Abstract
The conversion of an arginine residue in a protein to a citrulline residue, a reaction carried out by enzymes called peptidylarginine deiminases (PADs), is rather subtle. One of the terminal imide groups in arginine is replaced by oxygen in citrulline, thus resulting in the loss of positive charge and the gain of 1 dalton. This post-translational modification by PAD enzymes is conserved in vertebrates and affects specific substrates during development and in various mature cell lineages. Citrullination offers a unique perspective on autoimmunity because PAD activity is stringently regulated, yet autoantibodies to citrullinated proteins predictably arise. Autoantigens recognized by anti-citrullinated protein antibodies (ACPA) include extracellular proteins such as filaggrin, collagen II, fibrinogen, and calreticulin; membrane-associated proteins such as myelin basic protein; cytoplasmic proteins such as vimentin and enolase; and even nuclear proteins such as histones. Some ACPA are remarkably effective as diagnostics in autoimmune disorders, most notably rheumatoid arthritis (RA). Several ACPA can be observed before other clinical RA manifestations are apparent. In patients with RA, ACPA may attain a sensitivity that exceeds 70 % and specificity that approaches 96–98 %. The biological context that may account for the induction of ACPA emerges from studies of the cellular response of the innate immune system to acute or chronic stimuli. In response to infections or inflammation, neutrophil granulocytes activate PAD, citrullinate multiple autoantigens, and expel chromatin from the cell. The externalized chromatin is called a neutrophil extracellular “trap” (NET). Citrullination of core and linker histones occurs prior to the release of chromatin from neutrophils, thus implicating the regulation of citrullinated chromatin release in the development of autoreactivity. The citrullination of extracellular autoantigens likely follows the release of NETs and associated PADs. Autoantibodies to citrullinated histones arise in RA, systemic lupus erythematosus, and Felty’s syndrome patients. The citrullination of linker histone H1 may play a key role in NET release because the H1 histone regulates the entry and exit of DNA from the nucleosome. Juxtaposition of citrullinated histones with infectious pathogens and complement and immune complexes may compromise tolerance of nuclear autoantigens and promote autoimmunity.
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Introduction
The initial discovery of citrulline residues in proteins seemed to be a biochemical anomaly as it contradicted the central dogma of molecular biology. Since there is no codon or tRNA for citrulline, this amino acid residue cannot be translationally incorporated into newly synthesized proteins. However, decades ago, proteins containing citrulline were unambiguously identified in several mammalian tissues [1]. Thus, enzymes that convert arginine residues to citrulline residues in a protein were predicted to exist, and indeed, distinct enzymes were subsequently discovered in vertebrate species ranging from fish to humans and they were successfully purified from skin, muscle, and hair follicles [2, 3]. In total, higher eukaryotes express five peptidyl arginine deiminases (PADs), which modify proteins with important tissue-specific functions. However, the significance of this arginine modification remained uncertain until additional substrates were identified.
Among the first deimination substrates that were identified in the epidermis were keratin and filaggrin, two autoantigens targeted in disorders as diverse as pemphigus and rheumatoid arthritis (RA) [4, 5]. These studies identified filaggrin as the target of the previously unexplained reactivity of RA sera with rat epithelia [6]. These results prompted the search for the specific molecular determinants that account for RA reactivity. The search was directed toward a specific sequence contained within filaggrin, a protein that contains a high number of citrulline residues. Positive “hits” were recorded against peptides matching the sequence of filaggrin provided that the peptides incorporated citrulline residues in place of arginine residues during synthesis [7]. Such peptides proved to be particularly useful substrates in assays used to diagnose RA [2, 8–12]. The usefulness of the peptides was increased by joining their ends to form a loop. Such cyclic citrullinated peptides (CCP) were used for the development of an ELISA that attains a high sensitivity and exhibits remarkable specificity for RA over other autoimmune conditions [8]. The assay has since been optimized, and it is included among the revised 2010 classification criteria for RA [13].
Preceding studies on autoimmunity in multiple sclerosis (MS) patients had indicated that myelin basic protein (MBP) exists in alternative isoforms, which contain variable numbers of citrulline residues and differ by their isoelectric points, and that these isoforms differentially react with T cell lines from MS patients [14]. The number of citrulline residues was observed to change during development and during progression of MS [15]. Thus, deimination was shown to be regulated during development and to dictate immunoreactivity of autoantibodies in diverse disorders. These discoveries have had profound impacts on the diagnosis and clinical evaluation of RA and related autoimmune disorders.
Autoantibodies Recognize Citrulline in Diverse Autoantigens
The remarkable success of the anti-CCP assay in the detection of RA led to a sustained, worldwide effort to identify additional citrullinated autoantigens (Table 1). These efforts also shed light on the biological mechanisms that drive the conversion of arginine residues in proteins to citrulline residues. Early on, it was recognized that filaggrin is not the only extracellular matrix protein that is modified by PADs. Other important proteins that are deiminated by PADs and whose citrulline residues form part of the epitopes recognized by RA autoantibodies include collagen type II and fibrinogen. Elevated levels of citrullinated collagen II in the synovium of RA patients [16] suggested that PAD-mediated modification of cartilage in RA joints may directly contribute to the induction of autoantibodies. In parallel, a citrullinated peptide derived from the primary sequence of fibrin (residues 60 to 74) was shown to be useful as a clinical diagnostic for RA with a sensitivity of 74 % and a specificity of 95 % [12].
Interestingly, calreticulin, a plasma protein that is involved in binding to apoptotic cells [17] and that contributes to innate system activation, can recognize the conserved domain of RA-predisposing HLA molecules (the so-called “shared epitope”). Furthermore, the binding is enhanced by deimination of calreticulin [18]. This observation suggests that deimination of certain plasma proteins may play a regulatory role in the clearance of cell remnants. A similar anti-inflammatory role may be ascribed to the deimination of cytokines such as CXCL8, CXCL10, and TNF [19–21]. The deiminated cytokines have a reduced chemotactic potency and a decreased stimulatory effect on other immune cells. Perhaps, autoantibodies to citrullinated autoantigens may also have a beneficial effect by enhancing clearance of the modified antigens.
Conversely, increased citrullination of autoantigens such as myelin basic protein is associated with impaired function of the affected organs [22]. Similarly, the deimination of filaggrin promotes the unfolding and degradation of this structural protein [23], and the deimination of fibronectin decreases its function as ligand for adhesion receptors [24]. Deimination also has destabilizing effects on cytoplasmic proteins. Citrullinated vimentin is found at elevated levels in the synovial fluid and in circulating immune complexes of RA patients [25], and vimentin deimination is linked to the disassembly of the cytoskeleton [26]. By analogy, the deimination of F-actin capping protein presumably deregulates the formation of actin fibers [27].
Three additional proteins are preferentially recognized in their citrullinated form. The citrullinated form of the immunoglobulin chaperone BiP is preferentially bound by RA patients’ sera, and treatment of mice with citrullinated BiP promotes experimental arthritis [28]. The deiminated HSP90 heat shock protein was identified as a useful diagnostic autoantigen in interstitial lung disease that is a potentially serious manifestation of RA [29]. The deimination of enolase, a glycolytic enzyme, may play a role during the infection of gingival epithelial cells [30]. This enzyme is one of the substrates of the bacterial PAD, which is expressed by the periodontal pathogen Porphyromonas gingivalis. The discovery of infection-induced autoantigen deimination supports the intriguing possibility that an oral pathogen could induce autoantigen modifications and thus break immune tolerance. Clearly, analysis of anti-citrullinated protein autoantibodies (ACPA) has fostered the emergence of productive new areas of research (see [31, 32] as examples).
