[Show abstract][Hide abstract] ABSTRACT: Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and the activation of different types of cells. This altered state of cell activation, termed leukocyte reprogramming, contributes to patient outcome. However, the understanding of the process underlying sepsis and the role of regulatory T cells (Tregs) in sepsis remains to be elucidated. In this study, we investigated the role of CCR4, the CCL17/CCL22 chemokine receptor, in the innate and acquired immune responses during severe sepsis and the role of Tregs in effecting the outcome. In contrast with wild-type (WT) mice subjected to cecal ligation and puncture (CLP) sepsis, CCR4-deficient (CCR4-/-) septic mice presented an increased survival rate, significant neutrophil migration toward the infection site, a low bacterial count in the peritoneum, and reduced lung inflammation and serum cytokine levels. Thus, a better early host response may favor an adequate long-term response. Consequently, the CCR4-/- septic mice were not susceptible to secondary fungal infection, in contrast with the WT septic mice. Furthermore, Tregs cells from the CCR4-/- septic mice showed reduced suppressive effects on neutrophil migration (both in vivo and in vitro), lymphocyte proliferation and ROS production from activated neutrophils, in contrast with what was observed for Tregs from the WT septic mice. These data show that CCR4 is involved in immunosuppression after severe sepsis and suggest that CCR4+ Tregs negatively modulate the short and long-term immune responses.
PLoS ONE 07/2015; 10(7):e0133227. DOI:10.1371/journal.pone.0133227 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is involved in eosinophil biology and in type 2 inflammation, contributing to allergic and helminthic diseases. We hypothesized that MIF participates in the pathogenesis of eosinophilic esophagitis (EoE), an allergic condition characterized by esophageal eosinophilic inflammation. MIF is highly expressed in esophageal mucosa of patients with EoE, compared with gastro-esophageal reflux disease and control patients, where it co-localizes predominantly with eosinophils. In vitro, recombinant MIF promotes human eosinophil chemotaxis, while MIF antagonist and CXCR4 antagonist, AMD3100, revert this effect. In a model of EoE induced by ovalbumin, Mif-deficient mice have reduced inflammation and collagen deposition compared with wild-type (WT) mice. Importantly, treatment of WT mice with anti-MIF or with AMD3100 during the challenge phase prevents accumulation of eosinophils and tissue remodeling. Conversely, recombinant MIF promoted tissue eosinophil inflammation in allergic mice. Together, these results implicate MIF in the pathogenesis of esophageal inflammation and suggest that targeting MIF might represent a novel therapy for EoE.Mucosal Immunology advance online publication, 25 February 2015; doi:10.1038/mi.2015.6.
[Show abstract][Hide abstract] ABSTRACT: The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain con-taining 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K + efflux, whereas it was independent of heme in-ternalization, lysosomal damage, ATP release, the purinergic re-ceptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome compo-nents, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic tar-gets for diseases that have heme release. inflammation | mitochondria | ROS | NOX2 | Syk
Proceedings of the National Academy of Sciences 09/2014; 11(39). DOI:10.1073/pnas.1405023111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heme is an essential molecule expressed ubiquitously all through our tissues. Heme plays major functions in cellular physiology and metabolism as the prosthetic group of diverse proteins. Once released from cells and from hemeproteins free heme causes oxidative damage and inflammation, thus acting as a prototypic damage-associated molecular pattern. In this context, free heme is a critical component of the pathological process of sterile and infectious hemolytic conditions including malaria, hemolytic anemias, ischemia-reperfusion, and hemorrhage. The plasma scavenger proteins hemopexin and albumin reduce heme toxicity and are responsible for transporting free heme to intracellular compartments where it is catabolized by heme-oxygenase enzymes. Upon hemolysis or severe cellular damage the serum capacity to scavenge heme may saturate and increase free heme to sufficient amounts to cause tissue damage in various organs. The mechanism by which heme causes reactive oxygen generation, activation of cells of the innate immune system and cell death are not fully understood. Although heme can directly promote lipid peroxidation by its iron atom, heme can also induce reactive oxygen species generation and production of inflammatory mediators through the activation of selective signaling pathways. Heme activates innate immune cells such as macrophages and neutrophils through activation of innate immune receptors. The importance of these events has been demonstrated in infectious and non-infectious diseases models. In this review, we will discuss the mechanisms behind heme-induced cytotoxicity and inflammation and the consequences of these events on different tissues and diseases.
