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The complement alternative pathway in health and disease—activation or amplification?

2022; Wiley; Volume: 313; Issue: 1 Linguagem: Inglês

10.1111/imr.13172

1600-065X

R. A. Harrison, Claire L. Harris, Joshua M. Thurman,

Immune responses and vaccinations

In late 2020, one of us (RAH) was approached by the late Professor Sir Peter Lachmann and invited to join him in co-editing a collection of articles focused on the alternative pathway (AP), or amplification loop (AL), of C3 activation. He had recognized that much had changed since his first proposal of "C3 tickover." New controversies had arisen, significant advances in our understanding of complement involvement in disease had been made, and, with this, there had been a considerable stimulation of interest in complement-directed pharmaceutical intervention. Re-evaluation of the pathway was required for effective exploitation of new therapeutic opportunities. Sadly, Peter passed away while the collection was still in the content planning stage,1 but, joined by CLH and JMT, and together with Immunological Reviews, we have driven the project forward. Issue 313 is the culmination of this effort. We made a conscious decision not to make this a comprehensive collection of articles covering every aspect of the AP/AL, but instead to focus on aspects that either remain controversial, or which have re-ignited controversy, or whose understanding is still in its infancy. Thus, of the components of the AP/AL, only Factor D (FD), Properdin (P), and Factor H (FH) and its related proteins Factor H-like 1 (FHL-1) and the Factor H-related (FHRs) proteins are discussed in detail. That said, Factor B (FB) and C3 are more than adequately covered in other contributions. Component-specific contributions lead to fundamental questions regarding basic mechanisms of the AP/AL of complement. The focus then moves to diseases in which there is high probability of the AP/AL being a key driver of pathogenesis, starting with discussion of how animal studies have helped our understanding of these. Finally, the collection concludes with consideration of complement-directed therapeutics, with a focus on those that directly address AP/AL dysregulation. While Peter (Figure 1) made many contributions to our understanding of complement, that for which he is best recognised is his proposal, in 1973, of the "C3 tickover" hypothesis.2 The tickover hypothesis was elegant in its simplicity—it merely stated that C3b was continually generated in blood, but without addressing the mechanism by which this occurred. In making this hypothesis, he leaned very heavily on a marriage between his understanding of complement and, in particular, the properdin pathway as it was then known, and analysis of complement involvement in rare diseases. The C3 tickover hypothesis gave a key insight into how the AP/AL functioned in vivo, providing essential surveillance and an immediate innate immune response to invading pathogens in the absence of any adaptive immunity. It was not immediately accepted, with much effort in many laboratories spent in subsequent years in attempts to provide a more conventional mechanism for AP activation and "firing" of the AL (Figure 2). However, to quote from Pangburn's contribution later in this issue: "50 years after it was proposed nothing has replaced the tickover hypothesis, attesting to Peter Lachmann's unique insight and scientific boldness",3 and it is for this contribution in particular that many complementologists will best remember him. At its simplest, the alternative pathway for C3 activation requires just three proteins, C3, FB, and FD (Figure 3). Proteolytic activation of C3 to C3b, or generation of C3(H2O) by hydrolysis of its internal thioester bond,3 leads to assembly of C3bB or C3(H2O)B proenzyme complexes. FB bound to C3b or to C3(H2O) is the substrate for FD, and activation by FD generates the C3bBb or C3(H2O)Bb complexes, both of which can then cleave further C3 to C3b, creating a positive amplification loop for C3b generation. Unchecked, this would rapidly lead to exhaustion of C3, and hence the system is tightly regulated by the protease FI, which inactivates C3b, generating iC3b, which can no longer bind FB and participate in amplification of C3b generation. The FI-dependent inactivation of C3b requires a cofactor and, in the fluid phase, this is primarily provided by FH. In addition to providing cofactor activity, FH also accelerates the decay of C3bBb, C3(H2O)Bb, and their precursor complexes, limiting their potential to participate in amplified C3b generation. In the fluid-phase, in healthy individuals, down-regulation, not amplification, predominates. However, C3 has a further property, shared with C4 (and the α2-macroglobulin family), of an internal thioester.4 Proteolytic activation exposes the thioester, which has a transient ability to react with hydroxyl- or amino-functions on other proteins or carbohydrates, providing a long-lived "C3-tag".5 This initial C3b-binding to an adjacent surface is non-discriminatory, and