Abstract
Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.
Highlights
The complement system is an essential part of the innate and adaptive immune system to eliminate foreign antigens and pathogens as part of the normal host response [1]
C3aR transfer from the plasma membrane to the mitochondria via endosomal particles was demonstrated in oxidatively stressed H2O2-treated cells
Mitochondrial C3a receptor activation produced two critical responses; enhanced Ca2+ uptake leading to increased inner mitochondrial Ca2+concentration, and a reduction of mitochondrial respiration resulting in reduced ATP production and impaired maximal respiratory capacity
Summary
The complement system is an essential part of the innate and adaptive immune system to eliminate foreign antigens and pathogens as part of the normal host response [1]. The complement system is activated by various types of stimuli, such as oxidative stress, inflammatory factors, and ischemia [2]. Depending on the pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), the complement system can activate 3 distinct pathways: the classical pathway (CP), lectin pathway (LP), and alternative pathway (AP). The 3 pathways all participate in the formation of C3 convertases that will catalyze the proteolytic cleavage of complement component 3 (C3) into C3a and C3b. C3b subsequently participates in the formation of a C5 convertase that cleaves complement component 5 (C5) into C5a and C5b. Anaphylatoxins have multiple roles, including vasodilation and enhanced vascular permeability, as well as the mediation of chemotaxis and inflammation, and the generation of cytotoxic oxygen radicals [4], which are mediated by their receptors C3aR, C5aR, and C5L2 [5]
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