Inhibition of calcium/calmodulin (Ca2+ /CaM)-Calcium/calmodulin-dependent protein kinase II (CaMKII) axis reduces in vitro and ex vivo arrhythmias in experimental Chagas disease.

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.

Colonization by Enterobacteriaceae is crucial for acute inflammatory responses in murine small intestine via regulation of corticosterone production.

Although dysbiosis in the gut microbiota is known to be involved in several inflammatory diseases, whether any specific bacterial taxa control host response to inflammatory stimuli is still elusive. Here, we hypothesized that dysbiotic indigenous taxa could be involved in modulating host response to inflammatory triggers. To test this hypothesis, we conducted experiments in germ-free (GF) mice and in mice colonized with dysbiotic taxa identified in conventional (CV) mice subjected to chemotherapy-induced mucositis. First, we report that the absence of microbiota decreased inflammation and damage in the small intestine after administration of the chemotherapeutic agent 5-fluorouracil (5-FU). Also, 5-FU induced a shift in CV microbiota resulting in higher amounts of Enterobacteriaceae, including E. coli, in feces and small intestine and tissue damage. Prevention of Enterobacteriaceae outgrowth by treating mice with ciprofloxacin resulted in diminished 5-FU-induced tissue damage, indicating that this bacterial group is necessary for 5-FU-induced inflammatory response. In addition, monocolonization of germ-free (GF) mice with E. coli led to reversal of the protective phenotype during 5-FU chemotherapy. E. coli monocolonization decreased the basal plasma corticosterone levels and blockade of glucocorticoid receptor in GF mice restored inflammation upon 5-FU treatment. In contrast, treatment of CV mice with ciprofloxacin, that presented reduction of Enterobacteriaceae and E. coli content, induced an increase in corticosterone levels. Altogether, these findings demonstrate that Enterobacteriaceae outgrowth during dysbiosis impacts inflammation and tissue injury in the small intestine. Importantly, indigenous Enterobacteriaceae modulates host production of the anti-inflammatory steroid corticosterone and, consequently, controls inflammatory responsiveness in mice.

Intestinal microbiota regulates tryptophan metabolism following oral infection with Toxoplasma gondii.

INTRODUCTION: The intestinal microbiota plays an important role in modulating host immune responses. Oral Toxoplasma gondii infection can promote intestinal inflammation in certain mice strains. The IDO-AhR axis may control tryptophan (Trp) metabolism constituting an important immune regulatory mechanism in inflammatory settings. AIMS: In the present study, we investigated the role of the intestinal microbiota on Trp metabolism during oral infection with T gondii. METHODS AND RESULTS: Mice were treated with antibiotics for four weeks and then infected with T gondii by gavage. Histopathology and immune responses were evaluated 8 days after infection. We found that depletion of intestinal microbiota by antibiotics contributed to resistance against T gondii infection and led to reduced expression of AhR on dendritic and Treg cells. Mice depleted of Gram-negative bacteria presented higher levels of systemic Trp, downregulation of AhR expression and increased resistance to infection whereas depletion of Gram-positive bacteria did not affect susceptibility or expression of AhR on immune cells. CONCLUSION: Our findings indicate that the intestinal microbiota can control Trp availability and provide a link between the AhR pathway and host-microbiota interaction in acute infection with T gondii.

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.

The Aryl Hydrocarbon Receptor Modulates Production of Cytokines and Reactive Oxygen Species and Development of Myocarditis during Trypanosoma cruzi Infection.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in controlling several aspects of immune responses, including the activation and differentiation of specific T cell subsets and antigen-presenting cells, thought to be relevant in the context of experimental Trypanosoma cruzi infection. The relevance of AhR for the outcome of T. cruzi infection is not known and was investigated here. We infected wild-type (WT) mice and AhR knockout (AhR KO) mice with T. cruzi (Y strain) and determined levels of parasitemia, myocardial inflammation and fibrosis, expression of AhR/cytokines/suppressor of cytokine signaling (SOCS) (spleen/heart), and production of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) (spleen). AhR expression was increased in the heart of infected WT mice. Infected AhR KO mice displayed significantly reduced parasitemia, inflammation, and fibrosis of the myocardium. This was associated with an anticipated increased immune response characterized by increased levels of inflammatory cytokines and reduced expression of SOCS2 and SOCS3 in the heart. In vitro, AhR deficiency caused impairment in parasite replication and decreased levels of ROS production. In conclusion, AhR influences the development of murine Chagas disease by modulating ROS production and regulating the expression of key physiological regulators of inflammation, SOCS1 to -3, associated with the production of cytokines during experimental T. cruzi infection.

