Phagocytosis of Leishmania enhances macrophage activation by IFN-gamma and lipopolysaccharide

This investigation describes the ability of Leishmania promastigotes to enhance activation of bone marrow-derived murine macrophages in vitro if added together with rIFN-gamma in the presence or absence of LPS. Activation was defined as the capacity for arginine-derived NO2- production and the killing of intracellular Leishmania. Enhanced NO2- production was observed for either CBA or C3H/HeJ macrophages undergoing phagocytosis at the time of activation. Other phagocytic stimuli including inert polystyrene latex beads were as effective as Leishmania. No correlation could be demonstrated between the enhanced NO2- release and secretion of products of the respiratory burst or PGE2. However, TNF-alpha secretion was elevated in cultures undergoing phagocytosis and a relationship between hexosemonophosphate shunt activity and NO2- levels was evident. These studies confirm and extend previous reports that phagocytosis plays an important role in the regulation of macrophage physiology.     

Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2-during phagocytosis of opsonized promastigotes. Addition of the O2- scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2- enhances macrophage leishmanicidal activity. The importance of NO. produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO. in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor N(G)-monomethyl-L-arginine inhibited IFN-gamma-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-gamma. These data suggest that O2- and NO. each contribute to intracellular killing of L. chagasi in human and murine macrophages.     

Dual role for nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility

Using a murine model of susceptibility and resistance to paracoccidioidomycosis, we have previously demonstrated that immunosuppression occurs in susceptible (B10.A), but not in resistant (A/Sn), mouse strains. Accumulating evidence shows that NO is involved in the induction of T cell immunosuppression during infection as well as in the killing of Paracoccidioides brasiliensis. In the present work, we focused on NO and other macrophage products that could be associated with resistance or susceptibility to paracoccidioidomycosis. A striking difference was related to NO and TNF production. Macrophages from B10.A mice produced high and persistent NO levels, while in A/Sn animals, TNF production predominated. In in vitro cultures, P. brasiliensis-infected macrophages from A/Sn mice also produced large amounts of TNF, while B10.A macrophages only produced NO. TNF production by B10.A macrophages appeared to be suppressed by NO, because the addition of aminoguanidine sulfate, an inducible NO synthase (NOS2) inhibitor, resulted in TNF production. These results suggested that enhanced TNF or NO production is associated with resistance and susceptibility, respectively. However, regardless of the mouse strain, NOS2-deficient or aminoguanidine sulfate-treated mice presented extensive tissue lesions with increased fungal load in lungs and liver compared with their controls. We conclude that NOS2-derived NO is essential for resistance to paracoccidioidomycosis, but overproduction is associated with susceptibility.     

Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major

Amastigotes of Leishmania major have a great ability to evade destruction in host cells. This study investigated the activation in resident, inflammatory macrophages and J774 cells in vitro treated with lipopolysaccharide (LPS), soluble Leishmania antigen (SLA), calcium ionophore (CaI) and magnesium (Mg2+) alone or combined. An increase in nitric oxide (NO) production was observed in J774 or inflammatory macrophages treated with LPS alone or in combination with SLA and CaI. The same treatments did not affect the NO release by resident macrophages. There was no interference in uptake of L. major but CaI decreased intracellular proliferation of the parasite. This study demonstrated the importance of CaI in decreasing L. major proliferation inside murine macrophages while Mg2+ seemed to increase parasite proliferation. These finding may help to understand the events involved in host cells' clearance of this pathogen.     

Nitric oxide production by Leishmania-infected macrophages and modulation by cytokines and prostaglandins

Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS or NOS2) plays a major microbicidal role in murine macrophages and its importance is now emerging also in the dog and human models. In dogs we demonstrated that macrophages in vitro infected with Leishmania infantum produced NO, after stimulation with cytokine-enriched peripheral blood mononuclear cell supernatants. In addition, parasite killing was reduced by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine blood monocytes before macrophage differentiation did not release NO, and their leishmanicidal activity was instead correlated with superoxide anion and interferon (IFN)-gamma production. In human macrophage cultures, after infection with Leishmania infantum, we showed both iNOS expression by immunofluorescence and western blotting and NO release by the Griess reaction for nitrites. Various cytokines and prostaglandins can differently modulate NO synthesis. In our experiments, stimulation by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly enhanced iNOS expression and NO production in human macrophages. In addition, the prostaglandin E2 increased NO release in activated, Leishmania-infected human macrophages. These results are interesting in the light of a possible immunological or pharmacological regulation of NO synthesis and microbicidal functions of macrophages.     

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.     

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.     

An effect of parasite-encoded arginase on the outcome of murine cutaneous leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Deltaarg), as well as wild-type and complemented Deltaarg controls (Deltaarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p<0.05); 2) poorer survival of Deltaarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Deltaarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Deltaarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Deltaarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Deltaarg parasites produced more IFN-gamma and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.     

Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette-Guérin involves class II transactivator and depends on the Nramp1 gene.

The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mphi), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-gamma-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Abeta mRNA expression, which correlates with a higher level of IFN-gamma-induced phosphorylation of the STAT1-alpha protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Abeta gene expression in M. bovis BCG-infected macrophages activated with IFN-gamma.     

Leukotrienes are essential for the control of Leishmania amazonensis infection and contribute to strain variation in susceptibility

Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.     

Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: implications for the treatment of cutaneous leishmaniasis.

