IL-4 reduces resistance of mice to Trypanosoma cruzi infection

The role of IL-4 has often been studied, especially in the Leishmania major infection model, but not in Trypanosoma cruzi infection. In the present study, the role of IL-4 in host defense against infection with the Tulahuén strain of T. cruzi was examined by depleting IL-4 with an anti-IL-4 monoclonal antibody in vivo. In both IL-4 depleted and control C57BL/6 mice, the parasitemia showed peaks on the 21st day of infection. Both parasitemia and mortality were decreased in IL-4 depleted mice compared with control mice when IFN-γ and nitric oxide productions were increased in IL-4 depleted mice compared with control mice. The mice treated with N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, showed increased susceptibility to T. cruzi infection to the same level in both IL-4 depleted and control mice. Thus, it is suggested that endogenous IL-4 induces susceptibility to T. cruzi mainly by suppressing the production of IFN-γ and nitric oxide, which has trypanocidal activity. Received: 2 October 2000 / Accepted: 5 October 2000     

MIF-driven activation of macrophages induces killing of intracellular Trypanosoma cruzi dependent on endogenous production of tumor necrosis factor,nitric oxide and reactive oxygen species

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) is a key player in innate immunity. MIF has been considered critical for controlling acute infection by the protozoan Trypanosoma cruzi, but the underlying mechanisms are poorly understood. Our study aimed to analyze whether MIF could favor microbicidal activity of the macrophage, a site where T. cruzi grows and the initial effector cell against this parasite. Using murine macrophages infected in vitro, we examined the effect of MIF on their parasiticidal ability and attempted to identify inflammatory agents involved in MIF-induced protection. Our findings show that MIF is readily secreted from peritoneal macrophages upon T. cruzi infection. MIF activates both primary and J774 phagocytes boosting the endogenous production of tumor necrosis factor-alpha via mitogen-activated protein kinase p38 signaling, as well as the release of nitric oxide and reactive oxygen species, leading to enhanced pathogen elimination. MIF can also potentiate the effect of interferon-gamma on T. cruzi killing by J774 and mouse peritoneal macrophages, rendering these cells more competent in reducing intracellular parasite burden. The present results unveil a novel innate immune pathway that contributes to host defense and broaden our understanding of the regulation of inflammatory mediators implicated in early parasite containment that is decisive for resistance to T. cruzi infection.     

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine-producing cells, the production of type 1 [interleukin-2 (IL-2) and interferon (IFN)-γ], type 2 (IL-4, IL-5, IL-10), inflammatory [tumor necrosis factor (TNF)-α, IL-1α, IL-6] and regulatory (transforming growth factor-β) cytokines were examined. With the exception of IL-4 and IL-5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL-4 and IL-5 were not detected until later in infection in the resistant mice (>34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL-2 and IFN-γ and an earlier appearance of TNF-producing cells, in comparison to resistant mice. Cells producing IL-2 or IL-10 were transient in their appearance in the spleen while cells producing IL-1, IL-4, IL-5, IL-6, IFN-γ, TNF, or TGF-β were first detectable in either the acute or post-acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN-γ, TNF-α and TGF-β were particularly numerous even very late in the infection. Double-staining techniques were used to show that the vast majority of the IFN-γ-producing cells in the spleen were CD4^?, CD8^? α/β TCR⁺ T cells. This study confirms the transience of IL-2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late-acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine-producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.     

Cyclic AMP decreases the production of NO and CCL2 by macrophages stimulated with Trypanosoma cruzi GPI-mucins

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins (tGPI-mucin) present on the surface of the cellular membrane of Trypanosoma cruzi forms activate toll-like receptors 2 (TLR2) on the surface of immune cells and induce the release of several mediators of inflammation which may be relevant in the context of Chagas disease. Here, we evaluated the ability of tGPI-mucins to activate murine peritoneal macrophages to induce nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1/CCL2). We also investigated the ability of compounds which increase or mimic cyclic adenosine monophosphate (AMP) to modulate the production of NO and CCL2. Our data show that elevation of intracellular levels of cyclic AMP prevents the release of NO and CCL2 induced by tGPI-mucins in macrophages. Overall, the release of CCL2 was decreased to a greater extent and at lower concentrations of cyclic AMP-modifying agents than the production of NO. It is suggested that the elevation of cyclic AMP during T. cruzi infection may modify the release of pro-inflammatory mediators and alter significantly the course of T. cruzi infection. Andre Talvani and Sibele Ferreira Coutinho present equal contribution to this research.     