Absence of gut microbiota influences lipopolysaccharide-induced behavioral changes in mice.

Changes in the microbiota composition of gastrointestinal tract are emerging as potential players in the physiopathology of neuropsychiatric disorders. In the present work we evaluated the relationship between the absence of gut microbiota and neuroinflammatory mechanisms in a murine model of LPS-induced behavioral alterations. Germ-free (GF) or conventional male mice received a single i.p. injection of lipopolysaccharide (LPS i.p.; 0.83mg/Kg) or PBS, and after 24h they were tested for depressive-like behaviors (forced swimming test, tail suspension test - TST, or sucrose preference test - SPT). After behavioral evaluation, animals were analyzed for possible changes in neuroplasticity by means of BDNF, NGF and cytokines levels in prefrontal cortex and hippocampus, and the expression of Iba-1 (microglial activation marker) in the hippocampus, and the cellular activity marker, ΔFosB, in the dorsal raphe nucleus. In conventional mice, LPS induced depressive-like behaviors. LPS-induced changes were followed by up-regulation of the expression of TNF and Iba-1 in the hippocampus. The same effects were not observed in GF mice. Behavioral effects of LPS were not observed in GF mice submitted to TST. GF mice present a lower response to the anhedonia-like effect induced by LPS when compared to conventional animals (SPT). There was up-regulation of ΔFosB in the dorsal raphe nucleus in the absence of gut microbiota, events not influenced by LPS treatment. Our results suggest that gut-microbiota interactions influence depressive-like behaviors, raphe nucleus activation and activation of pro-inflammatory mechanisms within the hippocampus.

Trypanosoma cruzi Needs a Signal Provided by Reactive Oxygen Species to Infect Macrophages.

BACKGROUND: During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo. CONCLUSIONS: Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells.

Cytokine Expression in Patients Hospitalized for Severe Odontogenic Infection in Brazil.

INTRODUCTION: Severe odontogenic infections remain an important public health concern and a significant economic burden to public health care facilities. Despite this, several aspects of the disease, such as its immune response profile, remain poorly understood. The aim of this study was to search for an association between mRNA levels of the cytokines interferon-γ, interleukin (IL)-1ß, tumor necrosis factor-α, IL-17A, IL-10, and transforming growth factor-ß and the chemokines IL-8, CCL2/MCP-1, and CCL5 and odontogenic infection. METHODS: The case group was composed of 12 patients hospitalized in consequence of severe odontogenic infection, and our control group included 12 individuals with healthy periapical tissues. Clinical samples were taken from the case (drainage site) and control (periapical interstitial fluid) groups with the aid of paper points. Total RNA was extracted, complementary DNA was synthesized, and mRNA levels were determined by quantitative polymerase chain reaction. Data analysis was performed by using SPSS, and the Wilcoxon signed rank test was used to determine statistical significance (P < .05). RESULTS: Data generated showed a significantly increased expression of proinflammatory cytokines (interferon-γ, IL-1ß, tumor necrosis factor-α, and IL-17A), IL-8, and CCL2/MCP-1 in odontogenic infection patients. The mRNA levels of IL-10, transforming growth factor-ß, and CCL5 were similar in both study groups. CONCLUSIONS: In general, individuals presenting with odontogenic infections exhibited extraordinary proinflammatory cytokine profiles paralleled with unaltered expression of regulatory mediators.

