In Situ versus Systemic Immune Response in the Pathogenesis of Cutaneous Leishmaniasis.

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1ß, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1ß, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1ß and GzmB, IL-17 participates in the pathology of CL.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Multiomic profiling of cutaneous leishmaniasis infections reveals microbiota-driven mechanisms underlying disease severity.

Leishmania braziliensis is a parasitic infection that can result in inflammation and skin injury with highly variable and unpredictable clinical outcomes. Here, we investigated the potential impact of microbiota on infection-induced inflammatory responses and disease resolution by conducting an integrated analysis of the skin microbiome and host transcriptome on a cohort of 62 patients infected with L. braziliensis. We found that overall bacterial burden and microbiome configurations dominated with Staphylococcus spp. were associated with delayed healing and enhanced inflammatory responses, especially by IL-1 family members. Quantification of host and bacterial transcripts on human lesions revealed that high lesional S. aureus transcript abundance was associated with delayed healing and increased expression of IL-1ß. This cytokine was critical for modulating disease outcomes in L. braziliensis-infected mice colonized with S. aureus, given that its neutralization reduced pathology and inflammation. These results highlight how the human microbiome can shape disease outcomes in cutaneous leishmaniasis and suggest pathways toward host-directed therapies to mitigate the inflammatory consequences.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

The skin microbiome enhances disease through IL-1b and delays healing in cutaneous leishmaniasis patients.

Leishmania braziliensis infection results in inflammation and skin injury, with highly variable and unpredictable clinical outcomes. Here, we investigated the potential impact of microbiota on infection-induced inflammatory responses and disease resolution by conducting an integrated analysis of the skin microbiome and host transcriptome on a cohort of 62 L. braziliensis -infected patients. We found that overall bacterial burden and microbiome configurations dominated with Staphylococcus spp. were associated with delayed healing and enhanced inflammatory responses, especially by IL-1 family members. Dual RNA-seq of human lesions revealed that high lesional S. aureus transcript abundance was associated with delayed healing and increased expression of IL-1ß. This cytokine was critical for modulating disease outcome in L. braziliensis -infected mice colonized with S. aureus , as its neutralization reduced pathology and inflammation. These results implicate the microbiome in cutaneous leishmaniasis disease outcomes in humans and suggest host-directed therapies to mitigate the inflammatory consequences.

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.

Diabetes Modifies the Clinic Presentation of Cutaneous Leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) caused by L. braziliensis is characterized by 1 or multiple well-limited ulcerated lesions. Diabetes mellitus (DM) impairs neutrophil and monocyte function, and there is a report of vegetative lesions in a patient with both diseases in Morocco. Here we evaluate the influence of DM on clinical manifestations, immune response, and in the treatment of CL. METHODS: The participants were 36 DM patients with CL and 36 patients with CL without DM, matched by age and gender. The diagnosis of CL was performed by documentation of DNA of L. braziliensis by polymerase chain reaction in the lesion biopsy and histopathologic findings. All patients were treated with Glucantime (Sanofi-Aventis) 20 mg/kg of weight per day for 20 days. RESULTS: There was no difference in the majority of the clinical variables between the groups, and the cure rate in patients with CL and DM (67%) was similar to that observed in CL patients (56%; P ˃ .05). The most important finding was the documentation that 36% of the patients with DM and CL had atypical cutaneous lesions characterized by large superficial ulcers without defined borders. High levels of interferon-γ, tumor necrosis facor, and interleukin-1ß were detected in the supernatants of mononuclear cells stimulated with Leishmania antigen in patients with DM and atypical CL. Moreover, while cure was observed in only 33% of the patients with DM and atypical CL lesions, it was observed in 85% of patients with typical lesions (P < .05). CONCLUSIONS: DM modifies the clinical presentation of CL, enhances pro-inflammatory cytokine production, and impairs response to antimony therapy.

Unbalanced production of LTB4/PGE2 driven by diabetes increases susceptibility to cutaneous leishmaniasis.