it provides a "fixed" location on which C3b generation can be amplified. The stability of surface-bound C3 convertases is also enhanced by the recruitment of properdin, until recently regarded as the only positive regulator of the AP/AL. Clearly, such non-discriminatory deposition requires that host cells are adequately protected against damaging complement attack. This is provided both by the membrane-bound regulators, CD46 (Membrane Cofactor Protein, MCP), CD55 (Decay Accelerating Factor, DAF), and CD35 (Complement Receptor 1, CR1), and by FH recruited to polyanionic surfaces. The interaction of FH with C3b and/or polyanionic surfaces is complicated by the more recently discovered Factor H-related (FHRs) proteins.6 FHRs compete with FH in binding both to C3b and to surfaces and have been termed FH-deregulators. As such they too can act as positive regulators of the AP/AL. Their discovery also means that some of the earlier work on AP/AL activation and regulation might need to be re-interpreted to take into account their possible interference in assays. Nevertheless, despite these complexities, under normal circumstances host cells exposed in blood or plasma to a fully competent AP are fully protected against the damaging effects of amplified C3 activation and C3b deposition. In contrast, pathogens lack these regulatory mechanisms, and are subjected to an amplified AP/AL-dependent response, with ensuing recruitment of C3- and C5-dependent effector functions of complement. The importance of this "first-line" defense against infection is evidenced both by the increased infection risk in complement-deficient individuals, particularly in early years when the repertoire of adaptive responses will be limited, and by the considerable spectrum of complement evasion mechanisms that pathogens have evolved.7 In their review of FD, Sekine et al8 address a fundamental challenge that has recently given rise to some controversy, the nature of circulating FD. For the AP/AL to function as an "oven-ready" surveillance mechanism as envisaged in the tickover hypothesis, sufficient FD must circulate in the active form for its activity not to be rate limiting. Recent discoveries regarding the involvement of the mannan-activated serine protease 3 (MASP-3) in the activation of FD cast doubt on this; these are addressed in detail, with reference both to the consequences of FD deficiency and to evolutionary aspects of the AP/AL. One conclusion from their manuscript is that FD circulates in man predominantly in the active form, sufficient to enable tickover surveillance, removing doubts that had been raised over this fundamental function of complement. Related to this are recent data that suggest that FD might not be the rate-limiting enzyme of the AP/AL that it has long been assumed to be.9 The Factor H related proteins (FHRs) are the most recent "additions" to the AP/AL regulator family. In their article, de Jorge et al give a comprehensive overview of this family of proteins, discussing their relationship to each other and their functional interactions, with a timely update of current understanding of their mechanism of action. In this they draw not just on biochemical and molecular biological analysis, but also on disease associations of the different members of the family.6 They also discuss the intriguing possibility that one driver of their evolution and divergence could be a need to counter a complement evasion mechanism adopted by some pathogens, that of recruiting FH to their surface to counter AP/AL attack. In some bacteria it has been demonstrated that specific FHRs can compete with FH binding to the cell membrane, diminishing the bacterium's ability to mimic host cells. This might account for the considerable differences between FHRs in mice and humans (and other species), with FHRs evolving to counter species-specific pathogens. Despite it being the first component of the AP/AL to be purified (at that time C3' comprised not just C3, but also many of the lytic pathway components), properdin has proven one of the most difficult components to analyze. In part, this is because of its propensity to self-aggregate during isolation, aggregated properdin having properties that are not found in the native protein.10 Pedersen and co-workers present an elegant description of properdin function, largely drawn from structural insights, with novel data.11 They also bring a structural focus to the interactions of properdin with pathogens and some of their complement-evasion strategies. The role of the unusual C-mannosyl glycosylation of tryptophan residues remains enigmatic however. This glycosylation is seen too in the thrombospondin repeats of C6, C7, C8, and C9, and it has been suggested that glycosylation plays a role in the folding and stability of the thrombospondin domain.12 It would interesting to know whether these glycans interact with MBL; recruitment of MBL by surface-bound properdin, and hence MASP-3, could provide a local environment for enhanced FD activation, ensuring that it does not become rate limiting at high C3bB density. In typically erudite fashion, Liszewski and Atkinson13 set the scene for discussion of basic AP/AL mechanisms. They start with a timely reminder that a primitive surveillance system probably first arose as an intracellular system stored in intracellular vesicles, and go on to consider its evolution, not just the role in defense against pathogens, but also in maintenance of the healthy host, as a "vacuum cleaner" removing cell debris. In recent years, a number of authors have questioned current dogma over the role that water-hydrolysed C3 (now known as C3(H2O)) plays in triggering AP/AL activation.14-16 Over 40 years on from his discovery that C3(H2O) has C3b-like properties,17 Pangburn revisits the narrative.3 In particular, he addresses, through kinetic considerations, the extremely low probability that nascent C3b generated from a fluid-phase C3(H2O)Bb C3 convertase will engage with a surface, and suggests that other facilitatory mechanisms, for example, distortion of C3 when it interacts with specific surfaces such that the thioester is exposed and can react with surface groups,18 or "trace activation" of C3 through the LP or CP C3 convertases, might play a part. Interestingly, non-canonical proteolytic activation, for example by neutrophil proteases released at inflammatory sites, is not considered. Rodriguez de Cordoba19 brings clarity to a highly complex subject, that of genetics of the AP/AL components. In his wide-ranging contribution, he discusses not only "classical" aspects, but also exploration of rare variants, and the contribution that they make to disease both uniquely and through inherited complotype associations.20 He also reminds us of the relevance of eQTLs, a topic he first engaged with over 20 years ago in analyzing the basis for wide individual differences in FH levels.21 In their contribution, Ekdahl et al18 give a highly focused consideration of the impact of biomaterials such as those encountered in extracorporeal circuits on activation of the AP/AL. Despite much work to improve the compatibility of biomaterials, these remain "non-self" to complement, and the effects of extracorporeal activation are not restricted to the extracorporeal circuit, but spill over into the patient, with adverse clinical consequences. One of the greatest controversies that has arisen in the complement field in recent years has been that of intracellular complement. Much of this controversy has arisen because of a lack of clarity over what is being proposed, and what, in this context, is meant by "intracellular." As has been noted above, a primitive AP/AL system probably evolved in primordial organisms lacking a circulation, possibly within intracellular vesicles released into the environment when the organism was challenged. From this perspective, the recently described intracellular functions of complement are less radical than they might otherwise appear. King and Blom bring a much needed structure to discussion of possible canonical and non-canonical activities of complement in the intracellular environment.22 The immediate consequence of amplified C3 activation is enhanced C3-dependent effector function potential. In this context it is worth noting that while the FI-mediated cleavage of C3b to iC3b, and subsequently to C3c and C3dg, is often referred to as an inactivation event, it is merely the inactivation of the amplification potential of C3b. Surface-bound iC3b and C3dg remain very much active, but with different activities. These, as well as the properties and functions of the initial activation products of C3, C3a and C3b, are discussed in detail by Zarantonello et al.23 That C3 is important in innate immune defence is demonstrated by the consequences of inherited or acquired C3 deficiency and by defective regulation of C3 activation and breakdown. That C3-dependent activities are non-essential is demonstrated by the existence of colonies of C3-deficient guinea pigs and the normal development and function of C3−/− mice. This has significant implications for AP/AL-directed therapeutic intervention. It has long been known that there is extensive cross-talk between the complement and coagulation pathways. While this occurs at many different points in both proteolytic activation cascades, it is particularly apparent at the level of C3 activation. Beginning with an overview of both hemostasis and amplified C3 activation, Noris and Galbusera drive home the inter-relationship of the two systems, and its malfunction in thrombotic conditions.24 One interesting aspect of their discussion is that thrombotic thrombocytopenic purpura (TTP), a disease not commonly regarded as a disease of dysregulated complement activation, has an AP/AL activation component. This again has implications relevant to AP/AL-directed therapeutics. In their contribution, Shaughnessy et al7 focus on what has always been a key question not just for complementologists, but also more widely for immunologists and clinicians, the critical role of complement, and particularly amplified C3 activation, in innate immune defence against