Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.

BACKGROUND: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection

BACKGROUND Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

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.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

Intranasal vaccination with killed Leishmania amazonensis promastigotes antigen (LaAg) associated with CAF01 adjuvant induces partial protection in BALB/c mice challenged with Leishmania (infantum) chagasi.

The CAF01 adjuvant has previously been shown to be safe for human use and to be a potent adjuvant for several vaccine antigens. In the present work, we sought to optimize the Leishmania amazonensis antigens (LaAg) intranasal vaccine in an attempt to enhance the protective immune responses against Leishmania (infantum) chagasi by using the CAF01 association. LaAg/CAF01 vaccinated mice that were challenged 15 days after booster dose with L. (infantum) chagasi showed a significant reduction in their parasite burden in both the spleen and liver, which is associated with an increase in specific production of IFN-γ and nitrite, and a decrease in IL-4 production. In addition, LaAg/CAF01 intranasal delivery was able to increase lymphoproliferative immune responses after parasite antigen recall. These results suggest the feasibility of using the intranasal route for the delivery of crude antigens and of a human-compatible adjuvant against visceral leishmaniasis.