REVIEWS Lungs, joints and immunity against citrullinated proteins in rheumatoid arthritis Anca I. Catrina, A. Jimmy Ytterberg, Gudrun Reynisdottir, Vivianne Malmström and Lars Klareskog Abstract | Rheumatoid arthritis (RA) is a prototype for a criterion-defined inflammatory disease, for which the aetiology and initial molecular pathogenesis has been elusive for a long time. We describe in this Review how studies on the interplay between specific immunity, alongside genetic and environmental predisposing factors, provide new tools to understand the molecular basis of distinct subsets of the disease. A particular emphasis is on the possibility that pathogenic immune reactions might be initiated at other sites than the joints, and that the lungs could harbour such sites. New data strengthen this concept, showing that local immunity towards citrullinated proteins and accompanying inflammation might be present in the lungs early during disease development. This progress makes RA an interesting case for the future development of therapies that might be directed against disease-inducing immunity even before inflammation and destruction of joints has begun. Catrina, A. I. et al. Nat. Rev. Rheumatol. advance online publication 29 July 2014; doi:10.1038/nrrheum.2014.115

Introduction Research into autoimmune diseases covers two critical questions: first, which immunity is present and how is it related to disease phenotype; and second, how and when is that immunity triggered (that is, when tolerance is broken). In rheumatoid arthritis (RA), many aspects of these questions remained unanswere­d for many years, whilst, at the same time, development of therapeutics targeting different pro­inflammatory pathways advanced rapidly.1–4 This Review will describe some of the latest advances that have been made in the understanding of the aforementioned questions regarding autoimmunity and disease in RA. Notably, this progress has resulted in RA being considered a prototype autoimmune disease in which our understanding of the interplay between environ­mental triggers, susceptibility genes and potentially diseaseinducing autoimmunity exceeds that of most other human autoimmune conditions. We will put a special focus on mechanisms whereby tolerance to citrul­linated proteins can be broken and our prototypical example of an organ in which this phenomenon might happen is the lung. Other aspects on how systemic autoimmunity leads to joint inflammation and damage have already been published in this journal.5,6

Autoimmune reactions in RA Rheumatology Unit, Department of Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm S-17176, Sweden (A.I.C., A.J.Y., G.R., V.M., L.K.).

Over the years, a large number of antibody reactivities have been described in RA, beginning with rheumatoid fac­tor (RF) in the late 1930s, and followed by description of anti-perinuclear, anti-collagen II, anti-hnRNP (RA33), anti-glucose phosphate isomerase and anti-binding immunoglobulin (BiP) antibodies7–12 and, since 1998, a long series of antibodies towards various citrullinated

Correspondence to: A.I.C. [email protected]

Competing interests The authors declare no competing interests.

self-proteins and self-peptides.13–18 Antibodies against homo­citrullinated (carbamylated) proteins have also been identified in patients with RA.19 These differ­ent auto­ antibodies are found at varying frequencies in distinct subsets of RA.20–22 By contrast, reports on T‑cell reacti­ vities against self-proteins have been more sparse, but some T‑cell reactivity has been described against colla­ gen type II,23 hnRNP‑A224 and BiP,25 as well as citrul­ linated fibrinogen26 and vimentin.27 Incorporation of this knowledge on antibody and T‑cell reactivities into a more complete picture of RA aetiology and pathogenesis has, however, proven to be more difficult. Some interesting progress has now been made though, including the combination of these descriptive studies on autoimmune reactions in RA with other key features of the aetiology of the diseases, that is, genetic and environmental risk factors of importance in different disease subsets. Anti-citrullinated protein antibodies (ACPAs), RF and some other RA‑related antibodies, including antibodies to carbamylated proteins, can be present many years before onset of disease28–34—before any evidence of inflammation and immunity can be detected in the joints. Triggering of these immune reactions must, therefore, take place somewhere other than in the joints. The more interesting questions in RA pathogenesis are therefore which immune reactions are initiated, and when and how such reactions are initiated and subsequently spread also to the joints. One study that has addressed these questions was performed in a cohort of 400 individuals for whom one or more blood samples (700 blood samples in total) were obtained before onset of RA.35 An important observation was that antibody development followed a distinct pattern in which, in most cases, only one citrullinated epitope was initially recognized in each individual, and in which an increasing diversity in antibody reactivity to a series of different epitopes

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REVIEWS Key points ■■ Autoantibodies against post-translational modified citrullinated proteins, so-called anti-citrullinated protein antibodies (ACPAs), define a distinct clinical RA phenotype; this phenotype is characterized by an increased frequency of early inflammatory lung changes ■■ The presence of ACPAs before signs of inflammation in joints suggests that immunity against citrullinated proteins is initiated outside the joint ■■ Changes in the lung and enrichment of ACPAs in the lungs (bronchoalveolar lavage fluid) occur in both individuals at risk of developing RA as well as patients with early RA ■■ The lung, therefore, might be a site of initiation of immunity to citrullinated proteins ■■ Early targeting of the immune reactions in the lung might be a new approach to modulate disease

on several different citrullinated proteins followed as the time to disease onset approached. The nature of this first appearance of reactivity was, however, different between individuals. This observation indicates that break of tolerance to one citrullinated epitope predisposes to break of tolerance to additional citrullinated epitopes, and also that the mechanisms that contribute to the initial break of tolerance might be more related to recognition of citrul­ linated antigens per se than to a particular citrul­linated autoantigen. Distinct patterns of autoantibody development immediately before onset of clinical disease might be informative as to which aspects of the pre-­existing RA‑associated immunity might be causally involved in disease pathogenesis. Interestingly, levels of antibody responses towards some, but not all, of the epitopes (that is, antibodies against citrullinated enolase, vimentin and fillagrin) were specifically found to remarkably increase just before disease onset.35–37 One interesting question related to these very early auto­immune reactions is the relationship between the emer­gence of ACPA and other autoantibodies, in particular RF. RF was detected in healthy individuals before they developed RA, in a classic study by Aho and colleagues in Finland.28 A subsequent more detailed study on ACPAs and RF indicate that the occurrence of ACPAs might indeed be the first sign of autoimmunity, which is rapidly followed by the occurrence of RF.38 An attractive potential explanation to this series of events would be that T cells reactive to citrullinated auto­antigens and immune complexes containing citrullinated antigens might trigger RF production, according to the traditional model of RF formation.39,40 However, other possible explanations exist and there is obviously still much more to discover regarding both T‑cell and B‑cell reactivity repertoires preceding onset of clinical signs of RA. Such studies would enable further identification of additional specificities of auto­ antibodies occurring before clinical onset of RA, similar to autoantibodies to carbamylated proteins.19

Immunity against citrullinated proteins Events triggering RA Identification of exogenous triggers of autoimmunity and autoimmune diseases is a notoriously difficult task; illustrated in RA by the many years of search for infectious RA‑triggering agents, so far without reproducible results. However, the revolution in genome sequencing and the

improved characterization of disease-specific antibodies contributed greatly to new insights into how environmental agents, in particular smoking, interact with genetic risk factors in providing risk of developing ACPA-positive, but not ACPA-negative RA. This observation was first made in a Swedish case–control study of genetic and environmental determinants of RA41,42 and subsequently reproduced in a series of different case–control and cohort studies in white43–45 and Asian populations.46 Notably, these studies have shown that distinct major histocompatibility complex (MHC) class II alleles interact with smoking in providing high risk of ACPA-positive RA, and that certain other environmental exposures to the lung, such as silica47 and textile dust,48 could replace smoking in this interaction, suggesting that nicotine is not the inducing component. The possibility that air pollution also might represent such an exposure contributing to the risk of developing RA is still debated.49–51 Further support to the notion that the RA‑inducing component of smoke is most probably the irritants, and not nicotine, has come from data showing that oral nicotine-containing tobacco is not associated with any increased risk of RA.52 Taken together, these data suggest that environmental exposures in the lung might be driving forces for the production of pathogenic immunity in individuals carrying certain MHC class II genes. Interestingly, smoke exposure increases the risk of ACPA-positive RA even when smoking was discontinued up to 10–19 years before disease onset, providing an important piece of information for the question of when these exposures might exert their disease-promoting effects on the immune system.45,53 More knowledge is needed on the longitudinal sequ­ ences of these events and the determinants (genetic and environmental) that are responsible for the triggering of anti-citrulline immunity in healthy individuals and for transforming a healthy, but antibody-positive indi­vidual, to an antibody-positive patient with arthritis. These questions were addressed in a large population-based study that examined the interaction between genetic and environ­mental determinants for RA in twins.54 Using a blood biobank with specimens from more than 12,000 twins it was demonstrated that environment, lifestyle and stochastic factors (including smoking) might be more important than genetics in determining which indi­ viduals develop ACPAs. Genetic factors (and in particular HLA genes) might have a fairly large effect in determining which ACPA-positive individuals that will ultimately develop arthritis. Notably though, the case-wise concordance rates among twins were low for both being an ACPApositive healthy individual (3.7% for monozygotic versus 2.8% for same-sex dizygotic twins) and being an ACPApositive individual with RA (8.3% for monozygotic versus 4.4% for same-sex dizygotic twins), thus demonstrating that only a limited number of determinants for these two phenotypes have yet been identified. We conclude that current available data demonstrate the existence of a gradual development of immune processes driven by combinations of genetic and environ­mental factors, but in which only a limited number of these factors and mechanisms have yet been identified. The data that

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Tissue proteins

particular antigens. For autoantigens, the issue of peripheral immune activation will also be critically dependent on the status of negative selection of T cells with potential to recognize this particular auto­antigen. If any of the immune responses against citrul­linated antigens are directly pathogenic for RA (discussed in the next section), understanding of this activation and its precise mechanisms will be of critical importance for future efforts to treat and prevent RA by interfering with specific immunity. Here, we will scrutinize the presently available data for the three critical events in the context of the lung: presence and presentation of antigen; innate stimulation; and genetics. A schema­tic representation of our current understanding of these events is given in Figure 1.

Susceptibility genes

BAL proteins

APC Smoke Silica Irritants Activated T cells Lung changes B cells Citrulline ACPA Citrullinated proteins

Time to disease onset

ACPA levels

Figure 1 | A schematic model of initiation of RA‑associated immunity against citrullinated proteins in the lungs. Smoking, silica exposure and probably other air pollutants induce molecular changes in the lungs and the BAL fluid, resulting in increased expression of citrullinated proteins and/or peptides that can be mapped and characterized by unbiased liquid chromatography trace and tandem mass spectrometry analysis. APCs, such as dendritic cells, are activated by adjuvant effects and upregulate surface expression of MHC class. When a susceptibility MHC is present in a host, distinct citrullinated proteins are presented in the context of distinct MHC molecules to activate T cells that, in turn, will promote B‑cell maturation and activation with consecutive production of ACPAs. Distinct ACPA specificities appear in distinct individuals long before disease onset, disseminate systemically and undergo epitope spreading. This process leads to a mixed pool of ACPA specificities with increasing ACPA titres as the individual at risk approaches the time of disease onset. Abbreviations: ACPA, anti-citrullinated protein antibody; APC, antigen-presenting cell; BAL, bronchoalveolar lavage; MHC, major histocompatibility complex; RA, rheumatoid arthritis.

exist, however, strongly suggest that environ­mental factors directly and primarily affecting the lungs that are associated with risk of RA (such as smoke or other noxious inhaling agents) might trigger the first immune events in the lungs. Further triggering of joint inflammation might occur in a second step, probably needing additional stimuli. However, more knowledge is needed on the longitudinal sequences of these events and the determinants (genetic and environ­mental) that are responsible for the triggering of anti-citrulline immunity in healthy indi­ viduals and for transforming a healthy, but ACPA-positive, individual to an antibody-positive patient with arthritis.

Events in the lungs Triggering of an immune response normally requires the presence of the antigen in a form that allows efficient uptake in antigen-presenting cells, an innate signal that activates the antigen-presenting cells, typically dendritic cells, and that these events happen in a host with a genetic constitution that allows immune activation against the