pathogens. While their focus is on bacterial infection, the relevance of complement-dependent killing mechanisms is apparent when one considers the many different complement-evasion strategies that pathogens utilize to evade complement effector mechanisms. One of the less appreciated aspects of these are bacterial proteases designed to inactivate C3, possibly highlighting the importance of countering "close-proximity" proteolytic activation by host proteases at sites of infection and inflammation. It has long been known that the complement system is a downstream effector mechanism of antibody-mediated injury in autoimmune diseases. Greater understanding of the AP/AL, however, has also revealed that the AP/AL is centrally involved in many diseases not driven by autoantibodies. In many cases, the first evidence of a role for the AP/AL was the discovery of genetic variants in AP/AL genes in patients with particular diseases ("experiments of nature").25-29 These genetic associations provided important clues that the AP/AL plays in both health and disease, including a critical role in fighting infection but also as a cause of tissue inflammation. As outlined above, the tickover theory itself owes its origin to close observation of a patient with congenital deficiency of factor I. Professor Lachmann's insights—that the AP/AL is always activated through tickover and that insufficient AP/AL regulation allows the system to proceed unchecked—capture the unique biology of the AP/AL. These features of the AP/AL also explain its role in diseases of widely varying etiologies. Unlike the other cellular and molecular components of the immune system, the AP/AL does not require a recognition molecule, but rather activation is based on the interplay of AP/AL proteins with each surface they encounter and the local balance of positive and negative regulators. Close study of human genetics has shown that variations in essentially all AP/AL-related genes can shift this balance and have a major influence on infectious diseases, autoimmune and inflammatory diseases, tissue homeostasis and metabolism, and cancer. In his introduction to AP/AL-driven diseases, Peters, gives a highly personal view of a journey that takes us from the initial tickover hypothesis through basic mechanistic understanding and roles in disease to seminal and ground-breaking therapeutic strategies to target complement-mediated disease.30 It is worth noting just how much of our current understanding has come from the study of patients with rare diseases. As reviewed by Shaughnessy et al,7 deficiencies of AP/AL activating proteins (FB, FD, or properdin) are strong risk factors for bacterial infections. Clearly, therefore, this arm of the immune system provides a key line of defence against pathogens. Dysregulated activation of the AP/AL, on the other hand, is a key driver of multiple different inflammatory diseases. Thus, the net balance of AP/AL activation/regulation may be a zero-sum game: a decrease in AP/AL regulation can be advantageous in protecting the body from infection, but this comes at the cost of increasing the tendency towards inflammatory diseases. Michael Holers shows how animal models of complement-mediated pathology have revealed the importance of amplification through the AP/AL, even when activation is initiated by autoantibodies through the classical and/or lectin pathways.31 Indeed, tissue damage in multiple autoantibody-mediated diseases is almost completely dependent on AP/AL amplification; these diseases include rheumatoid arthritis and systemic lupus erythematosus. Perhaps one of the most unexpected—and significant—discoveries regarding the role of the AP/AL in disease is that the AP/AL is critical to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Complement proteins are not abundantly deposited in the kidneys of affected patients, and plasma C3 levels do not significantly change with active disease.32 However, Jennette et al demonstrated that AP/AL-deficient mice are protected in a mouse model of disease, and complement inhibition was also protective.33 These studies led to clinical trials of complement inhibitory drugs in this disease, and complement inhibition is now an approved treatment for ANCA-associated vasculitis. This is a major success story in biomedical research, as basic and pre-clinical studies led to development of a new treatment for a life-threatening disease. In addition to detailed analyses of samples from patients with complement-mediated conditions, elegant animal models have helped investigators to explore the mechanistic role of the AP/AL in disease. As outlined by Gibson et al34 in this issue, many animal models have been developed in response to observations in human patients. While mice are not men, particularly with respect to AP/AL regulatory proteins, these models have allowed investigators to confirm whether defects in the complement system that are identified in patients are causally related to disease. It has been shown, for example, that disease associated genetic variants first identified in patients with C3G