Immunomodulatory Role of Capsular Polysaccharides Constituents of Cryptococcus neoformans.

Cryptococcosis is a systemic fungal infection caused by Cryptococcus neoformans. In immunocompetent patients, cryptococcal infection is often confined to the lungs. In immunocompromised individuals, C. neoformans may cause life-threatening illness, either from novel exposure or through reactivation of a previously acquired latent infection. For example, cryptococcal meningitis is a severe clinical disease that can manifest in people that are immunocompromised due to AIDS. The major constituents of the Cryptococcus polysaccharide capsule, glucuronoxylomannan (GXM), and galactoxylomannan (GalXM), also known as glucuronoxylomanogalactan (GXMGal), are considered the primary virulence factors of Cryptococcus. Despite the predominance of GXM in the polysaccharide capsule, GalXM has more robust immunomodulatory effects on host cellular immunity. This review summarizes current knowledge regarding host-Crytococcus neoformans interactions and the role of capsular polysaccharides in host immunomodulation. Future studies will likely facilitate a better understanding of the mechanisms involved in antigenic recognition and host immune response to C. neoformans and lead to the development of new therapeutic pathways for cryptococcal infection.

Antibody Repertoires Identify ß-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi.

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and ß-tubulin. The major protein band recognized by host IgG was T. cruzi ß-tubulin. The T. cruzi ß-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi ß-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi ß-tubulin. A single immunization of mice with recombinant T. cruzi ß-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.

Role of Inactive and Active Trypanosoma cruzi Trans-sialidases on T Cell Homing and Secretion of Inflammatory Cytokines.

Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection.

Dependency of B-1 Cells in the Maintenance of Splenic Interleukin-10 Producing Cells and Impairment of Macrophage Resistance in Visceral Leishmaniasis.

Visceral leishmaniasis is a neglected disease caused by Leishmania protozoa parasites transmitted by infected sand fly vectors. This disease represents the second in mortality among tropical infections and is associated to a profound immunosuppression state of the host. The hallmark of this infection-induced host immunodeviation is the characteristic high levels of the regulatory interleukin-10 (IL-10) cytokine. In the present study, we investigated the role of B-1 cells in the maintenance of splenic IL-10 levels that could interfere with resistance to parasite infection. Using an experimental murine infection model with Leishmania (L.) infantum chagasi we demonstrated an improved resistance of B-1 deficient BALB/XID mice to infection. BALB/XID mice developed a reduced splenomegaly with diminished splenic parasite burden and lower levels of IL-10 secretion of purified splenocytes at 30 days post-infection, as compared to BALB/c wild-type control mice. Interestingly, we found that resident peritoneal macrophages isolated from BALB/XID mice were more effective to control the parasite load in comparison to cells isolated from BALB/c wild-type mice. Our findings point to a role of B-1 cells in the host susceptibility to visceral leishmaniasis.

B-1 cells modulate the murine macrophage response to Leishmania major infection.

AIM: To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major (L. major) in vitro. METHODS: Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE2) were determined using a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major. RESULTS: We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION: Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.

Implication of Apoptosis for the Pathogenesis of Trypanosoma cruzi Infection.

Apoptosis is induced during the course of immune response to different infectious agents, and the ultimate fate is the recognition and uptake of apoptotic bodies by neighboring cells or by professional phagocytes. Apoptotic cells expose specific ligands to a set of conserved receptors expressed on macrophage cellular surface, which are the main cells involved in the clearance of the dying cells. These scavenger receptors, besides triggering the production of anti-inflammatory factors, also block the production of inflammatory mediators by phagocytes. Experimental infection of mice with the parasite Trypanosoma cruzi shows many pathological changes that parallels the evolution of human infection. Leukocytes undergoing intense apoptotic death are observed during the immune response to T. cruzi in the mouse model of the disease. T. cruzi replicate intensely and secrete molecules with immunomodulatory activities that interfere with T cell-mediated immune responses and secretion of pro-inflammatory cytokine secretion. This mechanism of immune evasion allows the infection to be established in the vertebrate host. Under inflammatory conditions, efferocytosis of apoptotic bodies generates an immune-regulatory phenotype in phagocytes, which is conducive to intracellular pathogen replication. However, the relevance of cellular apoptosis in the pathology of Chagas' disease requires further studies. Here, we review the evidence of leukocyte apoptosis in T. cruzi infection and its immunomodulatory mechanism for disease progression.