Presence and presentation of citrullinated antigens The first evidence that risk factors identified from epidemiology could also contribute to the emergence of citrul­linated antigens in the lungs, was provided from immuno­staining experiments of bronchoalveolar lavage (BAL) cells, for which smoking was shown to increase the pres­ence of citrullinated proteins in these BAL cells.42 In parallel with these experiments, it was also shown that the lungs of patients with established RA might contain ecto­ pic lymphoid tissues, in which B cells were present that were able to bind citrullinated proteins, thus sug­gesting that local production of ACPAs can occur in lungs of patients with RA.55 Subsequent preliminary studies have demonstrated that peribronchial inflammation is a feature also in patients with early RA and, in this context, mainly restricted to ACPA-positive patients with RA.56,57 Similar findings were reported in ACPA-positive individuals with chronic lung disease and no signs of joint inflamma­tion.58 Signs of such inflammation have also been described by high-resolution CT investigation of ACPA-positive healthy individuals at high risk of development of RA.59 Further molecular studies on bronchial biopsy sam­ples, BAL fluid and cells from untreated ACPA-positive patients with RA at disease onset have demonstrated enrichment of ACPAs in BAL fluid as compared with blood (when adjusting for total Ig concentrations) accompanied by increased expression of citrullinated proteins.56 This finding is in a similar manner to the previous report of increased expression of citrullinated proteins and citrul­ linating enzymes in lungs and BAL fluid of healthy smokers.60 Notably, though, ACPA enrichment in BAL and peribronchial inflammation in early RA was not exclusively associated with smoking, but also present in nonsmoking ACPA-positive patients with RA,56,57 indicating that factors other than smoking might also drive the development of these early inflammatory changes in the lungs in individuals developing RA. In a similar manner, local enrichment of ACPA and RF has been des­cribed in provoked sputum of arthritis-free individuals at risk of developing RA,61 in some cases even in the absence of the same antibodies in the blood, further suggesting specific initiation in the lungs. This information inspired efforts to more precisely characterize which sets of proteins and/or peptides in the lungs are citrullinated because of smoking. Such efforts are ongoing and, so far,

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REVIEWS a few citrullinated proteins and/or peptides—notably v­imentin—have been identified in lungs of smokers by means of mass spectrometry. 62 In 2013, citrullinated heat-shock protein 90 in the lungs and antibodies against citrullinated heat-shock protein 90 in serum were associated with interstitial lung disease in RA.63 Further­more, single-walled carbon nanotubes promoted citrullination of cytokeratins and plectins in the lung when inhaled by mice.64 To get a more complete picture of citrullinated pro­teins and/or peptides that might trigger immunity in lungs, there is a need to produce a complete so-called citrulli­nome for the lung (an inventory of all the citrulli­ nated proteins present) to be compared with a ci­trullinome of the joint 65,66 and, eventually, citrullinomes from other sites where citrullination is present, but which are not affected in RA.67 Innate stimulation Three major mechanisms have been suggested for innate stimulation that could enhance antigen presentation and activate adaptive immunity. First, exogenous agents such as substances in cigarette smoke, silica and coal dust are all able to activate dendritic cells as well as other cells via pattern-recognition molecules.68 Second, these irritants, in particular smoke, are known to enhance bacterial colonization of the lungs and hence alter the local lung micro­ biome.69 Subtle changes of the microbiome, with increased rela­t ive abundance of Haemophilus and Streptococ­ cus species and decreased relative abundance for Prevo­ tella species, have been reported in the sputum of a few indi­viduals at risk of developing RA.70 Third, fragments of citrullinated proteins (induced by smoke) can themselves bind to pattern-recognition molecules and cause activation of several cell types including antigen-presenting cells.71 All three possibilities have so far been investigated rather superficially, and further detailed investigations of innate triggering in the lungs might provide us with important insights on initiating mechanisms and how to interfere with such mechanisms. This option is of high therapeutic interest as the lung is an organ that is accessible not only for external triggers but also for potential drugs that might interfere with such triggering. Also, the possibility that other mucosal sites (such as oral mucosa72–76 and the gut 77,78) might substitute the pathogenic role of the lungs in disease development warrants further investigations. Genetics The idea of a potential trigger in the lung of disease-­ specific, RA‑relevant immunity against citrullinated pro­teins emerged from the original description of the inter­action between smoking and HLA class II antigens occurring only in ACPA-positive RA (described earlier). Fur­ther refining of this original observation has demonstrated that HLA class II restrictions responsible for triggering of different ACPAs are quite different, with distinct relationships between different patterns of reactivities to epitopes of citrullinated autoantigens (such as vimentin, fibrinogen, enolase or collagen II) and different shared epitope alleles of the HLA-DR class II molecule.21 A detailed, fine mapping (based on single nucleotide

