The role of soluble leishmania proteins and the induction of inflammatory responses in American tegumentary leishmaniasis

PIMENTEL SOLÁ, J; GARCÍA BUSTOS, MF; RAGONE, P; MESIAS, A; MARCO JD; BARROSO PA; BONO M; BONO MC; PERÉZ-BRANDÁN C; PARODI C

Simposio; LXXI Reunión Anual de la Sociedad Argentina de Inmunología; 2023

American tegumentary leishmaniasis (ATL) displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the inflammatory response of the host may be an important factor determining the evolution of the disease. The aims of this work are to assess the specificity of soluble Leishmania proteins (SLP)for CL and ML patients in the in vitro induction of IFNγ. We also pretend to evaluate if cytokine induction varies depending on the Leishmania spp. used, pointed also to the relation of the induction to the clinical form of the disease and analyzing if ATL patients are capable to maintain longtime specific responses. Methods: We prepared a Leishmania lysate (SLP) from L. (V.) braziliensis (MHOM/BR/75/M2903) and L. (L.) amazonensis (MHOM/VE/84/MEL) massive cultures of promastigotes in the exponential phase of growth. Then we cultured freshly isolated PBMCs from the studied groups (1x106 cells/ml; 37°C, 5% CO2) in 24 wells plates in presence or absence of SLP mix composed by equalconcentrations (20 μg/ml) of L. (V.) braziliensisand L. (L.) amazonensis. Additionally PBMCs were stimulated with each strain separately. We collected culture supernatants after 7 days to assess the levels of IFNγ by ELISA. Studied groups: CL (n=9), ML (n=11), HS (healthy subjects, n=14) and DD (patients with differential diagnosis as vasculitis, psoriasis, n=16). Results: We determined that SLP specifically induced the production of IFNγ of CL (2360±596) and ML patients (3475±1717), with scarce concentrations obtained from the control groups (p˂ 0,0001). The addition of both speciesseparately showed that L. (V.) braziliensis (3487±785) was responsible for higher IFNγ amounts than L. (L.) amazonensis (1344±641) (p=0,0093) analyzing CL cases, but this effect was not observed in ML, with similar concentrations of the cytokine recorded for both strains (1867±1258; 1299±1056 respectively). Next, we performed the follow up of 4 CL patients with good response to therapy during 1, 3 and 6-10 months post-treatment obtaining similar levels of IFNγ ineach studied point. Conclusions: The specific induction of IFNγ produced by SLP in vitro on PBMCsfrom ATLpatients could be a useful tool to accompany classical methods in difficult diagnosis cases. L. (V.) braziliensisis responsible for the majority (>90%) of the infections found in the Northwest of Argentina, but we reported here than L. (V.) braziliensis proteins only induced increased concentration of IFNγ in CL patients. Possibly, T cell exhaustion produced in the chronic ML form could account for less capacity to mount stronger responses against the causative agent. In this regard, the results obtained during CL follow up indicate that at least 6-10 months after healing of the lesions, strong inflammatory specific responses are still maintained.*Results expressed as Media±SE(pg/ml)