FK506 impairs neutrophil migration that results in increased polymicrobial sepsis susceptibility.

OBJECTIVE: This study aimed to investigate the effects of FK506 on experimental sepsis immunopathology. It investigated the effect of FK506 on leukocyte recruitment to the site of infection, systemic cytokine production, and organ injury in mice with sepsis. METHODS: Using a murine cecal ligation and puncture (CLP) peritonitis model, the experiments were performed with wild-type (WT) mice and mice deficient in the gene Nfat1 (Nfat1-/-) in the C57BL/6 background. Animals were treated with 2.0 mg/kg of FK506, subcutaneously, 1 h before the sepsis model, twice a day (12 h/12 h). The number of bacteria colony forming units (CFU) was manually counted. The number of neutrophils in the lungs was estimated by the myeloperoxidase (MPO) assay. The expression of CXCR2 in neutrophils was determined using flow cytometry analysis. The expression of inflammatory cytokines in macrophage was determined using ELISA. The direct effect of FK506 on CXCR2 internalization was evaluated using HEK-293T cells after CXCL2 stimulation by the BRET method. RESULTS: FK506 treatment potentiated the failure of neutrophil migration into the peritoneal cavity, resulting in bacteremia and an exacerbated systemic inflammatory response, which led to higher organ damage and mortality rates. Failed neutrophil migration was associated with elevated CXCL2 chemokine plasma levels and lower expression of the CXCR2 receptor on circulating neutrophils compared with non-treated CLP-induced septic mice. FK506 did not directly affect CXCL2-induced CXCR2 internalization by transfected HEK-293 cells or mice neutrophils, despite increasing CXCL2 release by LPS-treated macrophages. Finally, the CLP-induced response of Nfat1-/- mice was similar to those observed in the Nfat1+/+ genotype, suggesting that the FK506 effect is not dependent on the NFAT1 pathway. CONCLUSION: Our data indicate that the increased susceptibility to infection of FK506-treated mice is associated with failed neutrophil migration due to the reduced membrane availability of CXCR2 receptors in response to exacerbated levels of circulating CXCL2.

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology.

BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

Inflammatory effect of Bothropstoxin-I from Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor.

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1ß and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1ß production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5'-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

IL-33 and ST2 as predictors of disease severity in children with viral acute lower respiratory infection.

BACKGROUND: Mechanisms influencing severity of acute lower respiratory infection (ALRI) in children are not established. We aimed to assess the role of inflammatory markers and respiratory viruses in ALRI severity. METHODS: Concentrations of interleukin(IL)-33, soluble suppression of tumorigenicity (sST)2, IL-1ß, tumor necrosis factor α, IL-4, IL-6 and IL- 8 and types of respiratory viruses were evaluated in children at the first and fifth days after hospital admission. Disease severity was defined as need for mechanical ventilation. RESULTS: Seventy-nine children <5 years-old were included; 33(41.8%) received mechanical ventilation. No associations between virus type, viral load or co-detections and severity of disease were observed. Detection of IL-33 and sST2 in nasopharyngeal aspirates (NPA) on admission were associated with higher risk for mechanical ventilation (RR = 2.89 and RR = 4.57, respectively). IL-6 and IL-8 concentrations were higher on Day 5 in mechanically ventilated children. IL-6 NPA concentrations decreased from Day 1 to Day 5 in children who did not receive mechanical ventilation. Increase in sST2 NPA concentrations from Day 1 to Day 5 was associated with longer hospital length of stay (p < 0.01). CONCLUSIONS: An exacerbated local activation of the IL-33/ST2 axis and persistently high sST2 concentrations over time were associated with severity of viral ALRI in children.

P2x7 Receptor Signaling Blockade Reduces Lung Inflammation and Necrosis During Severe Experimental Tuberculosis.

The risk of developing severe forms of tuberculosis has increased by the acquired immunodeficiency syndrome (AIDS) epidemic, lack of effective drugs to eliminate latent infection and the emergence of drug-resistant mycobacterial strains. Excessive inflammatory response and tissue damage associated with severe tuberculosis contribute to poor outcome of the disease. Our previous studies using mice deficient in the ATP-gated ionotropic P2X7 receptor suggested this molecule as a promising target for host-directed therapy in severe pulmonary tuberculosis. In this study, we assessed the effects of P2X7 pharmacological blockade on disease severity. First, we observed an increase in P2RX7 gene expression in the peripheral blood of tuberculosis patients compared to healthy donors. Lung leukocytes of mice infected with hypervirulent mycobacteria also showed increased expression of the P2X7 receptor. P2X7 blockade in mice with advanced tuberculosis recapitulated in many aspects the disease in P2X7-deficient mice. P2X7-directed therapy reduced body weight loss and the development of inflammatory and necrotic lung lesions, as well as delayed mycobacterial growth. Lower TNF-α production by lung cells and a substantial reduction in the lung GR-1+ myeloid cell population were observed after P2X7 inhibition. The effector CD4+ T cell population also decreased, but IFN-γ production by lung cells increased. The presence of a large population with characteristics of myeloid dendritic cells, as well as the increase in IL-6 production by lung cells, also indicate a qualitative improvement in the pulmonary immune response due to P2X7 inhibition. These findings support the use of drugs that target the P2X7 receptor as a therapeutic strategy to improve the outcome of pulmonary tuberculosis.

Mycobacterium tuberculosis-infected alveolar epithelial cells modulate dendritic cell function through the HIF-1α-NOS2 axis.

