Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.

Prostaglandin E2 contributes to L. braziliensis survival and therapeutic failure in cutaneous leishmaniasis.

Patients with cutaneous leishmaniasis (CL) present an exacerbated inflammatory response associated with tissue damage and ulcer development. In recent years, higher rates of failure to pentavalent antimoniate therapy have been observed, yet the underlying reason remains poorly understood. We hypothesize that the eicosanoid PGE2 favours the establishment of infection by L. braziliensis, which contributes to therapeutic failure. The aim of the present study was to investigate the influence of PGE2 on the survival of L. braziliensis in macrophages and rates of therapeutic failure in CL patients. PGE2, an eicosanoid derived from the metabolism of arachidonic acid by the COX-2 enzyme, plays several roles in immune response. We found that increased PGE2 decreases the microbicidal function of macrophages and is associated with disease severity and therapeutic failure. Additionally, the neutralization of COX-2 by NS398, a selective NSAID, increases the ability of macrophages to kill L. braziliensis and protects against the pathological inflammatory response. Our data suggest that NS398 may serve as an adjunct treatment for CL patients.

TNF-induced metalloproteinase-9 production is associated with neurological manifestations in HTLV-1-infected individuals.

HTLV-1-infected individuals may develop a neurologic inflammatory condition known as HTLV-1-associated myelopathy (HAM/TSP), in which the high production of TNF is observed. These patients exhibit higher proviral loads, enhanced production of proinflammatory cytokines and lymphocyte proliferation in comparison to asymptomatic HTLV-1 carriers and those presenting overactive bladder (OAB-HTLV-infected). Metalloproteinases (MMPs) are known to degrade the components of the blood-brain barrier, favoring the migration of infected cells into the central nervous system. Moreover, the unbalanced production of MMPs and their inhibitors (TIMPs) has also been associated with tissue damage. The present work studied the production of MMP-9 and TIMPs in HTLV-1-infected individuals with and without neurological manifestations. HAM/TSP patients presented higher concentrations of MMP-9 in peripheral blood mononuclear cell (PBMC) culture supernatants, as well as a higher MMP-9/TIMP-3 ratio when compared to the other groups studied. MMP-9 levels positively correlated with proviral load and TNF in OAB-HTLV-infected individuals, and the in vitro neutralization of TNF significantly decreased MMP-9 levels in PBMC culture supernatants. Our findings indicate an association between MMP-9 production and the proinflammatory state associated with HTLV-1 infection, as well as HAM/TSP.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor-γ Agonist, Downregulates the Inflammatory Response in Cutaneous Leishmaniasis Patients Without Interfering in Leishmania braziliensis Killing by Monocytes.

Patients with cutaneous leishmaniasis (CL) due to Leishmania braziliensis infection have an exacerbated inflammatory response associated with tissue damage and ulcer development. An increase in the rate of patients who fail therapy with pentavalent antimony has been documented. An adjuvant therapy with an anti-inflammatory drug with the potential of Leishmania killing would benefit CL patients. The aim of the present study was to investigate the contribution of peroxisome proliferator-activated receptor-γ (PPAR-γ) activation by pioglitazone in the regulation of the inflammatory response and L. braziliensis killing by monocytes. Pioglitazone is an oral drug used in the treatment of diabetes, and its main mechanism of action is through the activation of PPAR-γ, which is expressed in many cell types of the immune response. We found that activation of PPAR-γ by pioglitazone decreases the inflammatory response in CL patients without affecting L. braziliensis killing by monocytes. Our data suggest that pioglitazone may serve as an adjunctive treatment for CL caused by L. braziliensis.

The Influence of Infection by Different Leishmania (Viannia) braziliensis Isolates on the Pathogenesis of Disseminated Leishmaniasis.

Disseminated Leishmaniasis (DL) is an emerging and severe form of Leishmania (Viannia) braziliensis infection defined by the presence of 10 and up to more than 1,000 skin lesions. The mechanisms underlying parasite dissemination remain unknown. Genotypic differences among species of L. braziliensis have been associated with different clinical forms of disease. The present work compared the function of monocytes obtained from patients with cutaneous leishmaniasis (CL) and DL in response to infection with L. braziliensis isolates of both these two clinical forms of disease. Mononuclear cells obtained from DL and CL patients were infected with different L. braziliensis isolates, and numbers of infected cells, parasite load, respiratory burst, TLR2 and TLR4 expression and cytokine production were evaluated. DL isolates infected more monocytes, induced greater respiratory burst, and more cytokine production compared to isolates from CL patients regardless of the origin of monocytes (DL or CL). However, greater parasite multiplication and higher TLR2 and TLR4 expression were seen in monocytes from DL patients compared to CL following infection with DL isolates. Our results indicate the participation of both parasite genotype and host factors in the pathogenesis of DL.

