Lucas M. Kangussu

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.

Role of the Aryl Hydrocarbon Receptor in the Immune Response Profile and Development of Pathology during Plasmodium berghei Anka Infection

ABSTRACT Infection with Plasmodium falciparum may result in severe disease affecting various organs, including liver, spleen, and brain, resulting in high morbidity and mortality. Plasmodium berghei Anka infection of mice recapitulates many features of severe human malaria. The aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by ligands important in the modulation of the inflammatory response. We found that AhR-knockout (KO) mice infected with P. berghei Anka displayed increased parasitemia, earlier mortality, enhanced leukocyte-endothelial cell interactions in the brain microvasculature, and increased inflammation in brain (interleukin-17 [IL-17] and IL-6) and liver (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) compared to infected wild-type (WT) mice. Infected AhR-KO mice also displayed a reduction in cytokines required for host resistance, including TNF-α, IL-1β, and IFN-γ, in the brain and spleen. Infection of AhR-KO mice resulted in an increase in T regulatory cells and transforming growth factor β, IL-6, and IL-17 in the brain. AhR modulated the basal expression of SOCS3 in spleen and brain, and P. berghei Anka infection resulted in enhanced expression of SOCS3 in brain, which was absent in infected AhR-KO mice. These data suggest that AhR-mediated control of SOCS3 expression is probably involved in the phenotype seen in infected AhR-KO mice. This is, to our knowledge, the first demonstration of a role for AhR in the pathogenesis of malaria.

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.

Anxiolytic- and antidepressant-like effects of angiotensin-(1-7) in hypertensive transgenic (mRen2)27 rats

Angiotensin-(1-7) [Ang-(1-7)], a counter-regulatory peptide of the renin-angiotensin system (RAS) exerts its effects through the G-protein-coupled receptor Mas, which is expressed in different tissues, including the brain. Ang-(1-7) has a broad range of effects beyond the well-described cardiovascular and renal actions, including the modulation of emotional and behavioural responses. In the present study we tested the hypothesis that Ang-(1-7) could attenuate the anxiety- and depression-like behaviours observed in transgenic hypertensive (mRen2)27 rats (TGRs). We also hypothesized that Ang-(1-7) could be involved in the anxiolytic-like effect induced by ACE (angiotensin-converting enzyme) treatment in these hypertensive rats. Therefore, TGRs and Sprague-Dawley rats were subjected to the Elevated Plus Maze (EPM) test, Forced Swimming Test (FST) and Novelty Suppressed Feeding (NSF). TGRs presented a decreased percentage of entries in the open arms of the EPM test, a phenotype reversed by systemic treatment with enalapril or intracerebroventricular infusion of Ang-(1-7). It is interesting that pre-treatment with A779, a selective Mas receptor antagonist, prevented the anxiolytic-like effect induced by the ACE inhibitor. In the NSF test, TGRs showed increased latency to eating, an indicative of a higher aversion in response to a new environment. These animals also showed increased immobility in the FST. Again, Ang-(1-7) reversed this phenotype. Thus, our data showed that Ang-(1-7) can modulate anxiety- and depression-like behaviours in TGRs and warrant further investigation as a new therapy for certain psychiatric disorders.

Platelet-activating factor receptor (PAFR) plays a crucial role in experimental global cerebral ischemia and reperfusion.

Stroke is one of the most frequent causes of death and disability worldwide leading to a significant clinical and socioeconomic burden. Although different mechanisms are involved in the pathogenesis of stroke, inflammatory response occurs after ischemia and contributes to the expansion of brain injury. Platelet-activating factor receptor (PAF) plays crucial roles in both physiological and pathological conditions in the brain. PAF receptor (PAFR) may be expressed on cellular and nuclear membranes of various cell types, especially leukocytes, platelets, endothelial cells, neuronal cells and microglia. Herein, using mice lacking the PAFR receptor (PAFR(-/-)), we investigate a potential role for this receptor during experimental transient global cerebral ischemia and reperfusion (BCCAo). In PAFR deficiency, we observed a significant improvement in the neurological deficits, which were associated with a reduction of brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, a decrease in the percentage of necrotic cavities areas and in the frequency of ischemic neurons was also found by employing histometric analysis. In addition, in PAFR(-/-) mice there was prevention of caspase-3 activation and decreased vascular permeability and brain edema. Decreased brain levels of the cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and the chemokine (C-X-C motif) ligand 1 (CXCL1) by ELISA were also detected in PAFR(-/-) BCCAo animals. Taken together, our results suggest that PAFR activation might be crucial for the global brain ischemia and reperfusion injury.

Up-regulation of brain cytokines and chemokines mediates neurotoxicity in early acute liver failure by a mechanism independent of microglial activation

The neurological involvement in acute liver failure (ALF) is characterized by arousal impairment with progression to coma. There is a growing body of evidence that neuroinflammatory mechanisms play a role in this process, including production of inflammatory cytokines and microglial activation. However, it is still uncertain whether brain-derived cytokines and glial cells are crucial to the pathophysiology of ALF at the early stage, before coma development. Here, we investigated the influence of cytokines and microglia in ALF-induced encephalopathy in mice as soon as neurological symptoms were identifiable. Behavior was assessed at 12, 24, 36 and 48 h post-injection of thioacetamide, a hepatotoxic drug, through locomotor activity by an open field test. Brain concentration of cytokines (TNF-α and IL-1β) and chemokines (CXCL1, CCL2, CCL3 and CCL5) were assessed by ELISA. Microglial activation in brain sections was investigated through immunohistochemistry, and cellular ultrastructural changes were observed by transmission electron microscopy. We found that ALF-induced animals presented a significant decrease in locomotor activity at 24 h, which was accompanied by an increase in IL-1β, CXCL1, CCL2, CCL3 and CCL5 in the brain. TNF-α level was significantly increased only at 36 h. Despite marked morphological changes in astrocytes and brain endothelial cells, no microglial activation was observed. These findings suggest an involvement of brain-derived chemokines and IL-1β in early pathophysiology of ALF by a mechanism independent of microglial activation.