Poorly controlled diabetes mellitus leads to several comorbidities, including susceptibility to infections. Hyperglycemia increases phagocyte responsiveness, however immune cells from people with diabetes show inadequate antimicrobial functions. We and others have shown that aberrant production of leukotriene B4 (LTB4) is detrimental to host defense in models of bacterial infection. Here, we will unveil the consequences of high glucose in the outcome of Leishmania braziliensis skin infection in people with diabetes and determine the role of LTB4 in human phagocytes. We show that diabetes leads to higher systemic levels of LTB4, IL-6 and TNF-α in cutaneous leishmaniasis. Only LTB4 correlated with blood glucose levels and healing time in diabetes comorbidity. Skin lesions of people with leishmaniasis and diabetes exhibit increased neutrophil and amastigote numbers. Monocyte-derived macrophages from these individuals showed higher L. braziliensis loads, reduced production of Reactive Oxygen Species and unbalanced LTB4/PGE2 ratio. Our data reveal a systemic inflammation driven by diabetes comorbidity in opposition to a local reduced capacity to resolve L. braziliensis infection and a worse disease outcome.

The Elderly Respond to Antimony Therapy for Cutaneous Leishmaniasis Similarly to Young Patients but Have Severe Adverse Reactions.

There is evidence that elderly patients with cutaneous leishmaniasis (CL) have more mucosal and disseminated diseases than young patients and their cells produce less antigen-induced interferon (IFN)-γ. Herein, we compared the roles of interleukin (IL)-10 and IL-15 as modulators of antigen-induced immune responses and the incidence of adverse reaction and response to therapy in young versus elderly patients with CL. Study participants included 35 senior (60-85 years) and 35 young (18-40 years) patients who had a diagnosis of CL documented by typical cutaneous lesions containing Leishmania braziliensis DNA. Elderly patients had less lymph node enlargement. Antigen-induced blood cell cytokine responses were studied in the absence or presence of IL-10 antibody or exogenously added recombinant IL-15. The ratio of IFN-γ/IL-10 was lower in elderly patients, and IFN-γ production was enhanced by either neutralization of IL-10 or exogenous recombinant IL-15 in blood cells from elderly but not young patients. Patients were treated three times weekly with antimony at 20 mg/kg/day for 20 doses. Although there was no difference in response to therapy between the two groups, two young patients needed rescue therapy with amphotericin B. Ventricular arrhythmias and ventricular overload were more frequent in elderly patients. We conclude that elderly patients have alterations in the immune response that may influence clinical manifestations, but we did not find that they had a higher failure rate than young subjects to antimony therapy. However, because of the high rate of electrocardiographic abnormalities during therapy, antimony should not be used in elderly patients with CL.

Age modifies the immunologic response and clinical presentation of American tegumentary leishmaniasis.

Leishmania (Viannia) braziliensis is the main causal agent of American tegumentary leishmaniasis (ATL) that may present as cutaneous, mucosal, or disseminated cutaneous leishmaniasis. The disease is highly prevalent in young males and there is a lack of studies of ATL in the elderly. Herein, we compared clinical manifestations, immunologic response, and response to antimony therapy between patients > 60 years of age (N = 58) and patients who were 21-30 years of age (N = 187). The study was performed in Corte de Pedra, Bahia, Brazil, a well-known area of L. braziliensis transmission. Cytokine production by cultured peripheral blood mononuclear cells stimulated with soluble Leishmania antigen was performed. Elderly subjects more frequently had a previous history of cutaneous leishmaniasis, large lesions, or mucosal leishmaniasis, and they were less likely to have lymphadenopathy. There was no difference regarding gender and response to therapy. Peripheral blood mononuclear cells from elderly subjects produced a similar amount of tumor necrosis factor than young patients but they produced less interferon-gamma and more interleukin-10 than young subjects. We concluded that elderly patients with cutaneous leishmaniasis should be searched for mucosal or disseminated leishmaniasis. The decreased interferon-gamma production and increase in interleukin-10 observed in elderly patients may contribute to parasite persistence and L. braziliensis infection dissemination.