Transcriptomic landscape of skin lesions in cutaneous leishmaniasis reveals a strong CD8+ T cell immunosenescence signature linked to immunopathology.

The severity of lesions that develop in patients infected by Leishmania braziliensis is mainly associated with a highly cytotoxic and inflammatory cutaneous environment. Recently, we demonstrated that senescent T and NK cells play a role in the establishment and maintenance of this tissue inflammation. Here, we extended those findings using transcriptomic analyses that demonstrate a strong co-induction of senescence and pro-inflammatory gene signatures in cutaneous leishmaniasis (CL) lesions. The senescence-associated signature was characterized by marked expression of key genes such as ATM, Sestrin 2, p16, p21 and p38. The cell type identification from deconvolution of bulk sequencing data showed that the senescence signature was linked with CD8+ effector memory and TEMRA subsets and also senescent NK cells. A key observation was that the senescence markers in the skin lesions are age-independent of patients and were correlated with lesion size. Moreover, a striking expression of the senescence-associated secretory phenotype (SASP), pro-inflammatory cytokine and chemokines genes was found within lesions that were most strongly associated with the senescent CD8 TEMRA subset. Collectively, our results confirm that there is a senescence transcriptomic signature in CL lesions and supports the hypothesis that lesional senescent cells have a major role in mediating immunopathology of the disease.

CCR5 promotes the migration of CD8+ T cells to the leishmanial lesions.

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

Pathogenic CD8 T cell responses are driven by neutrophil-mediated hypoxia in cutaneous leishmaniasis.

Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8 T cell responses mediate disease. While these responses originate in the lymph node, we find that expression of the cytolytic effector molecule granzyme B is restricted to lesional CD8 T cells in Leishmania - infected mice, suggesting that local cues within inflamed skin induce cytolytic function. Expression of Blimp-1 ( Prdm1 ), a transcription factor necessary for cytolytic CD8 T cell differentiation, is driven by hypoxia within the inflamed skin. Hypoxia is further enhanced by the recruitment of neutrophils that consume oxygen to produce reactive oxygen species, ultimately increasing granzyme B expression in CD8 T cells. Importantly, lesions from cutaneous leishmaniasis patients exhibit hypoxia transcription signatures that correlate with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8 T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as other inflammatory skin diseases where cytolytic CD8 T cells contribute to pathogenesis.

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.

Variable gene expression and parasite load predict treatment outcome in cutaneous leishmaniasis.

Patients infected with Leishmania braziliensis develop chronic lesions that often fail to respond to treatment with antiparasite drugs. To determine whether genes whose expression is highly variable in lesions between patients might influence disease outcome, we obtained biopsies of lesions from patients before treatment with pentavalent antimony and performed transcriptomic profiling on these clinical samples. We identified genes that were highly variably expressed between patients, and the variable expression of these genes correlated with treatment outcome. Among the most variable genes in all the patients were components of the cytolytic pathway, and the expression of these genes correlated with parasite load in the skin. We demonstrated that treatment failure was linked to the cytolytic pathway activated during infection. Using a host-pathogen marker profile of as few as three genes, we showed that eventual treatment outcome could be predicted before the start of treatment in two separate cohorts of patients with cutaneous leishmaniasis (n = 21 and n = 25). These findings raise the possibility of point-of-care diagnostic screening to identify patients at high risk of treatment failure and provide a rationale for a precision medicine approach to drug selection in cutaneous leishmaniasis. This work more broadly demonstrates the value of identifying genes of high variability in other diseases to better understand and predict diverse clinical outcomes.

In Vitro Immunomodulatory Activity of a Transition-State Analog Inhibitor of Human Purine Nucleoside Phosphorylase in Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) is the most common clinical form of American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis. CL is associated with a strong Th1 immune response. This exacerbated inflammatory response is correlated with severity of disease and delays the healing time of the ulcer. The fourth-generation immucillin derivative (DI4G), a potent inhibitor of purine nucleoside phosphorylase, has been proposed as a promising agent in the treatment of diseases associated with T cell activation. Herein, we evaluated the in vitro immunomodulatory activity of DI4G in cells of patients presenting with CL. Peripheral blood mononuclear cells (PBMC) from CL patients were stimulated with soluble leishmania antigen (SLA), in the presence or absence of DI4G, and IFN-γ, TNF, CXCL9, and CXCL10 levels were determined by ELISA. Lymphocyte proliferation in the presence or absence of DI4G was also evaluated, using flow cytometry. DI4G was able to decrease (p < 0.05) IFN-γ production but did not change the TNF, CXCL9, and CXCL10 levels. DI4G decreased (p < 0.05) the lymphoproliferative response mediated by CD8+ T cells, but not that by CD4+ T cells. DI4G is able to attenuate the exaggerated immune response in CL, exhibiting immunomodulatory activity in IFN-γ production and in CD8+ T cell proliferation.