Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection.
ABSTRACT: The causative agent of Chagas' disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR)-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO) were not involved, we observed a significant increase in IL-6, TNF-?, and IL-1? production. Taking into account that IL-1? is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of mitochondrial ROS (mtROS) compared with control cells. Moreover, inhibition of mtROS production partially reversed the effect of rapamycin on parasite replication, with there being a significant increase in parasite load in rapamycin pretreated and infected macrophages from NLRP3 KO mice compared to wild-type control cells. Our findings strongly suggest that mTOR inhibition during T. cruzi infection induces NLRP3 inflammasome activation and mtROS production, resulting in an inflammatory-like macrophage profile that controls T. cruzi replication.
Project description:Background: The NLRP3 inflammasome is one of the key contributors to impaired wound healing in diabetes. In this study, we assessed the role of rapamycin on high glucose-induced inflammation in THP-1-derived macrophages and investigated the underlying signaling mechanisms. Methods: THP-1-derived macrophages were treated with high glucose to induce NLRP3 inflammasome activation. The cells were pretreated with rapamycin, BAY 11-7082, or PDTC before exposure to HG. mTOR, NF-?B, and NLRP3 inflammasome expression were measured by western blotting. Results: We found that rapamycin reduced NLRP3 inflammasome activation in macrophages. Rapamycin reduced NLRP3 inflammasome activation by inhibiting mTOR phosphorylation and NF-?B activation. Moreover, mTOR siRNA inhibited NF-?B activation, leading to the suppression of NLRP3 inflammasome activation. Conclusion: Rapamycin can ameliorate high glucose-induced NLRP3 inflammasome activation by attenuating the mTOR/NF-?B signaling pathway in macrophages. Rapamycin may act as a possible therapeutic option for high glucose-induced inflammatory response in impaired wound healing in the future.
Project description:In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1? production in m?s in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1?, or NADPH oxidase, we found that IL-1? production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1? regulated NF-?B signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1? activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1?-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1?/ROS and NF-?B signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1?/NF?B activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.
Project description:Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1? production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1? secretion. NLRP3(-/-) and caspase1(-/-) mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/-) and iNOS(-/-) mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-? were found in spleen cells from NLRP3(-/-) and caspase1(-/-) mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1? and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88(-/-) macrophages and rendered them as susceptible to T. cruzi infection as NLRP3(-/-) and caspase-1(-/-) macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.
Project description:Excessive and prolonged activation of macrophages underlies many inflammatory and autoimmune diseases. To regulate activation and maintain homeostasis, macrophages have multiple intrinsic mechanisms, one of which is modulation through autophagy. Here we demonstrate that autophagy induction by rapamycin suppressed the production of IL-1? and IL-18 in lipopolysaccharide- and adenosine triphosphate-activated macrophages at the post-transcriptional level by eliminating mitochondrial ROS (mtROS) and pro-IL1? in a p62/SQSTM1-dependent manner. In addition, rapamycin activated Nrf2 through up-regulation of p62/SQSTM1, which further contributed to the reduction of mtROS. Reduced IL-1? subsequently diminished the activation of p38 MAPK-NF?B pathways, leading to transcriptional down-regulation of IL-6, IL-8, MCP-1, and I?B? in rapamycin-treated macrophages. Therefore, our results suggest that rapamycin negatively regulates macrophage activation by restricting a feedback loop of NLRP3 inflammasome-p38 MAPK-NF?B pathways in autophagy- and p62/SQSTM1-dependent manners.
Project description:Neutrophils are involved in the initial steps of most responses to pathogens and are essential components of the innate immune response. Due to the ability to produce and release various soluble mediators, neutrophils may participate in the regulation of the inflammatory response. Little is known about the role of neutrophils during protozoan infections including infection by Trypanosoma cruzi. In the present study we investigated the importance of inflammatory neutrophils on macrophage activation and T. cruzi replication in vitro, in cells obtained from BALB/c mice and C57Bl/6 mice. Co-cultures of BALB/c apoptotic or live neutrophils with infected peritoneal macrophages resulted in increased replication of the parasites and in the production of TGF-? and PGE2. The treatment with anti-TGF-? neutralizing antibody and COX inhibitor blocked the parasite replication in vitro. On the other hand, co-cultures of T. cruzi infected macrophages with live neutrophils isolated from C57BL/6 mice resulted in decreased number of trypomastigotes in culture and increased production of TNF-? and NO. The addition of anti-TNF-? neutralizing antibody or elastase inhibitor resulted in the abolishment of macrophage microbicidal effect and increased parasite replication. Addition of elastase to infected macrophages reduced the replication of the parasites, and on the other hand, addition of a selective inhibitor of iNOS increased parasite growth, suggesting the role of NO in this system. Our findings reveal that neutrophils may regulate T. cruzi experimental infection and determine susceptibility and resistance to infection.
Project description:Mycobacterium abscessus (MAB) is a rapidly growing mycobacterium (RGM), and infections with this pathogen have been increasing worldwide. Recently, we reported that rough type (MAB-R) but not smooth type (MAB-S) strains enhanced type 1 interferon (IFN-I) secretion via bacterial phagosome escape, contributing to increased virulence. Here, we sought to investigate the role of mitochondrial oxidative stress in bacterial survival, IFN-I secretion and NLRP3 inflammasome activation in MAB-infected murine macrophages. We found that live but not heat-killed (HK) MAB-R strains increased mitochondrial ROS (mtROS) and increased release of oxidized mitochondrial DNA (mtDNA) into the cytosol of murine macrophages compared to the effects of live MAB-S strains, resulting in enhanced NLRP3 inflammasome-mediated IL-1? and cGAS-STING-dependent IFN-I production. Treatment of the infected macrophages with mtROS-modulating agents such as mito-TEMPO or cyclosporin A reduced cytosolic oxidized mtDNA, which inhibited the MAB-R strain-induced production of IL-1? and IFN-I. The reduced cytosolic oxidized mtDNA also inhibited intracellular growth of MAB-R strains via cytosolic escape following phagosomal rupture and via IFN-I-mediated cell-to-cell spreading. Moreover, our data showed that mtROS-dependent IFN-I production inhibited IL-1? production, further contributing to MAB-R intracellular survival in murine macrophages. In conclusion, our data indicated that MAB-R strains enhanced IFN-I and IL-1? production by inducing mtROS as a pathogen-associated molecular pattern (PAMP). These events also enhance bacterial survival in macrophages and dampen inflammation, which contribute to the pathogenesis of MAB-R strains.
Project description:Neospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes.Neospora caninum-infected wild-type (WT) macrophages and Nlrp3 -/- macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed.Many NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage.The NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum.
Project description:During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-? in plasma and decreased production of TNF-? and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-? and IL-6 24?h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs.
Project description:During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), ?-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/?-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/?-catenin pathway and then activating the Wnt/Ca+2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of ?-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.
Project description:The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease.