To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007 Presse Med. 2015; //: ///
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Quarterly Medical Review
Recent pathogenetic advances in ANCA-associated vasculitis William F. Pendergraft III1,2, Patrick H. Nachman 1
Available online:
1. University of North Carolina (UNC), kidney center, division of nephrology, department of medicine, Chapel Hill, NC 27599-7155, United States 2. Broad Institute of Harvard and MIT, Cambridge, MA, United States
Correspondence: Patrick H. Nachman, university of North Carolina (UNC), kidney center, division of nephrology, department of medicine, Chapel Hill, NC 27599-7155, United States. [email protected]
Vasculitis research: don't slow down and plan for a life-time commitment Christian Pagnoux et al., Toronto, Canada Recent pathogenetic advances in ANCAassociated vasculitis William F. Pendergraft et al., Chapel Hill, NC, United States Biologics in vasculitides: where do we stand, where do we go from now? Giulia Pazzola et al., Reggio Emilia, Italy Update on the treatment of ANCA associated vasculitis Rona M. Smith, Cambridge, United Kingdom Risks of treatments and long-term outcomes of systemic ANCA associated vasculitis Oliver Floßmann, Berkshire, United Kingdom Update on Takayasu's Arteritis Fatma Alibaz-Oner et al., Istanbul, Turkey What matters for patients with vasculitis? Elaine Novakovich et al., Bethesda, MD, United States
Summary Since the discovery of anti-neutrophil cytoplasmic autoantibodies (ANCA), great strides have been made in elucidating the etiology and pathogenesis of disease. In this article, we review recent published key breakthroughs in understanding the pathogenesis of ANCA vasculitis, including some that may lead to novel therapeutics. These breakthroughs have occurred in multiple areas of investigation. A European genome-wide association study (GWAS) revealed the importance of the genetic contribution of proteinase 3 (PR3) and its endogenous inhibitor, alpha (1)-antitrypsin as well as HLA risk. Epigenetic modification of autoantigen genes appears to contribute to perpetuation of disease and possibly relapse risk. Autoantigen excision, a novel method to detect autoantibody epitopes using mass spectrometry, not only revealed pathogenic epitopes in myeloperoxidase (MPO)-ANCA vasculitis and identified unique MPO-ANCA responsible for the majority of ANCA-negative small vessel vasculitis, but has vast applicability to other autoantibodymediated diseases. An explosion of biomarker studies has revealed circulating cytokines and alternative complement pathway products that may predict active disease. Interestingly, alternative complement pathway blockade in the murine model of disease is protective and a clinical trial in humans using an oral alternative complement pathway inhibitor is underway. Increasing clarity of the role of B and T cells in disease pathogenesis is ongoing. B cell depleting agents have shown great utility in remission induction and maintenance, and monitoring specific B cell subsets during the disease course may have predictive power for remission maintenance. Despite these substantial advances, more research is needed including, but not limited to, validation of existing discoveries. As additional novel discoveries emerge, so will novel therapies, and it is with great hope that these collective insights will ultimately lead to prevention and cure.
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nti-neutrophil cytoplasmic autoantibodies (ANCA), directed against the neutrophil granule proteins myeloperoxidase (MPO) and/or proteinase 3 (PR3), are pathogenic and cause small and medium vessel vasculitis [1]. Since the discovery of ANCA, great strides have been made in
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In this issue
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
W.F. Pendergraft III, P.H. Nachman
understanding the etiology and pathogenesis of disease. In this article, we review recently published key breakthroughs in understanding the pathogenetic mechanisms in ANCA-associated vasculitis that have opened the way to novel therapeutic approaches and interventions.
Genetics
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The recent advent of human whole genome sequencing has resulted in exceptional new insight into the role of genetics across all aspects of human health [2]. It has long been proposed that patients with ANCA-associated vasculitis have a genetic predisposition for disease, and investigators in the field have taken full advantage of this expanding technological breakthrough to identify genetic underpinnings. An analysis of 168 single nucleotide polymorphisms (SNPs) previously associated with Crohn's disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), celiac disease and ulcerative colitis was performed using genotype data from 2 different cohorts comprising a total of 880 patients with granulomatosis with polyangiitis (GPA) and 1969 controls [3]. SNPs within CTLA4, which encodes a T cell surface molecule termed cytotoxic T-lymphocyte antigen-4 (CTLA4) that down-regulates T-cell activation, were significantly associated with GPA (OR 0.79, 95% CI 0.70–0.89, P = 9.8 10 5). This result confirms previous genetic evidence implicating CTLA4 in the susceptibility to ANCA-associated vasculitis. One study from the United Kingdom identified a significant association of two SNPs within CTLA4 (exon 1 [+49] and exon 4 [CT60]) by PCR and restriction fragment length polymorphism (RFLP) of DNA from 222 patients with small vessel vasculitis (SVV) and 670 controls [4]. A separate study from the Netherlands identified that the +49 allele was significantly associated with patients with small vessel vasculitis [5]. Genotyping of the CT60 SNP (rs3087243) in CTLA4 using DNA from 641 patients with ANCA vasculitis confirmed a significant association (P = 6.4 10 3) and showed that the minor A allele was protective (OR 0.84), as compared to the risk-conferring G allele [6]. The contribution of CTLA4 polymorphisms to risk of ANCA-associated vasculitis was further confirmed by a recent meta-analysis of 7 different CTLA4 studies [7] suggesting that these genetic polymorphisms lead to T cell hyper-reactivity thereby contributing to the pathogenesis of disease. Importantly, however, all of the studies associating CTLA4 with ANCA-associated vasculitis were performed in cohorts of Caucasian patients of European descent. No association of CTLA4 was found in a small cohort of 69 Japanese patients with predominantly MPO-ANCA vasculitis [8]. A SNP (R620 W, rs2476601) in PTPN22, another gene encoding a negative regulator of T-cell activation, has also been associated with ANCA vasculitis in Caucasian European cohorts [6,9]. A recent meta-analysis of this risk-conferring polymorphism tested in four studies comprising 1399 Caucasians with
