Formyl peptide receptor 2 orchestrates mucosal protection against Citrobacter rodentium infection.

Citrobacter rodentium is an attaching and effacing intestinal murine pathogen which shares similar virulence strategies with the human pathogens enteropathogenic- and enterohemorrhagic Escherichia coli to infect their host. C. rodentium is spontaneously cleared by healthy wild-type (WT) mice whereas mice lacking Muc2 or specific immune regulatory genes demonstrate an impaired ability to combat the pathogen. Here we demonstrate that apical formyl peptide receptor 2 (Fpr2) expression increases in colonic epithelial cells during C. rodentium infection. Using a conventional inoculum dose of C. rodentium, both WT and Fpr2-/- mice were infected and displayed similar signs of disease, although Fpr2-/- mice recovered more slowly than WT mice. However, Fpr2-/- mice exhibited increased susceptibility to C. rodentium colonization in response to low dose infection: 100% of the Fpr2-/- and 30% of the WT mice became colonized and Fpr2-/- mice developed more severe colitis and more C. rodentium were in contact with the colonic epithelial cells. In line with the larger amount of C. rodentium detected in the spleen in Fpr2-/- mice, more C. rodentium and enteropathogenic Escherichia coli translocated across an in vitro mucosal surface to the basolateral compartment following FPR2 inhibitor treatment. Fpr2-/- mice also lacked the striated inner mucus layer that was present in WT mice. Fpr2-/- mice had decreased mucus production and different mucin O-glycosylation in the colon compared to WT mice, which may contribute to their defect inner mucus layer. Thus, Fpr2 contributes to protection against infection and influence mucus production, secretion and organization.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice.

BACKGROUND: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO). METHODS: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO. RESULTS: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45(+) leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4(+) and CD8(+) T cells, as well as macrophages, was significantly decreased. CONCLUSIONS: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation.

The phenotype of macrophages in atherosclerotic lesions can vary dramatically, from a large lipid laden foam cell to a small inflammatory cell. Classically, the concept of macrophage heterogeneity discriminates between two extremes called either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Polarisation of plaque macrophages is predominantly determined by the local micro-environment present in the atherosclerotic lesion and is rather more complex than typically described by the M1/M2 paradigm. In this review we will discuss the role of various polarising factors in regulating the phenotypical state of plaque macrophages. We will focus on two main levels of phenotype regulation, one determined by differentiation factors produced in the lesion and the other determined by T-cell-derived polarising cytokines. With foam cell formation being a key characteristic of macrophages during atherosclerosis initiation and progression, these polarisation factors will also be linked to lipid handling of macrophages.

Scavenger receptor class A type I/II determines matrix metalloproteinase-mediated cartilage destruction and chondrocyte death in antigen-induced arthritis.

OBJECTIVE: Scavenger receptor class A type I (SR-AI) and SR-AII are expressed by macrophages in particular and bind and internalize a broad range of molecules (including endotoxins, apoptotic bodies, and oxidized low-density lipoprotein). This study was undertaken to investigate the role of SR-AI/II in mediating severe cartilage destruction in antigen-induced arthritis (AIA). METHODS: AIA was induced in the knee joints of SR-AI/II(-/-) mice and wild-type (WT) controls. Joint inflammation and cartilage destruction (chondrocyte death) were measured by examining the histology of total knee joints. Matrix metalloproteinase (MMP)-mediated neoepitopes were measured by immunolocalization using anti-VDIPEN antibodies and chondrocyte activation with anti-S100A8 antibodies. Messenger RNA (mRNA) levels were determined in inflamed synovium using microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. In synovial washouts, cytokines (interleukin-1beta [IL-1beta], IL-10, and tumor necrosis factor alpha) and S100A8/S100A9 were measured using Luminex and enzyme-linked immunosorbent assay. RESULTS: Levels of SR-AI/II mRNA were strongly elevated in inflamed synovium in AIA. On days 2, 8, and 14 after AIA induction, joint inflammation (exudates/infiltrate) was similar between the 2 groups. In WT mice, severe cartilage destruction was found in multiple cartilage surfaces of the inflamed knee joint on day 14 after AIA induction. MMP-mediated matrix destruction ranged between 40% and 60%, and chondrocyte death was prominent in 40-75% of the cartilage surfaces. In striking contrast, in SR-AI/II(-/-) mice, despite comparable joint inflammation, pronounced cartilage destruction was almost completely absent. Levels of IL-1beta and S100A8/S100A9 were significantly lower on days 7 and 14 after AIA induction, but levels of mRNA for various MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) were comparable. CONCLUSION: Our findings indicate that SR-AI and SR-AII are crucial receptors involved in mediating severe cartilage destruction in AIA.

