Phosphatidylserine exposure on the surface of Leishmania amazonensis amastigotes modulates in vivo infection and dendritic cell function

Abstract

Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persistent lesions in most inbred strains of mice. In both cases, the infection is characterized by a marked immunosuppression of the host. We previously showed that amastigote forms of the parasite make use of surface-exposed phosphatidylserine (PS) molecules to infect host cells and promote alternative macrophage activation, leading to uncontrolled intracellular proliferation of the parasites. In this study, we demonstrated that treatment of infected mice with a PS-targeting monoclonal antibody ameliorated parasite loads and lesion development, which correlated with increased proliferative responses by lymphocytes. In addition, we observed an enhanced dendritic cell (DC) activation and antigen presentation in vitro. Our data imply that the recognition of PS exposed on the surface of amastigotes plays a role in down-modulating DC functions, in a matter similar to that of apoptotic cell clearance. This study provides new information regarding the mechanism of immune suppression in Leishmania infection.

Figure 1. PS-targeting treatment of infected mice ameliorates the disease

C57BL/6 mice were infected with 0.5–1 × 10⁶ parasites in the ear dermis or footpad and received i. p. injections of 100 μg of mAb mch1N11 (αPS) or isotype control every 2 days, starting one day prior to infection. (A) Parasite loads in the ear were determined by real-time PCR after 3 weeks of infection, and (B) footpad lesion sizes were determined by using a Vernon caliper. Draining LN cells from mAb-treated mice were obtained at 3 weeks post-infection and re-stimulated with SLA for 3 d. (C) A thymidine pulse was made, and, after 12 h, cell proliferation was evaluated or (D) SNs were collected and cytokine production measured by using a Bioplex kit. (A, C, D) Graph represents data from 3 pooled experiments. (B, C, D) Asterisks indicate statistical difference between αPS with isotype control (B) Graph represents one experiment with 5 mice per group. * p < 0.05, ** p < 0.01, *** p <0.001.

Figure 2. Blocking PS recognition boosts DC activation

BMDCs were infected with lesion-derived amastigotes that were pre-treated with 9 μg/ml of mAb mch1N11 (αPS) or isotype control. After 24 h of infection, (A, B) DCs were harvested for the analysis of activation markers by flow cytometry, and (C) the production of cytokines in the SN was measured by a bioplex assay. (A) Graph representative of 3 independent experiments. Numbers at the upper-right of each plot represents the total population MFI of the indicated labeling. Numbers at the bottom of each plot indicate the percentage of positive/negative cells (B, C) Graph represents data from 3 pooled experiments. (B) Asterisks indicate statistical difference when compared to isotype control. Am – amastigotes. * p < 0.05, ** p < 0.01, *** p <0.001.

Figure 3. PS-targeting treatment does not alter amastigote uptake by DCs

BMDCs were co-cultured with different amounts of lesion-derived, CFSE-labeled amastigotes that were pre-treated with 9 μg/ml of mAb mch1N11 (αPS). (A, B) After 24h of infection, BMDCs were harvested, stained for CD11c, and subjected to FACS analysis. Numbers in the dot plots indicate the percentage of CFSE⁺, CD11c⁺ BMDCs. (B) Graph represents data from 3 independent experiments.

Figure 4. Blocking PS recognition enhances overall antigen presentation capacity of infected DCs

(A, B) C57BL/6 WT and (C, D) FcR KO BMDCs were infected with lesion-derived amastigotes that were pre-treated with 9 μg/ml of mAb mch1N11 (PS-targeting) or isotype antibody in the presence or absence of different concentrations of OVA protein. After 24 h of infection, DCs were harvested and co-cultured with CFSE-labeled OTII CD4⁺ T cells for 3 days. The percentage of proliferating T cells was determined by CFSE dilution. (A, C) Graphs are representative of 3 independent experiments. (B, D) Graphs represent data from 3 pooled experiments. ** p <0.01, *** p <0.001.

Figure 5. Blocking PS recognition enhances parasite antigen presentation by infected DCs

(A) BMDCs were infected with lesion-derived amastigotes that were pre-treated with 9 μg/ml of mAb mch1N11 (PS-targeting) or isotype control. After 24 h of infection, DCs were harvested and co-cultured for 3 days with CFSE-labeled CD4⁺ T cells that were purified from draining LNs of infected mice. The percentage of proliferating cells was determined by CFSE dilution. (B) Graph represents data from 2 pooled experiments. *** p < 0.001.