ANCA-associated vasculitis and 9934 controls revealed a significant association of the A allele (OR 1.44, 95% CI 1.26–1.64, P < 0.00001) not only among all patients, but also within the disease subsets GPA (OR 1.72, 95% CI 1.35–2.20, P < 0.00001) and microscopic polyangiitis (MPA, OR 1.53, 95% CI 1.08–2.15, P = 0.02). The allele was also significantly associated with PR3ANCA (OR 1.74, 95% CI 1.25–2.430, P = 0.001) when compared to controls. Furthermore, there was allelic significance with lung (OR 1.69, 95% CI 1.21–2.36, P = 0.002), skin (OR 2.55, 95% CI 1.69–3.84, P < 0.0001) and peripheral nerve (OR 2.12, 95% CI 1.39–3.22, P = 0.0005) involvement when compared to other organ involvement [10]. Mechanistic examination of this gain of function variant in leukocytes from patients with ANCA vasculitis revealed persistently elevated basal phosphatase activity that acted as a dominant negative regulator of ERK activity to thereby blunt interleukin-10 production, an immunoprotective cytokine [11]. The CTLA4 and PTPN22 mutations have been implicated in many other autoimmune diseases, which would suggest their dysfunctional contribution to global immune dysregulation. The first ever genome-wide association study (GWAS) in ANCAassociated vasculitis was performed recently using DNA samples from 1233 patients in the United Kingdom with ANCA-associated vasculitis and 5884 controls and a replication cohort of 1454 Northern European case patients and 1666 controls [12]. Patients with PR3-ANCA had significant associations with HLA-DP and genes encoding alpha (1)-antitrypsin (SERPINA1, the endogenous inhibitor of PR3) and PR3 (PRTN3) (P = 6.2 10 89, P = 5.6 10 12 and P = 2.6 10 7, respectively). Patients with MPO-ANCA had a significant genome-wide association with HLA-DQ (P = 2.1 10 8). Interestingly, genetic associations were made most strongly with respect to ANCA serotype rather than disease phenotype. Furthermore, neither CTLA4 nor PTPN22 were identified by this GWAS, which could be explained at least in part by demographic makeup of participants studied and/or a low frequency of association with these two previous genes of interest. There appear to be clear racial and geographic differences that affect genetic predisposition. Reports of ANCA-associated vasculitis in Africa are rare and ANCA vasculitis is notably rare in African Americans; however, an association of the HLA-DRB1*15 allele with PR3-ANCA-positive disease was found by MHC class II genotyping, which confers a 73.3-fold higher risk in African American patients (OR 73.3, 95% CI 9.1–591.0, P = 2.3 10 9) than in community-based controls [13]. Examination of a validation cohort comprised of DNA samples collected from patients within the Vasculitis Clinical Research Consortium (VCRC) confirmed this strong association. Interestingly, the DRB1*1501 allelic variant, which is of Caucasian descent, was found in 50% of African American patients, whereas the DRB1*1503, of African descent, was under-represented in the discovery group examined from the South-eastern
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007 Recent pathogenetic advances in ANCA-associated vasculitis
Phenotype versus serotype An emerging concept in ANCA-associated vasculitis founded on the genetic discoveries listed above relates to the relationship between disease serotype and phenotype. In other words, is more pathogenetic insight gained by comparing patients with differing phenotypes (GPA, MPA, renal-limited disease) or serotypes (MPO, PR3, dual-positive, ANCA-negative)? Collectively, many of the findings described in this review lend credence to the concept that MPO- and PR3-AAV are genetically distinct diseases with phenotypic overlap and that translational studies and clinical trials may benefit from clustering patients by serotype.
Epigenetics Epigenetics, the study of durable alterations in the transcriptional potential of a cell that cannot be attributed to nucleotide sequence variations, is a field that will clearly provide valuable insight into the pathogenesis and perhaps etiology of ANCAassociated vasculitis. Despite a burst of activity related to understanding DNA methylation and histone modification in other forms of human disease [15,16], only one study specifically addressing epigenetics in ANCA-associated vasculitis has been published thus far [17], although investigations based on genome-wide methylation analyses of healthy human
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leukocyte subsets are currently ongoing by investigators in the field, which is encouraging [18]. To set the stage, our group has scientifically promoted the notion that patients with ANCA-associated vasculitis harbor peripheral leukocytes, primarily monocytes and neutrophils, which aberrantly express the ANCA autoantigens PR3 and MPO. This was first discovered by microarray analyses of leukocytes from patients with ANCA-associated vasculitis who also had glomerulonephritis [19]. Interestingly, in addition to these two genes, a global granulopoiesis signature was identified and in situ staining identified PR3 transcripts in mature monocytes and neutrophils [20]. MPO and PR3 transcriptional upregulation correlated with disease activity and absolute neutrophil counts, but did not correlate with immunosuppression, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender or age. Furthermore, this transcriptional aberration was not associated with disease-control patients with end-stage kidney disease (ESKD) or SLE. This was a surprising discovery since the MPO and PRTN3 genes are located on different chromosomes yet their expression is coordinately regulated. It was subsequently postulated that epigenetic modifications associated with gene silencing were dysfunctional and contributed to aberrant transcription of MPO and PR3 in mature monocytes and neutrophils. Our group examined MPO and PR3 loci for chromatin modifications in neutrophils from patients with ANCA vasculitis and healthy controls [17]. It was found that histone H3K27me3 is depleted at PR3 and MPO loci due to increased expression of Jumonji domain-containing 3 (JMJD3) demethylase as well as failure of the transcription factor RUNX3 to recruit EZH2, which is responsible for H3K27me3 methylation. These epigenetic changes may explain why MPO and PR3 are aberrantly upregulated in leukocytes of patients with ANCA vasculitis, which likely sparks and/or perpetuates the disease process if left unchecked. Numerous questions have yet to be answered in this area. Kurz et al. recently hypothesized that the granulopoiesis transcriptional signature discussed above could predict relapse, but found that MPO and PR3 mRNA levels correlated with corticosteroid therapy instead [21]. This is a clear discrepancy in the field that needs to be reconciled in order to make progress from both a genetic and epigenetic standpoint. An important question pertains as to whether the observed epigenetic changes are pathogenetically causative of ANCA vasculitis or merely a reflection of ongoing disease processes. If the former is true, then therapies could be conceived to specifically counteract or modify this epigenetic dysregulation.
Autoantigens, autoantibodies and biomarkers Patients with clear clinical and histopathologic evidence of small vessel vasculitis and/or pauci-immune necrotizing and crescentic glomerulonephritis but with negative ANCA serology have
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United States. Further support of this association was made by the demonstration that DRB1*1501 protein bound with high affinity to PR3 protein in vitro and on the surface of activated healthy human neutrophils [13]. It should be noted that while SNP analyses and GWAS provide important clues to genetic risk by revealing candidate loci, each locus identified explains only a very small fraction of phenotypic variance and all loci identified to date in ANCA-associated vasculitis explain only slightly more variance. As an example using a simple trait much more common than risk of ANCA-associated vasculitis, it has been proposed that roughly 93,000 SNPs are needed to explain 80% of the population variation in height [14]. This argues against the contention that the genetic associations gleaned thus far are likely to play a significant role in identifying populations at sufficiently higher risk of disease to institute a surveillance or preventive intervention. In addition to genetic risk loci, including those yet to be identified, it is clear that other factors contribute to the development and perpetuation of ANCA-associated vasculitis, which include but are not limited to copy number variations, epigenetic modifications and their relationship to environmental exposures. Overall, several risk alleles have been identified that are associated with ANCA vasculitis. Some risk alleles are specifically associated with the MPO- or PR3-ANCA serotypes, suggesting that MPO-ANCA vasculitis and PR3-ANCA vasculitis may be genetically distinct diseases with phenotypic overlap.
