Authors

HI Hammi³; AK Akarid¹; AD Arnoult²;  ¹ Hassan II University of Casablanca, Morocco;  ² INSERM U1197, France;  ³ INSERM U1197, University Paris Saclay/ University Hassan II, France

Discussion

The problem of leishmaniasis is becoming more urgent as its epidemiology is climate sensitive. Among the 3 forms of leishmaniasis, cutaneous leishmaniasis (CL) is the most spread form worldwide. This form causes skin lesions and ulcerations on different body parts, which can leave long-lasting scars and serious disability. The first signal in the establishment of an effective immune system is the recognition of the intruder by receptors such as Fc Receptors which are found mostly on immune cells. This family of receptors harbors a tyrosine-based activation motifs (ITAM), which initiates several important pathways and which may have a duality in response against intruders, either fighting (ITAM activator) or helping the invasion of the intruder (ITAM inhibitor). In our project we have chosen to study profoundly the interaction between the receptor CD32a or FcRIIA and two different strains of Moroccan Leishmania (L.major and L.tropica). Our experimental setup involved utilizing both murine and human cell lines. Bone marrow-derived macrophages (BMDMs) were isolated from femurs extracted of C57BL/6 Wild type (Wt) and CD32a transgenic (Tg) mice. Additionally, we used the human monocytic cell line THP-1 in which CD32a expression has been knocked down with shRNA. We infected those cells with L.major or L.tropica and their production of pro-inflammatory cytokines was assessed with ELISA tests. No significant production of IL-1β or TNF-a by THP-1 was noticed. Curiously, western blot of cells infected with both strains revealed a cleavage of CD32a happening with the strain L.tropica but not L.major. A Kinetic of the infection showed that the cleavage was happening 1h post-infection. The expression of the CD32a was assessed after the infection with both strains through flow cytometry and no significant difference was observed, suggesting that the cleavage was intracellular. We tried several protease inhibitors to prevent the cleavage of CD32a with no success. Furthermore, we tried to understand the pathways by which the receptor may play a role in the infection by analyzing the expression of different proteins involved in the ITAM pathways after infecting Wt or Tg BMDMs. We first investigated the proteins that play a role in the ITAM activator such as Syk and Fyn. Syk seems to be underexpressed in infected cells of both wt and transgenic mice. Fyn seem to be more underexpressed in BMDM from Tg mice infected with both strains. On the other side, proteins involved in the ITAM inhibitor, SHP1 and LYN seem to be activated as their phosphorylated form were observed in BMDM from Tg mice infected with both strains. It appears that the balance ITAMa/i tends towards the activation of an inhibitory ITAM in cellulo. Finally, we infected with both strains wild type and transgenic C57BL/6 mice in the ear. After a first experiment with no lesions, we have conducted a second experiment using Leishmania freidlin. Tg mice developed lesions at the second week post-infection, reaching its maximum after 5 weeks and then dropping right after next healing after 8 weeks. Wt mice start developing lesions after 3 weeks and reached a maximum at 5 weeks but still not healed after 8 weeks. The investigation of CD32a receptor interaction with Moroccan Leishmania strains sheds light on potential immune mechanisms in combating cutaneous leishmaniasis, with findings suggesting a tendency towards inhibitory ITAM activation in transgenic mice.