Frontiers in Pharmacology 05/2014; 5(115):1-20. DOI:10.3389/fphar.2014.00115 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) are deadly weapons used by phagocytes and other cell types, such as lung epithelial cells, against pathogens. ROS can kill pathogens directly by causing oxidative damage to biocompounds or indirectly by stimulating pathogen elimination by various nonoxidative mechanisms, including pattern recognition receptors signaling, autophagy, neutrophil extracellular trap formation, and T-lymphocyte responses. Thus, one should expect that the inhibition of ROS production promote infection. Increasing evidences support that in certain particular infections, antioxidants decrease and prooxidants increase pathogen burden. In this study, we review the classic infections that are controlled by ROS and the cases in which ROS appear as promoters of infection, challenging the paradigm. We discuss the possible mechanisms by which ROS could promote particular infections. These mechanisms are still not completely clear but include the metabolic effects of ROS on pathogen physiology, ROS-induced damage to the immune system, and ROS-induced activation of immune defense mechanisms that are subsequently hijacked by particular pathogens to act against more effective microbicidal mechanisms of the immune system. The effective use of antioxidants as therapeutic agents against certain infections is a realistic possibility that is beginning to be applied against viruses. Antioxid. Redox Signal. 20, 1000-1037.
[Show abstract][Hide abstract] ABSTRACT: Aims:
Glucuronoxylomannan (GXM) is the major polysaccharide component of Cryptococcus neoformans. We evaluated in this study whether GXM fractions of different molecular masses were functionally distinct.
Materials & methods:
GXM samples isolated from C. neoformans cultures were fractionated to generate polysaccharide preparations differing in molecular mass. These fractions were used in experiments focused on the association of GXM with cell wall components of C. neoformans, as well as on the interaction of the polysaccharide with host cells.
Results & conclusion:
GXM fractions of variable molecular masses bound to the surface of a C. neoformans acapsular mutant in a punctate pattern that is in contrast to the usual annular pattern of surface coating observed when GXM samples containing the full molecular mass range were used. The polysaccharide samples were also significantly different in their ability to stimulate cytokine production by host cells. Our findings indicate that GXM fractions are functionally distinct depending on their mass.
[Show abstract][Hide abstract] ABSTRACT: Today there are approximately 8 million cases of Chagas disease only in the Southern Cone and about 100 million people living with the risk of becoming infected. The present pharmacotherapy is sometimes ineffective and has serious side effects. Here we report a series of 4,5-dihydroisoxazoles incorporating hydroxamate moieties, which act as effective inhibitors of the carbonic anhydrase (CA) from Trypanosoma cruzi (TcCA). One compound (5g) was evaluated in detail and shows promising features as an antitrypanosomal agent. Excellent values for inhibition of growth for all three developmental forms of the parasite were observed at low concentrations of 5g (IC50 values of 7.0 µM to < 1 µM). The compound has a selectivity index (SI) of 6.7, and no cytotoxicity to macrophage cells. Preliminary in vivo data showed that 5g reduces bloodstream parasites and all treated mice survived; also it was more effective than the standard drug benznidazole.
[Show abstract][Hide abstract] ABSTRACT: Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate IL-1β synthesis, processing and secretion in platelets during DV infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1β in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to dengue virus in vitro. We demonstrated that dengue virus infection leads to assembly of NLRP3 inflammasomes, activation of caspase-1 and caspase-1-dependent IL-1β secretion. Our findings also indicate that platelet-derived IL-1β is chiefly released in microparticles through mechanisms dependent on mitochondrial ROS-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1β-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from dengue virus stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in dengue virus infection by inflammasome-dependent release of IL-1β.
[Show abstract][Hide abstract] ABSTRACT: Significance: Reactive oxygen species (ROS) are deadly weapons, used by phagocytes and other cell types, such as lung epithelial cell, against pathogens. ROS can kill pathogens directly by causing oxidative damage to biocompounds or indirectly, by stimulating pathogen elimination by various non-oxidative mechanisms, including pattern recognition receptors (PRR) signaling, autophagy, NET formation, and T lymphocyte responses. Thus, one should expect that inhibition of ROS production promote infection. Recent advances: Increasing evidences support that in certain particular infections, antioxidants decrease and prooxidants increase pathogen burden. Critical issues: Here, we review the classic infections that are controlled by ROS and the cases in which ROS appear as promoters of infection, challenging the paradigm. We discuss the possible mechanisms by which ROS could promote particular infections. These mechanisms are still not completely clear but include metabolic effects of ROS on pathogen physiology, ROS-induced damage to the immune system, and ROS-induced activation of immune defense mechanisms that are subsequently hijacked by particular pathogens to act against more effective microbicidal mechanisms of the immune system. Future directions: The effective use of antioxidants as therapeutic agents against certain infections is a realistic possibility that is beginning to be applied against viruses.