One immediate obvious implication of autoantibodies to citrullinated epitopes is that the post-translational modification (PTM) itself is the protagonist in converting the autoantigens into stimuli for the adaptive immune system. Tolerance is a strong force that normally prevents the activation of autoreactive B and T lymphocytes in individuals who remain free of autoimmune disease. Therefore, the central question in autoimmune disease research is to account for the initial events that break tolerance and lead to the specific recognition of autoantigens. One possibility, suggested by the prevalent occurrence of ACPA, was that the conversion of specific arginine residues into citrulline residues alters the recognition of B cell receptors and/or T cell receptors to such a degree that tolerance is evaded and lymphocytes to the modified autoantigens proliferate [33]. However, it remained a matter of speculation what conditions were likely to lead to a drastic change in the amount of citrullinated proteins. Possible candidates for these conditions were discovered in the course of studies into the regulation of PADs.
Histone Deimination
Deeper understanding of autoantigen deimination came from careful analysis of stimuli that causes the activation of PAD4, the only PAD that is localized to the nucleus and abundantly expressed in granulocytes and monocytes [2, 3]. Immunofluorescence with an anti-PAD4 monoclonal antibody demonstrated the highly variable expression levels and heterogeneous cellular distribution of PAD4 in human blood neutrophils (Fig. 1). Because PAD activity strictly depends on calcium, Hagiwara et al. induced granulocyte differentiation in HL-60 cells and then exposed them to calcium ionophore [34]. This treatment raised intracellular calcium levels and induced deimination. Antibodies to modified citrulline were used in two-dimensional protein gel electrophoresis to identify nucleophosmin and three of the four core histones (H2A, H3, and H4) as abundant substrates of PAD4 [34]. This and subsequent studies implicated apoptosis in the induction of histone deimination. Because granulocyte apoptosis is induced during the resolution phase of an inflammatory response, it was proposed that increased amounts of citrullinated autoantigens may be generated during an inflammatory response in vivo.
However, the idea that apoptosis induces deimination proved incorrect. This was concluded from studies showing that classic stimuli for apoptosis fail to induce PAD activation, and caspase inhibitors are unable to block deimination [35]. The solution to this dilemma was provided by experiments that identified a new form of neutrophil cell death. Brinkman and Zychlinski discovered that neutrophils initiate a programmed cell death quite distinct from apoptosis when in contact with bacteria, yeast, or viruses [36–38]. This program proceeds through stages of nuclear and granule membrane dissolution, chromatin unwinding, and the release of chromatin from the cell [39]. Because the extracellular chromatin may capture and immobilize microbes, the authors coined the expression “neutrophil extracellular traps” (NETs), and nowadays, this form of cell death is known as NETosis [37, 38].
Neeli et al. were the first to connect histone deimination with NET release by showing that HL-60 granulocytes and primary human blood neutrophils respond to numerous stimuli associated with infections or inflammation by rapidly inducing histone deimination [35]. The stimuli can be as diverse as lipopolysaccharide (LPS), tumor necrosis factor (TNF), hydrogen peroxide, or lipoteichoic acid. The specific induction of histone deimination can be visualized by immunofluorescence with antibodies that react with deiminated histone H3. Neeli et al. used this technique and discovered that deiminated histones are incorporated into NETs [35]. Alternatively, citrulline residues can be visualized with a chemical probe that reacts with the functional groups on citrulline (Fig. 2). This approach indicates that a majority of protein deimination occurs in the granulocyte nucleus. The direct relation between histone deimination and NETosis was confirmed by Wang and colleagues who showed PAD4-mediated histone PTM in cells undergoing NET release in response to calcium ionophore or TNF [40]. Later, examination of PAD4-deficient mice revealed that PAD4 activity is required for NETosis [41]. Therefore, evidence of deiminated histones has become synonymous with an inflammatory process [42–45]. Once conditions for physiologically induced histone deimination were identified, it became important to test whether deiminated histones serve as preferential substrates for disease-associated autoantibodies.
