Canonical and non-canonical autophagy modulation in human primary macrophages and its effect on the adaptive immune system
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Abstract
In this study we focused on the modulation of autophagy mechanisms in human monocyte derived macrophages (hMDM) focusing on (i) modulation of autophagy in pro- (hMDM-1) and anti-inflammatory (hMDM-2) macrophages as prototypic immunomodulatory cells, (ii) the role of autophagy in antigen processing and presentation, (iii) the impact of the Leishmania virulence factor GP63 on the host cells’ autophagy machinery and adaptive immunity and (iv) LC3-associated phagocytosis (LAP) as immune evasion mechanism for Leishmania.
Autophagy in hMDM-1 and hMDM-2 was induced by the chemicals Rapamycin, AZD8055 and PI-103 as well as by the peptide Tat-Beclin. In general, hMDM-2 were more susceptible for autophagy induction. Autophagy inhibition was achieved by Spautin-1 and Wortmannin treatment in both phenotypes and with LY294002 only in hMDM-1. Using RNA interference to achieve autophagy inhibition, a time- and target-dependent efficiency of protein reduction was observed. Interestingly, autophagy could be induced independently of ULK-1 and Beclin-1 in hMDM-1. Autophagy modulation in hMDM had no impact on Tetanus Toxoid induced T cell proliferation. Surprisingly, we found hMDM-2 to be superior to hMDM-1 in activating lymphocytes. Proteome and surface marker analysis revealed higher expression of proteins being involved in antigen processing and presentation in hMDM-2.
Based on the hypothesis that apoptotic Leishmania (Lm) induce the non-canonical autophagy pathway LAP resulting in a reduced T cell proliferation, we aimed to modulate LAP and analyzed the effect on the adaptive immune response. Replacing apoptotic parasites by LAP inducing stimuli such as zymosan and phosphatidylserine coated beads suppressed the Leishmania induced T cell proliferation and consequently enhanced intracellular parasite survival. Analyzing the underlying mechanisms of LAP in hMDM, inhibition of the NADPH oxidase by DPI blocked LAP induction. In addition, infection of hMDM with Staurosporine-treated Lm, being ROS positive, increased LC3 conversion which suggests ROS-dependent LAP induction in hMDM. Infection with apoptotic Lm and zymosan leads to enhanced phagolysosomal acidification, which might be a potential mechanism for altered antigen processing resulting in a reduced T cell proliferation.
In conclusion, these data provide a better understanding of autophagy in hMDM being a potential immune evasion mechanism for Leishmania. The obtained results may contribute in the development of safe and efficient therapeutic interventions in humans for the treatment of autophagy-related diseases.