Disruption of hedgehog signalling in ApoE - /- mice reduces plasma lipid levels, but increases atherosclerosis due to enhanced lipid uptake by macrophages.

Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression.

Gene profiling of cathepsin K deficiency in atherogenesis: profibrotic but lipogenic.

Recently, we showed that cathepsin K deficiency reduces atherosclerotic plaque progression, induces plaque fibrosis, but aggravates macrophage foam cell formation in the ApoE -/- mouse. To obtain more insight into the molecular mechanisms by which cathepsin K disruption evokes the observed phenotypic changes, we used microarray analysis for gene expression profiling of aortic arches of CatK -/-/ApoE -/- and ApoE -/- mice on a mouse oligo microarray. Out of 20 280 reporters, 444 were significantly differentially expressed (p-value of < 0.05, fold change of > or = 1.4 or < or = - 1.4, and intensity value of > 2.5 times background in at least one channel). Ingenuity Pathway Analysis and GenMAPP revealed upregulation of genes involved in lipid uptake, trafficking, and intracellular storage, including caveolin - 1, - 2, - 3 and CD36, and profibrotic genes involved in transforming growth factor beta (TGFbeta) signalling, including TGFbeta2, latent TGFbeta binding protein-1 (LTBP1), and secreted protein, acidic and rich in cysteine (SPARC), in CatK -/-/ApoE -/- mice. Differential gene expression was confirmed at the mRNA and protein levels. In vitro modified low density lipoprotein (LDL) uptake assays, using bone marrow derived macrophages preincubated with caveolae and scavenger receptor inhibitors, confirmed the importance of caveolins and CD36 in increasing modified LDL uptake in the absence of cathepsin K. In conclusion, we suggest that cathepsin K deficiency alters plaque phenotype not only by decreasing proteolytic activity, but also by stimulating TGFbeta signalling. Besides this profibrotic effect, cathepsin K deficiency has a lipogenic effect owing to increased lipid uptake mediated by CD36 and caveolins.

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Fifty-four new gene-based canine microsatellite markers.

Fifty-four new markers were developed to fill in gaps in the current map of canine microsatellites and to complement existing markers that may not be sufficiently informative in highly inbred canine pedigrees. Canine genes contained on the radiation hybrid map were used to obtain the sequence of the human homolog. A BLAST search versus the canine whole genome shotgun (wgs) sequence resource was used to obtain the sequence of the canine genomic contigs containing the homolog of the corresponding human gene. Canine sequences that contained microsatellites and mapped back to the correct location in the human genome were used to design primers for amplification of the microsatellites from canine genomic DNA. Heterozygosities of the markers were tested by genotyping grandparental DNAs obtained from the Nestle Purina Reference family DNA distribution center plus DNAs from unrelated Bouviers and Irish wolfhounds. Canine map positions of markers on the July 2004 freeze of the canine genome assembly were determined by in silico PCR or BLAST.

Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.

Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines.

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2),

Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on cholesterol levels and atherosclerosis in ApoE-deficient mice.

In the arterial wall, scavenger receptor class A (SRA) is implicated in pathological lipid deposition. In contrast, in the liver, SRA is suggested to remove modified lipoproteins from the circulation, thereby protecting the body from their pathological action. The role of SRA on bone marrow-derived cells in lipid metabolism and atherogenesis was assessed in vivo by transplantation of bone marrow cells overexpressing human SRA (MSR1) to apoE-deficient mice. In vitro studies with peritoneal macrophages from the transplanted mice showed that macrophage scavenger receptor function, as measured by cell association and degradation studies with acetylated LDL, was approximately 3-fold increased on overexpression of MSR1 in bone marrow-derived cells as compared with control mice. Despite the increased macrophage scavenger receptor function in vitro, no significant effect of MSR1 overexpression in bone marrow-derived cells on the in vivo atherosclerotic lesion development was found. In addition to arterial wall macrophages, liver sinusoidal Kupffer cells also overexpress MSR1 after bone marrow transplantation, which may scavenge atherogenic particles more efficiently from the blood compartment. Introduction of bone marrow cells overexpressing human MSR1 in apoE-deficient mice induced a significant reduction in serum cholesterol levels of approximately 20% (P:<0.001, 2-way ANOVA) as the result of a decrease in VLDL cholesterol. It is suggested that the reduction in VLDL cholesterol levels is due to increased clearance of modified lipoproteins by the overexpressed MSR1 in Kupffer cells of the liver, thereby protecting the arterial wall against the proatherogenic action of modified lipoproteins.

Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice.

Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in early atherogenesis, we investigated the role of scavenger receptor class A (SR-A) overexpression, especially on macrophages in lipoprotein metabolism and atherosclerosis. Bone marrow from human SR-A (MSR1)-overexpressing mice was transplanted into irradiated low density lipoprotein receptor knockout [LDLR(-/-)] mice. The transplantation resulted in an increase in total serum cholesterol (approximately 15 to 25%), especially in the VLDL fraction, when compared with LDLR(-/-) mice that were transplanted with bone marrow of wild-type littermates. Quantification of atherosclerotic lesions in the mice that were fed a "Western-type" diet for 3 months revealed that there were no differences in mean lesion area between LDLR(-/-) mice transplanted with MSR1 overexpressing and wild-type littermate bone marrow, despite increased scavenger receptor activity in vitro. The presence or absence of the LDLR in the transplanted bone marrow did not influence these results.In conclusion, introduction of MSR1-overexpressing bone marrow in LDLR(-/-) mice via bone marrow transplantation resulted in a slight increase in lipoprotein levels, but had no effect on the atherosclerotic lesion area, despite increased scavenger receptor activity in vitro.

Transgenic mouse models to study the role of the macrophage scavenger receptor class A in atherosclerosis.

Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice. The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the athero-susceptible background. To study the role of SR-A in a different model, we generated a transgenic mouse model with high level expression of the human SR-A gene using a 180 Kb yeast artificial chromosome (MSR1 transgenic mice). These mice show increased expression of SR-A according to the natural expression pattern. The MSR1 transgenic mice were crossed onto a low-density lipoprotein receptor deficient background and were fed a high fat diet for 10 weeks. After this period, the size of the atherosclerotic lesions in the proximal aorta was measured. Surprisingly, atherosclerosis was significantly reduced in the MSR1 transgenic mice. In a second study, the effect of SR-A was examined in APOE-3 Leiden mice providing a different athero-susceptible background. To exclude nonmacrophage effects, bone marrow was transplanted from MSR1 mice and wild-type littermates to APOE-3 Leiden transgenic mice. After 8 weeks on a high fat diet, atherosclerosis in the mice that had received MSR1 bone marrow was reduced compared with mice that had received wild-type bone marrow. This difference reached statistical significance when individual cholesterol exposure of the mice was taken into account. Both experiments indicated an antiatherogenic role of the SR-A. This observation cannot be explained easily by SR-A function in foam cell formation because in MSR1 macrophages in vitro foam cell formation is increased. Alternatively, however, SR-A may affect the activation of macrophages. Hence the response to lipopolysaccharide was measured in MSR1-transgenic macrophages. These macrophages showed a reduction in their activation in response to lipopolysaccharide, as measured by nitric oxide production. These data show that an elevated level of SR-A expression reduces atherosclerosis, potentially by modifying the response of macrophages to activation signals in the plaque.

Macrophage scavenger receptor class A: A multifunctional receptor in atherosclerosis.

In atherogenesis, elevated plasma levels of low density lipoprotein (LDL) lead to the chronic presence of LDL in the arterial wall. There, LDL is modified (eg, oxidized), and these modified lipoproteins activate endothelial cells, which attract circulating monocytes. These monocytes enter the vessel wall, differentiate into macrophages, and subject the modified lipoproteins to endocytosis through scavenger receptor pathways. This unrestricted uptake, which is not limited by intracellular cholesterol levels, eventually leads to the formation of lipid-filled foam cells, the initial step in atherosclerosis. Macrophage scavenger receptor class A (SRA) is thought to be one of the main receptors involved in foam cell formation, mediating the influx of lipids into the macrophages. In addition to this role in modified lipoprotein uptake by macrophages, the SRA has been shown to be important in the inflammatory response in host defense, cellular activation, adhesion, and cell-cell interaction. Given the importance of these processes in atherogenesis, these latter functions may prove to make the SRA a multifunctional player in the atherosclerotic process.