Tuberculosis kills more than 1 million people every year, and its control depends on the effective mechanisms of innate immunity, with or without induction of adaptive immune response. We investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by this interaction is critical for the early innate response against mycobacteria. We found that AEC-II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1α-inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expression and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that HIF-1α-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection. Such modulation might be useful for the exploitation of molecular targets to develop new therapeutic strategies against tuberculosis.

Immunomodulatory effect of fructose-1,6-bisphosphate on T-lymphocytes.

Sepsis remains an important and life-threatening problem, and is the most common cause of death in the intensive care unit. One promising therapeutic candidate for protection against injury in sepsis is fructose-1,6-bisphosphate (FBP), a high-energy glycolytic pathway intermediate. The objective of the study was to establish a role for FBP on the immune system, especially in lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy humans by gradient centrifugation. T-lymphocytes were stimulated for 96 h with phytohemagglutinin (PHA) and varying concentration of FBP. Fructose-1,6-bisphosphate at concentrations between 1.2 and 10 mM decreased proliferation of T-lymphocytes and reduced the viability only at concentrations 5.0 and 10 mM. The levels of soluble IL-2 receptor were reduced at FBP concentrations between 1.2 and 10 mM. In conclusion, this study demonstrates that FBP has important effect on immunomodulatory and this result can be correlated with the protection against injury in sepsis.

Alterations in the indexes of apoptosis and necrosis induced by galactosamine in the liver of Wistar rats treated with fructose-1,6-bisphosphate.

Galactosamine (GalN) is a hepatotoxic agent, which under determined situations provokes metabolic and energetic depletion as well as alterations in permeability, leading to cellular death. At the same time, it is known that fructose-1,6-bisphosphate (FBP) helps maintain cell energy levels and protects the cell against this lesive agent. We submitted two groups of male Wistar rats to the harmful intraperitoneal doses of GalN (400 mg/kg), one of which simultaneously received FBP (2 g/kg). Techniques were used in the analysis of the cellular components, adenosine triphosphate (ATP) and hepatic calcium and a close relationship between the types of cellular death unchained by these agents was established. The liver of the rats treated with GalN showed energy depletion and concomitant increase calcium in the hepatic tissue, which provoked higher levels of necrosis leading to reduce cellular viability. On the other hand, the group which had received GalN+FBP maintained calcium levels close to the control values and the energy rate did not decrease as much as in the GalN only group, but recovered the control values, within a period of 48 h. At the same time, the degree of apoptosis was greater than in the GalN group. This fact suggests that the FBP maintains cellular levels of ATP, thus protecting the cell from the toxic action of GalN, reducing the percentage of dead cells and causing an alteration in the pattern of the cell death, whereby there is an increase in the rate of apoptosis and a decrease in that of necrosis.

Intravenous toxicity of fructose-1,6-bisphosphate in rats.

Fructose-1,6-bisphosphate (FBP) is a bisphosphorilated sugar with a protective action against events that lead to cellular damage. The toxicity of the drug was assessed when administered intravenously in Wistar rats in doses of between 250 and 4000 mg/kg. Ionic calcium, total calcium, inorganic serum phosphate and the electrocardiographic profile of these animals were assessed. The lethal dose (LD(50)) was established by means of PROBIT processing. There was no reduction in the levels of total calcium, with the administration of increased doses of FBP, although there was a significant reduction in the levels of ionic calcium in those groups that received 250 mg/kg and over. The serum phosphate showed a significant statistical increase in those groups that received 750 mg/kg and over. The LD(50) obtained in 24 h was 1068 mg/kg. Though it was not possible to elucidate the toxic mechanism of FBP, the electrocardiogram (ECG) showed that all the rats died of cardiac arrest.

Anti-inflammatory effects of fructose-1,6-bisphosphate on carrageenan-induced pleurisy in rat.

In the present study, we evaluated the effect of fructose-1,6-bisphosphate (FBP), a high energy intermediate metabolite of glycolysis, in an acute model of lung injury. Injection of carrageenan into the pleural cavity of rats elicited an acute inflammation response characterized by a fluid accumulation in the pleural cavity which contained a large number of polymorphonuclear neutrophils. FBP (500mg/kg) attenuated the inflammation parameters: exudate volume, total leukocytes and the number of polymorphonuclear leukocytes, but the protein concentration in the exudate was not significantly affected by treatment with FBP. The precise site and mechanism of the anti-inflammatory effect was not addressed, considering the diverse pharmacological actions of FBP. This drug has anti-inflammatory actions suggesting that it may represent a novel strategy for the modulation of inflammatory response.

Physiopathological studies in septic rats and the use of fructose 1,6-bisphosphate as cellular protection.

OBJECTIVE: The aim of this research project was to test the ability of fructose 1,6-bisphosphate (FBP), which has anti-inflammatory effects and maintains cellular energy levels, to inhibit the septic process in an experimental model in rats. DESIGN: Prospective, controlled animal trial. SETTING: Research laboratory. SUBJECTS: Fed male Wistar rats. INTERVENTIONS: Three experimental groups were formed for the test: control group, untreated septic group, and septic group treated with FBP (500 mg/kg). MEASUREMENTS AND MAIN RESULTS: In the control group, there were no deaths; in the untreated septic group, the mortality rate was 100% within 15 hrs; in the septic group treated with FBP, the mortality rate reached 20% within 15 hrs. The blood cell tests revealed that concentrations of hematocrit, leukocytes, monocytes, and immature cells increased significantly in the untreated septic group compared with both the FBP-treated septic group and the control group. The histologic lesions verified in the heart, lungs, liver, and kidneys of septic animals were smaller and even absent in those treated with FBP. CONCLUSION: FBP reduced the mortality rate provoked by experimental sepsis and ameliorated hematologic and histologic alterations.