Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, presents an exaggerated Th1 response that is associated with ulcer development. Macrophages are the primary cells infected by Leishmania parasites and both reactive oxygen species (ROS) and nitric oxide (NO) are important in the control of Leishmania by these cells. The mechanism involved in the killing of L. braziliensis is not well established. In this study, we evaluate the role of ROS and NO in the control of L. braziliensis infection by monocytes from CL patients. After in vitro infection with L. braziliensis, the oxidative burst by monocytes from CL patients was higher when compared to monocytes from healthy subjects (HS). Inhibition of the ROS pathway caused a significant decrease in the oxidative burst in L. braziliensis infected monocytes from both groups. In addition, we evaluated the intracellular expression of ROS and NO in L. braziliensis-infected monocytes. Monocytes from CL patients presented high expression of ROS after infection with L. braziliensis. The expression of NO was higher in monocytes from CL patients as compared to expression in monocytes from HS. A strong positive correlation between NO production and lesion size of CL patients was observed. The inhibition of ROS production in leishmania-infected monocytes from CL patients allowed the growth of viable promastigotes in culture supernatants. Thus, we demonstrate that while production of ROS is involved in L. braziliensis killing, NO alone is not sufficient to control infection and may contribute to the tissue damage observed in human CL.

Cutaneous leishmaniasis (CL) caused byLeishmania braziliensisis characterized by a strong Th1 response that leads to skin lesion development. In areas whereL. braziliensistransmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8⁺T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4⁺IFN-γ⁺and CD8⁺IFN-γ⁺T cells. Monocytes from PBMC were infected withL. braziliensisand cocultured with CD8⁺T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8⁺IFN-γ⁺cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8⁺T cells induced more apoptosis of infected monocytes than did SC CD8⁺T cells. Granzyme B production in CD8⁺T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8⁺T cells are more cytotoxic and may be involved in pathology.

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.

Background: Leishmaniapreferentially infects macrophages, which allow the parasite to multiply but can also kill the parasite. Although the T cell response in human leishmaniasis is well-characterized, little is known about the concomitant macrophage behavior. The aim of this study was to characterize the macrophage immune response afterLeishmania braziliensisinfection in cells derived from cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML) patients, subclinical individuals (SC) and healthy control subjects (HS).Methods: Peripheral blood mononuclear cell-derived macrophages from the different groups were exposed toL. braziliensis in vitroand were evaluated for susceptibility toLeishmaniainfection, ability to killLeishmaniaand chemokine/cytokine production. Nitric Oxide (NO) and superoxide (O₂^-) levels in the supernatant of infected macrophage cultures were monitored.Results: After exposure toL. braziliensis, peripheral blood mononuclear cell-derived macrophages from SC individuals showed a lower infection rate and a smaller number of intracellular amastigotes compared to cells from CL and ML patients. Macrophages from CL and ML patients produced more chemokines and TNF-α than those from the SC group. Production of NO and O₂^-were detected but did not vary significantly among the different groups.Conclusions: Our data indicate that macrophages play a pivotal role in controllingL. braziliensisinfection and in leishmaniasis pathology by secreting pro-inflammatory chemokines/cytokines that activate and recruit T cells, overwhelming the inflammatory response.

Cutaneous leishmaniasis (CL) caused by infection with Leishmania braziliensis is characterized by an exaggerated inflammatory response that controls the parasite burden, but also contributes to pathology. While myeloid cells are required to eliminate the parasite, recent studies indicate that they may also participate in the inflammatory response driving disease progression. The innate immune response to leishmania is driven in part by the Toll-like receptors (TLRs) TLR2, TLR4, and TLR9. In this study, we used flow cytometric analysis to compare TLR2 and TLR4 expression in monocyte subsets (classical, intermediate, and non-classical) from CL patients and healthy subjects (HS). We also determined if there was an association of either the pro-inflammatory cytokine TNF or the anti-inflammatory cytokine IL-10 with TLR2 or TLR4 expression levels after L. braziliensis infection. In vitro infection with L. braziliensis caused CL monocytes to up-regulate TLR2 and TLR4 expression. We also found that intermediate monocytes expressed the highest levels of TLR2 and TLR4 and that infected monocytes produced more TNF and IL-10 than uninfected monocytes. Finally, while classical and intermediate monocytes were mainly responsible for TNF production, classical monocytes were the main source of IL-10. Collectively, our studies revealed that up-regulated TLR2/4 expression and TNF production by intermediate/inflammatory subsets of monocytes from patients correlates with detrimental outcome of cutaneous leishmaniasis.

Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects. Leishmania spp. are protozoan parasites that cause a spectrum of human diseases, and L. braziliensis causes chronic inflammatory skin lesions in residents of endemic regions of Latin America. Leishmania are obligate intracellular parasites in mammalian hosts, found in macrophages throughout infection. Nonetheless, other cell types including neutrophils also take up the parasite, but the role of neutrophils throughout chronic leishmaniasis remains unclear. We analyzed circulating neutrophils from patients in northeast Brazil with cutaneous leishmaniasis (CL) caused by L. braziliensis, compared to healthy controls from the same region. Our data revealed that neutrophils from both infected and healthy hosts took up comparable numbers of parasites, and parasite phagocytosis induced similar degrees of neutrophil activation. However, CL patient neutrophils produced more reactive oxidants than control neutrophils, and increased amounts of the chemokines CXCL8 and CXCL9 after parasite exposure. Interestingly, according to surface markers of PMN activation (CD62L, CD66b), we found that L. braziliensis activates both infected and uninfected “bystander” neutrophils from both patients and controls. Importantly, repeated measures showed the production of reactive oxidants and chemokine release were significantly decreased after therapeutic cure of infection. These data suggest that CL promotes a heightened inflammatory state in circulating neutrophils during active infection.