Enhancement on reactive oxygen species and COX-1 mRNA levels modulate the vascular relaxation induced by sodium nitroprusside in denuded mice aorta.

This study aimed to investigate the modulation of nitric oxide/reactive oxygen species in sodium nitroprusside relaxation in mice aorta. Sodium nitroprusside induced relaxation in endothelium‐intact (e+) and endothelium‐denuded (e−) aortas with greater potency in e+ than in e−. The nitric oxide synthase inhibitor did not alter the sodium nitroprusside relaxation in both e+ and e− aortas. However, the superoxide anion scavenger abolished the difference in sodium nitroprusside potency between e+ and e−. Sodium nitroprusside reduced dihydroethidium‐derived fluorescent products in both groups; however, the difference between intact and denuded mice aorta remains. The glutathione levels and basal antioxidant activity of superoxide dismutase were reduced in e− aorta when compared with e+, and these values were not altered by sodium nitroprusside. Confirming these results, the levels of lipid peroxidation in e+ were significantly lower when compared to e−, and these values were not altered by sodium nitroprusside. The sodium nitroprusside potency in the presence of a nonselective COX inhibitor or the EP/DP prostaglandin receptor antagonist in endothelium denuded was similar to that in intact mice aorta. Based on these results, we performed the COX‐1 and COX‐2 mRNA level studies, and in denuded mice aorta, there was an upregulation in COX‐1 mRNA levels. Taken together, our findings show that in the absence of endothelium, there is an enhancement of superoxide levels, leading to GSH consumption and higher levels of lipid peroxidation, showing an intense redox status. Furthermore, in denuded mice aorta, there was an upregulation of COX‐1 mRNA expression, leading to vasoconstrictor prostanoids synthesis. The interaction of vasoconstrictor prostanoids with its receptors EP/DP negatively modulates the vascular relaxation induced by SNP in denuded mice aorta.

Eccentric Overload Muscle Damage is Attenuated By a Novel Angiotensin- (1-7) Treatment

The development of new strategies to attenuate exercise-induced muscle damage may be helpful for training regimens. The aim of this study was to determine whether a oral formulation of angiotensin Ang-(1-7)[HPβCD/Ang-(1-7)] is effective to reduce pain, and muscle damage markers after eccentric-overload exercise. HPβCD (Placebo) and HPβCD/Ang-(1-7) (Ang-(1-7) group were treated for 7 days (one capsule/day). The pain was measured by visual analogue scale, maximal strength (MS) using force platform. Blood samples were collected for cytokines and creatine kinase (CK) analysis. The Ang-(1-7)-treated group reported less pain immediately (3.46±0.64 vs. placebo 3.80±0.77 cm) and 24 h after exercise (3.07±0.71 vs. 3.73±0.58 cm placebo) and higher MS at 24 h (24±12 N) and 48 h (30±15 N) vs. placebo (-8±9 N and -10±9 N). The CK for Ang-(1-7) (0.5±0.1 and 0.9±0.2 U/L) were lower at 48 and 72 h vs. placebo (fold changes of 1.7±0.5 and 1.5±0.3 U/L). The TNF-α level was lower in the treated group post-exercise (38±2.5 pg/ml) vs. placebo (45±2.9 pg/ml) but no significant changes were observed for IL-6 and IL-10. Our data indicate that treatment with Ang-(1-7) may attenuate pain, some of the muscle damage markers and improves performance following eccentric exercise.

Cardiovascular effects of small peptides of the renin angiotensin system

The renin‐angiotensin system (RAS) is a unique hormonal cascade which is composed by multiple enzymes and effector peptides. Recently, new peptides presenting biological activity have been discovered, increasing the complexity of the RAS. Here, we evaluated the effects of small peptides of the RAS in coronary bed of rats. Firstly, we examined the direct effect of small angiotensinergic peptides [Angiotensin (Ang) ‐(1–5), Ang‐(1–4) Ang‐(1–3), and Ang‐(1–2)] in coronary vessels. Noteworthy, it was observed that Ang‐(1–4), Ang‐(1–3), and Ang‐(1–2) caused a significant reduction in pressure perfusion. Because Ang‐(1–2) was the smallest peptide tested and presented the major effect, we decided to investigate its mechanisms of action. The effect of Ang‐(1–2) was partially dependent on the Mas receptor, nitric oxide release and angiotensin‐converting enzyme. Importantly, Ang‐(1–2) reduced the blood pressure of Wistar rats and SHR. Interestingly, SHR presented a more pronounced decrease in blood pressure levels than Wistar rats. Altogether, these data showed that angiotensinergic small peptides hold biological activities in coronary bed of rats.