Intracellular co-localization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): evidence for dual pathway of iNOS induction

Evidence is presented from studies in vitro and in vivo for a dual pathway of inducible nitric oxide synthase (iNOS) induction during Trypanosoma cruzi infection, one of which is interferon (IFN)-γ dependent and the other not. In vitro, the IFN-γ-dependent iNOS induction decreases parasite multiplication, and is in vivo associated with protection. iNOS induced by this pathway mediated a high NO output and showed a diffuse, cytoplasmic immunostaining in IFN-γ-activated macrophages in vitro as well as in cell infiltrates or infected tissues. Surprisingly, in such tissues, iNOS co-localized with parasite nests, and by immunoelectromicroscopy, iNOS was demonstrated on the parasite surface. iNOS co-localization with parasites was also seen in tissues from T. cruzi-infected IFN-γ receptor (R) knockout mice suggesting an IFN-γ-independent pathway of induction. However, no cytoplasmic iNOS was seen in inflammatory infiltrates of these tissues. IFN-γR^−/− mice displayed a dramatically enhanced susceptibility to infection with T. cruzi, diminished accumulation of iNOS mRNA in skeletal muscle and spleen cells, and reduced release of NO and per-oxynitrite. Expression of iNOS around intracellular parasites was also observed after infection of peritoneal macrophages or L-929 fibroblasts in vitro in the absence of other exogenous stimuli. A time-dependent NO release and enhanced accumulation of iNOS mRNA also was observed in infected peritoneal cells and fibroblasts. Cultured T. cruzi amastigotes, trypomastigotes, and epimastigotes were not labeled by the anti-iNOS antibodies and contained no iNOS mRNA, indicating that the iNOS detected actually originated from the mammalian cell. A pathogenic effect of low NO levels is suggested by the arresting effect of NOS inhibitors and the enhancing consequences of low concentrations of NO donors on intracellular parasite multiplication.     

Antigenic polymorphism of Trypanosoma cruzi: clonal analysis of trypomastigote surface antigens

The surface membrane antigens of infectious Trypanosoma cruzi trypomastigotes were studied at the levels of the strain and of individual trypomastigote clones. Blood trypomastigotes from three T. cruzi strains, Y, CL and Tehuantepec (“Teh”), were grown in vitro by weekly infection of J774 mouse macrophage tumor cells. Each T. cruzi strain was subsequently cloned by infection of J774 cells at limited trypomastigote dilution, and antisera were produced in mice against a selection of trypomastigote clones. Criss-cross panel analyses indicated the existence of a large degree of polymorphism among trypomastigote surface antigens. Various trypomastigote surface antigens were cross-reactive, appeared to be highly conserved, and were common to the three strains considered and to all the clones derived from each strain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that numerous trypomastigote antigenic proteins were precipitated by mouse antisera generated against cloned trypomastigotes. Some of these proteins were commonly distributed, while others were polymorphic. Finally, a state of cross-reactive immunity could be induced in C3H/He mice by infection with a cloned T. cruzi trypomastigote population. Immune mice resisted subsequent infections with lethal doses of wild-type bloodstream trypomastigotes from any one of the three T. cruzi strains.     

Effects of interleukin-4 deprivation and treatment on resistance to Trypanosoma cruzi

Trypanosoma cruzi (Y strain)-infected interleukin-4(-/-) (IL-4(-/-)) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-gamma) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4(-/-) mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-gamma synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-gamma synthesis levels; in contrast, IL-10 neutralization increased IFN-gamma production and addition of rIL-4 and/or rIL-10 diminished IFN-gamma synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruzi infection but that IL-4 can, in association with IL-10, modulate IFN-gamma production and resistance.     

Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice

Immunity to mycobacterial infection is closely linked to the emergence of T cells that secrete cytokines, gamma interferon (IFN-gamma), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), resulting in macrophage activation and recruitment of circulating monocytes to initiate chronic granuloma formation. The cytokine that mediates macrophage activation is IFN-gamma, and, like IL-12, IL-18 was shown to activate Th1 cells and induce IFN-gamma production by these cells. In order to investigate the role of IL-18 in mycobacterial infection, IL-18-deficient mice were infected with Mycobacterium tuberculosis and Mycobacterium bovis BCG Pasteur, and their capacities to control bacterial growth, granuloma formation, cytokine secretion, and NO production were examined. These mice developed marked granulomatous, but not necrotic, lesions in their lungs and spleens. Compared with the levels in wild-type mice, the splenic IFN-gamma levels were low but the IL-12 levels were normal in IL-18-deficient mice. The reduced IFN-gamma production was not secondary to reduced induction of IL-12 production. The levels of NO production by peritoneal macrophages of IL-18-deficient and wild-type mice did not differ significantly. Granulomatous lesion development by IL-18-deficient mice was inhibited significantly by treatment with exogenous recombinant IL-18. Therefore, IL-18 is important for the generation of protective immunity to mycobacteria, and its main function is the induction of IFN-gamma expression.     

Insights into IL-33 on inflammatory response during in vitro infection by Trypanosoma cruzi

Inflammatory and regulatory cytokines play an important role in the immunopathogenesis of Trypanosoma cruzi infection. Interleukin (IL)-33 is a member of the IL-1 superfamily of cytokines whose expression/production is upregulated following pro-inflammatory stimulation to alert the immune system in response to tissue stress or damage. The aim of this study was to evaluate the inflammatory profile induced in cultured J774 cells stimulated or not with IL-33 (10 ng/mL), with live parasites (1 × 10⁶ metacyclic trypomastigote forms) and/or total antigen, TcAg (100 µg/mL) and with both, IL-33 and TcAg/T. cruzi. The cultures were evaluated at 24 h and 48 h after addition of the stimuli. For this, the supernatants were collected for the measurement of TNF, IL-17, CCL2, and IL-10 by ELISA and of nitrite by the Griess method. TNF, IL-17, and CCL2 concentrations were elevated in the presence of TcAg or live T. cruzi parasites at 24 h, and the addition of IL-33 potentiated these effects at 48 h. In addition, the T. cruzi-amastigote forms reduced in those infected J774 cells stimulated with IL-33 at 48 h. In conclusion, the IL-33 elevated the production of the TNF, IL-17, and CCL2 in cultured J774 cells stimulated with T. cruzi and/or its antigen and reduced the intracellular parasites, providing impetus to new investigations on its potential actions on the parasite-induced inflammation.     

Interleukin-12 stimulation of lymphoproliferative responses in Trypanosoma cruzi infection.

The cytokine interleukin-12 (IL-12) is essential for resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma), a major activator of the parasiticidal effect of macrophages. A less studied property of IL-12 is its ability to amplify the proliferation of T or natural killer (NK) lymphocytes. We investigated the role of endogenously produced IL-12 in the maintenance of parasite antigen (T-Ag)-specific lymphoproliferative responses during the acute phase of T. cruzi infection. We also studied whether treatment with recombinant IL-12 (rIL-12) would stimulate T-Ag-specific or concanavalin A (Con A)-stimulated lymphoproliferation and abrogate the suppression that is characteristic of the acute phase of infection. Production of IL-12 by spleen-cell cultures during T. cruzi infection increased in the first days of infection (days 3-5) and decreased as infection progressed beyond day 7. The growth-promoting activity of endogenous IL-12 on T-Ag-specific proliferation was observed on day 5 of infection. Treatment of cultures with rIL-12 promoted a significant increase in Con A-stimulated proliferation by spleen cells from normal or infected mice. Enhanced T-Ag-specific proliferation by rIL-12 was seen in spleen cell cultures from infected mice providing that nitric oxide production was inhibited by treatment with the competitive inhibitor NG-monomethyl-L-arginine (NMMA). Enhancement of proliferation promoted by IL-12 occurred in the presence of neutralizing anti-interleukin-2 (IL-2) antibody, suggesting that this activity of IL-12 was partly independent of endogenous IL-2. Thymidine incorporation levels achieved with rIL-12 treatment of the cultures were approximately 50% of those stimulated by rIL-2 in the same cultures.     

