Complement: The Road Less Traveled

The complement system is an ancient pathogen recognition receptor system discovered by Jules Bordet in the late 19th century (1). It consists of over 50 proteins that are mostly generated by the liver and either circulate in the fluid phase or are membrane bound. Most complement core proteins exist in a preenzymatic form and are activated in a sequential and cascade-like fashion (2–4). There are three activation pathways: the classical pathway, the alternative pathway, and the lectin pathway (4). The three pathways are triggered by distinct pathogens or noxious (self) Ags but then cumulate at the activation of the complement core proteins C3 (C3a and C3b) and C5 (C5a and C5b) via the formation of C3/C5 convertases. C3 and C5 activation leads to the formation of the membrane attack complex that forms pores on the pathogen surface and induces its lytic killing (2, 5). The anaphylatoxins C3a and C5a signal through G protein–coupled receptors C3aR and C5aR, leading to the recruitment and activation of immune cells. C3b (and further breakdown products) are strong opsonins and mediate the phagocytic uptake and clearance of pathogens via scavenger cells (6). The complement system is regulated by a group of proteins under the umbrella “regulators of complement activation” that are placed at strategic locations along the pathways (7).

Overall, complement is recognized as the surveillance mechanism and the first line of host defense against pathogenic invasion (Fig. 1), and complement deficiencies are thus often associated with recurrent infections (8). Importantly, because complement is central to the detection and removal of self-derived danger (for example, apoptotic cells and immune complexes [ICs]), complement deficiencies can also cause autoimmune diseases such as systemic lupus erythematosus (9, 10). Furthermore, unwanted, uncontrolled, and prolonged complement activation are all connected with a range of highly prevalent inflammatory disease conditions, including arthritis and cardiovascular disease (11, 12). Thus, complement has been known for decades to be an important therapeutic target, and much time and effort have been invested in “resetting” hypercomplement activation in acute and chronic inflammation.

Over the last decade, unexpected additional locations of complement activation and function have been identified. Liver-derived and serum-circulating complement proteins are accepted as key in fighting blood-borne pathogens (2, 4, 13). However, studies from as early as the 1980s have indicated that many cell types can synthesize and secrete a range of complement components into the close environmental space (14–17) across tissues and organs ranging from brain (18, 19) and eye (20) to kidney (21, 22) and intestine (23). Such local complement production is biologically important for protection against infections because, for example, monocyte- and macrophage-derived C3, C4, and C5 components are required additional drivers in tissue protection against viruses and bacteria (14, 24, 25). An additional layer of complexity with regard to complement activity was added by the finding that complement can also be activated within cells and can engage complement activation receptors in subcellular compartments. The activity of intracellular complement (the “complosome”) (26) is associated with noncanonical complement functions because it controls basic cellular processes such as cell metabolism and autophagy. For example, C3a generated intracellularly by human CD4⁺ T cells engages the lysosomal C3aR and sustains T cell survival via the activation of tonic mammalian target of rapamycin (27). The cell-autonomous engagement of the complement regulator/receptor CD46 during T cell activation by T cell–generated C3b induces metabolic reprogramming needed for Th1 induction and CTL activation (28, 29). Interestingly, such complement-driven cell-autonomous immunity can also be triggered by C3 fragments that have been carried into the cells’ interior by pathogens that were opsonized with C3 fragments in serum (30).

Thus, the novel insights into complement biology across host tissues, in combination with the development of next-generation complement therapeutics, hold promise to tackle human inflammatory diseases more effectively. The International Complement Society (ICS) organizes an ICS Guest Symposium at the annual conference of the American Association of Immunologists (AAI) with the aim to inform about the progress and particularly the exciting findings in complement research and to engage immunologists across disciplines. At the 2022 AAI conference in Portland, OR (May 6–10), Jessy J. Alexander (University at Buffalo), and Jeanne T. Paz (Gladstone Institutes and University of California, San Francisco) presented unexpected new findings on complement activities in the kidney and in the brain, respectively. Furthermore, Michail S. Lionakis (National Institute of Allergy and Infectious Diseases, National Institutes of Health) gave an update on the role of complement in fungal infections and hinted at a new noncanonical complement activity in the protection against candidiasis. The ICS Guest Symposium session was concluded by V. Michael Holers (University of Colorado), who provided an overview of the complex roles played by complement during the natural history of rheumatoid arthritis (RA). In this brief review, we provide a summary of these presentations with appropriate biological background information and discussion on their future implications for complement research and beyond.