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
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presented important challenges regarding the pathogenetic role of ANCA. The negative serology also frequently causes delays in establishing the diagnosis and initiating appropriate treatment of such patients. To address this issue, a multi-center study by Roth et al. recently reported the development of a novel assay termed "epitope excision'' or "autoantigen excision'' to identify the specific target epitopes for ANCA [22]. Briefly, autoantigen (in this case MPO) is incubated with protein A/G-bound IgG containing MPO-ANCA. This mixture is washed extensively and then incubated with trypsin to digest non-complementarity determining region (CDR)-bound antigen away. After additional washing, the CDR-bound piece of antigen is eluted and subjected to protein sequencing by mass spectrometry. First, this assay allowed for highly-sensitive epitope mapping of murine and human MPOANCA and revealed that patients with ANCA-associated vasculitis harbor autoantibodies to pathogenic epitopes. Antibodies to MPO could also be detected in sera of healthy controls, but their epitope specificities differed significantly when compared to those from patients with MPO-ANCA vasculitis. Importantly, some patients with small vessel vasculitis who are ANCA-negative by the standard clinical ELISA test, were in fact found to harbor autoantibodies to a pathogenic MPO epitope capable of in vitro neutrophil activation and induction of nephritis in mice. It was discovered that this antibody-epitope interaction was blocked by the presence of a fragment of ceruloplasmin. Ceruloplasmin is the circulating endogenous inhibitor of MPO, which is removed during the IgG purification step, thus explaining the negative result of the standard serum-based clinical ANCA test. Although these findings need independent validation, this study not only identified immunodominant and pathogenic epitopes using a novel epitope mapping assay, but also laid the groundwork for the development of a future clinical assay to use for patients with ANCA-negative disease that could expedite diagnosis and treatment. This may also pertain to an enhanced ability to identify individuals prior to disease development given the recent findings by Olson et al. that PR3-ANCA were detected in patients with GPA before disease onset [23]. Regardless, this work could provide a better understanding of pathogenesis and evolution of disease. Controversy persists with respect to ANCA target autoantigens other than MPO and PR3. Kain et al. reported in 2008 the presence of autoantibodies to lysosome-associated membrane protein-2 (LAMP-2) in the serum of the majority of patients tested with pauci-immune focal necrotizing and crescentic glomerulonephritis [24]. An epitope within LAMP-2 (p41-9) was shown to have 100% homology to FimH, a bacterial adhesion, and autoantibodies directly-cross reacted suggesting a molecular mimic. Furthermore, rats immunized with FimH developed autoantibodies to rat and human LAMP-2 and disease akin to what is observed in humans. A subsequent report from the same group revealed the presence of anti-LAMP-2
autoantibodies in patients from Austria, the Netherlands and the United Kingdom [25]. The same group later published that 8 of 11 (73%) of patients with ANCA-negative pauci-immune focal necrotizing glomerulonephritis harbored anti-LAMP-2 autoantibodies [26]. In a conflicting report, Roth et al. measured anti-LAMP-2 autoantibodies in 680 serum samples from two academic centers in the United States from patients with ANCA glomerulonephritis (n = 329), ANCA-negative glomerulonephritis (n = 104), patients with E. coli urinary tract infection (UTI, n = 104), disease controls (n = 19) and healthy volunteers (n = 124) [27]. Anti-LAMP-2 autoantibodies were detected in only 21% of sera rom patients with ANCA-associated vasculitits and 16% of patients with a UTI who did not have ANCA vasculitis. Thus, the relevance of LAMP-2 and anti-LAMP-2 antibodies in ANCA-associated vasculitis requires further investigation. Biomarkers, alternatively termed bioindicators, are needed to identify patients with ANCA-associated vasculitis prior to disease development and to identify predictors of treatment response as well as relapse. There have been numerous biomarker studies to date. The most robust measured 28 serum proteins based on hypothetical disease association in 186 patients who participated in the rituximab in ANCA-associated vasculitis (RAVE) trial before and six months after treatment in order to try and distinguish severe disease (BVAS/WG 3) from remission (BVAS/WG = 0) [28]. Active disease was best discriminated from remission (AUC > 0.8) and healthy controls (AUC > 0.9) by levels of CXCL13, MMP-3 and TIMP-1. Although yet to be confirmed, there is now preliminary evidence in humans for a contribution of the alternative complement pathway in ANCA-associated vasculitis. Increased plasma levels of C3a, C5a, soluble C5b-9 and Bb in plasma were identified in 66 patients with active disease as compared to 54 patients in remission [29]. Urinary Bb levels were also found to correlate with and serum creatinine in 27 patients with active disease, and correlated inversely with the proportion of normal glomeruli in matched kidney biopsy specimens [30]. Again, these findings require validation in other controlled clinical settings.
Immune cell dysregulation Immune and non-immune cells have perennially been the subject of intense analysis in the field of ANCA-associated vasculitis, and B and T-lymphocytes are most hotly studied at present. Recognizing the pathogenic role of ANCA has fostered the therapeutic use of B cell depleting agents. Ongoing investigations are focused on measuring circulating B cells numbers and proportions of B cell subsets to monitor disease activity and potentially as indicators of risk of relapse. Bunch et al. longitudinally investigated for 4–99 months the peripheral B cell phenotype in 54 patients with either active or quiescent ANCAassociated vasculitis as compared to 68 healthy controls [31]. Patients with active disease had a lower percentage of CD5+ B cells when compared to patients in remission or healthy
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007 Recent pathogenetic advances in ANCA-associated vasculitis
Animal models The well-characterized mouse model of MPO-ANCA vasculitis, whereby anti-mouse MPO autoantibodies purified from
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MPO-deficient mice immunized with murine MPO are passively transferred into wild-type mice and cause vasculitis and pauciimmune crescentic glomerulonephritis, has permitted exploration of the role of various inflammatory pathways. Based on this model, a previously unsuspected role of complement activation was recently demonstrated. Upon the passive transfer of antiMPO antibodies, disease induction failed with administration of cobra venom factor and in mice deficient in complement factors C5 and B, whereas mice deficient in C4 developed disease comparable to that observed in wild-type mice [36]. These findings point to the importance of the alternative, but not the classical or lectin binding pathways of complement activation in this animal model of ANCA-associated disease. Furthermore, disease could be also be completely abolished or markedly ameliorated by treating the mice with a C5-inhibiting monoclonal antibody either 8 hours before or 1 day after disease induction with anti-MPO IgG and lipopolysaccharide [37]. Conversely, C6 deficient mice are not protected from disease induction by anti-MPO antibodies, suggesting that the C5b-9 membrane attack complex formation is not essential in the pathogenetic pathway. It was subsequently demonstrated in humans that blockade of the C5a receptor on human neutrophils abrogated their stimulation in vitro [38]. More recent work confirmed the immunopathogenetic importance of the alternative complement pathway in that blockade of C5a receptor (C5aR) activity protected against disease development [39]. Transgenic mice expressing human C5aR were protected from disease when given an oral small molecule antagonist of human C5aR called CCX168. Overall, these results suggest an important role for alternative complement pathway activation in the pathogenesis of ANCA vasculitis and identify C5a and its receptor as a targetable therapeutic pathway. A clinical trial is underway to assess the safety and efficacy of CCX168 in patients with ANCA vasculitis (http://www. clinicaltrials.gov, NCT01363388). It is also important to note that the evidence for the role of the alternative complement pathway in ANCA-associated vasculitis emanates from a model of anti-MPO-mediated disease, and indirectly from patients with MPO-ANCA [29,30]. To date, there is no direct evidence in mice or men that this also applies to PR3-ANCA-mediated disease.