Safety and protective effectiveness of two strains of Lactobacillus with probiotic features in an experimental model of salmonellosis.

Two strains of Lactobacillus, previously isolated from bovine faeces and tested in vitro for properties desired in probiotics, were evaluated for their in vivo effectiveness in protecting against experimental salmonellosis. L. salivarius L38 and L. acidophilus L36 previously demonstrated the ability to successfully colonize the gastrointestinal tract of germ-free mice and stimulate the immune system associated with the intestinal mucosa. L38- or L36-feeding showed no detrimental effect on the general health indicators and did not induce changes in normal architecture of liver and small intestine, indicating that the use of these strains is apparently safe. In control animals fed L38 strain, several cytokines had augmented mRNA levels that can be associated with a homeostatic state of intestinal mucosa, while L36 had less diverse regulation. IgA production and secretion in the intestinal lumen induced by infection was abrogated by pretreating with both lactobacilli. In addition, liver and small intestine histological scores and, translocation of Salmonella cells to liver and spleen, indicated that these strains did not confer protection against the infection. So, the IL-12:IL-18àIFN-g axis, essential for an effective immune response against Salmonella, was not favored with L38 or L36 strains. However, increased expression of IL-10 in different portions of the gastrointestinal tract of L38-fed animals is indicative of anti-inflammatory effect to be explored furthermore.

A defective TLR4 signaling for IFN-ß expression is responsible for the innately lower ability of BALB/c macrophages to produce NO in response to LPS as compared to C57BL/6.

C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-ß and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-ß has a determinant role since 1) anti-IFN-ß neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2) exogenous rIFN-ß confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-ß-mediated STAT-1 activation pathway.

Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.

The endogenous cytokine profile and nerve fibre density in mouse ear Leishmania major-induced lesions related to nociceptive thresholds.

Several reports have shown that cutaneous leishmaniasis lesions are painless, suggesting that Leishmania infection interferes with pain perception. Comparisons of inflammation-induced hyperalgesia between BALB/c and C57BL/6 mice have been little explored in the literature, and comparative data regarding nociception in leishmaniasis are non-existent. In susceptible BALB/c mice and resistant C57BL/6 mice that were intradermally inoculated with a low dose of Leishmania major in the ear, we investigated the variation in nociception over a 12-wk period post-infection and this variation's association with the structure of nerve fibres and the presence of endogenous cytokines that are classically considered hyper- or hypo-nociceptive. Infected BALB/c mice presented susceptibility and severe lesions. Infected C57BL/6 mice exhibited resistance and healing lesions. The immune response involved pro- and anti-inflammatory cytokine secretion, respectively. The infection-induced hypoalgesia in BALB/c mice after wks 9 was accompanied by decreased levels of IL-6 and IL-10 in ear tissue with intact nerves. C57BL/6 mice showed short-lived hyperalgesia in wks 2, which was related to increased local levels of IL-6, KC/CXCL-1, TNF-α and IL-10 and a decrease in nerve density. The increase in pro-inflammatory cytokine IL-6, KC/CXCL-1 and TNF-α levels during hyperalgesia suggested a role for these mediators in afferent nerve sensitisation, which was secondary to the inflammatory damage of nerve fibres stained by PGP 9.5. In contrast, the mechanisms of hypoalgesia may include the downregulation of cytokines, the preservation of the structure of nerve endings, and as yet uninvestigated unidentified differences in neurotransmitter release or a direct role of the parasites in the context of the progressive and permissive inflammatory response of BALB/c mice.

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 SOCS2 in modulating heart damage and function in a murine model of acute Chagas disease.

