Complement and in Antineutrophil Cytoplasmic -Associated Vasculitis – Partners in Crime: Interviews with Two Key Opinion Leaders

Interviewees: Mårten Segelmark,1 YKO (Onno) Teng2

1. Department of Nephrology, Lund University, Lund, Sweden 2. Department of Nephrology, Leiden University Medical Center, Leiden, the Netherlands

Disclosure: Prof Segelmark has received consultancy fees from ChemoCentryx and research grants from Hansa Biopharma. Dr Teng has declared no conflicts of interest.

Support: The publication of this interview feature was supported and reviewed by Vifor Pharma.

Acknowledgements: Medical writing assistance was provided by Dr Juliet George, Chester, UK.

Disclaimer: The opinions expressed in this article belong solely to the two named interviewees.

Citation: EMJ Nephrol. 2020;8[Suppl 2]:2-10.

Interview Summary Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) comprises a group of rare, necrotising inflammatory diseases. AAV can cause damage to blood vessels throughout the body, including in the kidneys, lungs, and intestines, and is typically treated with immunosuppressive and immunomodulatory therapies to help induce remission. However, the pathophysiology underlying this multifactorial condition is not completely defined. During interviews conducted by EMJ in early 2020, two experts in the field of nephrology and , Prof Mårten Segelmark and Dr Onno Teng, discussed the current understanding of the pathological processes involved in the damage associated with AAV. This included key interactions in the immune pathways that lead from the initial stage of ANCA production to the resulting vessel . In particular, the experts highlighted the critical but often overlooked role of complement and neutrophils in this process. The importance of this knowledge for improving the treatment and care of patients with AAV was also emphasised.

THE MECHANISMS OF DAMAGE IN polyangiitis ([GPA] previously known as Wegener’s granulomatosis), and eosinophilic GPA (previously ANTINEUTROPHIL CYTOPLASMIC known as Churg–Strauss syndrome).1 The damage ANTIBODY-ASSOCIATED VASCULITIS and inflammation in AAV predominantly affects small blood vessels, with localised of AAV is characterised by distinct pathologic vessel walls observed across multiple body lesions linked to the production of ANCA. With systems. Because of this, , classification based on pathological as well as inflammation of the glomerular capillaries of clinical features, the main forms of AAV include the kidneys, is another common component of microscopic polyangiitis, granulomatosis with the AAV diseases. Whenever disease pathology