polymorphisms and imputation) of the MHC genes has added to existing knowledge of the shared epitope of HLADR,79 showing that at least four independent MHC class II structures (three in the HLA-DR and one in HLA-DP) as well as one MHC class I structure (in HLA‑B) associate with ACPA-positive RA.80 Taken together, these data strongly suggest that several different peptides binding to different HLA class II might be involved in the initial triggering of immunity to a number of different citrullinated autoantigens (and also noncitrullinated RA‑relevant antigens). Using protein crystals, it has been shown how distinct citrullinated peptides bind to RA‑associated HLA-DR.81 In addition to the MHC genes acting as major genetic determinants, a number of other genes are involved in setting thresholds for immune activation and in determining the character of the eventual immune response in RA. The best studied polymorphism in this respect is that in PTPN22, which codes for a tyrosine phosphatase involved downstream in signalling in T cells and B cells (and in other cells).82–84 An interesting feature of this poly­ morphism (a 1858C>T genotype) is that the suscepti­bility allele is associated with higher threshold for T‑cell and B‑cell activation than normal, not a lower threshold as might have been expected.85–88 A potential explanation to this somewhat paradoxical finding might be linked to the inherent and critical determinant for an auto­immune res­ ponse, namely the degree of negative selection of rele­vant T cells in the thymus, and the complementary restriction of the B‑cell repertoire that occurs in the bone marrow. We speculate, but have so far no experimental proof, that citrullinated proteins might not be so common in the thymus and its epithelial cells, as is the case for non­ modified self-molecules, and that this concept might to some extent explain the presence of T cells to citrullinated self-­peptides in healthy indi­viduals as well as in patients with RA. Nevertheless, it is feasible that some mechanisms for MHC-class-II-dependent negative selection might be present. If so, presence of the risk allele of PTPN22, enabling a broader repertoire of autoreactive T cells to enter into the periphery including the lungs, would fit into the model of MHC-class-II-dependent immunity in ACPApositive RA. Defects in peripheral immune tolerance might also be present. Interestingly, another gene, MHC class II transactivator (CIITA) associated with increased risk of RA (at least in some populations89) shows a similar pattern, that is, it is the gene variant associated with a low expression of MHC class II that associates with disease. Additional and informative genetic associations are between risk of ACPA-positive RA and polymorphisms in the promoter regions of protein-arginine deiminase type-4 (also known as peptidylarginine deiminase 4; PADI4).90,91 These polymorphisms were first found, and are mainly present, in Asian populations, but similar associations with certain PADI4 polymorphisms (SNPs rs2240336 and rs766449) have also been found in white populations.92 Although the functional effects of the polymorphisms have not yet been identified, except for a suggested effect on mRNA stability,90 the associations of RA with these genes provides an additional strong support for the

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Osteoclast activation B cell

Neutrophil

Citrullinated antigen

ACPA

NET release

FcγR Complement activation

Macrophage activation

Precursor cell

Complement Chondrocyte changes Fibroblast activation

Figure 2 | Summary of the pathogenic roles of ACPAs in RA. Antibodies can contribute to local perpetuation of synovial inflammation through several potential mechanisms: binding to citrullinated proteins located in the cellular membrane, binding to Fcγ receptors, binding to citrullinated proteins bound to other cellular receptors (such as TLR4) and binding to citrullinated proteins incorporated in NETs. Complement binding and activation by ACPAs might further contribute to local synovial inflammation. The inflamed rheumatoid synovial membrane and fluid are a reservoir of activated immune cells and locally produced antibodies. B cells can develop into plasma cells in the joint tissue. B cells (or plasma cells) can be retrieved from synovial fluid (or tissue) and RNA from individual cells can be used to make recombinant monoclonal antibodies. By this approach, a high proportion of the synovial IgG-expressing cells (memory and early plasmablasts) were shown to carry citrulline-reactive B‑cell receptors that could represent important APCs and largely contribute to propagation of the autoimmune responses within the affected joints. Abbreviations: ACPA, anti-citrullinated protein antibody; APC, antigen-presenting cell; FcγR, Fcγ receptor; NET, neutrophil extracellular trap; RA, rheumatoid arthritis; TLR4, Toll-like receptor 4.

involvement of citrullination and anti-citrulline immunity in the pathogenesis of ACPA-positive RA. Interestingly, antibodies against PADI4 might result in a feed-forward loop, leading to generation of citrullinated autoantigens by PADI4 activation.93 Taken together, we have an interesting scenario for triggering of specific immunity, with an initiator organ (the lung) and a series of triggers (in particular smoking), with a series of MHC genetic polymorphisms tentatively linked to different, but specific, anti-citrulline immunities, and with several additional genes that modify access to autoantigens and repertoires of autoreactive lymphocytes. This described scenario offers fascinating opportunities for basic studies of how autoimmunity might be triggered and regulated, and also offers a multitude of opportunities to define targets for organ-specific, cell-specific and/or antigen-specific therapies.