The kidney is an immunologically active organ (0.5–1% body weight) with 20–25% (1–1.2 L/min) of cardiac output traversing the kidney and the glomerulus filtering 90–120 ml/min (31). The kidney becomes a multipronged intersection where the pathogen induces complement activation and complement evasion strategies, and activated complement instructs local innate and adaptive immune system responses. Also, the kidney generates ammonia that interacts with C3 to activate the complement alternative pathway (32). The kidney is a major extrahepatic site of complement production with different kidney cells synthesizing both C3 and C5 and complement regulatory proteins such as CD46, CD55, CD59, and Crry (33, 34). Studies show that along with IC-mediated diseases such as lupus nephritis and IgA nephropathy, where complement levels are a part of the clinical evaluation, the complement system is also engaged and causes pathology in diseases including antineutrophil cytoplasmic Ab (ANCA)-associated vasculitis (35) and dense deposit disease (36). Complement inhibitors are effective in diseases such as atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, and ANCA-associated vasculitis but shows only limited benefit in IC-mediated diseases such as lupus and RA (37–41). ICs are present in 45–65% of patients with glomerular disease (42–44), and they have varying patterns of glomerular deposition.

As mentioned in the Introduction, complement activation occurs intracellularly across cells and dictates cellular behavior (26). However, its impact on tissue homeostasis is just beginning to emerge. Dr. Alexander addressed the question whether understanding complement compartmentalization, intrinsic complement, and its functions better could be helpful in increasing complement therapeutic effectiveness and disease management. To address this, she focused on the central complement pathway regulator factor H (FH) (4). FH synthesized by the liver restrains the complement cascade particularly at the alternative pathway and maintains C3 levels (2, 45). Here, Dr. Alexander presented novel data showing that FH could be serving as the “guardian” (21) from within the kidney endothelial cell (Fig. 1). She showed that both human and mouse kidney endothelial cells express FH. Also, reducing endothelial FH levels resulted in altered expression of MASP (mannan-binding lectin-associated serine protease), C1s, and C4, which are initiating proteins of the lectin and classical pathways, suggesting cross-communication or -regulation among the three complement pathways (46). Absence of FH also altered the actin cytoskeleton with the formation of stress fibers leading to increased endothelial layer permeability (46). The kidney endothelium is a part of the glomerular filtration apparatus (47). Once the endothelial layer loses its integrity, the glomerular basement membrane that has no complement regulators (48) becomes exposed to large complement proteins and other toxins. In addition, actin cytoskeletal remodeling influences proliferation and angiogenesis, and Dr. Alexander showed data that FH deficiency altered these parameters in kidney endothelial cells. Replenishing intracellular FH by transfection reverted cell proliferation close to normal levels. Importantly, earlier work from her laboratory showed that C5a, the breakdown product of C5, causes similar changes of cytoskeletal remodeling and proliferation of brain endothelial cells (49). Thus, controlled local and/or intracellular complement activation may regulate endothelial cells across tissues. Moreover, a recent publication from another group showed that intracellular FH can drive kidney tubular epithelial cell turnover, epithelial–mesenchymal transition, and malignant transformation (50). Dr. Alexander’s laboratory is now focusing on understanding the roles of downstream effectors in FH-induced changes of kidney endothelial cells. Some findings include the observation that FH deficiency causes the translocation of NF-κB into the nucleus, where this transcription factor then modulates different innate and adaptive immune responses and plays a critical role in mediating inflammatory responses. Overall, the results presented suggest that modulating FH in kidney endothelial cells could be an effective target for maintaining glomerular homeostasis.