2 NEPHROLOGY • June 2020 • Cover Image © Frederic Prochasson / 123RF.com EMJ is limited to glomerulonephritis alone, with no system? The standard way of thinking of this is evidence of systemic inflammation, physicians that there is an unknown trigger factor, classify the disease as renal-limited vasculitis.1,2 'X', that leads to massive release of the enzyme- Together, the effects of AAV can result in systemic containing granules. PR3 and MPO would then be organ damage and failure, with the kidneys and exposed outside the cell. This excessive quantity lungs as major targets.3 would mean the enzymes couldn’t be cleared quickly enough so, at some point, they would be Underlying these effects, AAV has a complex picked up by an immune cell as an autoantigen. pathophysiology that is generally divided into However, as most of the PR3 and MPO is displayed two stages: ANCA development (i.e., antibody on the plasma membranes of neutrophils, it’s response) and damage processes. In Stage 1 still a very difficult concept to understand how of AAV, the body loses its tolerance to ANCA these are presented to the immune antigens and B cells produce ANCA in an system.” Dr Teng continued: “About 15 years ago, autoimmune response. ANCA is most commonly it was discovered that neutrophils can produce directed against the neutrophil lysosomal enzyme neutrophil extracellular traps (NET), which are ([PR3] in GPA) and myeloperoxidase extracellular strands of DNA.6 If you analyse this 1 ([MPO] in microscopic polyangiitis), but other extracellular DNA you find that all the enzymes targets have been reported, such as lysosome- that are normally in the cytoplasm of neutrophils, associated membrane . Stage 2 comprises including MPO and PR3, are actually on the DNA of the active vasculitic process and associated these NET. So now you have a highly inflammatory damage or inflammation, which appearssituation in which the is triggered to be driven by the priming of neutrophils by extracellular DNA where it will also pick up all and their and interaction with the the MPO and PR3 displayed on these 4,5 complement system. NET. By harbouring the autoantigens, NET trigger Focussing on the roles of neutrophils and the a humoral autoimmune response, leading to complement pathway in the route to vasculitic ANCA development.” damage in AAV, the experts agreed that knowledge Dr Teng went on to explain that this process was incomplete. “It is well known that both of ‘NETosis’ is a physiological mechanism that neutrophils and complement are very important occurs in every healthy person when neutrophils for the disease process at different levels, but encounter , viruses, or fungi. “It is a very their involvement is not completely understood,” quick reaction, a sort of kamikaze response, in said Prof Segelmark. “Ultimately, we know that which the neutrophil throws out its DNA,” he are the end product of an autoimmune stated. “Because neutrophils are most abundantly response, where some type of autoantigen has present in the circulation, it seems plausible that triggered the immune system, leading to B-cell for this encounter to take place, the bacteria responses that prompt production. must be within the circulation, i.e., a very severe While we can measure those , the . When a person has a severe infection associated processes are less defined,” added with pathogens in the bloodstream, you want Dr Teng. your immune system (and, in this case, your neutrophils) to elicit a fast response. NETosis is one THE ROLE OF NEUTROPHILS of the neutrophil’s quickest responses.7 It extrudes its own DNA strands, which can trap bacteria and Discussing evidence for the involvement of contain the infection.” Crucially, in AAV, when neutrophils in initiating the disease process, Dr there are no bacteria to elicit this response, NET Teng explained: “We know that AAV is very much still appear to be activated. According to Dr Teng: related to neutrophils because the antigens that “If from AAV patients is added to healthy cause the ANCA autoantibodies (PR3 and MPO) neutrophils, they will form an excessive amount of are both enzymes that you find in the cytoplasmic NET. We don’t understand the exact trigger but granules of neutrophils. People ask why AAV we know that it’s not ANCA because the excessive patients make antibodies against these enzymes, NET formation is still induced when ANCA is taken some of which are enclosed in a granule, inside a out of the serum. Moreover, we’ve shown that neutrophil. How are they exposed to the immune when you take serum from a lot of AAV patients

