Antimony Resistant Leishmania donovani but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response

Abstract

In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions.

Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (Sb ^S-LD) or resistant (Sb ^R-LD) Leishmania donovani isolates.

At day 2, PBMC cultures exposed to Sb ^S-LD and Sb ^R-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4 ⁺CD25 ⁻CD127 ⁻ type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4 ⁺CD25 ⁺CD127 ^low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of Sb ^R-LD.

Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from Sb ^R-LD exposed PBMCs had more pronounced suppressive ability compared to Sb ^S-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to Sb ^S–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified Sb ^R-LD lacking unique terminal sugar in surface glycan.

Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that Sb ^R-LD have higher immunomodulatory capacity which may favour aggressive pathology.

Author Summary

The disease Kala-azar is caused by Leishmania donovani (LD). The disease is characterized by the depression of cellular immune response. In the Indian subcontinent LD parasites are mostly resistant to commonly used antileishmanial drug, like sodium antimony gluconate (SAG). It is known that infection with pentavalent antimony (Sb)-resistant parasites induces aggressive pathology- the cause is still not known. Sb-resistant parasites endowed with unique glycan which may also play an important role in the pathogenesis as following removal of terminal sugar of glycan these parasites behave like sensitive parasites. The diagnosis of the disease is possible after the disease sets in and therefore limited information is available on the host-parasite interaction at the onset of disease. As a surrogate of in vivo scenario we studied the interaction between normal human PBMC with Sb-sensitive and Sb-resistant parasites. The Sb-resistant parasites upon interaction with human peripheral blood mononuclear cells (PBMC) in vitro produced two distinct inhibitory cytokines, IL-10 and TGF-β. Similar experiment with Sb-sensitive LD induced much less amount of above cytokines. Thus aggressive pathology induced by Sb-resistant LD, may be, in part attributed to production of dual inhibitory cytokines where surface glycan of the parasite may play a decisive role.