Complement Receptor 3 and its role in the interaction of primary human macrophages with apoptotic Leishmania major promastigotes
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Abstract
Leishmania parasites are the causative agent of the neglected tropical disease leishmaniasis. The sand fly vector-borne, disease-initiating Leishmania promastigote life stage (Lm) is comprised of viable and apoptotic parasites. Targeting of the latter by LC3-associated phagocytosis (LAP) leads to a decreased activation of the adaptive immune system. Based on these findings, this study focuses on (1) the internalization of apoptotic Lm by human monocyte-derived pro- (hMDM1) and anti-inflammatory (hMDM2) macrophages, (2) evasion of viable Lm from degradation as well as (3) identification and (4) functional characterization of factors that lead to uptake of apoptotic Lm and LAP induction. For detailed analysis of the apoptotic Lm population we separated viable from apoptotic Lm. Phagocytosis assays by flow cytometry in which hMDMs were differentially infected with viable or apoptotic Lm showed that hMDMs ingested apoptotic Lm more rapidly than viable parasites. Investigations of supernatants from differentially infected hMDMs and hMDDCs by ELISA revealed not only a higher release of the anti-inflammatory cytokine IL-10 but also an increase in secretion of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8 in presence of apoptotic Lm. Live cell microscopy showed that LAP can be triggered by viable and apoptotic Lm. Phagosomes harboring apoptotic parasites were targeted more frequently and more rapidly by eGFP-LC3 in hMDM2 as compared to phagosomes containing viable parasites. To identify factors that initiate apoptotic parasite internalization and LC3 recruitment, magnetic isolation of apoptotic Lm-containing phagosomes was performed. Subsequent mass spectrometry and data analysis revealed the phagocytic receptors low-density lipoprotein receptor-related protein 1 (CD91) and Complement Receptor 3 (CR3) to be highly abundant in phagosomes harboring apoptotic Lm. Blocking of CD91 with a monoclonal antibody was not efficient and knockdown of CD91 with siRNA did not affect uptake of Lm, but the interaction still might be relevant and should be investigated in more detail. Inhibition of CR3 with a functional antibody significantly reduced uptake of apoptotic Lm by hMDMs. This receptor blocking in turn resulted in a decreased production of pro-inflammatory TNF-α by hMDM1 and a significantly diminished IL-10 secretion by hMDM2. Inhibition of CR3 strikingly reduced LC3 lipidation as a measure for LAP induction in hMDM2 challenged with apoptotic or stationary phase Lm. Intriguingly, decreased internalization of apoptotic Lm neither resulted in a higher T cell proliferation nor in an increased survival of viable Lm within hMDMs. In conclusion, the data for the first time describe CR3 to play a significant role in the interaction of hMDM2 with especially apoptotic Lm and indicate a role of the receptor in LAP. Control of receptor-mediated internalization and LAP of apoptotic Lm could serve as a novel therapeutic approach to combat the neglected tropical disease leishmaniasis.