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.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Mouse strain susceptibility to trypanosome infection: an arginase-dependent effect

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with N(omega)-hydroxy-nor-L-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.     

Immune evasion by Helicobacter pylori is mediated by induction of macrophage arginase II

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Decoy receptor 3 enhances tumor progression via induction of tumor-associated macrophages

Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.     

IFN-gamma-independent autocrine cytokine regulatory mechanism in reprogramming of macrophage responses to bacterial lipopolysaccharide.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-gamma, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-gamma knockout (IFN-gamma(-/-)) mice exhibit only marginal LPS-induced TNF-alpha, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-gamma. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-gamma(-/-) macrophage responses in a dose-dependent manner that manifested by an increased TNF-alpha and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12(-/-) and IL-10(-/-) mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-alpha and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-gamma-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-gamma-dependent induction of antimicrobial and tumoricidal activity in macrophages.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

Immunoregulation during disease progression in prediabetic NOD mice: inverse expression of arginase and prostaglandin H synthase 2 vs. interleukin-15.

Non-obese diabetic (NOD) mice spontaneously develop insulin dependent diabetes due to autoimmune destruction of beta-cells. The progression of insulitis can be accelerated and synchronized in the pancreas by a single injection of 250 mg/kg cyclophosphamide. In this study, we will report on three immune mediators that were not known to be expressed during insulitis until now. Early insulitis in ten-week-old female NOD mice was associated with strong expression of prostaglandin H synthase 2 in the pancreas and of arginase, an antagonist enzyme of the inducible NO synthase. After acceleration of insulitis progression by cyclophosphamide, expression of the two enzymes was downregulated within 24 h. There was strong concomitant upregulation of IL-15 gene expression that preceded lymphocyte invasion of islets and a rise of IFN-gamma mRNA levels by several days. The comparison of individual pancreata showed that the expression of IL-12 and IL-18 mRNA closely correlated with levels of IL-15 gene expression. We conclude that arginase and prostaglandin H synthase 2 expression is associated with peri-insulitis, while IL-15 is a candidate cytokine in driving destructive insulitis, as it elicits Th1-cytotoxic responses in lymphoid as well as in non-lymphoid immune cells and is unusually resistant to downregulation by antagonistic cytokines. This is the first report on arginase, prostaglandin H synthase 2 and IL-15 expression in pancreatic lesions of prediabetic NOD mice.     

Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease

More than 60% of STAT6(-/-) mice immunologically reject spontaneous metastatic mammary carcinoma and survive indefinitely if their primary tumors are removed, whereas 95% of STAT6-competent BALB/c mice succumb to metastatic disease. BALB/c and STAT6-deficient mice with primary tumors have elevated levels of Gr1(+)CD11b(+) myeloid suppressor cells (MSCs), which inhibit T cell activation. After removal of primary tumor, MSC levels revert to baseline in STAT6-deficient mice, but remain elevated in BALB/c mice. The decrease is IFN-gamma dependent, as is the reduction in metastatic disease. Neither BALB/c nor STAT6-deficient MSCs produce inducible NO synthase; however, both produce arginase and reactive oxygen species. STAT6-deficient mice produce M1 macrophages, which contain high levels of NO and are tumoricidal, whereas BALB/c mice produce M2 macrophages, which make arginase and are not tumoricidal. Immunity in STAT6-deficient mice requires the activation of NO-producing M1 macrophages that are tumoricidal, the reduction in MSC levels to baseline after surgical removal of primary tumor, and the activation of tumor-specific T cells. These mechanisms occur in STAT6(-/-) mice because STAT6 deficiency prevents signaling through the type 2 IL-4Ralpha, thereby blocking the production of arginase and promoting the synthesis of NO.     

Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.     

