Gut Microbiota-Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens.

The gut microbiota is compartmentalized in the intestinal lumen and induces local immune responses, but it remains unknown whether the gut microbiota can induce systemic response and contribute to systemic immunity. We report that selective gut symbiotic gram-negative bacteria were able to disseminate systemically to induce immunoglobulin G (IgG) response, which primarily targeted gram-negative bacterial antigens and conferred protection against systemic infections by E. coli and Salmonella by directly coating bacteria to promote killing by phagocytes. T cells and Toll-like receptor 4 on B cells were important in the generation of microbiota-specific IgG. We identified murein lipoprotein (MLP), a highly conserved gram-negative outer membrane protein, as a major antigen that induced systemic IgG homeostatically in both mice and humans. Administration of anti-MLP IgG conferred crucial protection against systemic Salmonella infection. Thus, our findings reveal an important function for the gut microbiota in combating systemic infection through the induction of protective IgG.

Microbiota-Induced Antibodies Are Essential for Host Inflammatory Responsiveness to Sterile and Infectious Stimuli.

The indigenous intestinal microbiota is frequently considered an additional major organ of the human body and exerts profound immunomodulating activities. Germ-free (GF) mice display a significantly different inflammatory responsiveness pattern compared with conventional (CV) mice, and this was dubbed a "hyporesponsive phenotype." Taking into account that the deposition of immune complexes is a major event in acute inflammation and that GF mice have a distinct Ig repertoire and B cell activity, we aimed to evaluate whether this altered Ig repertoire interferes with the inflammatory responsiveness of GF mice. We found that serum transfer from CV naive mice was capable of reversing the inflammatory hyporesponsiveness of GF mice in sterile inflammatory injury induced by intestinal ischemia and reperfusion, as well as in a model of lung infection by Klebsiella pneumoniae Transferring serum from Ig-deficient mice to GF animals did not alter their response to inflammatory insult; however, injecting purified Abs from CV animals restored inflammatory responsiveness in GF mice, suggesting that natural Abs present in serum were responsible for altering GF responsiveness. Mechanistically, injection of serum and Ig from CV mice into GF animals restored IgG deposition, leukocyte influx, NF-κB activation, and proinflammatory gene expression in inflamed tissues and concomitantly downregulated annexin-1 and IL-10 production. Thus, our data show that microbiota-induced natural Abs are pivotal for host inflammatory responsiveness to sterile and infectious insults.

Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.

UNLABELLED: Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. CONCLUSION: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue.

Suppressor of cytokine signaling 2 modulates the immune response profile and development of experimental cerebral malaria.

Plasmodium falciparum infection results in severe malaria in humans, affecting various organs, including the liver, spleen and brain, and resulting in high morbidity and mortality. The Plasmodium berghei ANKA (PbA) infection in mice closely recapitulates many aspects of human cerebral malaria (CM); thus, this model has been used to investigate the pathogenesis of CM. Suppressor of cytokine signaling 2 (SOCS2), an intracellular protein induced by cytokines and hormones, modulates the immune response, neural development, neurogenesis and neurotrophic pathways. However, the role of SOCS2 during CM remains unknown. SOCS2 knockout (SOCS2(-/-)) mice infected with PbA show an initial resistance to infection with reduced parasitemia and production of TNF, TGF-ß, IL-12 and IL-17 in the brain. Interestingly, in the late phase of infection, SOCS2(-/-) mice display increased parasitemia and reduced Treg cell infiltration, associated with enhanced levels of Th1 and Th17 cells and related cytokines IL-17, IL-6, and TGF-ß in the brain. A significant reduction in protective neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), was also observed. Moreover, the molecular alterations in the brain of infected SOCS2(-/-) mice were associated with anxiety-related behaviors and cognition impairment. Mechanistically, these results revealed enhanced nitric oxide (NO) production in PbA-infected SOCS2(-/-) mice, and the inhibition of NO synthesis through l-NAME led to a marked decrease in survival, the disruption of parasitemia control and more pronounced anxiety-like behavior. Treatment with l-NAME also shifted the levels of Th1, Th7 and Treg cells in the brains of infected SOCS2(-/-) mice to the background levels observed in infected WT, with remarkable exception of increased CD8(+)IFN(+) T cells and inflammatory monocytes. These results indicate that SOCS2 plays a dual role during PbA infection, being detrimental in the control of the parasite replication but crucial in the regulation of the immune response and production of neurotrophic factors. Here, we provided strong evidence of a critical relationship between SOCS2 and NO in the orchestration of the immune response and development of CM during PbA infection.

