness but not allergic inflammation

Pharmacological inhibition or genetic disruption of cyclooxygenase (COX)-1 or COX-2 exacerbates the inflammatory and functional responses of the lung to environmentally relevant stimuli. To further examine the contribution of COX-derived eicosanoids to basal lung function and to allergic lung inflammation, transgenic (Tr) mice were generated in which overexpression of human COX-1 was targeted to airway epithelium. Although no differences in basal respiratory or lung mechanical parameters were observed, COX-1 Tr mice had increased bronchoalveolar lavage fluid PGE(2) content compared with wild-type littermates (23.0 +/- 3.6 vs 8.4 +/- 1.4 pg/ml; p < 0.05) and exhibited decreased airway responsiveness to inhaled methacholine. In an OVA-induced allergic airway inflammation model, comparable up-regulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 Tr mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content (70 vs 76% of total cells, respectively), and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine (p > 0.05). To eliminate the presumed confounding effects of COX-2 up-regulation, COX-1 Tr mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation.     

COX-2 is not required for the development of murine chronic pancreatitis

Chronic pancreatitis is a severe inflammation of the pancreas associated with destruction of the parenchyma, fibrosis, and persistent abdominal pain. Cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins, key mediators of the inflammatory response, are elevated in patients with chronic pancreatitis. Previous studies investigated COX-2 as a therapeutic target. These reports showed a reduced pathology in COX-2-deficient mice with a better outcome. Here we compared the role of COX-2 in acute and chronic pancreatic inflammation using the same COX-2(-/-) mouse model of cerulein-induced pancreatitis. In a setting of acute pancreatitis, juvenile COX-2(-/-) mice exhibited a reduced histopathological score compared with wild-type littermates; on the contrary, adult mice did not show any difference in the development of the disease. Similarly, in a setting of chronic pancreatitis induced over a period of 4 wk, adult mice of the two strains showed comparable histological score and collagen deposition. However, the abundance of mRNAs coding for profibrotic genes, such as collagen, α-smooth muscle actin, and transforming growth factor-β was consistently lower in COX-2(-/-) mice. In addition, comparable histological scores and collagen deposition were observed in wild-type mice treated with a COX-2 inhibitor. We conclude that, in contrast to what was observed in the rat pancreatitis models, COX-2 has a limited and age-dependent effect on inflammatory processes in the mouse pancreas. These results suggest that COX-2 modulates the inflammatory process during the development of pancreatitis in a species-specific manner. Thus the pathophysiological roles of COX-2 and its therapeutic implications in patients with pancreatitis should be reexamined.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2(-/-) mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2(-/-) mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2(-/-) mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2(-/-) mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction.     

Deficiency of interleukin-1 receptor antagonist deteriorates fatty liver and cholesterol metabolism in hypercholesterolemic mice

Although the anti-inflammatory effect of interleukin-1 (IL-1) receptor antagonist (IL-1Ra) has been described, the contribution of this cytokine to cholesterol metabolism remains unclear. Our aim was to ascertain whether deficiency of IL-1Ra deteriorates cholesterol metabolism upon consumption of an atherogenic diet. IL-1Ra-deficient mice (IL-1Ra(-/-)) showed severe fatty liver and portal fibrosis containing many inflammatory cells following 20 weeks of an atherogenic diet when compared with wild type (WT) mice. Expectedly, the levels of total cholesterol in IL-1Ra(-/-) mice were significantly increased, and the start of lipid accumulation in liver was observed earlier when compared with WT mice. Real-time PCR analysis revealed that IL-1Ra(-/-) mice failed to induce mRNA expression of cholesterol 7alpha-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, with concurrent up-regulation of small heterodimer partner 1 mRNA expression. Indeed, IL-1Ra(-/-) mice showed markedly decreased bile acid excretion, which is elevated in WT mice to maintain cholesterol level under atherogenic diet feeding. Therefore, we conclude that the lack of IL-1Ra deteriorates cholesterol homeostasis under atherogenic diet-induced inflammation.     

Endothelial expression of endoglin in normocholesterolemic and hypercholesterolemic C57BL/6J mice before and after atorvastatin treatment

Endoglin (CD105) is a homodimeric transmembrane glycoprotein strongly related to transforming growth factor (TGF)-beta signaling and many pathological states. In this study, we wanted to evaluate whether endoglin is expressed in normocholesterolemic and hypercholesterolemic C57BL/6J mice as well as whether it is affected by atorvastatin treatment in these mice. C57BL/6J mice were fed with chow diet or an atherogenic diet for 12 weeks after weaning. In 2 atorvastatin-treated groups, mice were fed the same diets (chow or atherogenic) as described above except atorvastatin was added at the dosage of 10 mg x kg(-1) x day(-1) for the last 8 weeks before euthanasia. Biochemical analysis of blood samples revealed that administration of atherogenic diet significantly increased levels of total cholesterol, VLDL, LDL, and decreased levels of HDL. Atorvastatin treatment resulted in a significant decrease in total cholesterol and VLDL only in mice fed by atherogenic diet. Quantitative stereological analysis revealed that atorvastatin significantly decreased endothelial expression of endoglin in C57BL/6J mice fed the atherogenic diet. In conclusion, we demonstrated that endothelial expression of endoglin is upregulated by hypercholesterolemia and decreased by the hypolipidemic effect of atorvastatin in C57BL/6J mice, suggesting that endoglin expression could be involved in atherogenesis.     

