Leishmania amazonensiscontrols macrophage-regulated cell death to establish chronic infectionin vitroandin vivo

Abstract

Pathogenic protists of the genus Leishmania have evolved various strategies to exploit macrophages as host cells and subvert their immuno-metabolic functions to favour intracellular parasite survival. Surprisingly little is known on how Leishmania affects regulated cell death (RCD) pathways of its host cell, even though increased survival of in vitro infected macrophages has been reported, and chronic macrophage infection in vivo causes the devastating immunopathologies of leishmaniasis. To overcome this limitation and gain first systems-level insight into the interaction between intracellular Leishmania and the host cell RCD pathways, including apoptosis, pyroptosis and necroptosis, we applied transcriptomic analyses on L. amazonensis-infected, primary macrophages (termed LIMs) and used YO-PRO-1 to monitor cell death by fluorescent microscopy. RNAseq analyses at day 3 post-infection (PI) revealed dichotomic dysregulation of more than 60% of RCD-related genes in LIMs, characterized by up-regulation of anti-RCD and down-regulation of pro-RCD markers, including key regulators common to the three forms of cell death such as casp8, fadd, tradd, tnfaip3, tax1bp1, birc3, and itch. This profile correlated with expression changes of transcription factors known to regulate RCD, including AP1 and NF-κB family members, pparγ and cebpβ. Consequently, LIMs showed remarkable longevity in culture for at least 50 days, despite a constant increase of parasite burden to about 100 parasites per cell, while non-infected cells were cleared from the culture in just a few days. Longitudinal expression analysis of LIMs at days 0, 3, 15, and 30 PI by RT-qPCR confirmed stable maintenance of this high longevity profile with the dichotomic decrease and increase of RCD-activators and -inhibitors, respectively. LIMs further showed significant resistance to RCD-inducing signals compared to non-infected cells, including CSF-1 deprivation (intrinsic apoptosis), actinomycin D treatment (extrinsic apoptosis), LPS/ATP stimulation (pyroptosis). Significantly, we extended the anti-RCD expression pattern and RCD resistance phenotype to L. amazonensis-infected .

Investigating pyroptosis as a mechanism of L. major cell‐to‐cell spread in the human BLaER1 infection model

“…By harnessing the possibilities of reverse genetics by this cell line model, we could show that BLaER1 GSDMD −/− cells are more refractory to pyroptosis than wild‐type BLaER1 cells, which allowed us to study the involvement of this cell death mechanism in cell‐to‐cell spread of L. major parasites. This research is of high importance as the long‐held assumption that parasites egress via mechanical rupture due to high parasite burdens, has been challenged by the high parasite burdens tolerated by infected macrophages (Lecoeur et al, 2022) and by the implication of regulated cell death in parasite egress (Heyde et al, 2018). In line with a previous report conducted in the murine system, our live‐cell imaging data demonstrate that induction of pyroptosis in BLaER1 leads to a rapid release of intracellular parasites.…”

“…For a long time, cell‐to‐cell spread of Leishmania was attributed to mechanical rupture caused by high parasite burdens. However, this assumption has been challenged by the finding that macrophages can tolerate extremely high parasite burdens (Lecoeur et al, 2022). This raises the question how Leishmania egress from its host cell, an event crucial for Leishmania to maintain and progress the infection.…”