Skin wound healing is accelerated and scarless in the absence of commensal microbiota.

The commensal microbiota has a high impact on health and disease by modulating the development and homeostasis of host immune system. Immune cells are involved in virtually every aspect of the wound repair process; however, the impact of commensal microbiota on skin wound healing is largely unknown. In this study, we evaluated the influence of commensal microbiota on tissue repair of excisional skin wounds by using germ-free (GF) Swiss mice. We observed that macroscopic wound closure rate is accelerated in the absence of commensal microbiota. Accordantly, histologically assessed wound epithelization was accelerated in GF in comparison with conventional (CV) Swiss mice. The wounds of GF mice presented a significant decrease in neutrophil accumulation and an increase in mast cell and macrophage infiltration into wounds. Interestingly, alternatively activated healing macrophage-related genes were highly expressed in the wound tissue of GF mice. Moreover, levels of the anti-inflammatory cytokine IL-10, the angiogenic growth factor VEGF and angiogenesis were higher in the wound tissue of those mice. Conversely, scarring and levels of the profibrogenic factor TGF-ß1 were greatly reduced in GF mice wounded skin when compared with CV mice. Of note, conventionalization of GF mice with CV microbiota restored wound closure rate, neutrophil and macrophage accumulation, cytokine production, and scarring to the same extent as CV mice. Overall, our findings suggest that, in the absence of any contact with microbiota, skin wound healing is accelerated and scarless, partially because of reduced accumulation of neutrophils, increased accumulation of alternatively activated healing macrophages, and better angiogenesis at wound sites.

Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5.

Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice.

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, µMT(-/-) mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.

Platelet-activating factor receptor blockade ameliorates Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice.

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by oral biofilm-producing microorganisms, such as Aggregatibacter actinomycetemcomitans. The levels of the phospholipid platelet-activating factor (PAF) in the saliva, gingival crevicular fluid, and periodontal tissues are significantly increased during inflammatory conditions, such as PD, but the exact mechanism that links PAF to alveolar bone resorption is not well understood. In the current study, alveolar bone resorption was induced by experimental PD through the oral inoculation of A. actinomycetemcomitans in wild-type (WT) and PAF receptor knockout (Pafr(-/-)) mice. In vitro experiments using A. actinomycetemcomitans lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with a PAF receptor antagonist (UK74505) were also performed. The expression of lyso-PAF acetyltransferase in periodontal tissues was significantly increased 3 h after A. actinomycetemcomitans LPS injection in mice. WT and Pafr(-/-) mice that were subjected to oral inoculation of A. actinomycetemcomitans presented neutrophil accumulation and increased levels of CXCL-1 and tumor necrosis factor alpha (TNF-α) in periodontal tissues. However, Pafr(-/-) mice presented less alveolar bone loss than WT mice. The in vitro blockade of the PAF receptor impaired the resorptive activity of A. actinomycetemcomitans LPS-activated osteoclasts. In conclusion, this study shows for the first time that the blockade of PAF receptor may contribute to the progression of PD triggered by A. actinomycetemcomitans by directly affecting the differentiation and activity of osteoclasts.