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen.

Neutrophils increase or reduce parasite burden in Trypanosoma cruzi-infected macrophages, depending on host strain: role of neutrophil elastase.

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-ß and PGE2. The treatment with anti-TGF-ß 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-α and NO. The addition of anti-TNF-α 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.

Infection with Leishmania major induces a cellular stress response in macrophages.

We investigated early cellular responses induced by infection with Leishmania major in macrophages from resistant C57/BL6 mice. Infection increased production of reactive oxygen species by resident, but not inflammatory peritoneal macrophages. In addition, infection increased activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) in resident, but not in inflammatory peritoneal macrophages. Infection also increased expression of membrane and soluble FasL, but infected macrophages remained viable after 48 h. Infection increased secretion of cytokines/chemokines TNF-α, IL-6, TIMP-1, IL-1RA, G-CSF, TREM, KC, MIP-1α, MIP-1ß, MCP-1, and MIP-2 in resident macrophages. Addition of antioxidants deferoxamine and N-acetylcysteine reduced ROS generation and JNK activation. Addition of antioxidants or JNK inhibitor SP600125 reduced secretion of KC. Furthermore, treatment with antioxidants or JNK inhibitor also reduced intracellular parasite replication. These results indicated that infection triggers a rapid cellular stress response in resident macrophages which induces proinflammatory signals, but is also involved in parasite survival and replication in host macrophages.

Proinflammatory clearance of apoptotic neutrophils induces an IL-12(low)IL-10(high) regulatory phenotype in macrophages.

Clearance of apoptotic exudate neutrophils (efferocytosis) induces either pro- or anti-inflammatory responses in mouse macrophages depending on host genetic background. In this study, we investigated whether neutrophil efferocytosis induces a stable macrophage phenotype that could be recalled by late restimulation with LPS. Bone marrow-derived macrophages previously stimulated by pro- but not anti-inflammatory neutrophil efferocytosis expressed a regulatory/M2b phenotype characterized by low IL-12 and high IL-10 production following restimulation, increased expression of LIGHT/TNF superfamily 14, Th2-biased T cell responses, and permissive replication of Leishmania major. Induction of regulatory/M2b macrophages required neutrophil elastase activity and was partially dependent on TLR4 signaling. These results suggested that macrophage differentiation to a regulatory phenotype plays a role in resolution of inflammation but could contribute to increased humoral Ab responses and parasite persistence in the infected host.

Impaired innate immunity in Tlr4(-/-) mice but preserved CD8+ T cell responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-deficient mice.

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-gamma secreting CD8+ T cells specific for H-2K(b)-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2(-/-), Tlr4(-/-), Tlr9(-/) (-) or Myd88(-/-) mice generated both specific cytotoxic responses and IFN-gamma secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-gamma+CD4+ cells was diminished in infected Myd88(-/-) mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-gamma, TNF-alpha and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4(-/-) mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.

Apoptotic lymphocytes treated with IgG from Trypanosoma cruzi infection increase TNF-alpha secretion and reduce parasite replication in macrophages.