Future directions and conclusions Major pathogenetic inroads have been made recently, but it remains clear that there is more to do in the field of ANCAassociated vasculitis. Many of the recent discoveries discussed clearly require validation in separate cohorts, and confirmatory studies are required to resolve discrepant results. The field has been enlightened by the results of the European GWAS, which revealed the importance of the genetic contribution of PR3 and its endogenous inhibitor, alpha (1)-antitrypsin as well as HLA risk; however, the majority of patients included harbored PR3ANCA and the power to detect genetic associations with patients
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controls. Furthermore, patients treated with rituximab who were on low or no maintenance immunosuppression and who had a low or sharply declining repopulation percentage of CD5+ B cells relapsed sooner than patients maintained on high maintenance immunosuppression (median 17 versus 29 months, respectively, P = 0.002). With the presumed intention of validating these findings, Unizony et al. recently analyzed absolute and relative numbers of CD5+ B cells from PBMC samples collected from 197 participants in the RAVE trial over 18 months and were unable to identify a significant association with disease severity and/or failure of induction therapy; however, analytic methodologies may have been different [32]. CD5 is one of several cell surface markers that characterize regulatory B cells. Although there is currently controversy in the field about which set of markers characterizes regulatory B cells, it is generally agreed upon that a regulatory B cell must be functionally capable of producing interleukin-10 [33]. Given the discrepancy in the field regarding the role of monitoring B cells to assess disease activity as well as the utility of monitoring CD5 + B cells specifically, more work is needed to determine appropriate phenotypic methods to detect regulatory B cells and inclusion of these measurements in future clinical trials is warranted. With regards to recent advances in understanding the role of T cells in ANCA-associated vasculitis, Free et al. hypothesized that T cell dysregulation is involved in disease pathogenesis and tested this hypothesis by evaluating CD4+ T cell populations from 62 patients with ANCA-associated vasculitis compared to 19 healthy controls [34]. Patients with active disease had a higher frequency of regulatory T cells. However, a proportion of regulatory T cells were found to be lacking FoxP3 exon 2, associated with a decreased suppressive function. Furthermore, a pro-inflammatory CD4+ T cell population was identified that was also increased in frequency and was resistant to suppression by regulatory T cells. Recent systems-level transcriptional profiling of CD8+ T cells from patients with ANCA-associated vasculitis revealed a three gene signature (ITGA2, NOTCH1 and PTPN22) that identified patients who have a poor prognosis [35]. This signature had similar predictive prognostic value in patients with SLE, but was unable to differentiate between the two diseases (ANCA vasculitis vs. SLE). Unfortunately, this predictive signature was absent in total peripheral blood mononuclear cell preparations, but was detectable only from enriched CD8+ T cells, which makes the clinical application of this test less practical. In addition, as with many of the findings discussed in this review, these findings require validation in other cohorts of patients with ANCA-associated vasculitis.
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
W.F. Pendergraft III, P.H. Nachman
harboring MPO-ANCA was limited. A GWAS including a larger number of MPO-ANCA positive patients is greatly needed and North American investigators are poised to make this possible given the extensive collection of DNA samples across centers within the VCRC. As additional novel discoveries emerge, so too will novel therapies, and it is with great hope that these collective insights will ultimately lead to prevention and cure.
Acknowledgments: the authors wish to thank the patients with ANCAassociated vasculitis and the other scientists and health care providers dedicated to their care at UNC. WFP3 and PHN are supported by grants from the NIDDK (P01-DK058335-14 and 1UM1-DK100845-01). WFP3 was also supported in part by NIDDK grant # 5 F32 DK097891-02 and receives translational research funding from The Broad Institute. Disclosure of interest: the authors declare that they have no conflicts of interest concerning this article.
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[11] Cao Y, Yang J, Colby K, Hogan SL, Hu Y, Jennette CE, et al. High basal activity of the PTPN22 gain-of-function variant blunts leukocyte responsiveness negatively affecting IL-10 production in ANCA vasculitis. PLoS One 2012;7:e42783. [12] Lyons PA, Rayner TF, Trivedi S, Holle JU, Watts RA, Jayne DR, et al. Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med 2012;367:214–23. [13] Cao Y, Schmitz JL, Yang J, Hogan SL, Bunch D, Hu Y, et al. DRB1*15 Allele Is a Risk Factor for PR3-ANCA Disease in African Americans. J Am Soc Nephrol 2011;22:1161–7. [14] Goldstein DB. Common genetic variation and human traits. N Engl J Med 2009;360:1696–8. [15] Johansson A, Enroth S, Gyllensten U. Continuous aging of the human DNA methylome throughout the human lifespan. PLoS One 2013;8:e67378. [16] Ziller MJ, Gu H, Muller F, Donaghey J, Tsai LT, Kohlbacher O, et al. Charting a dynamic DNA methylation landscape of the human genome. Nature 2013;500:477–81. [17] Ciavatta DJ, Yang J, Preston GA, Badhwar AK, Xiao H, Hewins P, et al. Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis. J Clin Invest 2010;120:3209–19. [18] Zilbauer M, Rayner TF, Clark C, Coffey AJ, Joyce CJ, Palta P, et al. Genome-wide methylation analyses of primary human leukocyte subsets identifies functionally important celltype-specific hypomethylated regions. Blood 2013;122:e52–60. [19] Alcorta DA, Preston GA, Munger WE, Sullivan P, Yang JJ, Waga I, et al. Microarray studies of gene expression in circulating leukocytes in kidney diseases. Exp Nephrol 2002;10:139–49. [20] Yang JJ, Pendergraft WF, Alcorta DA, Nachman PH, Hogan SL, Thomas RP, et al. Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody-associated glomerulonephritis. J Am Soc Nephrol 2004;15:2103–14.
[21] Kurz T, Weiner M, Skoglund C, Basnet S, Eriksson P, Segelmark M. A myelopoiesis gene signature during remission in anti-neutrophil cytoplasm antibody-associated vasculitis does not predict relapses but seems to reflect ongoing prednisolone therapy. Clin Exp Immunol 2014;175:215–26. [22] Roth AJ, Ooi JD, Hess JJ, van Timmeren MM, Berg EA, Poulton CE, et al. Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis. J Clin Invest 2013;123:1773–83. [23] Olson SW, Owshalimpur D, Yuan CM, Arbogast C, Baker TP, Oliver D, et al. Relation between asymptomatic proteinase 3 antibodies and future granulomatosis with polyangiitis. Clin J Am Soc Nephrol 2013;8:1312–8. [24] Kain R, Exner M, Brandes R, Ziebermayr R, Cunningham D, Alderson CA, et al. Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis. Nat Med 2008;14:1088– 96. [25] Kain R, Tadema H, McKinney EF, Benharkou A, Brandes R, Peschel A, et al. High prevalence of autoantibodies to hLAMP-2 in antineutrophil cytoplasmic antibody-associated vasculitis. J Am Soc Nephrol 2012;23:556–66. [26] Peschel A, Basu N, Benharkou A, Brandes R, Brown M, Dieckmann R, et al. Autoantibodies to hLAMP-2 in ANCA-negative pauciimmune focal necrotizing GN. J Am Soc Nephrol 2014;25:455–63. [27] Roth AJ, Brown MC, Smith RN, Badhwar AK, Parente O, Chung H, et al. Anti-LAMP-2 antibodies are not prevalent in patients with antineutrophil cytoplasmic autoantibody glomerulonephritis. J Am Soc Nephrol 2012;23:545–55. [28] Monach PA, Warner RL, Tomasson G, Specks U, Stone JH, Ding L, et al. Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis. Ann Rheum Dis 2013;72:1342–50. [29] Gou SJ, Yuan J, Chen M, Yu F, Zhao MH. Circulating complement activation in patients with anti-neutrophil cytoplasmic antibodyassociated vasculitis. Kidney Int 2013;83: 129–37.