Autoantibodies to Deiminated Histones
Numerous observations had indicated that activated neutrophils present a more suitable target for certain types of autoantibodies [46]. However, Dwivedi et al. systematically tested the idea that deiminated histones are the preferred antigens of human autoantibodies [47]. First, the authors compared binding of systemic lupus erythematosus (SLE), RA, and Felty’s syndrome patients’ sera to unstimulated versus LPS-treated neutrophils by confocal microscopy and determined that activated neutrophils and their NETs react more avidly with patient IgG. Second, Dwivedi et al. prepared deiminated histones by incubating purified histones with recombinant PAD4 and observed preferential binding to deiminated histones in ELISA and Western blots. Subsets of SLE and RA sera and essentially all Felty’s syndrome sera showed preferential binding. Felty’s syndrome is a rare but severe variant of RA. Third, these authors also showed that patients’ sera contained substances leading to increased levels of spontaneous NETosis. Thus, a potential vicious cycle of NETosis induction and autoantibodies to NET-associated antigens was discovered [33]. Pratesi and colleagues extended the work by Dwivedi et al. by using citrullinated peptides derived from histone H4 and showing that the peptides exhibit equal or better discrimination between RA and control sera than citrullinated peptides derived from filaggrin do [48].
Fundamental insights into the relation between NETosis and autoimmunity were derived in a notable series of papers published in 2011. Lupus neutrophils were shown to often include a population of low density granulocytes that exhibit an increased tendency for NETosis [49]. In vivo, these cells potentially account for the observed NET DNA in affected kidneys and skin, along with the increased abundance of NET components in blood that may act as lupus autoantigens [49]. The consequences of an increased NET release in vivo may lead to elevated levels of pro-inflammatory cytokines such as interferon-α (IFN-α) and interleukin-17. The relation between NETs and the production of IFN-α was made explicit by showing that NET components consisting of DNA and the cationic defensin LL37 activate plasmacytoid dendritic cells via toll-like receptor 9 (TLR9) to secrete IFN-α [50]. Additional experiments suggested that SLE patients’ neutrophils are primed by IFN-α to release NETs in response to autoantibody-ribonucleoprotein complexes [51]. These experiments cast previously known roles of TLR9 and type I IFN in a new light.
An activated neutrophil subset was also characterized in patients with RA. Studies by Khandpur et al. [52] identified NETting neutrophils in synovial tissues of RA patients and showed that neutrophils are stimulated to undergo NETosis by incubation with ACPA reacting with vimentin. Such NETs had a distinct composition of NET components, as demonstrated by RA NET purification followed by mass spectrometry [52]. In conjunction, these studies strengthened the argument that neutrophils play an important role in the pathogenesis of SLE and RA. Additional evidence supporting the idea that neutrophil activation is responsible for introducing modifications into autoantigens and that these PTM, in turn, stimulate autoantibody production is provided by the prevalence of RA autoantibodies to oxidatively modified autoantigens [53]. However, studies in a mouse model of lupus could not confirm the contribution of NETs to the development of autoimmunity, as breeding to mice incapable of NET release failed to ameliorate the typical pathology or autoantibody production [54]. It is unclear whether differences in Fc receptors between mice and humans may account for the observed differences in experimental outcomes [55]. Additional parallels between PAD deimination and human pathology exist and promise exciting applications of PAD inhibitors in the treatment of cardiovascular diseases [45] and cancer [56].