Phagocytic removal of apoptotic lymphocytes exacerbates replication of Trypanosoma cruzi in macrophages. We investigated the presence of Ab against apoptotic lymphocytes in T. cruzi infection and the role of these Ab in parasite replication. Both control and chagasic serum contained IgG Ab that opsonized apoptotic lymphocytes. Treatment of apoptotic lymphocytes with purified IgG from chagasic, but not control serum, reduced T. cruzi replication in macrophages. The protective effect of chagasic IgG depended on Fcgamma receptors, as demonstrated by the requirement for the intact Fc portion of IgG, and the effect could be abrogated by treating macrophages with an anti-CD16/CD32 Fab fragment. Chagasic IgG displayed increased reactivity against a subset of apoptotic cell Ag, as measured by flow cytometry and immunoblot analyses. Apoptotic lymphocytes treated with chagasic IgG, but not control IgG, increased production of TNF-alpha, while decreasing production of TGF-beta1 by infected macrophages. Increased control of parasite replication required TNF-alpha production. Previous immunization with apoptotic cells or injection of apoptotic cells opsonized with chagasic IgG reduced parasitemia in infected mice. These results indicate that Ab raised against apoptotic cells could play a protective role in control of T. cruzi replication by macrophages.

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages.

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-gamma or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE(2) produced. Exogenous PGE(2) increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.

Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand.

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF-alpha, NO and iNOS expression, while GXM predominantly induced TGF-beta secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.

Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase.

We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.

Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection.

In experimental Chagas' disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan-caspase blocker peptide inhibited expression of the active form of effector caspase-3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi-infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN-gamma by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF-alpha and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan-caspase blocker peptide improves protective type-1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas' disease.

Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors.

We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100microM) and also to inhibit T-cell proliferation (66% at 10microM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.

Turnover of neutrophils mediated by Fas ligand drives Leishmania major infection.

Apoptosis mediated by Fas ligand (FasL) initiates inflammation characterized by neutrophilic infiltration. Neutrophils undergo apoptosis and are ingested by macrophages. Clearance of dead neutrophils leads to prostaglandin- and transforming growth factor-beta-dependent replication of Leishmania major in macrophages from susceptible mice. How L. major induces neutrophil turnover in a physiological setting is unknown. We show that BALB/c FasL-sufficient mice are more susceptible to L. major infection than are FasL-deficient mice. FasL promotes the apoptosis of infected resident macrophages and attracts neutrophils. Furthermore, FasL-sufficient neutrophils exacerbate L. major replication in macrophages, whereas FasL-deficient neutrophils induce parasite killing. These contrasting effects are due to delaying apoptosis and the clearance of FasL-deficient neutrophils. The transfer of neutrophils exacerbates infection in FasL-sufficient mice but reduces infection in FasL-deficient mice. Depletion of neutrophils abolishes the susceptibility of FasL-sufficient mice. These data illustrate a deleterious role of the FasL-mediated turnover of neutrophils on L. major infection.

The importance of aberrant T-cell responses in Chagas disease.

Persistence of Trypanosoma cruzi is associated with damage to the heart, which is a characteristic of Chagas disease. In this article, we discuss recently identified mechanisms of aberrant T-cell activation that are responsible for persistence of T. cruzi and cardiac injury. Among them, apoptosis of host cells drives T. cruzi replication in macrophages and is present in cardiac inflammation. It is proposed that phagocytic removal of infected apoptotic cardiomyocytes, combined with signaling through innate immune receptors, is required to initiate immune responses that damage the heart.

CD40 signaling induces reciprocal outcomes in Leishmania-infected macrophages; roles of host genotype and cytokine milieu.

We investigated the influence of CD40-CD40 ligand-mediated signaling on induction of microbicidal activity against Leishmania major in macrophages from resistant (B6) and susceptible (BALB) mouse strains. CD40 engagement induced leishmanicidal activity in resistant macrophages, but increased parasite replication in susceptible macrophages. CD40 engagement induced comparable TNF-alpha production in macrophages from both strains. However, increased IL-10 production was restricted to susceptible macrophages. Increased parasite replication in susceptible macrophages was prevented by a neutralizing anti-IL-10 antibody. In the presence of IFN-gamma, CD40 engagement induced Leishmania killing by macrophages from both strains. Therefore, the outcome of CD40 signaling on effector responses against L. major depends on host genotype and the cytokine milieu, and a source of IFN-gamma is required for a protective response.