Further evidence for an anti-inflammatory role of artesunate in experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a clinical syndrome resulting from Plasmodium falciparum infection. A wide range of clinical manifestations follow the disease including cognitive dysfunction, seizures and coma. CM pathogenesis remains incompletely understood and without treatment this condition is invariably fatal. Artesunate has been accepted as the most effective drug for treating severe malaria. Besides its antiparasitic activity, an anti-inflammatory property has also been reported. In the current study, the immunomodulatory role of artesunate was investigated using a Plasmodium berghei ANKA model of CM, trough evaluation of behavioural changes and cytokines expression in hippocampus and in frontal cortex. METHODS: C57Bl/6 mice were infected with P. berghei by intraperitoneal route, using a standardized inoculation of 106 parasitized erythrocytes. Memory function was evaluated using the step-down inhibitory avoidance test. The mRNA expression of IFN-γ, IL-1ß, IL-6 and TNF in the frontal cortex and hippocampus of control and infected mice on day 5 post-infection were estimated by quantitative real time PCR. Plasmodium berghei -infected mice also received intraperitoneally a single dose of artesunate (32 mg/kg) on day 4 post-infection, and 24 hours after treatment behavioural and immunological analysis were performed. The protein levels of cytokines IL-2, IL-6, IL-10, IL-17, IFN-γ, TNF in the serum, frontal cortex and hippocampus of controls and P. berghei -infected mice treated or not treated with artesunate were determined using a cytometric bead array (CBA) kit. The survival and neurological symptoms of CM were also registered. RESULTS: CM mice presented a significant impairment of aversive memory compared to controls on day 5 post-infection. A higher mRNA expression of pro-inflammatory cytokines was found in the hippocampus and frontal cortex of infected mice. A single dose of artesunate was also able to decrease the expression of inflammatory cytokines in the hippocampus and frontal cortex of P. berghei-infected mice. In parallel, a significant improvement in neurological symptoms and survival were observed in artesunate treated mice. CONCLUSIONS: In summary, the current study provided further evidence that CM affects key brain areas related to cognition process. In addition, different patterns of cytokine expression during the course of CM could be modulated by a single administration of the anti-malarial artesunate.

Pro-inflammatory effects of the mushroom Agaricus blazei and its consequences on atherosclerosis development.

PURPOSE: Extracts of the mushroom Agaricus blazei (A. blazei) have been described as possessing immunomodulatory and potentially cancer-protective activities. However, these effects of A. blazei as a functional food have not been fully investigated in vivo. METHODS: Using apolipoprotein E-deficient (ApoE(-/-)) mice, an experimental model of atherosclerosis, we evaluated the effects of 6 or 12 weeks of A. blazei supplementation on the activation of immune cells in the spleen and blood and on the development of atherosclerosis. RESULTS: Food intake, weight gain, blood lipid profile, and glycemia were similar between the groups. To evaluate leukocyte homing and activation, mice were injected with (99m)Tc-radiolabeled leukocytes, which showed enhanced leukocyte migration to the spleen and heart of A. blazei-supplemented animals. Analysis of the spleen showed higher levels of activation of neutrophils, NKT cells, and monocytes as well as increased production of TNF-α and IFN-γ. Circulating NKT cells and monocytes were also more activated in the supplemented group. Atherosclerotic lesion areas were larger in the aorta of supplemented mice and exhibited increased numbers of macrophages and neutrophils and a thinner fibrous cap. A. blazei-induced transcriptional upregulation of molecules linked to macrophage activation (CD36, TLR4), neutrophil chemotaxy (CXCL1), leukocyte adhesion (VCAM-1), and plaque vulnerability (MMP9) were seen after 12 weeks of supplementation. CONCLUSIONS: This is the first in vivo study showing that the immunostimulatory effect of A. blazei has proatherogenic repercussions. A. blazei enhances local and systemic inflammation, upregulating pro-inflammatory molecules, and enhancing leukocyte homing to atherosclerosis sites without affecting the lipoprotein profile.

Role of the chemokine receptor CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis.

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection.

BACKGROUND: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. METHODS: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91(phox-/-)) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. RESULTS: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91(phox-/-) mice, indicating a larger inflammatory response in gp91(phox-/-) even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91(phox-/-) at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. CONCLUSION: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

IL-1ß Is Involved with the Generation of Pain in Experimental Autoimmune Encephalomyelitis.

Pain is one of the main symptoms of multiple sclerosis, a demyelinating disease of the central nervous system that affects millions of people worldwide. The experimental autoimmune encephalomyelitis (EAE) is considered an experimental model of multiple sclerosis, and besides motor weakness, hypernociception is one of the clinical signs of animals with EAE. In this study, we investigated the influence of some cytokines in the generation of the hypernociceptive response in a mouse model of EAE using MOG35-55. We measured some cytokines in the dorsal root ganglia (DRG), an important anatomical structure involved in pain. We found increased levels of the cytokines TNF-α, IL-1ß, and Kc in DRGs of animals with EAE. We used the antibody IL-1ra to antagonize the effects of IL-1ß, and animals presented a decrease in the hypernociceptive response. Thus, our results suggest that hypernociception in this experimental model of EAE may be a consequence of the increase in some cytokines in DRGs, especially IL-1ß.