Creative Commons Attribution-Non Commercial 4.0 June 2020 • NEPHROLOGY 3 and mix this with neutrophils, these neutrophils the disease manifestations of AAV, but it seems become massively activated, more so than if you logical that NETosis is important.”12 use serum from healthy controls. There is also an association with the level of disease activity, as THE ROLE OF COMPLEMENT the more active the patient’s disease, the more NET that are induced,” he said. Furthermore, Prof It has been suggested that the combination of Segelmark pointed out that it has been shown complement and neutrophils is a pivotal but under- that neutrophils from patients with AAV, including recognised partnership in the pathophysiology those without signs of ongoing inflammation, of AAV, and Prof Segelmark gave an overview of are intrinsically prone to undergo NETosis, both evidence for the involvement of complement in spontaneously8 and when stimulated with ANCA.9 AAV disease processes. “The first good models This is mirrored by a decreased tendency to of AAV used rodent knockouts to identify the undergo spontaneous apoptosis, a mode of cell various parts of the complement system that death that is not associated with tissue damage.10 were necessary for disease.13,14 It became apparent Prof Segelmark and Dr Teng then considered that it was not the terminal pathway (to vascular the current evidence for the involvement of damage) that was the most important element, neutrophils in the vasculitic damage observed in but rather the recruitment of the neutrophils by patients with AAV. “It is known that neutrophils the complement system that was essential,” he are extremely critical in all forms of small vessel explained. “In addition, there is circumstantial vasculitis; may also be involved but evidence. In the 1990s, before the genomic era, neutrophils are the key culprits in causing the we found that there were genetic variants in the damage, at least that’s my opinion,” began Prof effect of C3 component: C3F and C3S. The C3F Segelmark. He cited early studies using animal allele was shown to be more prone to activation models of glomerulonephritis that identified than C3S, which is the more common allele, and neutrophils as a necessary cellular component there was a higher-than-expected frequency of glomerular injury,11 and added: “In the of the C3F allele in patients with systemic glomerulonephritis in AAV, immune complexes vasculitis.15,16 Later, these alleles were also are present but it’s the neutrophils chewing up shown to be influential in the outcome of renal the basement membrane, leading to bleeding, transplantation17,18 and the occurrence of age- and entering the extravascular space that triggers related .”19,20 Components the process of their formation.” C3 and C5 are common to all three pathways that The experts then considered how the different activate the complement system: the classical, neutrophil-related mechanisms, including NETosis, , and alternative pathways (Figure 1). The may contribute to the vessel damage in AAV. “Of experts agreed that it is the self-sustaining course, without adhesion there wouldn’t be any alternative complement system that seems to be neutrophils there, so that is very important. Then chiefly involved in the pathophysiology of AAV. the neutrophils need to be activated and release Under nonactivated conditions, the alternative their contents by exocytosis or by NETosis,” pathway is maintained at a low level of C3 said Prof Segelmark. “Activated neutrophils will autoactivation, known as ‘tick-over', by control express all sorts of proteins that make them proteins. Once triggered, this autoactivation adhere to the vessel wall,” continued Dr Teng, loop is amplified, and C3 is converted to who also cited the generation of reactive oxygen and by a different factor from that used by species, including those derived from MPO, as the classical and lectin pathways. In turn, C5 is being a key mechanism in triggering NET release. activated to form the potent chemoattractant “We know that after a few hours of activation C5a, as well as the cytotoxic membrane attack these neutrophils will explode and extrude their complex C5b-9. The result is amplification of the NET. Once neutrophils go into NETosis they start inflammation response that continues until it is to adhere more to the vessel wall, as do a lot of specifically downregulated by other complement immune cells, eventually causing the small-vessel factors. Dr Teng explained: “C5 is the common destruction.” Prof Segelmark concluded: “I cannot effector pathway of the complement system and tell you what would happen if NETosis could be C5a, a by-product of C5 activation, is a highly blocked completely, whether that would prevent proinflammatory molecule.

4 NEPHROLOGY • June 2020 EMJ Classical pathway Alternative pathway (immune complexes) (microbes, mannose) (spontaneous, microbes)

Lectin pathway Classical pathway MBL, , Alternative pathway -11 C1q , C3(H20)

C1r MASP C1-INH Factor B C1s MASP C2, C4 C4BP C2b Bb Factor I C1-INH CR1 CR1 C4b C3b DAF DAF C3

C3a C3a C3b Factor H, MCP, C3b DAF, CR1, C2b Factor I C4b Bb P C3b C5 C3b C3b

C5a

C5b

C6, C7, C8, C9 CD59

C7 C8 C6 C5b C9

Figure 1: The three complement pathways. C1-INH: C1 inhibitor; CR1: 1; C4BP: C4b-binding protein; DAF: decay-accelerating factor; MASP: mannose-binding lectin-associated serine ; MBL: mannose-binding lectin; MCP: membrane cofactor protein. Adapted from Poppelaars F et al., 2018.21