Activation of the TLR4 signaling pathway and abnormal cholesterol efflux lead to emphysema in ApoE-deficient mice

Smokers with airflow obstruction have an increased risk of atherosclerosis, but the relationship between the pathogenesis of these diseases is not well understood. To determine whether hypercholesterolemia alters lung inflammation and emphysema formation, we examined the lung phenotype of two hypercholesterolemic murine models of atherosclerosis at baseline and on a high-fat diet. Airspace enlargement developed in the lungs of apolipoprotein E-deficient (Apoe(-/-)) mice exposed to a Western-type diet for 10 wk. An elevated number of macrophages and lymphocytes accompanied by an increase in matrix metalloproteinase-9 (MMP-9) activity and MMP-12 expression was observed in the lungs of Apoe(-/-) mice on a Western-type diet. In contrast, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice did not exhibit lung destruction or inflammatory changes. Most importantly, we revealed augmented expression of the downstream targets of the Toll-like receptor (TLR) pathway, interleukin-1 receptor-associated kinase 1, and granulocyte colony-stimulating factor, in the lungs of Apoe(-/-) mice fed with a Western-type diet. In addition, we demonstrated overexpression of MMP-9 in Apoe(-/-) macrophages treated with TLR4 ligand, augmented with the addition of oxidized LDL, suggesting that emphysema in these mice results from the activation of the TLR pathway secondary to known abnormal cholesterol efflux. Our findings indicate that, in Apoe(-/-) mice fed with an atherogenic diet, abnormal cholesterol efflux leads to increased systemic inflammation with subsequent lung damage and emphysema formation.     

Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates innate and adaptive immunity. Recent studies have shown that the activation of TLR-dependent signaling pathways plays important roles in the pathogenesis of ischemia-reperfusion (I/R) injuries in many organs. All TLRs, except TLR3, use a common adaptor protein, MyD88, to transduce activation signals. We investigated the role of MyD88 in I/R injury of the small intestine. MyD88 and cyclooxygenase-2 (COX-2) knockout and wild-type mice were subjected to intestinal I/R injury. I/R-induced small intestinal injury was characterized by infiltration of inflammatory cells, disruption of the mucosal epithelium, destruction of villi, and increases in myeloperoxidase activity and mRNA levels of TNF-α and the IL-8 homolog KC. MyD88 deficiency worsened the severity of I/R injury, as assessed using the histological grading system, measuring luminal contents of hemoglobin (a marker of intestinal bleeding), and counting apoptotic epithelial cells, while it inhibited the increase in mRNA expression of TNF-α and KC. I/R significantly enhanced COX-2 expression and increased PGE(2) concentration in the small intestine of wild-type mice, which were markedly inhibited by MyD88 deficiency. COX-2 knockout mice were also highly susceptible to intestinal I/R injury. Exogenous PGE(2) reduced the severity of injury in both MyD88 and COX-2 knockout mice to the level of wild-type mice. These findings suggest that the MyD88 signaling pathway may inhibit I/R injury in the small intestine by inducing COX-2 expression.     

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A(2) enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE(2) in the brain. These studies seek to determine the function of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in inflammatory PGE(2) production in the brain. We wondered whether cPLA(2)alpha functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA(2)alpha (cPLA(2)alpha(-/-)) after systemic administration of LPS. cPLA(2)alpha(-/-) mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE(2) concentration of untreated cPLA(2)alpha(-/-) mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE(2) was significantly less in cPLA(2)alpha(-/-) than in cPLA(2)alpha(+/+) mice (24.4 +/- 3.8 vs. 49.3 +/- 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA(2)alpha regulates COX-2 levels and modulates inflammatory PGE(2) levels. These results indicate that cPLA(2)alpha inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.     

Surfactant protein D is proatherogenic in mice

Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd-/-) mice. Atherogenesis involves both inflammation and lipid deposition, and we investigated the role of SP-D in the development of atherosclerosis. SP-D synthesis was localized to vascular endothelial cells. Atherosclerotic lesion areas were 5.6-fold smaller in the aortic roots in Spd-/- mice compared with wild-type C57BL/6N mice on an atherogenic diet. HDL cholesterol (HDL-C) was significantly elevated in Spd-/- mice. Treatment of Spd-/- mice with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (26%), and LDL cholesterol (28%). Plasma TNF-alpha was reduced in Spd-/- mice (45% difference). SP-D was proatherogenic in the mouse model used. The effect is likely to be due to the observed disturbances of plasma lipid metabolism and alteration of the inflammatory process, which underlie the reduced susceptibility to atherosclerosis in Spd-/- mice.     

Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.     

Toll-Like Receptor 2 Mediates Ischemia-Reperfusion Injury of the Small Intestine in Adult Mice

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.