Recombinant interleukin-12 enhances resistance of mice to Listeria monocytogenes infection

The effect of recombinant murine IL-12 (rIL-12) or anti-IL-12 antibody administration on resistance to murine listeriosis was investigated. Mice given a single 0.5 μg dose of rIL-12 had 1.5 log₁₀ fewer listeriae in their spleens and livers as compared with control infected mice 3 days after L. monocytogenes challenge. Conversely, administration of anti-IL-12 IgG caused an equivalent increase in the cfu of L. monocytogenes recovered from the spleens and livers as compared to control mice. This is the first report of such a protective effect from a single dose of rIL-12. Treatment of uninfected mice with rIL-12 induced IFN-γ mRNA production in their livers. Infection of mice with L. monocytogenes caused a similar increase in IFN-γ mRNA levels that was not increased further by concurrent treatment with rIL-12. Treatment of mice with an anti-IFN-γ MAb eliminated the protective effect of IL-12 on Listeria infection. Expression of TNF-γ, IL-10 and IL-12p40 mRNA in L. monocytogenes-infected mice were not significantly altered by administration of either anti-IL-12 IgG or rIL-12. rIL-12 administration was associated with increased serum AST levels, a measure of liver damage, 1 day after treatment in L. monocytogenes-infected mice. In addition, rIL-12 administration was associated with the increased presence of small inflammatory foci and necrotic hepatocytes in both infected and uninfected mice, suggesting a proinflammatory role for IL-12 in the liver.     

Apoptotic lymphocytes treated with IgG from Trypanosoma cruzi infection increase TNF‐α 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 Fcγ 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‐α, while decreasing production of TGF‐β1 by infected macrophages. Increased control of parasite replication required TNF‐α 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.     

Resistance to Trypanosoma cruzi infection in mice does not necessarily correlate with production of interferon-g in vivo

Interferon-γ (IFN-γ) is the most important mediator of inhibition of intracellular replication of Trypanosoma cruzi in vitro and has a protective effect against this parasite if administered in vivo. Here we have analyzed the importance of IFN-γ for resistance against a lethal infection with T. cruzi in a mouse model system. Resistant B6D2 mice survived the infection with a virulent strain of T. cruzi, whereas susceptible BALB/c mice died within 3 weeks. Both strains produced large amounts of IFN-γ after infection. Surprisingly, susceptible mice had higher serum concentrations of IFN-γ and showed, using in situ hybridization a stronger increase in IFN-γ mRNA-producing cells in their spleens than resistant mice. Moreover, this pattern was also found when immune spleen cells were stimulated with parasite antigens in vitro. However, a marked difference between these mice was found in the production of IL-4, which was much higher in susceptible mice in vivo and in vitro. No difference was found for IL-10. These data show that, at least in the mouse strain/parasite combination used, production of IFN-γ is not the decisive factor determining resistance or susceptibility to T. cruzi. Rather, it is possible that the balance between protective (e.g., IFN-γ) and exacerbative cytokines (e.g., IL-4) may decide over disease control or progression. Received: 10 April 1998     

Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-γ) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-γ production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-α gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-α production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.     

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2–10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0·01 to 100 μg/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.     

Effects of Strains of Lactococcus lactis on the Production of Nitric Oxide and Cytokines in Murine Macrophages

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host’s gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.     

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.     

Mice with Genetic Deletion of Group VIA Phospholipase A2β Exhibit Impaired Macrophage Function and Increased Parasite Load in Trypanosoma cruzi-Induced Myocarditis

Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A₂β (iPLA₂β) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA₂β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA₂β-knockout (iPLA₂β-KO) mice infected with T. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA₂β-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA₂β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA₂β-KO mice produced significantly less nitric oxide (NO) and caused less T. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA₂β activity does not influence myocardial inflammation, but iPLA₂β is essential for T. cruzi clearance.