Preclinical models showed that if you block C5, interesting part about properdin is that it is, itself, there is a lot of disease improvement.”13 a molecule of the complement system that is produced by myeloid-derived cells, , Reflecting on potential differences in the or neutrophils, which are the primary responders regulation of this process in AAV, Prof Segelmark for inflammation. So, in inflamed tissues, including commented: “Our current knowledge doesn’t in AAV, there are a lot of macrophages and point to anything being different in the mechanisms of the alternative pathway in AAV; neutrophils to produce properdin and ensure that rather, it might be that they are more active, i.e., C3 is bound to membranes locally in the tissue, appropriate activation of the alternative pathway and thus activate the alternative complement by dying neutrophils or vascular endothelial system locally.” This local activation aspect of the damage can still be detrimental if not controlled.” complement response is characteristic of AAV, Dr Teng expanded on this point: “One of the most and one which is highly interconnected. “Local important molecules that is needed for excessive activation of the alternative pathway by the complement pathway activation is a protein called complement system brings about C5a release. properdin. For the alternative pathway to be The main task of C5a is to induce of activated, C3 must be bound to a the macrophages and neutrophils to the inflamed and this binding is mediated by properdin. The area which, in turn, produce a lot of properdin

Creative Commons Attribution-Non Commercial 4.0 June 2020 • NEPHROLOGY 5 causing C3 activation, and then again, more C5a. in the serum] are not really useful in AAV.” Dr It is a vicious circle,” concluded Dr Teng. Teng agreed with this view. “In most patients, you don’t see complement systemically, so, for a COMPLEMENT IN TREATMENT AND clinician, this measurement is not relevant. For a research scientist it could be interesting to assess DIAGNOSIS the minority of patients who do show systemic activation of the alternative pathway. However, Given the clear evidence for involvement of even then, measuring systemic complement and complement, and its potential overactivation in activation is less informative than measuring AAV, a frequent question is how this fits with the complement at the tissue level,” he said. glomerulonephritis in AAV appearing to be pauci- immune, with little evidence of complement on biopsy. “First of all, pauci-immune does not mean OTHER CELL INVOLVEMENT that there is nothing,” explained Prof Segelmark. “It means that it is not abundant, or the prominent Concerning the involvement of other cells in element. Here we have a process where the the pathophysiology of AAV, Prof Segelmark complement system seems to be activated on commented: “It is likely that other cells come in the surface of neutrophils, but neutrophils are later, after the ‘early-ish’ initiation timepoint when not present at inflammatory loci for more than neutrophils or eosinophils are, I think, the key maybe 24 or 48 hours. So, this may be a transient players. After breakdown of the integrity of the activation that does not lead to a long-standing vascular wall, there will be bleeding and there will deposition, unlike immune complexes which seem be coagulation. As yet, we do not really know the to get stuck in the glomeruli,” he clarified. role of thrombocytes; they are present, they are activated under these circumstances, and they Adding to this reasoning, Dr Teng stated: “The have a lot of substances to release, so coagulation fact that there are not many antibodies seen in might play a part. However, at this early stage, the kidney tissue at biopsy in AAV, even though my view is that T cells or B cells are not that kidney inflammation is one of the hallmarks important locally,” he said. Dr Teng agreed that for diagnosing the disease, again points to the involvement of other cells comes later in the involvement of the alternative, as opposed to process, “Many different cell types are attracted the classical, complement pathway. Studies have to the damaged vascular wall. You get chemotaxis indeed confirmed that C3 deposition in kidney of and macrophages, local activation biopsies of AAV patients associates with worse of complement, more chemoattractants, and prognosis.22 However, then we have to ask how then you get a secondary with the alternative complement pathway is activated? T cells and eventually B cells going into the area Here, again, I think it’s more a case of local tissue of vascular damage. It is the start of a whole self- complement activation than systemic activation.” fulfilling mechanism,” he said. Both experts noted how this predominance of complement in the tissues presents a challenge THERAPEUTIC TARGETS in terms of measuring its levels during patient diagnosis or treatment in AAV. Prof Segelmark Having considered underlying evidence for explained: “The normal way that complement is the complement–neutrophil interaction in AAV, measured is testing for the level of lytic activity the experts moved on to discuss whether this in the serum. To make it more meaningful (in represents a relevant investigational target in AAV) we need other tools, other measures of AAV. “Both theoretically and also from the animal complement activation that could be more useful models and clinical data that have been generated, and here, I think, the jury is still out. There is a it seems logical to target the neutrophils in some lot of literature on complement products and way,” stated Prof Segelmark. “There are probably 23,24 breakdown products in AAV, but, as yet, we do lots of other ways to do this but targeting the not know how to use this information clinically. complement–neutrophil interaction is certainly Standard assays that have been developed one of them,” he suggested. for systemic erythematosus (SLE), for example [which measure complement levels