[Show abstract][Hide abstract] ABSTRACT: Parasites of the Leishmania genus are the causative agents of leishmaniasis in humans, a disease that affects more than 12 million people worldwide. These parasites replicate intracellularly in macrophages, and the primary mechanisms underlying host resistance involve the production of nitric oxide (NO). In this study we show that the Nlrp3 inflammasome is activated in response to Leishmania infection and is important for the restriction of parasite replication both in macrophages and in vivo as demonstrated through the infection of inflammasome-deficient mice with Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum chagasi. Inflammasome-driven interleukin-1β (IL-1β) production facilitated host resistance to infection, as signaling through IL-1 receptor (IL-1R) and MyD88 was necessary and sufficient to trigger inducible nitric oxide synthase (NOS2)-mediated production of NO. In this manuscript we identify a major signaling platform for host resistance to Leishmania spp. infection and describe the molecular mechanisms underlying Leishmania-induced NO production.
Nature medicine 06/2013; 19(7). DOI:10.1038/nm.3221 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.
[Show abstract][Hide abstract] ABSTRACT: Background
Staphylococcus aureus is unrestrictedly found in humans and in animal species that maintain thermal homeostasis. Inadequate cleaning of processing equipment or inappropriate handling can contaminate processed food and cause severe food poisoning. Staphylococcal enterotoxin B (SEB), a potent superantigenic exotoxin, is produced by 50% of clinical isolates of S. aureus and is associated with massive food poisoning and with the induction of toxic shock syndrome.
A gene sequence encoding a recombinant SEB (rSEB), devoid of superantigenic activity, was successfully cloned and expressed in a cytoplasmic or a secreted form in the food-grade lactic acid bacterium Lactococcus lactis. The recombinant protein detected in the cytoplasm or in the culture medium exhibited the expected molecular mass and was recognized by a SEB-polyclonal antibody. Oral immunization with the recombinant L. lactis strains induced a protective immune response in a murine model of S. aureus infection. Immunized mice survived intraperitoneal challenge with an S. aureus SEB-producer strain. Counts of S. aureus in the spleen of rSEB-immunized mice were significantly reduced. The rSEB-immunized mice showed significant titers of anti-SEB IgA and IgG in stools and serum, respectively. Both recombinant L. lactis strains were able to elicit cellular or systemic immune responses in mice, with no significant difference if rSEB was produced in its cytoplasmic or secreted form. However, recombinant L. lactis expressing the cytoplasmic rSEB increased the survival rate of the challenged mice by 43%.
These findings show the vaccine efficacy of L. lactis carrying an attenuated SEB, in a murine model, following lethal S. aureus challenge.
[Show abstract][Hide abstract] ABSTRACT: Wallerian degeneration (WD) comprises a series of events that includes activation of non-neuronal cells and recruitment of immune cells, creating an inflammatory milieu that leads to extensive nerve fragmentation and subsequent clearance of the myelin debris, both of which are necessary prerequisites for effective nerve regeneration. Previously, we documented accelerated axon regeneration in animals lacking galectin-3 (Gal-3), a molecule associated with myelin clearance. To clarify the mechanisms underlying this enhanced regeneration, we focus here on the early steps of WD following sciatic nerve crush in Gal-3(-/-) mice. Using an in vivo model of nerve degeneration, we observed that removal of myelin debris is more efficient in Gal-3(-/-) than in wild-type (WT) mice; we next used an in vitro phagocytosis assay to document that the phagocytic potential of macrophages and Schwann cells was enhanced in the Gal-3(-/-) mice. Moreover, both RNA and protein levels for the pro-inflammatory cytokines IL-1β and TNF-α, as well as for Toll-like receptor (TLR)-2 and -4, show robust increases in injured nerves from Gal-3(-/-) mice compared to those from WT mice. Collectively, these data indicate that the lack of Gal-3 results in an augmented inflammatory profile that involves the TLR-cytokine pathway, and increases the phagocytic capacity of Schwann cells and macrophages, which ultimately contributes to speeding the course of WD.