Nitric oxide (NO) is considered a key molecule in the defense against intracellular pathogens, particularly Leishmania. The expression of inducible nitric oxide synthase and consequent production of NO by infected macrophages has been shown to correlate with leishmaniasis resistance in the murine model as well as in human patients. Nitric oxide donors have been used successfully in the treatment of cutaneous leishmaniasis in humans, although their mechanisms of action are not fully understood. In the present work, the dose-dependent cytotoxic effects of the NO-donors S-nitroso-N-acetyl-l-cysteine (SNAC) and S-nitrosoglutathione (GSNO) against Leishmania were evaluated. GSNO inhibited the growth of Leishmania major and Leishmania amazonensis with in vitro 50% inhibitory concentrations (IC(50)) of 68.8+/-22.86 and 68.9+/-7.9 micromol L(-1), respectively. The IC(50) for SNAC against L. major and L. amazonensis were, respectively, 54.6+/-8.3 and 181.6+/-12.5 micromol L(-1). The leishmanicidal activity of GSNO, but not of SNAC, was reversed by ascorbic acid (AA) and dithiothreitol (DTT), suggesting that the mechanism of action of GSNO is related to the transnitrosation of parasite proteins. These results demonstrate that SNAC and GSNO have leishmanicidal activity, and are thus potential therapeutic agents against cutaneous leishmaniasis.     

Production of nitric oxide by murine macrophages induced by lipophosphoglycan of Leishmania major

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is the surface glycoconjugate lipophosphoglycan (LPG). In addition, LPG is shown to be useful as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we made attempts to characterize functions of the culture supernatant, and membrane LPG isolated from metacyclic promastigotes of Leishmania major. The purification scheme included anion-exchange chromatography, hydrophobic interaction chromatography and cold methanol precipitation. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by SDS-PAGE and thin layer chromatography. The effect of mLPG and sLPG on nitric oxide (NO) production by murine macrophages cell line (J774.1A) was studied. Both sLPG and mLPG induced NO production in a dose dependent manner but sLPG induced significantly higher amount of NO than mLPG. Our results show that sLPG is able to promote NO production by murine macrophages.     

Activation of the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase by macrophage-stimulating protein results in the induction of arginase activity in murine peritoneal macrophages.

Regulation of macrophage activities in response to inflammatory stimuli must be finely tuned to promote an effective immune response while, at the same time, preventing damage to the host. Our lab and others have previously shown that macrophage-stimulating protein (MSP), through activation of its receptor RON, negatively regulates NO production in response to IFN-gamma and LPS by inhibiting the expression of inducible NO synthase (iNOS). Furthermore, activated macrophages from mice harboring targeted mutations in RON produce increased levels of NO both in vitro and in vivo, rendering them more susceptible to LPS-induced endotoxic shock. In this study, we demonstrate that stimulation of murine peritoneal macrophages with MSP results in the RON-dependent up-regulation of arginase, an enzyme associated with alternative activation that competes with iNOS for the substrate L-arginine, the products of which are involved in cell proliferation and matrix synthesis. Expression of other genes associated with alternative activation, including scavenger receptor A and IL-1R antagonist, is also up-regulated in MSP-stimulated murine macrophages. Stimulation of cells with IFN-gamma and LPS blocks the ability of MSP to induce arginase activity. However, pretreatment of cells with MSP results in the up-regulation of arginase and inhibits their ability to produce NO in response to IFN-gamma and LPS, even in the presence of excess substrate, suggesting that the inhibition of NO by MSP occurs primarily through its ability to regulate iNOS expression.     

Successful therapy of lethal murine visceral leishmaniasis with cystatin involves up-regulation of nitric oxide and a favorable T cell response

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4(+) T cell differentiation. A 6-fold increase in NO production was observed with 0.5 microM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-gamma. In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-gamma treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistance.     

The change in leukotrienes and lipoxins in activated mouse peritoneal macrophages

The aim of this study was to investigate to what extent the generation of leukotrienes (LTs) and lipoxins (LXs) was affected by the expression of definite levels of macrophage activation. We used a system of murine peritoneal macrophages at different states of activation consisting in resident macrophages and FCS-, thioglycollate- or Corynebacterium parvum-elicited macrophages. The profile of lipoxygenase metabolites in resident macrophages was characterized by the presence of high levels of 12-HETE, followed by 15-HETE, 5-HETE, LTB(4) and 6-trans-LTB(4), 6-trans-12-epi-LTB(4). A comparable pattern was also found in FCS-elicited macrophages which appeared not to be responsive to the challenge with interferon gamma plus LPS, as measured by the generation of NO and tumor necrosis factor alpha. Resident as well as FCS-elicited macrophages also generated appreciable quantities of LXs (A(4) and B(4)). Thioglycollate-elicited macrophages, which expressed a state of 'responsive' macrophages, showed a block of the LT and LX synthesis. This block was also present in C. parvum-elicited macrophages which expressed a fully 'activated' phenotype, reflected by their capacity of releasing NO and tumor necrosis factor alpha even though they were not challenged. These results provide the first evidence that the level of 'responsive' as well as 'activated' macrophages was associated with of a simultaneous block of LTB(4) and LXs.     

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic's pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNgamma. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-kappaB activation and murine iNOS promoter activity in LPS and IFNgamma-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC(50) (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O(+) human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis.     

Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.