Marise P. Nunes

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-α. 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.

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‐α, NO and iNOS expression, while GXM predominantly induced TGF‐β 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.

INCREASED SUSCEPTIBILITY OF FAS LIGAND-DEFICIENT GLD MICE TO TRYPANOSOMA CRUZI INFECTION DUE TO A TH2-BIASED HOST IMMUNE RESPONSE

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation‐induced CD4+ T cell death (AICD), and in Fas  :  FasL‐mediated cytotoxicity. When CD4+ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas  :  FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.

Costimulation of Host T Lymphocytes by a Trypanosomal trans-Sialidase: Involvement of CD43 Signaling

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.

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.

A surgery for American cuteneous and visceral leishmaniasis among 1,342 dogs from areas in Rio de Janeiro (Brazil) where the human diseases occur

There are areas in the periphery of Rio de Janeiro city where human cases of Visceral and/or Cutaneous Leishmaniasis occur. The parasites have been identified as Leishmania donovani and Leishmania braziliensis braziliensis respectively. A survey for Leishmaniasis was done among 1,342 dogs from those areas using an indirect immunofluorescent test. From the dogs, 616 came from areas where only human cases of Visceral Leishmaniasis occurred, 373 from an area where all human cases were of Cutaneous Leishmaniasis and 353 from a third area (Campo Grande) where both visceral and cutaneous human cases were detected. The prevalence of parasite antibody titers among dogs from areas of Cutaneous Leishmaniasis was significantly higher than that of Visceral Leishmaniasis (8.6% vs. 4.3%, p less than 0.02). The highest prevalence was observed among dogs from the area where both diseases are present (12.7%).

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-γ secreting CD8+ T cells specific for H-2Kb-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-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+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-γ, TNF-α 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.

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.

Indirect immunofluorescence test in new world Leishmaniasis: serological and clinical relation-ship

The indirect immunofluorescence test (IF) for anti-Leishmania antibodies (IgG and IgM) was performed with sera from the following groups of individuals: 214 cutaneous leishmaniasis patients, 28 healthy subjects with positive Montenegros skin test (MST), 29 healthy subjects with negative MST and 16 visceral leishmaniasis patients. The first four groups came from a suburban area of Rio de Janeiro (Jacarepaguá) where cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis is endemic. It was observed that IF-IgM titers were significantly higher amongst the cutaneous leishmaniasis patients with less than four months of disease as compared to those with longer periods and that IF-IgG titers were significantly higher in patients with multiple lesions as compared to those with single lesions. The visceral leishmaniasis patients had IF-IgG titers significantly higher than those from cutaneous leishmaniasis patients. A group of 28 individuals selected amongst the 214 cutaneous leishmaniasis patients had their IF-titers (IgG and IgM) compared to those of the two control groups of healthy subjects from the endemic area, respectively with positive and negative MST. Significantly higher titers of IF-IgG and IF-IgM were found in the group with active disease. The same group of patients showed IF-IgG titers significantly lower at the end of the antimonial therapy than those observed during this treatment.

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes.

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT(1) and AT(2), have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4(+) and CD8(+) T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10(-8)M Ang II did not change any of the parameters evaluated. However, 10(-6)M losartan, an AT(1) receptor antagonist: (i) reduced the percentage of CD25(+) and CD69(+) cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10(-6)M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10(-8)M). None of these responses was modified by 10(-7)M PD123319, an AT(2) antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT(1) receptor activation. Moreover, AT(1) receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT(2) receptor expression did not change after T cell receptor triggering.