Of course, these are early insights, and understanding the complexities of and potential cross-communications between intracellular, cell-autonomous, and serum-derived complement will require substantial future work and research efforts. However, this will likely be rewarding because inflammation of the vasculature is common to the broadest range of diseases, unfortunately as recently demonstrated by the SARS-CoV-2–induced COVID-19 (51, 52), with local and/or widespread vessel inflammation being one of its cardinal features.

In her presentation, Dr. Jeanne T. Paz kept with the scheme of dissecting the roles of locally generated complement in tissue biology. The brain is a uniquely placed and immune-privileged organ, separated from systemic effects by the presence of the blood–brain barrier (53), in which endothelial cells with intricate junctional formations prevent the influx of immune cells and large proteins. The brain comprises a complex assembly of functionally different cell types, such as neurons that transmit impulses; oligodendrocyte cells that generate myelin; astrocytes that maintain homeostasis; and microglia, the critical immune cells in the brain (54). Rather unexpectedly, research on the complement system in the brain was and is one of the major drivers in our understanding of extrahepatic and local complement activity (55, 56). Because the brain is considered immune-privileged, one might expect any presence of complement to be a sign of disease/neuroinflammation. However, complement in the brain is actually a driver of normal development and homeostatic tissue activity (Fig. 1). So far, most cells assessed in the brain synthesize complement proteins. Complement produced in the brain is involved in neurogenesis (57–59), synaptic pruning (60, 61), scavenging apoptotic debris (62–64), and the response to and removal of inflammatory insults. However, excessive and prolonged complement activation does contribute to neurodegenerative disease and to neuropathies during aging (58, 65–67). In addition, complement activation in the periphery heavily impacts the brain: The anaphylatoxins C3a and C5a generated during systemic complement activation render the blood–brain barrier leaky (49) and can foster cancer metastasis (68). There is a substantial body of literature on these subjects, and we guide the reader to those for more detailed insights on the exciting and diverse roles of complement in the CNS (61, 65, 69).

Dr. Paz presented data that further substantiated our understanding that complement can indeed play a very sinister role in the brain, specifically during traumatic brain injury (TBI). TBI is a leading cause of disability in children and adults (70). Although TBI acutely disrupts the cortex, the outmost part of the brain, most TBI-related disabilities reflect secondary injuries that accrue over time as consequences of the initial impact. Understanding where, when, and how secondary injuries develop is critical for preventing disability after TBI. The Paz group applied spatial transcriptomics in a mouse model of TBI and found C1q and C4 among the top differentially upregulated genes 5 wk after TBI in a subcortical brain region called the thalamus, which is reciprocally connected with the impacted cortex (71). Using immunohistochemistry and in vivo electrophysiology, they noted that increased C1q expression colocalized with neuron loss and chronic inflammation and correlated with disruption in sleep spindles and emergence of epileptic activities. Mice treated with an Ab that blocks the complement-activating effect of C1q ameliorated these pathological features, suggesting that C1q is a disease modifier in TBI. Using single-nucleus RNA sequencing, the Paz group pinpointed microglia as the source for generating the detrimental C1q production and suggested overall that the activation of the classical complement pathway in the thalamus could be a target for treating TBI-related disabilities (71). The latter is a notion that the laboratory is now following up with further studies.