6 NEPHROLOGY • June 2020 EMJ Dr Teng also supported the therapeutic relevance the steroid side effects.” According to Dr Teng, of the complement–neutrophil interaction. “A year having an alternative would mean being able ago, we would have queried whether complement to consider the side effects of a treatment even inhibition would be beneficial for AAV patients,” when selecting therapy for patients at the most he began, “but now we have Phase II and Phase high-risk stages of disease. “This would obviously III studies showing the activity of a C5a-inhibiting be a benefit for patients, and also for clinicians compound in patients with AAV: the CLEAR who have to keep these patients on steroids and and ADVOCATE studies.25,26 So now we can say manage compliance, despite all the side effects,” that there is definitely complement activation he said. While Prof Segelmark shared this view, in AAV, and targeting complement, at least, he also cautioned about the consequences of complement proteins, is something that is useful completely blocking the complement pathway. for patients. However, do we know exactly how “The complement system is very important in this complement inhibition improves the disease many respects, removing debris and preventing mechanism? This is still an open question,” said , for instance. Every time you ‘tune Dr Teng. Continuing this point, Prof Segelmark down’ a system, there are side effects. In the commented: “We don’t even know, completely, acute situation you may be afraid of some type how current therapies like cyclophosphamide [an of opportunistic infection; if there is a long-term immunosuppressant] or rituximab [a monoclonal break in the system then impeding the clearance antibody to CD20] work in AAV. So, there are a of dead cells and debris in the body may cause lot of unknowns. Complement inhibition would issues, and could perhaps trigger SLE-like seem to stop the vasculitic process and prevent symptoms. There is always a danger when you new damage occurring. Of course, there are other start altering important systems in the body, but possible processes coming in later, after the first I think that halting the vasculitic process in this 24 hours, that could be important to target in way could be a very promising target,” he said. order to get the right kind of healing to occur, to prevent fibrosis, and prevent the accumulation of Dr Teng agreed that while having great potential, debris. However, I think that if we could prevent complement inhibition may not be the complete the interaction between the neutrophils and solution, giving his reasoning from a different complement, it is likely that the neutrophils would angle. “From the clinical point of view, of course be less numerous at the site, that they would be the benefit is that you have a more targeted less activated, and that they would produce much treatment. From a scientific point of view, the less damage,” he proposed. counterargument is also true. We are used to general immunosuppression where we just Dr Teng expanded on this by explaining, suppress the whole immune system; now we “Complement is part of the acute inflammatory have a targeted approach which might be very reaction we see in AAV and C5a is a mediator potent, but it may not suppress the immune of a lot of inflammation. As anti-inflammatory system well enough. For instance, it could be agents, steroids have been the backbone of our that once the inflammation is effectively reduced, treatment for AAV for decades. At this point, I another aspect of the immune system or another think that C5a inhibition could be as effective as mechanism that causes inflammation is not steroids in reducing that inflammatory reaction, targeted. So, I think you have to ask whether this as the Phase II CLEAR study showed that a is the solution for everything in AAV, or if some complement (C5a) inhibitor could replace steroid other part of the immune system is being missed.” use.25 Although we haven’t seen full Phase III data yet, findings appear positive versus standard Dr Segelmark added: “There are many steroid treatment.26 For the patient and the development studies in other [non-AAV] forms of clinician, I think it would be a major breakthrough glomerulonephritis that are focussing on different if you could treat AAV patients without using parts of the complement system. There is a lot steroids, especially at the beginning when the of ongoing research, and there are definitely inflammation in AAV is really severe. We tend aspects that would be of interest to evaluate to use very high doses of steroids at this point, also in relation to AAV.” “Complement inhibition when there is risk of pulmonary haemorrhage is progress, but it also raises a lot of questions,” and kidney failure, so there is less concern about concluded Dr Teng.