Events in the joints A major challenge in our efforts to understand the pathogenesis of ACPA-positive RA is to understand why joints are the primary targets of inflammation associated with immu­­nity against citrullinated proteins, and to what extent there might be a specific relationship between the ini­tial gen­eration of this immunity in the lungs and the su­bsequent inflammation in joints. Some interesting potential answers to these questions have been produced, mainly related to the effector functions of disease-related antibodies and, more specifically, effector functions of ACPAs. Antibodies to the citrullinated cartilage-specific protein collagen II have been identified in some patients with RA, and antibodies to the same epitopes have been shown to induce arthritis in experimental model systems.94 However, the anti-­citrullinated collagen II antibodies defined so far are not so prevalent (with only 37% of the ACPA-positive patients being also positive for anticitrullinated collagen antibodies21) to provide an overall explanation, and we do not know to what extent sufficient amounts of these antibodies are present in humans to enable triggering of arthritis. Moreover, antibodies to citrullinated vimentin have been shown to contribute to the activation of osteoclasts both in vitro and in vivo (in animal models), potentially through direct binding on the cellular surface of osteoclasts, and thus possibly contribute to joint erosions and osteoporosis.95 Another challenging hypothesis is that bone-marrow oedema, possibly caused by osteoclast activation, might constitute an early stage of joint inflammation96 and contribute to the triggering of a joint-specific inflammation that, in turn, would attract immune cells also recognizing other citrullinated proteins. Furthermore, immunity to additional citrullinated proteins, such as enolase, histones and BiP, might contribute to joint inflammation by as yet unidentified mechanisms. This alternative mechanism concerning anti-enolase immunity has been suggested97 and debated.98 These effector functions might be mediated through antibody binding to citrullinated proteins located in the cellular membrane (as demonstrated for anti-mutated citrullinated vimentin and osteoclasts95), to Fcγ receptors and/or to other cellular receptors such as Toll-like receptor‑4 (as demonstrated for citrullinated fibrinogen incorporated in immune complexes and macro­phages),71 and probably occur due to complement activation.99 An additional interesting option has been described after the demonstration that some ACPAs are able to induce the release of neutrophil extracellular traps (NETs), containing large amounts of citrullinated proteins (including histones), and that these NETs are able to promote inflammation.96 The potential joint-specific feature of this connection with NETs might be the well-known abundance of activated neutrophils in inflamed joints100 that might be relevant in focusing inflammation to joints. Figure 2 summarizes ACPA effector functions contributing to the perpetuation of chronic joint inflammation.

The lung–joint connection in RA Although all these hypotheses mentioned earlier give some clues as to why the joints are the primary targets of

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REVIEWS the inflammation mediated by immunity against citrul­ linated proteins, it is still unknown how the generation of this immunity in the lung specifically associates with arthritis-inducing immune events in the joints and not in other organs. Also, it is not known why, after initiation of the immune responses, the lungs are generally spared from more extensive immune damage. To answer these questions, focused investigations on com­monalities between immunity present in lungs and joints are needed to explore the more detailed specifi­city and function of B‑cell and T‑cell immunity to various citrul­linated proteins in the two compartments. So far, such studies have mainly focused on joint inflammation, dem­ onstrating that antibodies to several citrullinated auto­anti­ gens are produced locally in the joint. Higher titres of these antibodies have been found in synovial fluids than in serum in patients with RA,101,102 and also one report suggests an accumulation of ACPA-producing B cells in inflamed tissues, determined by means of immuno­histochemistry with biotinylated citrullinated peptides binding to B cells and/or plasma cells in the tissue.103 A more direct proof of the presence of high numbers of ACPA-producing B cells in the joints of ACPA-positive patients with RA has been provided from experiments in which monoclonal human antibodies have been generated from IgG+ B cells from synovial fluid of ACPA-positive patients with RA using single-cell-cloning technology.104 An amazing 20–35% of all such IgG+ B cells proved to pro­duce antibodies towards different citrullinated peptides. These clones with specifi­ city for citrullinated peptides displayed no cross­reactivity with the arginine version of the peptides. Further­more, the clones had undergone substantial somatic mutations in their variable parts indicative that B cells had received T-cell help. The monoclonal antibodies showed varying fine specificity with some of them displaying a wide cross­ reactivity between different citrulli­nated pep­tides, and others displaying a preference for cer­tain peptides—in our first small series of patients, the most common reactivity was towards α‑enolase.104 These monoclonal antibodies can now be used for detailed studies of the effector functions of various well-defined ACPAs. Similar and comparative studies are also ongoing to characterize the adaptive immune response in lungs of patients with early RA. The immune system has evolved to manage a delicate balance between maximal recognition of microbial antigens and minimal crossreactions to self-antigens. Given the dynamic nature of microbes, the immune system must be equally dynamic in changing its behaviour depending on which external stimuli it encounters. The organs that primarily encounter external stimuli are the skin, the mouth, the gut and the upper airways, including the lungs. The skin has its epithelial layer as a barrier and specific cells such epidermal Langerhans cells act as antigen-presenting cells in the epidermis. The gut has the acid-rich stomach as a first line of defence with a number of antigen-presenting cells present in the downstream intestinal mucosa, and also well-developed system for induction of tolerance to encountered antigens. The lung is more exposed than any other organ to the environment, including microbes, and it lacks a physical