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[30] Gou SJ, Yuan J, Wang C, Zhao MH, Chen M. Alternative complement pathway activation products in urine and kidneys of patients with ANCA-associated GN. Clin J Am Soc Nephrol 2013;8:1884–91. [31] Bunch DO, McGregor JG, Khandoobhai NB, Aybar LT, Burkart ME, Hu Y, et al. Decreased CD5+ B cells in active ANCA vasculitis and relapse after rituximab. Clin J Am Soc Nephrol 2013;8:382–91. [32] Unizony S, Lim N, Phippard DJ, Carey VJ, Miloslavsky EM, Tchao NK, et al. Peripheral CD5 B-cells in ANCA-associated vasculitis. Arthritis Rheumatol 2015;67:535–44. [33] Hilgenberg E, Shen P, Dang VD, Ries S, Sakwa I, Fillatreau S. Interleukin-10-producing B
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cells and the regulation of immunity. Curr Top Microbiol Immunol 2014;380:69–92. [34] Free ME, Bunch DO, McGregor JA, Jones BE, Berg EA, Hogan SL, et al. Patients with antineutrophil cytoplasmic antibody-associated vasculitis have defective treg cell function exacerbated by the presence of a suppression-resistant effector cell population. Arthritis Rheum 2013;65:1922–33. [35] McKinney EF, Lyons PA, Carr EJ, Hollis JL, Jayne DR, Willcocks LC, et al. A CD8+ T cell transcription signature predicts prognosis in autoimmune disease. Nat Med 2010;16:586– 91 [1p.]. [36] Xiao H, Schreiber A, Heeringa P, Falk RJ, Jennette JC. Alternative complement pathway in the pathogenesis of disease mediated
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by anti-neutrophil cytoplasmic autoantibodies. Am J Pathol 2007;170:52–64. [37] Huugen D, van EA, Xiao H, Peutz-Kootstra CJ, Buurman WA, Tervaert JW, et al. Inhibition of complement factor C5 protects against antimyeloperoxidase antibody-mediated glomerulonephritis in mice. Kidney Int 2007;71:646– 54. [38] Schreiber A, Xiao H, Jennette JC, Schneider W, Luft FC, Kettritz R. C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis. J Am Soc Nephrol 2009;20:289–98. [39] Xiao H, Dairaghi DJ, Powers JP, Ertl LS, Baumgart T, Wang Y, et al. C5a receptor (CD88) blockade protects against MPO-ANCA GN. J Am Soc Nephrol 2014;25:225–31.
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Recent pathogenetic advances in ANCA-associated vasculitis
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on line on www.em-consulte.com/revue/lpm www.sciencedirect.com
Quarterly Medical Review
Recent pathogenetic advances in ANCA-associated vasculitis William F. Pendergraft III1,2, Patrick H. Nachman 1
Available online:
1. University of North Carolina (UNC), kidney center, division of nephrology, department of medicine, Chapel Hill, NC 27599-7155, United States 2. Broad Institute of Harvard and MIT, Cambridge, MA, United States
Correspondence: Patrick H. Nachman, university of North Carolina (UNC), kidney center, division of nephrology, department of medicine, Chapel Hill, NC 27599-7155, United States. [email protected]
Vasculitis research: don't slow down and plan for a life-time commitment Christian Pagnoux et al., Toronto, Canada Recent pathogenetic advances in ANCAassociated vasculitis William F. Pendergraft et al., Chapel Hill, NC, United States Biologics in vasculitides: where do we stand, where do we go from now? Giulia Pazzola et al., Reggio Emilia, Italy Update on the treatment of ANCA associated vasculitis Rona M. Smith, Cambridge, United Kingdom Risks of treatments and long-term outcomes of systemic ANCA associated vasculitis Oliver Floßmann, Berkshire, United Kingdom Update on Takayasu's Arteritis Fatma Alibaz-Oner et al., Istanbul, Turkey What matters for patients with vasculitis? Elaine Novakovich et al., Bethesda, MD, United States
Summary Since the discovery of anti-neutrophil cytoplasmic autoantibodies (ANCA), great strides have been made in elucidating the etiology and pathogenesis of disease. In this article, we review recent published key breakthroughs in understanding the pathogenesis of ANCA vasculitis, including some that may lead to novel therapeutics. These breakthroughs have occurred in multiple areas of investigation. A European genome-wide association study (GWAS) revealed the importance of the genetic contribution of proteinase 3 (PR3) and its endogenous inhibitor, alpha (1)-antitrypsin as well as HLA risk. Epigenetic modification of autoantigen genes appears to contribute to perpetuation of disease and possibly relapse risk. Autoantigen excision, a novel method to detect autoantibody epitopes using mass spectrometry, not only revealed pathogenic epitopes in myeloperoxidase (MPO)-ANCA vasculitis and identified unique MPO-ANCA responsible for the majority of ANCA-negative small vessel vasculitis, but has vast applicability to other autoantibodymediated diseases. An explosion of biomarker studies has revealed circulating cytokines and alternative complement pathway products that may predict active disease. Interestingly, alternative complement pathway blockade in the murine model of disease is protective and a clinical trial in humans using an oral alternative complement pathway inhibitor is underway. Increasing clarity of the role of B and T cells in disease pathogenesis is ongoing. B cell depleting agents have shown great utility in remission induction and maintenance, and monitoring specific B cell subsets during the disease course may have predictive power for remission maintenance. Despite these substantial advances, more research is needed including, but not limited to, validation of existing discoveries. As additional novel discoveries emerge, so will novel therapies, and it is with great hope that these collective insights will ultimately lead to prevention and cure.
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nti-neutrophil cytoplasmic autoantibodies (ANCA), directed against the neutrophil granule proteins myeloperoxidase (MPO) and/or proteinase 3 (PR3), are pathogenic and cause small and medium vessel vasculitis [1]. Since the discovery of ANCA, great strides have been made in
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In this issue
tome xx > n8x > xx 2015 http://dx.doi.org/10.1016/j.lpm.2015.04.007 © 2015 Published by Elsevier Masson SAS.
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
W.F. Pendergraft III, P.H. Nachman
understanding the etiology and pathogenesis of disease. In this article, we review recently published key breakthroughs in understanding the pathogenetic mechanisms in ANCA-associated vasculitis that have opened the way to novel therapeutic approaches and interventions.