Creative Commons Attribution-Non Commercial 4.0 June 2020 • NEPHROLOGY 7 CONCLUSION Although these interactions are not yet clearly defined, both preclinical and clinical data support their involvement in the inflammation and AAV has an intricate pathophysiology that, damage that is characteristic of AAV. Moreover, consistent with its name, may be perceived as such studies have revealed evidence of highly predominantly related to the activities of ANCA interrelated pathways that can be blocked at key autoantibodies. However, as Prof Segelmark points to alleviate disease processes. The experts and Dr Teng described, the combination of the agreed that while the actions of complement and alternative complement system and neutrophils neutrophils in AAV are not yet fully understood, is integral to disease development, from the targeting this intricate partnership could triggering of ANCA production through to help deliver new, potentially better-tolerated, generation of vasculitic damage (Figure 2). approaches to treatment.

1 Loss of to ANCA antigens and the development of ANCA

2 Primed neutrophils present ANCA antigens (e.g., MPO and PR3 at their cell surface that bind to ANCA, resulting in neutrophil activation

3 Activated neutrophils adhere to and pen- etrate the blood vessel wall, and release mediators of inflammation and cell injury. e.g., NET

4 Activated neutrophils also release factors such as properdin that have an autocrine role in activating the alternative complement pathway, leading to the generation of C5a

5

Activation of C5a amplifies ANCA-induced inflammation and vascular damage

6

Necrotising vasculitis in small blood vessels. Over a few days, acute inflammation and necrosis are replaced by chronic inflammation and scarring

Figure 2: Key stages in antineutrophil cytoplasmic antibody-associated vasculitis and the role of complement and neutrophils. ANCA: Antineutrophil cytoplasmic antibody; MPO: myeloperoxidase; NET: neutrophil extracellular traps; PR3: proteinase 3.

8 NEPHROLOGY • June 2020 EMJ Biographies

Prof Mårten Segelmark Lund University, Lund, Sweden Prof Mårten Segelmark is a specialist in internal medicine and nephrology who trained at Lund University Hospital in Lund, Sweden, and completed postdoctoral training at the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. He is now Professor of Nephrology at the Linköping, Sweden, and Lund Universities. He served as President of the XIVth International ANCA and Vasculitis Workshop in 2009, and as President of the Swedish Society of Nephrology (SNF) 2012–2013. Since 2016, he has been the Vice President of the Immunonephrology Working Group of the European Renal Association–European Dialysis and Transplant Association (ERA–EDTA). Prof Segelmark has published 111 original articles and 37 reviews and book chapters.

Dr YKO (Onno) Teng Leiden University Medical Center, Leiden, the Netherlands Dr YKO (Onno) Teng is a nephrology clinician-scientist at the Department of Internal Medicine of the Leiden University Medical Center (LUMC) in Leiden, the Netherlands. He is head of the nephrology outpatient clinic and coordinator of the Leiden outpatient clinic for Lupus, Vasculitis, and Complement-mediated Systemic diseases (LuVaCs) which accommodates top referral, multidisciplinary, tertiary care on a regional and national level. He also leads and coordinates a clinical, multidisciplinary pathway aimed at counselling and guiding a pregnancy wish for patients with SLE or antiphospholipid syndrome.

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