No abstract

The complement system provides host defense against pathogens and environmental stress. C3, the central component of complement, is present in the blood and increases in the bronchoalveolar lavage fluid following injury. We recently discovered that C3 is taken up by certain cell types and cleaved intracellularly to C3a and C3b. C3a is required for CD4+T cell survival. These observations made us question if complement operates at environmental interfaces, particularly in the respiratory tract. We found that airway epithelial cells [AECs, represented by both primary human tracheobronchial cells (hTECs) and BEAS-2B (cell line)] cultured in C3-free media were unique from other cell types in that they contained large intracellular stores of de novo synthesized C3. A fraction of this protein reduced ("storage form") while the remainder did not, consistent with it being pro-C3 ("precursor form"). These two forms of intracellular C3 were absent in CRISPR knockout-induced C3-deficient AECs and decreased using C3 siRNA, indicating endogenous generation. Proinflammatory cytokine exposure increased both stored and secreted forms of C3. Further, AECs took up C3 from exogenous sources, which mitigated stress-associated cell death (e.g., oxidative stress, starvation). C3 stores were notably increased within AECs in lung tissues from individuals with different end-stage lung diseases. Thus, at-risk cells furnish C3 through biosynthesis and/or uptake to increase locally available C3 during inflammation, while intracellularly, these stores protect against certain inducers of cell death. These results establish the relevance of intracellular C3 to airway epithelial biology and suggest novel pathways for complement-mediated host protection in the airway.

The complement system is an evolutionarily ancient component of immunity that revolves around the central component C3. With the recent description of intracellular C3 stores in many types of human cells, our view of the complement system has expanded. In this article, we hypothesize that a primitive version of C3 comprised the first element of the original complement system and initially functioned intracellularly and on the membrane of single-cell organisms. With increasing specialization and multicellularity, C3 evolved a secretory capacity that allowed it to play a protective role in the interstitial space. Upon development of a pumped circulatory system, C3 was synthesized in large amounts and secreted by the liver to protect the intravascular space. Recent discoveries of intracellular C3 activation, a C3-based recycling pathway and C3 being a driver and programmer of cell metabolism suggest that the complement system utilizes C3 to guard not only extracellular but also the intracellular environment. We predict that the major functions of C3 in all four locations (i.e., intracellular, membrane, interstitium and circulation) are similar: opsonization, membrane perturbation, triggering inflammation and metabolic reprogramming.

A key component of both innate and adaptive immunity, new understandings of the complement system are expanding its roles beyond that traditionally appreciated. Evidence is accumulating that complement has an intracellular arsenal of components that provide not only immune defense, but also assist in key interactions for host cell functions. Although early work has primarily centered on T cells, the intracellular complement system likely functions in many if not most cells of the body. Some of these functions may trace their origins to the primitive complement system that began as a primeval form of C3 likely tasked for protection from intracellular pathogen invasion. This later expanded to include extracellular defense as C3 became a secreted protein to patrol the vasculature. Other components were added to the growing system including regulators to protect host cells from the indiscriminate effects of this potent system. Contemporary cells may retain some of these vestigial remnants. We now know that a) C3 serves as a damage-associated molecular pattern (in particular by coating pathogens that translocate into cells), b) most cells store C3 and recycle C3(H2O) for immediate use, and c) C3 assists in cellular survival and metabolic reprogramming. Other components also are part of this hidden arsenal including C5, properdin, factors H and B, and complement receptors. Importantly, better definition of the intracellular complement system may translate into new target discovery to assist in creating the next generation of complement therapeutics.

Summary Complement is traditionally thought of as a pro-inflammatory effector mechanism of anti-tumor immunity. However, complement is also important for effective clearance of apoptotic cells, which can be an anti-inflammatory and tolerogenic process. We show that localized fractionated radiation therapy (RT) of subcutaneous murine lymphoma results in tumor cell apoptosis and local complement activation. Co-treatment of mice with tumor-targeted complement inhibition markedly improved therapeutic outcome of RT, an effect linked to early increases in apoptotic cell numbers and increased inflammation. Improved outcome was dependent on an early neutrophil influx, and was characterized by increased numbers of mature dendritic cells and the subsequent modulation of T cell immunity. Appropriate complement inhibition may be a promising strategy to enhance a mainstay of treatment for cancer.