cause similar disease when transgenically expressed in rodents.35, 36 Studies in animal models have also allowed investigators to test whether complement inhibition is an effective approach for a particular disease, and these pre-clinical studies have been instrumental in bringing complement therapeutics to the clinic. The necessity for reliable and accurate assays for defining complement involvement in disease should not be underestimated—without them there is always the danger that patients will be inappropriately treated, and that treatment outcomes might be misinterpreted. Thurman and Fremeaux-Bacci37 give a comprehensive overview of today's armamentarium of AP/AL relevant assays. Daina et al38 review the extensive data linking the AP/AL with the pathogenesis of various kidney diseases. The two prototypical complement-mediated kidney diseases are atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). A wide range of underlying molecular defects in AP/AL regulation have been discovered in patients with these diseases. aHUS and C3G are associated with genetic variants in AP/AL genes, for example, and with autoantibodies to complement proteins. In both diseases, too, congenital or acquired defects in complement regulation by the alternative pathway regulator FH are among the strongest risk factors for disease, yet disease penetrance is incomplete. Patients who share the same molecular impairments in AP/AL regulation can present with either of these diseases, and family members with the same genetic variants often do not develop disease at all. Clearly, therefore, there are additional factors that modify the risk of AP-mediated kidney disease. A related observation is that risk factors that predispose patients to aHUS and C3G are systemic yet complement-mediated inflammation is often limited to the kidneys. Thurman and Harrison39 review the unique physiologic features of the kidney that make it particularly susceptible to pathologic AP/AL activation. One of the most striking instances of impaired complement regulation is seen in paroxysmal nocturnal hemoglobinuria (PNH). PNH is caused by the clonal expansion of erythrocytes that lack the surface complement regulators CD55 (decay accelerating factor; DAF) and CD59. The pathophysiology and clinical features of PNH are reviewed by Risitano et al.40 Complement inhibitory drugs block the underlying cause of PNH and are now clinically approved for treatment of PNH. Furthermore, the rapid response of PNH to complement inhibition and easily measured clinical biomarkers of this response (increase in hematocrit, decrease in lactate dehydrogenase levels), make PNH well-suited for testing new complement inhibitory drugs. The downside of this is that there are a limited number of patients available for clinical trials; this, plus the efficacy of drugs now available, makes trial recruitment difficult. The authors review the current landscape of clinical trials of complement inhibitors in PNH and show how these studies are also revealing new insights into complement biology. The kidney is commonly affected by dysregulated AP/AL activation, but it is not simply a passive target of AP/AL activation. Elegant work has shown that the kidney plays an active role in complement activation. Sacks et al have performed studies in transplant models demonstrating that production of complement proteins by kidney cells is a critical determinant of transplant rejection.41 Although the liver is generally considered the source of most complement, extrahepatic production of complement proteins, such as by kidney tubular cells, creates a microenvironment which favors AP/AL activation. Furthermore, these investigators have shown that collectin-11 is produced by stressed tubular epithelial cells, and it binds to L-fucose displayed on the surface of the epithelial cells and activates complement.42 This work is reviewed by Sacks and Nauser.43 The collectin-11-L fucose interaction may represent a mechanism by which complement is locally activated in the kidney. In addition to these fulminant diseases caused by dysregulation of the AP/AL, more subtle functional defects in AP/AL regulation contribute to common diseases, including age-related macular degeneration. As described by de Jong et al44 genetic variants in complement genes account for more than 50% of the risk of age-related macular degeneration, a major cause of vision loss. This has led to development of AP/AL-related biomarkers of AMD risk, and in several clinical trials of complement inhibitory drugs. Unfortunately, complement inhibitors have had only limited success in these ophthalmology trials. The authors point out that AMD is a complex and multifactorial disease, and there are multiple challenges conducting clinical trials in this disease, not least of which is that some of the drugs need to be administered intra-ocularly. Nevertheless, some of the trials have yielded promising results, and the use of complement inhibitors to treat AMD is still being actively investigated. Hinted at, but not developed, is the thought that the ARMS2/HTRA1 