Transdifferentiation of neutrophils facilitates the establishment of infection by Leishmania donovani parasites.

Abstract

Neutrophil transdifferentiation involves the acquisition of dendritic cell-like properties, challenging the traditional view of neutrophils being solely phagocytes. The presence of transdifferentiated neutrophils is established in Visceral Leishmaniasis, but not in its dermal sequel, Post Kala-azar Dermal Leishmaniasis. Accordingly, this study investigated the altered functionalities of neutrophils focusing on the acquisition of dendritic cell-like properties and its impact on infection establishment. In PKDL cases, immunophenotyping of neutrophil-dendritic cells (N-DC hybrids) was performed using flow cytometry, along with studying the status of N-DC hybrid inducing cytokines (TNF-α, IFN-γ) and growth factor (GM-CSF). Ex vivo infection of neutrophils with L. donovani was monitored by droplet digital PCR, employing A2; additionally, their frequency of transdifferentiation, oxidative and phagocytic status, as well as apoptosis potential were quantified by flow cytometry. Compared with healthy controls, neutrophils from PKDL cases demonstrated a significant upregulation of CD83 positivity, but the frequency of co-stimulation (HLA-DR, CD80/86) was unaltered. PKDL cases demonstrated raised levels of TNF-α and IFN-γ, but GM-CSF remained unchanged. Following ex vivo infection of neutrophils, infection was evident at 2 h and was accompanied by CD83 positivity. Furthermore, the CD66b⁺/CD83 vis-à-vis CD66b⁺/CD83^- subset exhibited heightened generation of reactive oxygen species (ROS), enhanced phagocytosis, and increased apoptosis. Taken together, neutrophils from PKDL cases demonstrated transdifferentiation with the absence of antigen-presenting function. Virulent Leishmania induced transdifferentiation in neutrophils, altering their functionalities and facilitating parasite uptake, along with heightened generation of intra-neutrophilic ROS and enhanced apoptosis, which possibly facilitated their engulfment by macrophages, thereby bolstering the "Trojan horse" mechanism of parasite transfer.