Host-Directed Therapies for Cutaneous Leishmaniasis.

Cutaneous leishmaniasis exhibits a wide spectrum of clinical presentations from self-resolving infections to severe chronic disease. Anti-parasitic drugs are often ineffective in the most severe forms of the disease, and in some cases the magnitude of the disease can result from an uncontrolled inflammatory response rather than unrestrained parasite replication. In these patients, host-directed therapies offer a novel approach to improve clinical outcome. Importantly, there are many anti-inflammatory drugs with known safety and efficacy profiles that are currently used for other inflammatory diseases and are readily available to be used for leishmaniasis. However, since leishmaniasis consists of a wide range of clinical entities, mediated by a diverse group of leishmanial species, host-directed therapies will need to be tailored for specific types of leishmaniasis. There is now substantial evidence that host-directed therapies are likely to be beneficial beyond autoimmune diseases and cancer and thus should be an important component in the armamentarium to modulate the severity of cutaneous leishmaniasis.

Granzyme B Inhibition by Tofacitinib Blocks the Pathology Induced by CD8 T Cells in Cutaneous Leishmaniasis.

In cutaneous leishmaniasis, the immune response is not only protective but also mediates immunopathology. We previously found that cytolytic CD8 T cells promote inflammatory responses that are difficult to treat with conventional therapies that target the parasite. Therefore, we hypothesized that inhibiting CD8 T-cell cytotoxicity would reduce disease severity in patients. IL-15 is a potential target for such a treatment because it is highly expressed in human patients with cutaneous leishmaniasis lesions and promotes granzyme B‒dependent CD8 T-cell cytotoxicity. Here we tested whether tofacitinib, which inhibits IL-15 signaling by blocking Jak3, might decrease CD8-dependent pathology. We found that tofacitinib reduced the expression of granzyme B by CD8 T cells in vitro and in vivo systemic and topical treatment, with tofacitinib protecting mice from developing severe cutaneous leishmaniasis lesions. Importantly, tofacitinib treatment did not alter T helper type 1 responses or parasite control. Collectively, our results suggest that host-directed therapies do not need to be limited to autoimmune disorders and that topical tofacitinib application should be considered a strategy for the treatment of cutaneous leishmaniasis disease in combination with antiparasitic drugs.

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis.

BACKGROUND: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood. METHODS: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells. RESULTS: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production. CONCCLUSIONS: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology.

CD8+ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1ß production.

Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1ß release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1ß significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1ß release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology.

Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion.

Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes.

Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major.

Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections.

Intermediate monocytes contribute to pathologic immune response in Leishmania braziliensis infections.

Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16(+) (both intermediate [CD14(+)CD16(+)] and nonclassical [CD14(dim)CD16(+)]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16(+) monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response.

Matrix metalloproteinase 9 production by monocytes is enhanced by TNF and participates in the pathology of human cutaneous Leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis (CL) due to L.braziliensis infection is characterized by a strong inflammatory response with high levels of TNF and ulcer development. Less attention has been given to the role of mononuclear phagocytes to this process. Monocytes constitute a heterogeneous population subdivided into classical, intermediate and non-classical, and are known to migrate to inflammatory sites and secrete inflammatory mediators. TNF participates in the induction of matrix metalloproteinases (MMPs). MMP-9 is an enzyme that degrades basal membrane and its activity is controlled by the tissue inhibitor of metalloproteinase. METHODS: Mononuclear cells were obtained from ex-vivo labeling sub-populations of monocytes and MMP-9, and the frequency was determined by flow cytometry. Culture was performed during 72 hours, stimulating the cells with SLA, levels of MMP-9 and TIMP-1 in the supernatants were determined by ELISA. RESULTS: We observed that cells from CL lesions secrete high amounts of MMP-9 when compared to healthy subjects. Although MMP-9 was produced by monocytes, non-classical ones were the main source of this enzyme. We also observed that TNF produced in high level during CL contributes to MMP-9 production. CONCLUSIONS: These observations emphasize the role of monocytes, TNF and MMP-9 in the pathogenesis of L. braziliensis infection.

Cytotoxic T cells mediate pathology and metastasis in cutaneous leishmaniasis.

Disease progression in response to infection can be strongly influenced by both pathogen burden and infection-induced immunopathology. While current therapeutics focus on augmenting protective immune responses, identifying therapeutics that reduce infection-induced immunopathology are clearly warranted. Despite the apparent protective role for murine CD8⁺ T cells following infection with the intracellular parasite Leishmania, CD8⁺ T cells have been paradoxically linked to immunopathological responses in human cutaneous leishmaniasis. Transcriptome analysis of lesions from Leishmania braziliensis patients revealed that genes associated with the cytolytic pathway are highly expressed and CD8⁺ T cells from lesions exhibited a cytolytic phenotype. To determine if CD8⁺ T cells play a causal role in disease, we turned to a murine model. These studies revealed that disease progression and metastasis in L. braziliensis infected mice was independent of parasite burden and was instead directly associated with the presence of CD8⁺ T cells. In mice with severe pathology, we visualized CD8⁺ T cell degranulation and lysis of L. braziliensis infected cells. Finally, in contrast to wild-type CD8⁺ T cells, perforin-deficient cells failed to induce disease. Thus, we show for the first time that cytolytic CD8⁺ T cells mediate immunopathology and drive the development of metastatic lesions in cutaneous leishmaniasis.

Polymerase chain reaction (PCR) is highly sensitive for diagnosis of mucosal leishmaniasis.

We evaluated the use of polymerase chain reaction (PCR) for diagnosis of mucosal leishmaniasis (ML) in an endemic area in Acre, Brazil, where Leishmania braziliensis is present. Leishmania DNA was detected 34 of 35 cases, yielding a positivity rate of 97.1%, which was higher than the positivity rates for all of the other diagnostic methods studied, namely Montenegro skin test (MST), anti-Leishmania serological testing and microscopic examination of lesion biopsy specimens. These findings have led us to propose guidelines for the diagnosis of ML that use PCR as the principal method of parasitological confirmation of cases.