CD4+ T Cell-Dependent Macrophage Activation Modulates Sustained PS Exposure on Intracellular Amastigotes of Leishmania amazonensis

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4+ T cell and MΦ activation status in vitro and in vivo. L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4+ T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4+ T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses

NOVEL 17-KILODALTON LEISHMANIA ANTIGEN REVEALED BY IMMUNOCHEMICAL STUDIES OF A PURIFIED GLYCOPROTEIN FRACTION RECOGNIZED BY MURINE LYMPHOCYTES-T

FED UNIV RIO DE JANEIRO,INST MICROBIOL,BR-21941 RIO DE JANEIRO,RJ,BRAZILFED UNIV RIO DE JANEIRO,CARLOS CHAGAS FILHO INST BIOPHYS,BR-21941 RIO DE JANEIRO,RJ,BRAZILWeb of Scienc

Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva

Immunohistochemistry for OPB, TXNPx and HSP70 in infected and non infected (small pictures) footpads from BALB/c and BALB/c nude mice.

<p>Immunohistochemistry for OPB, TXNPx and HSP70 in infected and non infected (small pictures) footpads from BALB/c and BALB/c nude mice.</p

Western blot for tryparedoxin peroxidase (TXNPx) expression in 5 pairs of lesion derived amastigotes from BALB/c and BALB/c nude mice.

<p>A. Western blot image showing tryparedoxin peroxidase (upper band) and GAPDH (lower band) in soluble extracts from BALB/c derived amastigotes (1–5) and BALB/c nude derived amastigotes (6–10). B. Expression of tryparedoxin peroxidase relative to GAPDH in the 10 samples. Statistical analysis by t-test. * = p<0.05</p

Western blot for OPB expression in 5 pairs of lesion derived amastigotes from BALB/c and BALB/c nude mice.

<p>A. Western blot image showing OPB (upper band) and GAPDH (lower band) in soluble extracts from BALB/c derived amastigotes (1–5) and BALB/c nude derived amastigotes (6–10). B. Expression of OPB relative to GAPDH in the 10 samples. Statistical analysis by t-test. * = p<0.05</p

Comparison of lymphoid, myeloid and dendritic cell populations in spleens (A), popliteal lymph nodes (B) and footpads (C) of infected and non infected BALB/c and BALB/c nude mice by flow cytometry.

<p>Results of one experiment with 3 animals of each for control condition and four of each for infection. Statistical analyses by ANOVA followed by Tukey´s multiple comparison test for all comparisons except for lymphoid and dendritic cells in footpads (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003411#pntd.0003411.g002" target="_blank">Fig. 2C</a> left), where t test was employed. * = p<0.05</p

Footpad lesion sizes (difference between infected and non-infected footpads) in mm of BALB/c (n = 6) and BALB/c nude (n = 5) mice infected with <i>L. amazonensis</i>, measured weekly for 13 weeks.

<p>Footpad lesion sizes (difference between infected and non-infected footpads) in mm of BALB/c (n = 6) and BALB/c nude (n = 5) mice infected with <i>L. amazonensis</i>, measured weekly for 13 weeks.</p

Identity of the differentially expressed spots.

<p>Identity of the differentially expressed spots.</p