Ameliorative effect of vanadyl(IV)–ascorbate complex on high-fat high-sucrose diet-induced hyperglycemia,insulin resistance,and oxidative stress in mice

There is mounting evidence demonstrating causative links between hyperglycemia, oxidative stress, and insulin resistance, the core pathophysiological features of type 2 diabetes mellitus. Using a combinational approach, we synthesized a vanadium–antioxidant (i.e., l-ascorbic acid) complex and examined its effect on insulin resistance and oxidative stress. This study was designed to examine whether vanadyl(IV)-ascorbate complex (VOAsc) would reduce oxidative stress, hyperglycemia, and insulin resistance in high-fat high-sucrose diet (HFSD)-induced type 2 diabetes in mice. Male C57BL/6J mice were fed a HFSD for 12 weeks to induce insulin resistance, rendering them diabetic. Diabetic mice were treated with rosiglitazone, sodium l-ascorbate, or VOAsc. At the end of treatment, fasting blood glucose, fasting serum insulin, homeostasis model assessment-insulin resistance index, and serum adipocytokine levels were measured. Serum levels of nitric oxide (NO) parameters were also determined. The liver was isolated and used for determination of malondialdehyde, reduced glutathione, and catalase levels, and superoxide dismutase and glutathione peroxidase activities. VOAsc groups exhibited significant reductions in serum adipocytokine and NO levels, and oxidative stress parameters compared to the corresponding values in the untreated diabetic mice. The results indicated that VOAsc is non-toxic. In conclusion, we identified VOAsc as a potentially effective adjunct therapy for the management of type 2 diabetes.     

Central pharmacological activity of a quaternary ammonium compound in streptozotocin diabetic mice

Possible changes in blood-brain barrier (BBB) function as a result of diabetes were investigated by assessing antagonism of morphine analgesia in diabetic mice by methylnaltrexone (MeNTX), an opioid receptor blocker that does not cross the BBB when administered subcutaneously (SC). In streptozotocin (STZ)-treated diabetic mice--but not vehicle-treated, non-diabetic mice--treatment with SC MeNTX significantly reduced morphine analgesia. In vehicle-treated, non-diabetic mice, morphine analgesia was antagonized by MeNTX administered intracerebroventricularly and by SC naltrexone, which crosses the BBB. Reduction of STZ-induced hyperglycemia by insulin reversed the effectiveness of SC MeNTX in antagonizing morphine analgesia. We hypothesize that in STZ diabetic mice, MeNTX was able to cross the BBB and block brain opioid receptors.     

Multidose streptozotocin induction of diabetes in BALB/cBy mice induces a T cell proliferation defect in thymocytes which is reversible by interleukin-4

Thymic T cell function in streptozotocin-treated (STZ) diabetic mice has been examined. STZ administration suppresses thymic T cell proliferation in response to mitogen stimulation in vitro. Secretion of IL-4 was dramatically reduced; however, secretion of IL-2 or IFN-gamma was not significantly inhibited. RT-PCR analysis of thymocyte RNA revealed that levels of IL-4 mRNA were dramatically decreased in STZ-treated mice. Levels of mRNA encoding IFN-gamma were similar, but the appearance was delayed in thymocytes derived from STZ-treated mice, implying differential regulation of IL-4 and IFN-gamma. Defective thymocyte proliferation was partially restored by exposure to IL-2 in vitro; however, IL-4 completely reversed the STZ-induced defect. Administration in vivo of IL-4 before STZ treatment reversed the STZ-induced thymocyte proliferation defect and prevented both pancreatic islet destruction and hyperglycemia. Thymocyte cell surface differentiation markers were not appreciably different from control mice. Collectively these experiments suggest that STZ treatment of mice reduces expression of IL-4 which is associated with development of autoimmune diabetes.     

Trypanosoma brucei: infection in murine diabetes

The course of infection due to Trypanosoma brucei infection was observed in genetically diabetic and streptozotocin-induced diabetic mice. A strain of T. brucei, TREU 667, was used which produces a chronic infection in C57BL/6(B6) mice lasting greater than 60 days. Genetic diabetic mice (+db/+db) are obese, and have elevated blood glucose levels, normal levels of insulin, and impaired cell-mediated immunity. Their littermates (m+/m+, m+/+db) are of normal weight, and are normoglycemic and immunocompetent. The infected +db/+db mice lived significantly longer than the nondiabetic littermates. In contrast to this finding, streptozotocin-induced diabetic B6 mice developed higher parasitemia and had shorter survival times than control B6 mice. Continuous treatment with insulin of these streptozotocin-induced diabetic mice led to normalization of blood glucose and a significant reduction of parasitemia. While hyperglycemia may be associated with higher parasitemia and death in streptozotozin-induced diabetes, genetic factors may play an additional role in the genetic models.     

Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production

The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.     

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.