boarder such as for the skin or the gut. Thus, it is feasible that the lung is the organ where the immune system faces its most difficult problems in determining when and towards which agents or molecules to raise an immune res­ponse. With this reasoning, it is not surprising that immune reactions against self-molecules might also be triggered in the lung after certain stimuli of innate immu­nity. In fact, this scenario seems to not be unique to immunity against citrullinated proteins in RA, but simi­lar effects of external stimuli, including smoking, has been seen in the context of multiple sclerosis associated with HLA-DRB1*15105 and in anti-Jo1 positive myositis asso­ciated with HLA-DRB1*03.106 In these three cases of auto­immune diseases, we can assume that external stimuli, such as smoking, both stimulates local innate immunity and activates antigen-presenting cells and provides an increased expression of different autoantigens. Depend­ ing on an individual’s MHC class II genes and other genes that modulate immunity, different immunities might subsequently be triggered and contribute to various organspecific autoimmune diseases. Detailed understanding of how these immunities occur in the lung and how they can be regulated, as is the case naturally in most individuals, would be key to efforts aimed at developing new strat­ egies and agents that can prevent these immune res­ponses emerging, or enable their re-regulation possibly by local treatment in the lungs.

Conclusions Progress in understanding of the complex interplay between specific autoimmunity and genetic and environ­ mental predisposing factors unravels the molecular basis of distinct clinical disease phenotypes in RA. One such clinical phenotype is ACPA-positive RA, in which a break of tolerance to citrullinated proteins might take place in extra-articular sites (such as the lungs) and result in generation of autoantibodies before disease onset. In a second step, so far poorly understood, events will target the immune reactions triggered in lungs to the joint compartment and initiate local chronic inflammation. Future studies to explore the relationships between inflammation in the lungs and in the joints are needed, with the goal to identify key events involved in the gradual development of arthritis-promoting immunity that can be targeted with preventive or therapeutic measures. Review criteria We searched MEDLINE with the terms “rheumatoid arthritis”, “pre-rheumatoid arthritis”, “at risk”, “lungs”, and “anti citrullinated protein antibodies”, as well as other specific terms when needed, alone and in combination and included all reports published in English between January 1974 and January 2014 (last updated on January 2014). We reviewed all abstracts (original work, review, case report, letter, and so on) and selected relevant papers. We aimed to select the most recent publications, but also refer to the first publication on a certain finding and include other comprehensive studies and reviews relevant to the subject according to authors’ opinions. Reference lists of identified papers were searched for further leads.

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Initiative Be The Cure (115142‑2) and the Swedish Research Council. A.J.Y. acknowledges grant support from the Swedish Foundation for Strategic Research. L.K. acknowledges support from the Swedish Foundation for Strategic Research, the European 7th framework programme (FP7/ 2007–2013) Euro-TEAM (305549), the Innovative Medicine Initiative Be The Cure (115142‑2) and the Swedish Research Council.

Acknowledgements A.I.C. acknowledges support from the Swedish Foundation for Strategic Research, the Initial Training Networks 7th framework programme Osteoimmune (289150), the European 7th framework programme (FP7/2007–2013) Euro-TEAM (305549), the Innovative Medicine

Author contributions All authors made substantial contributions to researching data for the article and made substantial contributions to discussions of content and writing the article. A.I.C. and L.K. had the main responsibility for reviewing and/or editing of the article before submission.

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