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.
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.
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.
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.
•Lack of gut microbiota alters LPS-induced depressive-like behaviors.•Germ-free mice present impaired increase of TNF and microglial cells activation within the hippocampus after LPS ...stimulus.•Animals lacking gut microbiota display an elevated basal level of dorsal raphe activation.
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.
The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by ...significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-alpha, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-alpha and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-alpha or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their "no inflammation" phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.
The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead ...to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-alpha production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-alpha production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.
Reactive oxygen species (ROS) produced by NADPH phagocyte oxidase isoform (NOX2) are critical for the elimination of intracellular pathogens in many infections. Despite their importance, the role of ...ROS following infection with the eukaryotic pathogen
has not been fully elucidated. We addressed the role of ROS in C57BL/6 mice following intradermal infection with
Despite equivalent parasite loads compared with wild-type (WT) mice, mice deficient in ROS production by NOX2 due to the absence of the gp91 subunit (gp91
) had significantly more severe pathology in the later stages of infection. Pathology in gp91
mice was not associated with alterations in CD4
T cell-mediated immunity but was preceded by enhanced neutrophil accumulation at the dermal infection site. Ex vivo analysis of infected versus uninfected neutrophils revealed a deficiency in infection-driven apoptosis in gp91
mice versus WT mice. gp91
mice presented with higher percentages of healthy or necrotic neutrophils but lower percentages of apoptotic neutrophils at early and chronic time points. In vitro infection of gp91
versus WT neutrophils also revealed reduced apoptosis and CD95 expression but increased necrosis in infected cells at 10 h postinfection. Provision of exogenous ROS in the form of H
O
reversed the necrotic phenotype and restored CD95 expression on infected gp91
neutrophils. Although ROS production is typically viewed as a proinflammatory event, our observations identify the importance of ROS in mediating appropriate neutrophil apoptosis and the importance of apoptosis in inflammation and pathology during chronic 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.
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.
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.
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.
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