The Leishmania antigen-specific pro-inflammatory response in cutaneous leishmaniasis is linked to disease progression but not to the therapeutic failure of pentavalent antimonials.

High levels of pro-inflammatory cytokines in cutaneous leishmaniasis patients are associated with tissue damage and ulcer development. We found higher levels of TNF and IL-1ß in peripheral blood mononuclear cell supernatants in response to soluble Leishmania antigen in individuals with a longer duration of disease. In addition, Leishmania braziliensis-infected patients with a longer disease progression before treatment presented a shorter time to cure after treatment onset. No associations were found between the levels of the pro-inflammatory cytokines IL-6, TNF and IL-1-ß and patients' response to pentavalent antimony treatment. Our data suggest that while the Leishmania antigen-specific pro-inflammatory cytokines investigated may lead to ulcer development, they do not influence therapeutic failure in cutaneous leishmaniasis patients.

Inhibition of gamma-secretase activity without interfering in Notch signalling decreases inflammatory response in patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

Localized skin inflammation during cutaneous leishmaniasis drives a chronic, systemic IFN-γ signature.

Cutaneous leishmaniasis is a localized infection controlled by CD4+ T cells that produce IFN-γ within lesions. Phagocytic cells recruited to lesions, such as monocytes, are then exposed to IFN-γ which triggers their ability to kill the intracellular parasites. Consistent with this, transcriptional analysis of patient lesions identified an interferon stimulated gene (ISG) signature. To determine whether localized L. braziliensis infection triggers a systemic immune response that may influence the disease, we performed RNA sequencing (RNA-seq) on the blood of L. braziliensis-infected patients and healthy controls. Functional enrichment analysis identified an ISG signature as the dominant transcriptional response in the blood of patients. This ISG signature was associated with an increase in monocyte- and macrophage-specific marker genes in the blood and elevated serum levels IFN-γ. A cytotoxicity signature, which is a dominant feature in the lesions, was also observed in the blood and correlated with an increased abundance of cytolytic cells. Thus, two transcriptional signatures present in lesions were found systemically, although with a substantially reduced number of differentially expressed genes (DEGs). Finally, we found that the number of DEGs and ISGs in leishmaniasis was similar to tuberculosis-another localized infection-but significantly less than observed in malaria. In contrast, the cytolytic signature and increased cytolytic cell abundance was not found in tuberculosis or malaria. Our results indicate that systemic signatures can reflect what is occurring in leishmanial lesions. Furthermore, the presence of an ISG signature in blood monocytes and macrophages suggests a mechanism to limit systemic spread of the parasite, as well as enhance parasite control by pre-activating cells prior to lesion entry.

Evaluation of the Ability of Miltefosine Associated with Topical GM-CSF in Modulating the Immune Response of Patients with Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) due to L. braziliensis is associated with an exaggerated inflammatory response and tissue damage. Miltefosine is more effective than pentavalent antimony (Sbv) in the treatment of CL, and here, we evaluate the ability of Sbv, miltefosine, and GM-CSF administered intravenously, orally, or topically, respectively, to modify the immune response. Patients were treated with miltefosine plus GM-CSF, miltefosine plus placebo, or Sbv. Mononuclear cells were stimulated with soluble Leishmania antigen (SLA) on day 0 and day 15 of therapy, and cytokine levels were determined in supernatants by ELISA. The lymphocyte proliferation and oxidative burst were evaluated by flow cytometry, and the degree of infection and Leishmania killing by optical microscopy. Proliferation of CD4+ T cells were enhanced in patients using miltefosine and in CD8+ T cells when GM-CSF was associated. Enhancement in the oxidative burst occurred in the miltefosine plus GM-CSF group on day 15 of therapy. Moreover, the number of L. braziliensis in infected monocytes on day 15 as well as the percentage of infected cells was lower after 48- and 72-hour culture in cells from patients treated with miltefosine plus GM-CSF. In addition to the ability of miltefosine to kill Leishmania, the changes in the immune response caused by miltefosine and GM-CSF may increase the cure rate of CL patients using these drugs.