Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

BackgroundSevere leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.Methodology/Principal findingsMale Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.Conclusion/SignificanceThis study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Resistance of Interleukin-1β-Deficient Mice to Fatal Sindbis Virus Encephalitis

Interleukin-1β (IL-1β) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1β in CNS viral pathogenesis, we compared the natural histories of IL-1β-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1β^−/− mice compared to NSV-infected wild-type mice (4 versus 88%, P < 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1β^−/− mice could be detected. These results suggest that IL-1β deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.     

Interleukin-6 Mediates Epithelial–Stromal Interactions and Promotes Gastric Tumorigenesis

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial–stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6–positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6^−/−) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6^−/− mice, compared with WT mice. Impaired tumor development in IL-6^−/− mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3–related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell–conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Galectin-3 Ablation Enhances Liver Steatosis,but Attenuates Inflammation and IL-33-Dependent Fibrosis in Obesogenic Mouse Model of Nonalcoholic Steatohepatitis

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3^−/−) and wild-type (LGALS3^+/+) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3^−/− mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3^−/− mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b⁺Ly6C^hi monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b⁺IL-13⁺ cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3^−/− mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3^−/− peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3^−/− mice, which was associated with less numerous hepatic IL-13-expressing CD11b⁺ cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.     

Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

P47phox?/? Mice Are Compromised in Expansion and Activation of CD8+ T Cells and Susceptible to Trypanosoma cruzi Infection

Macrophage activation of NAD(P)H oxidase (NOX2) and reactive oxygen species (ROS) is suggested to kill Trypanosoma cruzi that causes Chagas disease. However, the role of NOX2 in generation of protective immunity and whether these mechanisms are deregulated in the event of NOX2 deficiency are not known, and examined in this study. Our data showed that C57BL/6 p47^phox−/− mice (lack NOX2 activity), as compared to wild-type (WT) mice, succumbed within 30 days post-infection (pi) to low doses of T. cruzi and exhibited inability to control tissue parasites. P47^phox−/− bone-marrow and splenic monocytes were not compromised in maturation, phagocytosis and parasite uptake capacity. The deficiency of NOX2 mediated ROS was compensated by higher level of inducible nitric oxide synthase (iNOS) expression, and nitric oxide and inflammatory cytokine (TNF-α, IFN-γ, IL-1β) release by p47^phox−/− macrophages as compared to that noted in WT controls infected by T. cruzi. Splenic activation of Th1 CD4⁺T cells and tissue infiltration of immune cells in T. cruzi infected p47^phox−/− mice were comparable to that noted in infected control mice. However, generation and activation of type 1 CD8⁺T cells was severely compromised in p47^phox−/− mice. In comparison, WT mice exhibited a robust T. cruzi-specific CD8⁺T cell response with type 1 (IFN-γ+TNF-α>IL-4+IL-10), cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺) phenotype. We conclude that NOX2/ROS activity in macrophages signals the development of antigen-specific CD8⁺T cell response. In the event of NOX2 deficiency, a compromised CD8⁺T cell response is generated, leading to increased parasite burden, tissue pathogenesis and mortality in chagasic mice.     

T Cell Hypo-Responsiveness against Leishmania major in MAP Kinase Phosphatase (MKP) 2 Deficient C57BL/6 Mice Does Not Alter the Healer Disease Phenotype

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T_H1 response. By contrast, in the present study footpad infection of MKP-2^−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2^+/+ mice. Analysis of immune responses following infection demonstrated a reduced T_H1 response in MKP-2^−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4⁺ and CD8⁺ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2^−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2^+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T_H1/T_H2 balance was unaltered in MKP-2^−/− compared with wild-type mice. Although non-stimulated MKP-2^−/− macrophages were more permissive to L. major growth than macrophages from MKP-2^+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2^−/− and MKP-2^+/+ macrophages. Consequently, in the absence of any switch in the T_H1/T_H2 balance in MKP-2^−/− mice, no significant change in disease phenotype was observed.     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

Differential Modulation by IL-17A of Cholangitis versus Colitis in IL-2Rα Deleted Mice

IFN-γ is a signature Th1 cell associated cytokine critical for the inflammatory response in autoimmunity with both pro-inflammatory and potentially protective functions. IL-17A is the hallmark of T helper 17 (Th17) cell subsets, produced by γδT, CD8+ T, NK and NKT cells. We have taken advantage of our colony of IL-2Rα^−/− mice that spontaneously develop both autoimmune cholangitis and inflammatory bowel disease. In this model CD8+ T cells mediate biliary ductular damage, whereas CD4+ T cells mediate induction of colon-specific autoimmunity. Importantly, IL-2Rα^−/− mice have high levels of interferon γ (IFN-γ), and interleukin-17A (IL-17A). We produced unique double deletions of mice that were either IL-17A^−/−IL-2Rα^−/− or IFN-γ^−/−IL-2Rα^−/− to specifically address the precise role of these two cytokines in the natural history of autoimmune cholangitis and colitis. Of note, deletion of IL-17A in IL-2Rα^−/− mice led to more severe liver inflammation, but ameliorated colitis. In contrast, there were no significant changes in the immunopathology of double knock-out IFN-γ^−/− IL-2Rα^−/− mice, compared to single knock-out IL-2Rα^−/− mice with respect to cholangitis or colitis. Furthermore, there was a significant increase in pathogenetic CD8+ T cells in the liver of IL-17A^−/−IL-2Rα^−/− mice. Our data suggest that while IL-17A plays a protective role in autoimmune cholangitis, it has a pro-inflammatory role in inflammatory bowel disease. These data take on particular significance in the potential use of anti-IL-17A therapy in humans with primary biliary cirrhosis.     

Nippostrongylus-Induced Intestinal Hypercontractility Requires IL-4 Receptor Alpha-Responsiveness by T Cells in Mice

Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (T_H2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This T_H2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLck^creIL-4Rα^−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα^−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted T_H2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4⁺ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα^−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.     

Vascular Cellular Adhesion Molecule-1 (VCAM-1) Expression in Mice Retinal Vessels Is Affected by Both Hyperglycemia and Hyperlipidemia

Background

Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα).

Methodology/Principal Findings

Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE^−/−) and TNFα deficient (TNFα^−/−, ApoE^−/−/TNFα^−/−) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1β mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1β mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE^−/− than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE^−/− mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides.

Conclusions/Significance

Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE^−/− mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.     

Structural and Functional Characterization of an Orphan ATP-Binding Cassette ATPase Involved in Manganese Utilization and Tolerance in Leptospira spp.

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn²⁺, we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn²⁺, suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn²⁺ toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg²⁺-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.     

Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes

Introduction

Our objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli.

Methods

Cartilage explants from FrzB^−/− and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1β (IL-1β) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB^−/− and wild-type mice. The expression and release of MMP-3 and −13 were determined by RT-PCR, western blot and ELISA. The accumulation of β-catenin was assessed by RT-PCR and western blot.

Results

Cartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB^−/− cartilage explants compared to wild-type (P = 0.17). IL-1β dose-dependently induced Mmp-13 and −3 gene expression and protein release by cultured chondrocytes. IL-1β-mediated increase in MMP-13 and −3 was slightly enhanced in FrzB^−/− chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and β-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB^−/− chondrocytes to IL-1β and load may be associated with an over-stimulation of the canonical Wnt/β-catenin pathway.

Conclusions

Our results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/β-catenin pathway.     

The Tyrosine Kinase Btk Regulates the Macrophage Response to Listeria monocytogenes Infection

In this study we investigated the role of Bruton''s tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk ^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk ^−/− BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk ^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk ^−/− mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk ^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.