George A. DosReis

Macrophage Interactions with Neutrophils Regulate Leishmania major Infection

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE2 and TGF-β production by macrophages, while parasite killing depended on neutrophil elastase and TNF-α production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.

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.

TGF-β Mediates CTLA-4 Suppression of Cellular Immunity in Murine Kalaazar

Recent studies indicate important roles for CTLA-4 engagement in T cells, and for TGF-β production in the immunopathogenesis of murine kalaazar or visceral leishmaniasis, but a functional link between these two pathways in helping intracellular parasite growth is unknown. Here we report that Ag or anti-CD3 activation of splenic CD4+ T cells from visceral leishmaniasis leads to intense CTLA-4-mediated TGF-β1 production, as assessed either by CTLA-4 blockade or by direct CTLA-4 cross-linkage. Production of TGF-β1 accounted for the reciprocal regulation of IFN-γ production by CTLA-4 engagement. Following CD4+ T cell activation, intracellular growth of Leishmania chagasi in cocultured splenic macrophages required both CTLA-4 function and TGF-β1 secretion. Cross-linkage of CTLA-4 markedly increased L. chagasi replication in cocultures of infected macrophages and activated CD4+ T cells, and parasite growth could be completely blocked with neutralizing anti-TGF-β1 Ab. Exogenous addition of rTGF-β1 restored parasite growth in cultures protected from parasitism by CTLA-4 blockade. These results indicate that the negative costimulatory receptor CTLA-4 is critically involved in TGF-β production and in intracellular parasite replication seen in murine kalaazar.

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.

Interactions with apoptotic but not with necrotic neutrophils increase parasite burden in human macrophages infected with Leishmania amazonensis

Neutrophils are involved in the initial steps of most responses to pathogens. In the present study, we evaluated the effects of the interaction of apoptotic vs. necrotic human neutrophils on macrophage infection by Leishmania amazonensis. Phagocytosis of apoptotic, but not viable, neutrophils by Leishmania‐infected macrophages led to an increase in parasite burden via a mechanism dependent on TGF‐β1 and PGE2. Conversely, infected macrophages’ uptake of necrotic neutrophils induced killing of L. amazonensis. Leishmanicidal activity was dependent on TNF‐α and neutrophilic elastase. Nitric oxide was not involved in the killing of parasites, but the interaction of necrotic neutrophils with infected macrophages resulted in high superoxide production, a process reversed by catalase, an inhibitor of reactive oxygen intermediate production. Initial events after Leishmania infection involve interactions with neutrophils; we demonstrate that phagocytosis of these cells in an apoptotic or necrotic stage can influence the outcome of infection, driving either parasite survival or destruction.

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.

The central role of Fas-ligand cell signaling in inflammatory lung diseases.

Following inflammation and injury in the lung, loss of epithelial cell precursors could determine the balance between tissue regeneration and fibrosis. This review discusses evidence that proapoptotic Fas‐Fas ligand (FasL) signaling plays a central role in pulmonary inflammation, injury and fibrosis. FasL signaling induces inflammatory apoptosis in epithelial cells and alveolar macrophages, with concomitant IL‐1β and chemokine release, leading to neutrophil infiltration. FasL signaling plays a critical role in models of acute lung injury, idiopathic pulmonary fibrosis and silicosis; blockade of Fas‐FasL interactions either prevents or attenuates pulmonary inflammation and fibrosis. Serologic and immunohistochemical studies in patients support a major pathogenic role of Fas and FasL molecules in inflammatory lung diseases. Identification of the pathogenic role of FasL could facilitate the discovery of more effective treatments for currently untreatable inflammatory lung diseases.

Glycoinositolphospholipid from Trypanosoma cruzi: Structure, Biosynthesis and Immunobiology

The pathogenic protozoan parasite Trypanosoma cruzi expresses on its surface an unusual family of glycoinositolphospholipids (GIPLs) closely related to glycosylphosphatidylinositol (GPI) anchors. Different parasite isolates express distinct GIPLs which fall into two series, depending on the substitution of the third mannosyl residue in the conserved glycan sequence Man4-(AEP)-GlcN-InsPO4 by ethanolamine phosphate or beta-galactofuranose. Although the exact role of these molecules in the cell biology and pathogenicity of T. cruzi remains unknown, the lipid and glycan moieties impart distinct responses to host T and B lymphocytes and phagocytes, overall favouring an immune response permissive to the parasite. The biosynsthesis of GIPLs follows a pathway similar to that observed for GPI anchors. However, a more detailed understanding might enable the development of specific inhibitors of parasite-specific enzymes and lead to novel drugs to ameliorate Chagas disease.