Infection with Trypanosoma cruzi induces inflammation, which limits parasite proliferation but may result in chagasic heart disease. Suppressor of cytokine signaling 2 (SOCS2) is a regulator of immune responses and may therefore participate in the pathogenesis of T. cruzi infection. SOCS2 is expressed during T. cruzi infection, and its expression is partially reduced in infected 5-lipoxygenase-deficient [knockout (KO)] mice. In SOCS2 KO mice, there was a reduction in both parasitemia and the expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-10, SOCS1, and SOCS3 in the spleen. Expression of IFN-γ, TNF-α, SOCS1, and SOCS3 was also reduced in the hearts of infected SOCS2 KO mice. There was an increase in the generation and expansion of T regulatory (Treg) cells and a decrease in the number of memory cells in T. cruzi-infected SOCS2 KO mice. Levels of lipoxinA(4) (LXA(4)) increased in these mice. Echocardiography studies demonstrated an impairment of cardiac function in T. cruzi-infected SOCS2 KO mice. There were also changes in calcium handling and in action potential waveforms, and reduced outward potassium currents in isolated cardiac myocytes. Our data suggest that reductions of inflammation and parasitemia in infected SOCS2-deficient mice may be secondary to the increases in Treg cells and LXA(4) levels. This occurs at the cost of greater infection-associated heart dysfunction, highlighting the relevance of balanced inflammatory and immune responses in preventing severe T. cruzi-induced disease.

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection.

(•)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice.

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.

Host susceptibility to Brucella abortus infection is more pronounced in IFN-γ knockout than IL-12/ß2-microglobulin double-deficient mice.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. IFN-γ, IL-12, and CD8+ T lymphocytes are important components of host immune responses against B. abortus. Herein, IFN-γ and IL-12/ß2-microglobulin (ß2-m) knockout mice were used to determine whether CD8+ T cells and IL-12-dependent IFN-γ deficiency would be more critical to control B. abortus infection compared to the lack of endogenous IFN-γ. At 1 week after infection, IFN-γ KO and IL-12/ß2-m KO mice showed increased numbers of bacterial load in spleens; however, at 3 weeks postinfection (p.i.), only IFN-γ KO succumbed to Brucella. All IFN-γ KO had died at 16 days p.i. whereas death within the IL-12/ß2-m KO group was delayed and occurred at 32 days until 47 days postinfection. Susceptibility of IL-12/ß2-m KO animals to Brucella was associated to undetectable levels of IFN-γ in mouse splenocytes and inability of these cells to lyse Brucella-infected macrophages. However, the lack of endogenous IFN-γ was found to be more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies.

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.

Resolution of neutrophilic inflammation by H2O2 in antigen-induced arthritis.

OBJECTIVE: Neutrophil accumulation contributes to the pathogenesis of rheumatoid arthritis. This study was undertaken to examine the ability of H2O2 to influence neutrophilic inflammation in a model of antigen-induced arthritis (AIA) in mice. METHODS: AIA was induced by administration of antigen into the knee joints of previously immunized mice. Neutrophil accumulation was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity in the tissue surrounding the mouse knee joint. Apoptosis was determined by morphologic and molecular techniques. The role of H2O2 was studied using mice that do not produce reactive oxygen species (gp91phox-/- mice) and drugs that enhance the generation or enhance the degradation of H2O2. RESULTS: Antigen challenge of immunized mice induced neutrophil accumulation that peaked at 12-24 hours after challenge. H2O2 production peaked at 24 hours, after which time, the inflammation resolved. Neutrophil recruitment was similar in wild-type and gp91phox-/- mice, but there was delayed resolution in gp91phox-/- mice or after administration of catalase. In contrast, administration of H2O2 or superoxide dismutase (SOD) resolved neutrophilic inflammation. The resolution of inflammation induced by SOD or H2O2 was accompanied by an increase in the number of apoptotic neutrophils. Apoptosis was associated with an increase in Bax and caspase 3 cleavage and was secondary to phosphatidylinositol 3-kinase (PI3K)/Akt activation. CONCLUSION: Our findings indicate that levels of H2O2 increase during neutrophil influx and are necessary for the natural resolution of neutrophilic inflammation. Mechanistically, enhanced levels of H2O2 (endogenous or exogenous) inhibit p-Akt/NF-κB and induce apoptosis of migrated neutrophils. Modulation of H2O2 production may represent a novel strategy for controlling neutrophilic inflammation in the joints.

Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus.

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.