Genetics
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The recent advent of human whole genome sequencing has resulted in exceptional new insight into the role of genetics across all aspects of human health [2]. It has long been proposed that patients with ANCA-associated vasculitis have a genetic predisposition for disease, and investigators in the field have taken full advantage of this expanding technological breakthrough to identify genetic underpinnings. An analysis of 168 single nucleotide polymorphisms (SNPs) previously associated with Crohn's disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), celiac disease and ulcerative colitis was performed using genotype data from 2 different cohorts comprising a total of 880 patients with granulomatosis with polyangiitis (GPA) and 1969 controls [3]. SNPs within CTLA4, which encodes a T cell surface molecule termed cytotoxic T-lymphocyte antigen-4 (CTLA4) that down-regulates T-cell activation, were significantly associated with GPA (OR 0.79, 95% CI 0.70–0.89, P = 9.8 10 5). This result confirms previous genetic evidence implicating CTLA4 in the susceptibility to ANCA-associated vasculitis. One study from the United Kingdom identified a significant association of two SNPs within CTLA4 (exon 1 [+49] and exon 4 [CT60]) by PCR and restriction fragment length polymorphism (RFLP) of DNA from 222 patients with small vessel vasculitis (SVV) and 670 controls [4]. A separate study from the Netherlands identified that the +49 allele was significantly associated with patients with small vessel vasculitis [5]. Genotyping of the CT60 SNP (rs3087243) in CTLA4 using DNA from 641 patients with ANCA vasculitis confirmed a significant association (P = 6.4 10 3) and showed that the minor A allele was protective (OR 0.84), as compared to the risk-conferring G allele [6]. The contribution of CTLA4 polymorphisms to risk of ANCA-associated vasculitis was further confirmed by a recent meta-analysis of 7 different CTLA4 studies [7] suggesting that these genetic polymorphisms lead to T cell hyper-reactivity thereby contributing to the pathogenesis of disease. Importantly, however, all of the studies associating CTLA4 with ANCA-associated vasculitis were performed in cohorts of Caucasian patients of European descent. No association of CTLA4 was found in a small cohort of 69 Japanese patients with predominantly MPO-ANCA vasculitis [8]. A SNP (R620 W, rs2476601) in PTPN22, another gene encoding a negative regulator of T-cell activation, has also been associated with ANCA vasculitis in Caucasian European cohorts [6,9]. A recent meta-analysis of this risk-conferring polymorphism tested in four studies comprising 1399 Caucasians with
ANCA-associated vasculitis and 9934 controls revealed a significant association of the A allele (OR 1.44, 95% CI 1.26–1.64, P < 0.00001) not only among all patients, but also within the disease subsets GPA (OR 1.72, 95% CI 1.35–2.20, P < 0.00001) and microscopic polyangiitis (MPA, OR 1.53, 95% CI 1.08–2.15, P = 0.02). The allele was also significantly associated with PR3ANCA (OR 1.74, 95% CI 1.25–2.430, P = 0.001) when compared to controls. Furthermore, there was allelic significance with lung (OR 1.69, 95% CI 1.21–2.36, P = 0.002), skin (OR 2.55, 95% CI 1.69–3.84, P < 0.0001) and peripheral nerve (OR 2.12, 95% CI 1.39–3.22, P = 0.0005) involvement when compared to other organ involvement [10]. Mechanistic examination of this gain of function variant in leukocytes from patients with ANCA vasculitis revealed persistently elevated basal phosphatase activity that acted as a dominant negative regulator of ERK activity to thereby blunt interleukin-10 production, an immunoprotective cytokine [11]. The CTLA4 and PTPN22 mutations have been implicated in many other autoimmune diseases, which would suggest their dysfunctional contribution to global immune dysregulation. The first ever genome-wide association study (GWAS) in ANCAassociated vasculitis was performed recently using DNA samples from 1233 patients in the United Kingdom with ANCA-associated vasculitis and 5884 controls and a replication cohort of 1454 Northern European case patients and 1666 controls [12]. Patients with PR3-ANCA had significant associations with HLA-DP and genes encoding alpha (1)-antitrypsin (SERPINA1, the endogenous inhibitor of PR3) and PR3 (PRTN3) (P = 6.2 10 89, P = 5.6 10 12 and P = 2.6 10 7, respectively). Patients with MPO-ANCA had a significant genome-wide association with HLA-DQ (P = 2.1 10 8). Interestingly, genetic associations were made most strongly with respect to ANCA serotype rather than disease phenotype. Furthermore, neither CTLA4 nor PTPN22 were identified by this GWAS, which could be explained at least in part by demographic makeup of participants studied and/or a low frequency of association with these two previous genes of interest. There appear to be clear racial and geographic differences that affect genetic predisposition. Reports of ANCA-associated vasculitis in Africa are rare and ANCA vasculitis is notably rare in African Americans; however, an association of the HLA-DRB1*15 allele with PR3-ANCA-positive disease was found by MHC class II genotyping, which confers a 73.3-fold higher risk in African American patients (OR 73.3, 95% CI 9.1–591.0, P = 2.3 10 9) than in community-based controls [13]. Examination of a validation cohort comprised of DNA samples collected from patients within the Vasculitis Clinical Research Consortium (VCRC) confirmed this strong association. Interestingly, the DRB1*1501 allelic variant, which is of Caucasian descent, was found in 50% of African American patients, whereas the DRB1*1503, of African descent, was under-represented in the discovery group examined from the South-eastern
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007 Recent pathogenetic advances in ANCA-associated vasculitis
Phenotype versus serotype An emerging concept in ANCA-associated vasculitis founded on the genetic discoveries listed above relates to the relationship between disease serotype and phenotype. In other words, is more pathogenetic insight gained by comparing patients with differing phenotypes (GPA, MPA, renal-limited disease) or serotypes (MPO, PR3, dual-positive, ANCA-negative)? Collectively, many of the findings described in this review lend credence to the concept that MPO- and PR3-AAV are genetically distinct diseases with phenotypic overlap and that translational studies and clinical trials may benefit from clustering patients by serotype.
Epigenetics Epigenetics, the study of durable alterations in the transcriptional potential of a cell that cannot be attributed to nucleotide sequence variations, is a field that will clearly provide valuable insight into the pathogenesis and perhaps etiology of ANCAassociated vasculitis. Despite a burst of activity related to understanding DNA methylation and histone modification in other forms of human disease [15,16], only one study specifically addressing epigenetics in ANCA-associated vasculitis has been published thus far [17], although investigations based on genome-wide methylation analyses of healthy human
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leukocyte subsets are currently ongoing by investigators in the field, which is encouraging [18]. To set the stage, our group has scientifically promoted the notion that patients with ANCA-associated vasculitis harbor peripheral leukocytes, primarily monocytes and neutrophils, which aberrantly express the ANCA autoantigens PR3 and MPO. This was first discovered by microarray analyses of leukocytes from patients with ANCA-associated vasculitis who also had glomerulonephritis [19]. Interestingly, in addition to these two genes, a global granulopoiesis signature was identified and in situ staining identified PR3 transcripts in mature monocytes and neutrophils [20]. MPO and PR3 transcriptional upregulation correlated with disease activity and absolute neutrophil counts, but did not correlate with immunosuppression, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender or age. Furthermore, this transcriptional aberration was not associated with disease-control patients with end-stage kidney disease (ESKD) or SLE. This was a surprising discovery since the MPO and PRTN3 genes are located on different chromosomes yet their expression is coordinately regulated. It was subsequently postulated that epigenetic modifications associated with gene silencing were dysfunctional and contributed to aberrant transcription of MPO and PR3 in mature monocytes and neutrophils. Our group examined MPO and PR3 loci for chromatin modifications in neutrophils from patients with ANCA vasculitis and healthy controls [17]. It was found that histone H3K27me3 is depleted at PR3 and MPO loci due to increased expression of Jumonji domain-containing 3 (JMJD3) demethylase as well as failure of the transcription factor RUNX3 to recruit EZH2, which is responsible for H3K27me3 methylation. These epigenetic changes may explain why MPO and PR3 are aberrantly upregulated in leukocytes of patients with ANCA vasculitis, which likely sparks and/or perpetuates the disease process if left unchecked. Numerous questions have yet to be answered in this area. Kurz et al. recently hypothesized that the granulopoiesis transcriptional signature discussed above could predict relapse, but found that MPO and PR3 mRNA levels correlated with corticosteroid therapy instead [21]. This is a clear discrepancy in the field that needs to be reconciled in order to make progress from both a genetic and epigenetic standpoint. An important question pertains as to whether the observed epigenetic changes are pathogenetically causative of ANCA vasculitis or merely a reflection of ongoing disease processes. If the former is true, then therapies could be conceived to specifically counteract or modify this epigenetic dysregulation.