Histone Deimination and Chromatin Structure
Experiments with autoantibodies to deiminated histones show that studies in autoimmunity have the ability to illuminate basic principles in molecular biology. H1 linker histones are a family of seven isoforms of extranucleosomal histones that occupy the DNA between adjacent nucleosomes [57]. As such, linker histones are in an ideal position to regulate chromatin structure and gene expression [58]. Because it was not known whether H1 histones are deiminated, conditions that strongly induce PAD4 were used to stimulate neutrophils and linker histones were purified based on their unique solubility in 5 % perchloric acid [59]. Mass spectrometry revealed that only two of the three arginine residues in H1.2 are converted to citrulline residues in neutrophils (Fig. 3), a conclusion that was confirmed in vitro by incubation with the recombinant PAD4 [59]. Peptides containing either of these two citrulline residues were used to determine which citrullinated peptide is the preferred substrate of autoantibodies from SLE or Sjögren’s syndrome patients. Even though only about 6 % of SLE sera contained autoantibodies to deiminated H1 histones, the most prevalent binding was to the citrulline residue at position 53 (Fig. 3). This residue is located within the most conserved portion of the H1 helix-turn-helix domain [59]. Residue 53 plays a key role in regulating chromatin structure, a conclusion consistent with recent observations in pluripotent stem cells. Deimination of H1 linker histone by PAD4 is essential for the reprogramming of gene expression that is required during differentiation of pluripotent stem cells into separate cell lineages [60]. Autoantibodies to deiminated H1 linker histones thus identify a crucial switch in chromatin structure that is essential for stem cell gene reprogramming.
Deimination of core histones similarly has broader implications for gene expression. Now, in the classic experiments on histone deimination, Cuthbert et al. [61] and Wang et al. [62] identified hormone responsive genes whose promoters exhibit deiminated histones. Subsequent studies provided additional examples of gene regulation by histone deimination and linked PAD4 to diverse biological processes ranging from mammalian development to tumorigenesis [63–67]. Because PAD4 autocitrullinates and thereby alters autoantibody binding [68], it will be important to determine whether and to what extent autoantibodies that arise is systemic autoimmune disorders affect the function of PAD4 and its specific chromatin substrates.
Summary and Future Perspectives
From nearly a decade of research into autoantibodies to deiminated histones, it stands established that inflammatory conditions lead to a neutrophil cell death that is both antimicrobial and prone to induce autoantibodies to chromatin autoantigens. Autoantibodies to deiminated histones arise in distinct autoimmune disorders and thus provide arguments for the important role of neutrophils in the initial stimulation of the adaptive immune system that leads to autoimmunity. Clearly, it is imperative to pursue studies on deimination and its regulation during NETosis. Early successes of therapies for autoimmune disorders that are based on inhibition of PAD4 suggest that inhibitors of PAD4 will find broad applications in rheumatology clinics. Studies in animal models of autoimmunity have shown significant improvement in disease presentation upon administration of PAD4 inhibitors [3]. The remarkable list of clinical conditions that were improved by PAD4 inhibition includes experimental arthritis, lupus, MS-like disease, and colitis [69–72]. It is reasonable to expect that future efforts to understand and regulate PAD4 will continue to yield real benefits for patients suffering from diverse autoimmune disorders.
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Acknowledgments
Research in the SM’s laboratory is financially supported by the French Centre National de la Recherche Scientifique, Région Alsace, Laboratory of Excellence Medalis (ANR-10-LABX-0034), EquipEx program I2MC (ANR-11-EQPX-022), Initiative of Excellence (IdEx), Strasbourg University, and ImmuPharma France. Research in the MR’s laboratory is financially supported by The Lupus Research Institute of New York and the ORR Fund of Memphis, TN. We gratefully acknowledge the kind gift of the anti-PAD4 monoclonal antibody from Dr. Katsuhiko Nakashima of the National Cancer Center Institute of Japan. The authors thank Dr. Gilbert de Murcia, CNRS, who took the original images shown in Fig. 3b. We are grateful to Dr. Indira Neeli and Dr. Teruki Hagiwara for their helpful comments that improved this manuscript.
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Muller, S., Radic, M. Citrullinated Autoantigens: From Diagnostic Markers to Pathogenetic Mechanisms. Clinic Rev Allerg Immunol 49, 232–239 (2015). https://doi.org/10.1007/s12016-014-8459-2
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DOI: https://doi.org/10.1007/s12016-014-8459-2