The role of serum circulating “classic” complement in the protection against fungal infections is mostly understood among complementologists. However, the functions of cell-autonomous and intracellular complement activities during pathogen sensing and removal have not yet been explored, but this is an area of growing interest. Dr. Michail S. Lionakis gave an update on complement in fungal infections and shared some initial insights into new and ongoing work on complement and candidiasis in his laboratory. In his presentation, Dr. Lionakis first summarized the published work on the role of complement in host defense against systemic candidiasis (72–75). Fungal infections during hospitalization are a common occurrence, especially in critically ill patients in the intensive care unit. In a 2019 Centers for Disease Control and Prevention report, fungi were among the major pathogens that were drug resistant in the United States (76). Along with increased infections by nosocomial pathogens, fungi that are emerging due to climatic and other environmental changes enhance the risk further (77, 78). Improving fungal diagnostic modalities and identifying effective therapeutic targets are urgent needs. The recent introduction in the clinic of the C5-targeted mAb eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria and hemolytic uremic syndrome has uncovered a critical contribution of C5a signaling in antifungal host defense because eculizumab-treated patients were reported to develop meningococcal infections and systemic candidiasis with high mortality (79, 80), as well as invasive pulmonary aspergillosis (81). These observations in humans were unexpected because of the lack of reported invasive fungal disease in patients with inherited C5 deficiency (Fig. 1). Patients with C5 deficiency are typically at high risk for invasive infections by encapsulated bacteria (82). This susceptibility prompted the U.S. Food and Drug Administration to update the eculizumab package insert in 2018 to warn of the risk of invasive fungal infections in addition to pyogenic bacterial disease in vulnerable patients receiving the drug.

Prior work had examined inbred DBA/2 and A/J mouse strains, which are C5-deficient, and had found these strains to have significantly greater mortality after systemic challenge with Candida albicans (72–74) in a model that relies heavily on phagocyte-mediated innate, not lymphocytic, responses for effective host defense (77). These inbred mice had greater fungal burden in several but not all examined tissues after infection associated with enhanced proinflammatory responses in infected organs (72–74). Yet, the mechanisms of impaired phagocyte-dependent immune responses were not thoroughly examined in those studies, and the inbred DBA/2 and A/J mouse strains exhibit additional immunological defects beyond C5 deficiency (83, 84). Another study examined C3-deficient mice after systemic challenge with C. albicans and Candida glabrata and found them to exhibit increased mortality after infection with associated enhanced levels of proinflammatory cytokines in infected tissues; yet, the precise mechanisms of impaired host defense in the absence of C3 were not defined (75). Recent work from the Lionakis laboratory dissected the mechanisms by which complement critically contributes to host defense against invasive fungal disease. This is particularly timely because the rising infections by systemic candidiasis and the multidrug-resistant Candida auris (78, 85) infections in response to treatment with eculizumab (79, 86) and other novel complement pathway inhibitors have become a major clinical problem during treatment of various vulnerable patient populations. Dr. Lionakis presented unpublished data that shed light on the mechanisms by which C5a-C5aR1 signaling promotes protective phagocyte-dependent host defense against invasive candidiasis in mice and humans (J.V. Desai, D. Kumar, T. Freiwald, D. Chauss, M.D. Johnson, M.S. Abers, J. Steinbrink, J.R. Perfect, B. Alexander, V. Matzaraki, M.A. Zarakas, V. Oikonomou, L. Wang, J.K. Lim, D. Launder, H.R. Conti, M. Swamydas, M.T. McClain, M. Kazemian, M.G. Netea, V. Kumar, J. Köhl, C. Kemper, B. Afzali, and M.S. Lionakis, submitted for publication). The importance of complement activation in antifungal immunity was highlighted and underscores the need for careful surveillance of patients treated with various complement pathway inhibitors for the development of opportunistic fungal disease.

The three presentations detailed above are examples of changes in our thinking about complement biology and complement in human disease. Among the key realizations in our field is the understanding that complement operates at different and unexpected locations, that is, in circulation (liver), locally (cell derived), and intracellularly (cell autonomous). In addition, a given role of complement in a specific cell manner also changes with the activation state of that cell and often in a temporal fashion (87, 88). Consequently, we need to understand the distinct roles of complement at different locations and over the course of complement engagement (onset, progression, resolution) to fully comprehend its role in normal biology and to tackle the system optimally in diseases. This notion was then brought beautifully forward and applied to the pathogenesis of RA by the presentation of V. Michael Holers.