Autoantigens, autoantibodies and biomarkers Patients with clear clinical and histopathologic evidence of small vessel vasculitis and/or pauci-immune necrotizing and crescentic glomerulonephritis but with negative ANCA serology have
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United States. Further support of this association was made by the demonstration that DRB1*1501 protein bound with high affinity to PR3 protein in vitro and on the surface of activated healthy human neutrophils [13]. It should be noted that while SNP analyses and GWAS provide important clues to genetic risk by revealing candidate loci, each locus identified explains only a very small fraction of phenotypic variance and all loci identified to date in ANCA-associated vasculitis explain only slightly more variance. As an example using a simple trait much more common than risk of ANCA-associated vasculitis, it has been proposed that roughly 93,000 SNPs are needed to explain 80% of the population variation in height [14]. This argues against the contention that the genetic associations gleaned thus far are likely to play a significant role in identifying populations at sufficiently higher risk of disease to institute a surveillance or preventive intervention. In addition to genetic risk loci, including those yet to be identified, it is clear that other factors contribute to the development and perpetuation of ANCA-associated vasculitis, which include but are not limited to copy number variations, epigenetic modifications and their relationship to environmental exposures. Overall, several risk alleles have been identified that are associated with ANCA vasculitis. Some risk alleles are specifically associated with the MPO- or PR3-ANCA serotypes, suggesting that MPO-ANCA vasculitis and PR3-ANCA vasculitis may be genetically distinct diseases with phenotypic overlap.
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
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presented important challenges regarding the pathogenetic role of ANCA. The negative serology also frequently causes delays in establishing the diagnosis and initiating appropriate treatment of such patients. To address this issue, a multi-center study by Roth et al. recently reported the development of a novel assay termed "epitope excision'' or "autoantigen excision'' to identify the specific target epitopes for ANCA [22]. Briefly, autoantigen (in this case MPO) is incubated with protein A/G-bound IgG containing MPO-ANCA. This mixture is washed extensively and then incubated with trypsin to digest non-complementarity determining region (CDR)-bound antigen away. After additional washing, the CDR-bound piece of antigen is eluted and subjected to protein sequencing by mass spectrometry. First, this assay allowed for highly-sensitive epitope mapping of murine and human MPOANCA and revealed that patients with ANCA-associated vasculitis harbor autoantibodies to pathogenic epitopes. Antibodies to MPO could also be detected in sera of healthy controls, but their epitope specificities differed significantly when compared to those from patients with MPO-ANCA vasculitis. Importantly, some patients with small vessel vasculitis who are ANCA-negative by the standard clinical ELISA test, were in fact found to harbor autoantibodies to a pathogenic MPO epitope capable of in vitro neutrophil activation and induction of nephritis in mice. It was discovered that this antibody-epitope interaction was blocked by the presence of a fragment of ceruloplasmin. Ceruloplasmin is the circulating endogenous inhibitor of MPO, which is removed during the IgG purification step, thus explaining the negative result of the standard serum-based clinical ANCA test. Although these findings need independent validation, this study not only identified immunodominant and pathogenic epitopes using a novel epitope mapping assay, but also laid the groundwork for the development of a future clinical assay to use for patients with ANCA-negative disease that could expedite diagnosis and treatment. This may also pertain to an enhanced ability to identify individuals prior to disease development given the recent findings by Olson et al. that PR3-ANCA were detected in patients with GPA before disease onset [23]. Regardless, this work could provide a better understanding of pathogenesis and evolution of disease. Controversy persists with respect to ANCA target autoantigens other than MPO and PR3. Kain et al. reported in 2008 the presence of autoantibodies to lysosome-associated membrane protein-2 (LAMP-2) in the serum of the majority of patients tested with pauci-immune focal necrotizing and crescentic glomerulonephritis [24]. An epitope within LAMP-2 (p41-9) was shown to have 100% homology to FimH, a bacterial adhesion, and autoantibodies directly-cross reacted suggesting a molecular mimic. Furthermore, rats immunized with FimH developed autoantibodies to rat and human LAMP-2 and disease akin to what is observed in humans. A subsequent report from the same group revealed the presence of anti-LAMP-2
autoantibodies in patients from Austria, the Netherlands and the United Kingdom [25]. The same group later published that 8 of 11 (73%) of patients with ANCA-negative pauci-immune focal necrotizing glomerulonephritis harbored anti-LAMP-2 autoantibodies [26]. In a conflicting report, Roth et al. measured anti-LAMP-2 autoantibodies in 680 serum samples from two academic centers in the United States from patients with ANCA glomerulonephritis (n = 329), ANCA-negative glomerulonephritis (n = 104), patients with E. coli urinary tract infection (UTI, n = 104), disease controls (n = 19) and healthy volunteers (n = 124) [27]. Anti-LAMP-2 autoantibodies were detected in only 21% of sera rom patients with ANCA-associated vasculitits and 16% of patients with a UTI who did not have ANCA vasculitis. Thus, the relevance of LAMP-2 and anti-LAMP-2 antibodies in ANCA-associated vasculitis requires further investigation. Biomarkers, alternatively termed bioindicators, are needed to identify patients with ANCA-associated vasculitis prior to disease development and to identify predictors of treatment response as well as relapse. There have been numerous biomarker studies to date. The most robust measured 28 serum proteins based on hypothetical disease association in 186 patients who participated in the rituximab in ANCA-associated vasculitis (RAVE) trial before and six months after treatment in order to try and distinguish severe disease (BVAS/WG 3) from remission (BVAS/WG = 0) [28]. Active disease was best discriminated from remission (AUC > 0.8) and healthy controls (AUC > 0.9) by levels of CXCL13, MMP-3 and TIMP-1. Although yet to be confirmed, there is now preliminary evidence in humans for a contribution of the alternative complement pathway in ANCA-associated vasculitis. Increased plasma levels of C3a, C5a, soluble C5b-9 and Bb in plasma were identified in 66 patients with active disease as compared to 54 patients in remission [29]. Urinary Bb levels were also found to correlate with and serum creatinine in 27 patients with active disease, and correlated inversely with the proportion of normal glomeruli in matched kidney biopsy specimens [30]. Again, these findings require validation in other controlled clinical settings.