Lack of platelet-activating factor receptor protects mice against diet-induced adipose inflammation and insulin-resistance despite fat pad expansion.

OBJECTIVE: The role of platelet-activating factor (PAF) on diet-induced inflammatory and metabolic dysfunction is unknown. The effects of diet-induced metabolic and inflammatory dysfunction in mice with deletion of the PAF receptor (PAFR(-/-) ) were evaluated in this study. METHODS: Wild-type and PAFR(-/-) mice were fed chow (WT-C and PAFR(-/-) -C) or high-refined carbohydrate-containing diet (WT-HC and PAFR(-/-) -HC). PAFR(-/-) - RESULTS: HC mice gained more weight and adiposity than PAFR(-/-) -C and WT-HC mice. Lipogenesis increased and hormone-sensitive lipase expression decreased in PAFR(-/-) -HC compared to WT-HC mice. WT-HC mice had impaired glucose tolerance and insulin sensitivity compared to WT-C mice. In contrast, glucose tolerance and insulin sensitivity in PAFR(-/-) -HC mice were similar to that of lean littermates. PAFR(-/-) -HC mice expressed significantly more peroxisome proliferator-activator receptor gamma (PPARγ) than PAFR(-/-) -C and WT-C mice. Resistin increased in WT-HC mice compared to WT-C mice. However, the levels of resistin were 35% lower in PAFR(-/-) -HC mice than WT-HC mice. PAFR(-/-) presented with less HC diet-induced adipose tissue inflammation than WT mice. Adipocytes isolated from PAFR(-/-) mice incubated in media containing normal or high levels of glucose secreted less interleukin-6 and tumor necrosis factor alpha and presented lower rate of lipolysis than WT mice. CONCLUSION: PAFR deficiency resulted in less inflammation in adipose tissue and improvement in glucose homeostasis when fed the HC diet. The higher adiposity observed in PAFR(-/-) mice fed HC diet could be owing to the maintenance of insulin sensitivity, decreased adipocyte lipolysis rate, high lipogenesis and PPARγ expression, and lower inflammatory milieu in adipose tissue.

Evaluation of laboratory tests for dengue diagnosis in clinical specimens from consecutive patients with suspected dengue in Belo Horizonte, Brazil.

BACKGROUND: Dengue is a widely spread arboviral disease in tropical and subtropical regions of the world. Dengue fever presents clinical characteristics similar to other febrile illness. Thus laboratory diagnosis is important for adequate management of the disease. OBJECTIVES: The present study was designed to evaluate the diagnostic performance of real-time PCR and serological methods for dengue in a real epidemic context. STUDY DESIGN: Clinical data and blood samples were collected from consecutive patients with suspected dengue who attended a primary health care unit in Belo Horizonte, Brazil. Serologic methods and real-time PCR were performed in serum samples to confirm dengue diagnosis. RESULTS: Among the 181 consecutive patients enrolled in this study with suspected dengue, 146 were considered positive by serological criteria (positive NS1 ELISA and/or anti-dengue IgM ELISA) and 138 were positive by real-time PCR. Clinical criteria were not sufficient for distinguishing between dengue and non-dengue febrile illness. The PCR reaction was pre-optimized using samples from patients with known viral infection. It had similar sensitivity compared to NS1 ELISA (88% and 89%, respectively). We also evaluated three commercial lateral flow immunochromatographic tests for NS1 detection (BIOEASY, BIORAD and PANBIO). All three tests showed high sensitivity (94%, 91% and 81%, respectively) for dengue diagnosis. CONCLUSION: According to our results it can be suggested that lateral flow tests for NS1 detection are the most feasible methods for early diagnosis of dengue.

Beneficial effects of the activation of the angiotensin-(1-7) MAS receptor in a murine model of adriamycin-induced nephropathy.

Angiotensin-(1-7) [Ang-(1-7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-ß. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas(+/+) ) and Mas knockout (Mas (-/-)) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas(+/+) , but not in Mas (-/-) mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1-7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.

Platelet-activating factor receptor plays a role in the pathogenesis of graft-versus-host disease by regulating leukocyte recruitment, tissue injury, and lethality.