RA is a chronic autoimmune disease whose exact etiology is not completely understood. The triggers include mucosal inflammation and dysbiosis (89). RA most often affects the peripheral small joints, causing the synovium to thicken by accumulating and incorporating immune cells to form the pannus that destroys cartilage and bone (90). The lectin pathway, classical pathway, and alternative pathway are likely all activated in RA by diverse triggers (11, 91) such as autoantibodies, necrotic cells, or exposed collagen and thus important contributors to the disease pathology in humans and in mouse models of the disease. The activation of major complement components C3 and C5 (92) are increased in the synovium in RA, indicating perpetuated local complement activation. The presentation by Dr. Holers provided a somewhat different angle on complement and RA: Instead of paying attention, as most do, to the symptomatic site, the inflamed joint, his talk focused on the role of the complement system throughout the evolution of RA (Fig. 1). Dr. Holers stressed the fact that RA exhibits a prolonged preclinical period of time that involves the development of chronic mucosal inflammation, especially prominent in the lung and intestine, which is associated with the local production of RA-related autoantibodies designated anticitrullinated protein Abs (93) and rheumatoid factor (94). His team helped spearhead detailed analyses of intact and cleaved complement activation components in the sputum of individuals with preclinical RA (95). This work detailed the presence of complement activation fragments that are associated with neutrophil extracellular trap formation and elevated cytokines and chemokines (96). Furthermore, the observed complement activation during this earlier preclinical phase is causally associated with the development of neutrophil extracellular traps and autoantibody generation. Transition of the disease to involve the synovium was also associated with informative and specific patterns of complement gene expression and localization of activation fragments and receptors. As a major outcome, the team noted positive associations in early RA between a subset of synovial but not peripheral blood cell complement gene expression levels encoding factors such as factor B and C5aR1 with clinical disease activity. Conversely, and somewhat unexpectedly, C5 gene expression itself is inversely associated with disease activity. Immunohistochemical analyses of the inflamed tissue sites demonstrated regional differences in complement factor localization and inverse relationships between activation fragments and regulatory molecules. Therefore, RA is a disease with substantial opportunities for therapeutic impact, and specific attention to early disease and regulation of the C3/C5 convertases may be particularly worthy.

The complement system was thought to be fully functionally defined and well understood for many years. Novel complement insights and discoveries over the last two decades, however, have proved us wrong. Complement is not only a proinflammatory pathogen fighter but also at the heart of normal cell and tissue development and function. The system not only actively operates body-wide in serum but also participates on a cellular and subcellular level in basic cell physiology and regenerative processes. The exact modes of specifically the latter, noncanonical complement activities and their regulation are incompletely understood and therefore need to be explored further. Much progress has been made in several areas of complement therapeutics through the development of drugs such as eculizumab (41), avacopan (97), and C1 inhibitor, among others, as well as ravulizumab (40) (a long-lasting C5a inhibitor) and sutimlimab (an inhibitor for C1s), that are approved for diseases such as paroxysmal nocturnal hemoglobinuria, ANCA vasculitis, and cold agglutinin disease, rendering these diseases more manageable. However, a larger number of complement therapeutics have not delivered in the clinical and/or more common disease settings. Understanding the recently discovered noncanonical complement activities on a molecular level and their interplay with the classic, circulating components as well as with other pathogen recognition receptor systems may provide new opportunities for drug development. Areas that need more in-depth exploration include the role of cell-autonomous complement in stromal and parenchymal cells in RA (98), the impact of the mucosal microbiota on local complement activities in health and disease, and the role of complement in pain perception and illness behavior. Finally, one of the hottest subjects in the complement field is its clear impact on cancer (for better or for worse), and we expect substantial new insights here soon (50, 99–104).

We thank the patients, researchers, and clinicians who have added to our understanding of complement biology over time. This brief review is about the recent advances in the complement field presented at the AAI and is not meant to be an in-depth assessment of complement. Thus, we apologize to the many researchers whose work should be acknowledged but was not cited because of space constraints.

The authors have no financial conflicts of interest.