European Journal of Neuroscience 02/2013; 37(10). DOI:10.1111/ejn.12161 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) are deadly weapons used by phagocytes and other cell types, such as lung epithelial cells, against pathogens. ROS can kill pathogens directly by causing oxidative damage to biocompounds or indirectly by stimulating pathogen elimination by various nonoxidative mechanisms, including pattern recognition receptors signaling, autophagy, neutrophil extracellular trap formation, and T-lymphocyte responses. Thus, one should expect that the inhibition of ROS production promote infection. Increasing evidences support that in certain particular infections, antioxidants decrease and prooxidants increase pathogen burden. In this study, we review the classic infections that are controlled by ROS and the cases in which ROS appear as promoters of infection, chal-lenging the paradigm. We discuss the possible mechanisms by which ROS could promote particular infections. These mechanisms are still not completely clear but include the metabolic effects of ROS on pathogen physiology, ROS-induced damage to the immune system, and ROS-induced activation of immune defense mechanisms that are subsequently hijacked by particular pathogens to act against more effective microbicidal mechanisms of the immune system. The effective use of antioxidants as therapeutic agents against certain infections is a realistic possibility that is beginning to be applied against viruses. Antioxid. Redox Signal. 00, 000–000. I. Introduction II. Antioxidants and Their Mode of Action III. ROS Can Promote Pathogen Elimination by Direct Oxidative Damage or by a Variety of Innate and Adaptive Mechanisms A. Direct oxidative damage to microbes B. O 2 -c promotes enzymatic elimination of microorganisms indirectly C. ROS promote autophagy D. ROS inhibit mTOR kinase, triggering an antiviral response E. ROS promote NETosis F. ROS promote cell death of infected reservoirs G. PRRs use ROS as signaling intermediaries in inflammation H. ROS are chemoattractors to phagocytes I. ROS can activate NRF2-target genes, a part of the antioxidant defense response that interferes with innate immunity J. ROS interfere with iron storage and tissue mobilization, influencing iron availability to pathogens K. ROS interfere with lipid metabolism and foam cell formation L. ROS influence phagosomal proteolysis through cathepsin inactivation M. ROS interfere with protein immunogenicity, antigenic presentation, Th polarization, and co-stimulation by dendritic cells IV. Pathogens That ROS Contribute to Eliminating A. Bacterial infections combated by ROS B. Viral infections combated by ROS
[Show abstract][Hide abstract] ABSTRACT: Bacterial clearance is one of the most important beneficial consequences of the innate immune response. Chemokines are important mediators controlling leukocyte trafficking and activation, whereas reactive oxygen and nitrogen species are effectors in bacterial killing. In the present work, we used in vivo and in vitro models of infections to study the role of monocyte chemoattractant protein 1 (MCP-1)/CCL2 and nitric oxide (NO) in the bacterial clearance in sepsis. Our results show that MCP-1/CCL2 and NO levels are increased in the peritoneal cavity of mice 6 h after sepsis induced by cecal ligation and puncture. Pretreatment with anti-MCP-1/CCL2 monoclonal antibodies increased the number of colony-forming units (CFUs) recovered in the peritoneal lavage fluid. Moreover, CFU counts were increased in the peritoneal fluid of CCR2 mice subjected to cecal ligation and puncture. In vitro stimulation of peritoneal macrophages with recombinant MCP-1/CCL2 reduced CFU counts in the supernatant after challenge with Escherichia coli. Conversely, treatment with anti-MCP-1/CCL2 increased CFU counts under the same experimental condition. Stimulation of cultured macrophages with MCP-1/CCL2 and interferon had a synergistic effect on NO production. Macrophages from CCL2 mice showed a consistent decrease in NO production when compared with wild-type controls after stimulation with LPS + interferon. Finally, we showed incubation of macrophages with E. coli, and the ERK inhibitor U0126 increased CFU numbers and decreased intracellular levels of NO. In conclusion, we demonstrated for the first time that MCP-1/CCL2 has a crucial role in the clearance of bacteria by mechanisms involving increased expression of inducible NO synthase and production of NO by ERK signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND PURPOSE: The mechanism of immunosuppression induced by severe sepsis is not fully understood. The production of prostaglandin E2 (PGE2) during sepsis is wellknown, but its role in long-term consequences of sepsis has not been explored. The current study evaluates the role of PGE2 in the development of immunosuppression secondary to sepsis, and its potential as therapeutic target. EXPERIMENTAL APPROACH: Cecal ligation and puncture (CLP) was used as an experimental model for sepsis induction in Balb/c and C57BL/6 mice. Immunosuppression was evaluated by the response to secondary infection with Aspergillus fumigatus in sepsis survivors. The role of prostanoids was evaluated in vivo and in vitro by treatment with the cyclooxygenase (COX)-inhibitor ketoprofen. KEY RESULTS: Balb/c mice were more susceptible than C57BL/6 to severe sepsis and to secondary infection, with a greater mortality rate. PGE2 concentrations found in bronchoalveolar lavage (BAL) in Sham and CLP group after fungal challenge were much higher in Balb/c than in C57BL/6 mice. Ketoprofen treatment improved survival of septic Balb/c mice subjected to secondary infection, while also enhancing macrophage phagocytosis and neutrophil recruitment to the lungs. We identified a pivotal role for PGE2 acting on EP4 receptors in modulating cytokine production differentially by sham and septic macrophages. Furthermore, sepsis also altered key enzymes in PGE2 synthesis and degradation. CONCLUSIONS: Our results indicate the involvement of PGE2 in severe sepsis-induced immunosuppression. Inhibition of PGE2 production represents an attractive target to improve innate immune response against secondary infection in the immunocompromised host.