PAF is a potent lipid mediator involved in several manifestations of acute inflammation, including leukocyte influx, leukocyte interaction with endothelium, and production of inflammatory cytokines. The present study evaluated the relevance of PAFR for the pathogenesis of acute GVHD using a model of adoptive transfer of splenocytes from WT or PAFR(-/-) C57BL/6J to B6D2F1 mice. Mice, which received PAFR(-/-) splenocytes or treatment with the PAFR antagonist, showed reduced clinical signs of disease and no mortality. In GVHD mice receiving PAFR(-/-) splenocytes, there was deceased bacterial translocation and tissue injury. Furthermore, production of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL2, CCL3, and CCL5) and accumulation of CD8(+) cells in intestine and liver were reduced in mice transplanted with the PAFR(-/-) splenocyte. Mechanistically, an absence or pharmacological blockade of PAFR was associated with decreased rolling and adhesion of leukocytes to the mesenteric microcirculation, as assessed by intravital microscopy. Despite decreased GVHD, there was maintained GVL activity when PAFR(-/-) leukocytes were transferred into WT mice. In conclusion, PAFR on donor leukocytes plays a critical role in GVHD by mediating leukocyte influx and cytokine production in target tissues. PAFR antagonist may potentially be useful in the treatment of GVHD in bone marrow-transplanted patients.

Dengue-3 encephalitis promotes anxiety-like behavior in mice.

Dengue virus is a human pathogen that may cause meningoencephalitis and other neurological syndromes. The current study investigated anxiety-like behavior and expression of proinflammatory cytokines and pro-apoptotic caspase-3 in the hippocampus of C57BL/6 mice infected with non-adapted Dengue virus 3 genotype I (DENV-3) inoculated intracranially with 4×10(3) (plaque-forming unit) PFU. Anxiety-like behavior was assessed in control and DENV-3 infected mice using the elevated plus maze. The open field test was performed to evaluate locomotor activity. Histopathological changes in CA regions of the hippocampus were assessed by haematoxylin and eosin staining. Immunoreactive and protein levels of cleaved caspase-3 were also analyzed in the hippocampus. The mRNA expression of IL-6 and TNF-α in the hippocampus were estimated by quantitative real time (polymerase chain reaction) PCR. All procedures were conducted on day 5 post-infection. We found that DENV-3 infected mice presented higher levels of anxiety in comparison with controls (p≤0.05). No difference in motor activity was found between groups (p=0.77). The infection was followed by a significant increase of TNF-α and IL-6 mRNA expression in the hippocampus (p≤0.05). Histological analysis demonstrated meningoencephalitis with formation of perivascular cuffs, infiltration of immune cells and loss of neurons at CA regions of hippocampus. Numerous caspase-3 positive neurons were visualized at CA areas in DENV-3 infected mice. Marked increase of cleaved caspase-3 levels were observed after infection. This study described anxiety-like behavior, hippocampal inflammation and neuronal apoptosis associated with DENV-3 infection in the central nervous system.

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection.

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.

IFN-γ production depends on IL-12 and IL-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner.

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Severe dengue infection in humans is characterized by thrombocytopenia, increased vascular permeability, hemorrhage and shock. However, there is little information about host response to DENV infection. Here, mechanisms accounting for IFN-γ production and effector function during dengue disease were investigated in a murine model of DENV-2 infection. IFN-γ expression was greatly increased after infection of mice and its production was preceded by increase in IL-12 and IL-18 levels. In IFN-γ(-/-) mice, DENV-2-associated lethality, viral loads, thrombocytopenia, hemoconcentration, and liver injury were enhanced, when compared with wild type-infected mice. IL-12p40(-/-) and IL-18(-/-) infected-mice showed decreased IFN-γ production, which was accompanied by increased disease severity, higher viral loads and enhanced lethality. Blockade of IL-18 in infected IL-12p40(-/-) mice resulted in complete inhibition of IFN-γ production, greater DENV-2 replication, and enhanced disease manifestation, resembling the response seen in DENV-2-infected IFN-γ(-/-) mice. Reduced IFN-γ production was associated with diminished Nitric Oxide-synthase 2 (NOS2) expression and NOS2(-/-) mice had elevated lethality, more severe disease evolution and increased viral load after DENV-2 infection. Therefore, IL-12/IL-18-induced IFN-γ production and consequent NOS2 induction are of major importance to host resistance against DENV infection.