Immune cell dysregulation Immune and non-immune cells have perennially been the subject of intense analysis in the field of ANCA-associated vasculitis, and B and T-lymphocytes are most hotly studied at present. Recognizing the pathogenic role of ANCA has fostered the therapeutic use of B cell depleting agents. Ongoing investigations are focused on measuring circulating B cells numbers and proportions of B cell subsets to monitor disease activity and potentially as indicators of risk of relapse. Bunch et al. longitudinally investigated for 4–99 months the peripheral B cell phenotype in 54 patients with either active or quiescent ANCAassociated vasculitis as compared to 68 healthy controls [31]. Patients with active disease had a lower percentage of CD5+ B cells when compared to patients in remission or healthy
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007 Recent pathogenetic advances in ANCA-associated vasculitis
Animal models The well-characterized mouse model of MPO-ANCA vasculitis, whereby anti-mouse MPO autoantibodies purified from
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MPO-deficient mice immunized with murine MPO are passively transferred into wild-type mice and cause vasculitis and pauciimmune crescentic glomerulonephritis, has permitted exploration of the role of various inflammatory pathways. Based on this model, a previously unsuspected role of complement activation was recently demonstrated. Upon the passive transfer of antiMPO antibodies, disease induction failed with administration of cobra venom factor and in mice deficient in complement factors C5 and B, whereas mice deficient in C4 developed disease comparable to that observed in wild-type mice [36]. These findings point to the importance of the alternative, but not the classical or lectin binding pathways of complement activation in this animal model of ANCA-associated disease. Furthermore, disease could be also be completely abolished or markedly ameliorated by treating the mice with a C5-inhibiting monoclonal antibody either 8 hours before or 1 day after disease induction with anti-MPO IgG and lipopolysaccharide [37]. Conversely, C6 deficient mice are not protected from disease induction by anti-MPO antibodies, suggesting that the C5b-9 membrane attack complex formation is not essential in the pathogenetic pathway. It was subsequently demonstrated in humans that blockade of the C5a receptor on human neutrophils abrogated their stimulation in vitro [38]. More recent work confirmed the immunopathogenetic importance of the alternative complement pathway in that blockade of C5a receptor (C5aR) activity protected against disease development [39]. Transgenic mice expressing human C5aR were protected from disease when given an oral small molecule antagonist of human C5aR called CCX168. Overall, these results suggest an important role for alternative complement pathway activation in the pathogenesis of ANCA vasculitis and identify C5a and its receptor as a targetable therapeutic pathway. A clinical trial is underway to assess the safety and efficacy of CCX168 in patients with ANCA vasculitis (http://www. clinicaltrials.gov, NCT01363388). It is also important to note that the evidence for the role of the alternative complement pathway in ANCA-associated vasculitis emanates from a model of anti-MPO-mediated disease, and indirectly from patients with MPO-ANCA [29,30]. To date, there is no direct evidence in mice or men that this also applies to PR3-ANCA-mediated disease.
Future directions and conclusions Major pathogenetic inroads have been made recently, but it remains clear that there is more to do in the field of ANCAassociated vasculitis. Many of the recent discoveries discussed clearly require validation in separate cohorts, and confirmatory studies are required to resolve discrepant results. The field has been enlightened by the results of the European GWAS, which revealed the importance of the genetic contribution of PR3 and its endogenous inhibitor, alpha (1)-antitrypsin as well as HLA risk; however, the majority of patients included harbored PR3ANCA and the power to detect genetic associations with patients
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controls. Furthermore, patients treated with rituximab who were on low or no maintenance immunosuppression and who had a low or sharply declining repopulation percentage of CD5+ B cells relapsed sooner than patients maintained on high maintenance immunosuppression (median 17 versus 29 months, respectively, P = 0.002). With the presumed intention of validating these findings, Unizony et al. recently analyzed absolute and relative numbers of CD5+ B cells from PBMC samples collected from 197 participants in the RAVE trial over 18 months and were unable to identify a significant association with disease severity and/or failure of induction therapy; however, analytic methodologies may have been different [32]. CD5 is one of several cell surface markers that characterize regulatory B cells. Although there is currently controversy in the field about which set of markers characterizes regulatory B cells, it is generally agreed upon that a regulatory B cell must be functionally capable of producing interleukin-10 [33]. Given the discrepancy in the field regarding the role of monitoring B cells to assess disease activity as well as the utility of monitoring CD5 + B cells specifically, more work is needed to determine appropriate phenotypic methods to detect regulatory B cells and inclusion of these measurements in future clinical trials is warranted. With regards to recent advances in understanding the role of T cells in ANCA-associated vasculitis, Free et al. hypothesized that T cell dysregulation is involved in disease pathogenesis and tested this hypothesis by evaluating CD4+ T cell populations from 62 patients with ANCA-associated vasculitis compared to 19 healthy controls [34]. Patients with active disease had a higher frequency of regulatory T cells. However, a proportion of regulatory T cells were found to be lacking FoxP3 exon 2, associated with a decreased suppressive function. Furthermore, a pro-inflammatory CD4+ T cell population was identified that was also increased in frequency and was resistant to suppression by regulatory T cells. Recent systems-level transcriptional profiling of CD8+ T cells from patients with ANCA-associated vasculitis revealed a three gene signature (ITGA2, NOTCH1 and PTPN22) that identified patients who have a poor prognosis [35]. This signature had similar predictive prognostic value in patients with SLE, but was unable to differentiate between the two diseases (ANCA vasculitis vs. SLE). Unfortunately, this predictive signature was absent in total peripheral blood mononuclear cell preparations, but was detectable only from enriched CD8+ T cells, which makes the clinical application of this test less practical. In addition, as with many of the findings discussed in this review, these findings require validation in other cohorts of patients with ANCA-associated vasculitis.
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To cite this article: Pendergraft III WF., Nachman PH.. Recent pathogenetic advances in ANCA-associated vasculitis. Presse Med. (2015), http://dx.doi.org/10.1016/j.lpm.2015.04.007
W.F. Pendergraft III, P.H. Nachman
harboring MPO-ANCA was limited. A GWAS including a larger number of MPO-ANCA positive patients is greatly needed and North American investigators are poised to make this possible given the extensive collection of DNA samples across centers within the VCRC. As additional novel discoveries emerge, so too will novel therapies, and it is with great hope that these collective insights will ultimately lead to prevention and cure.
Acknowledgments: the authors wish to thank the patients with ANCAassociated vasculitis and the other scientists and health care providers dedicated to their care at UNC. WFP3 and PHN are supported by grants from the NIDDK (P01-DK058335-14 and 1UM1-DK100845-01). WFP3 was also supported in part by NIDDK grant # 5 F32 DK097891-02 and receives translational research funding from The Broad Institute. Disclosure of interest: the authors declare that they have no conflicts of interest concerning this article.
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