risk might also, through extracellular matrix remodeling, impact on surface regulation of AP/AL activation, and lead to eye pathology consequent on dysregulated complement activation. Consideration of complement involvement in pregnancy-associated disorders is dealt with by Smith-Jackson and Harrison.45 This review illustrates the dilemma that is faced when treating complement-dependent disease. Not only is complement involved in pathology, but it can also be involved in critical development processes, and the issue of how and when to treat will be crucial for successful therapy. In 1990, Fearon et al published a seminal paper in the journal Science where they described the utility of the extracellular domain of Complement Receptor 1 (CR1, CD35), expressed as a soluble recombinant protein, to ameliorate complement-mediated tissue injury following reperfusion injury of ischemic rat myocardium.46 This study was driven by the potency of soluble CR1 (sCR1) in controlling both C3 and C5 convertases of all pathways of the complement system at concentrations which were orders of magnitude lower than physiological, soluble counterparts, FH and C4BP. Soluble CR1 (TP10) had a remarkable effect in the rat model of ischemia/reperfusion (I/R) injury, reducing infarct size, inhibiting MAC formation on the vascular endothelium, and decreasing inflammatory cell infiltration. TP10 showed high therapeutic potential in many animal models of AP/AL-driven disease and its favorable safety profile marked the start of an era for anti-complement therapeutics. The clinical development path for the next generation of therapeutics was greatly facilitated by the identification of diseases that were driven by the alternative pathway or amplification loop, where clearly defined mechanisms could be identified. In this issue, Risitano et al40 take us through the development path of drugs with potential to treat PNH. The simplicity of the disease mechanism has resulted in its utility as a test-bed for many drugs developed over the years and indeed, in development today. One of the central cell types impacted in this disease is the erythrocyte and the target cell and mechanism are easily translated to the laboratory in the form of hemolysis assays. Many potential AP-blocking drugs have been tested in vitro, including the anti-C3b antibody H1747 and mini-FH,48 and a significant number have entered the clinic. The first complement drug to be approved for PNH was the anti-C5 monoclonal antibody, eculizumab (FDA approval 2007), introduced in this issue by Risitano et al40 and extensively explored from a mechanistic point of view by Schmidt and Smith.49 Although the disease is driven by the AP/AL, blockade of C5 has a profound effect on the course of disease. However, the clinical use of eculizumab in PNH unexpectedly provided further insight into the disease mechanism and the molecular mechanisms of the AP/AL convertases. The phenomenon of extravascular hemolysis in PNH patients treated with eculizumab was described in 2009 by Risitano et al50 and this phenomenon is expanded on here by Schmidt and Smith.49 Due to the lack of CD55 on PNH erythrocytes, in the face of C5 blockade the AP/AL continues to drive opsonization of erythrocytes, resulting in a coating of C3 fragments that facilitate erythro-phagocytosis in the extravascular spaces in the liver and spleen. Treatment over time has also resulted in expansion of the PNH clone sizes as those cells are no longer lysed by the MAC; the clinical implications of this remain unclear. Schmidt and Smith go on to describe a series of studies suggesting that strong AP/AL activation, due either to either the nature of the surface or the complement activating potential, can drive C5 conformational changes (termed "priming") and activation, even in the absence of proteolytic cleavage and in the presence of C5-blockers.49 While their article focuses on anti-C5, other potential therapeutic protein modulators of the AP/AL include those that target the FHR axis6, 19 and the natural control proteins of the AP/AL, FH and FI. As the potential utility of complement blockade has become better understood and drugs approved, attention has turned to "proximal" complement inhibition with a keen focus on the AP/AL. The pathogenic role of the AP/AL in a plethora of diseases, including those driven by the classical and/or lectin pathways fueled the study of AP/AL inhibitors in a wide range of animal models of disease.31, 34 The development of these molecules occurred at a time when there was a huge explosion in interest in anti-complement therapeutics, driven by the clear demonstration that several renal diseases, such as aHUS and C3G, were primarily consequent on AP/AL dysregulation. The genetic association of multiple proteins involved in activation or control of the AP/AL with the common blinding disease, age-related macular degeneration (AMD),44 also drove keen interest from the industrial sector. Kolev et al describe the evolution of molecules that block complement at the level of C3 with a particular focus on pegcetacoplan, the only C3-blocker currently approved for clinical use (EMPAVELI™ for PNH).51 Pegcetacoplan binds to native and activated C3, blocking amplification, but it does not block C3(H2O) generation nor the proteolytic cleavage of C3 by non-convertase enzymes. Intriguingly, the authors suggest that pegcetacoplan modulates disease while retaining significant hemolytic activity, as measured by ex vivo hemolysis assays; residual activity of the classical pathway is striking (CH50). This finding is particularly pertinent from the point of view of limiting infection risk. Pegcetacoplan is currently administered subcutaneously, but other small molecule inhibitors of the AP/AL that are orally bioavailable are now in development. These include LMW inhibitors of FB and FD. Schubart et al52 eloquently take us through the rationale for specific blockade of the amplification loop and the evolution of the small molecule inhibitors. Supplementation of FH and FI is a strategy that has been trialled in the clinic in recent years, initially through protein supplementation and more recently through gene therapy approaches. Dreismann et al53 take us through emerging strategies that target complement at the genetic rather than the protein level. Gene therapy has potential to deliver additional complement control to localized spaces, such as the outer retina of the eye, where compartmentalization and continuous supply of the molecule from a local "biofactory" might deliver sufficient control to ameliorate diseases such as AMD. Complement was first discovered as an effector mechanism of antibody-mediated elimination of bacteria, and more than 50 years ago pre-clinical data suggested that therapeutic complement inhibitors might be effective for treating immune-complex diseases.54 It is interesting, therefore, that the complement inhibitors that were first approved are for AP/AL-mediated diseases (PNH and aHUS). The fact that complement is the primer driver of these diseases makes them suitable for clinical trials of complement-inhibitory drugs. In addition, standard immunosuppressive drugs target the primary disease process in antibody-mediated diseases, yet these drugs may have little effect on the AP/AL. As discussed in several of the contributions in this issue, drugs that specifically block the AP/AL are now in clinical development. Specific AP/AL inhibitors are categorically different than the currently available immunosuppressive drugs. In this context, the entry of complement inhibitory drugs into the clinic will likely provide a major advance in our understanding of disease mechanisms. Unlike many animal models, few human diseases are monogenic, the majority have a complex etiology. Even for diseases in which there is compelling rationale for AP/AL involvement, the complex AP genotype will only be a part of the story. Phenotype will be compounded by non-complement factors, both genetic and environmental. The efficacy (or lack of efficacy) of a complement inhibitory drug in a particular disease will provide more compelling evidence for the pathogenic role of complement in disease than any pre-clinical experiments or clinical associations. As experience with these new drugs mounts, we will undoubtedly gain important new insights into the role of the AP/AL in disease, much as was seen with anti-C5 treatment of PNH. Importantly, as more experience is gained with these drugs, the "elephant in the room", infection risk associated with complement inhibition, will become better defined. In this context, it is worth noting that the objective of AP/AL therapy in chronic diseases such as AMD is to restore "normal" regulatory function, not to block the activity totally. Where infection risk is real, targeted therapies may well provide a solution. The roles of the AP/AL in both healthy individuals and disease are highlighted in this issue of Immunological Reviews and take us on a journey from the initial tickover hypothesis through understanding of basic mechanistic and disease pathology, something that continues today, to seminal and ground-breaking therapeutic strategies. We believe that exciting times lie ahead, and hope that this collection of articles will benefit not just complementologists, but also specialists in other fields who wish to gain insight into how the AP/AL might impact their specialty. RAH is the owner and director of RAH Pharma Consulting Ltd. He also was employed by Novartis Pharma AG from 2001 to 2013, and between 2013 and 2022 held various consultancy agreements with Novartis. CLH is an employee of Gyroscope Therapeutics, a Novartis company; the opinions expressed are entirely her own or those of co-authors and are not necessarily those of Gyroscope Therapeutics nor Novartis. CLH has consulted for other companies developing anti-complement drugs and has received research funding from companies developing anti-complement drugs. JMT is a consultant for Q32 Bio, Inc., a company developing complement inhibitors. He holds stock in and may receive royalty income from Q32 Bio, Inc. No original data is contained in this manuscript.