Objectives Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA.
Patients and methods Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA.
Results Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA.
Conclusions Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.
- Early Rheumatoid Arthritis
- Rheumatoid Arthritis
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Rheumatoid arthritis (RA) is a chronic, inflammatory joint disorder. The presence of antibodies to proteins/peptides in which arginine is modified to citrulline (anti-citrullinated protein antibodies (ACPAs)) is an early feature in the development of a major subset of RA.1–2 However, the place of origin, fine specificity and targets of these antibodies in early phases of the disease are still not completely elucidated despite recent progress in the field.3–5 The fact that these antibodies appear in the serum before any clinical signs of inflammation in the joints6 indicates that the initial triggering of ACPA production may take place somewhere other than the joints. The lungs have been suggested to be a major candidate for this site based on the fact that smoking is a major risk factor for ACPA-positive RA,7–9 and that smoking can induce both citrullination of proteins in lungs8 and activation of antigen-presenting cells in the same tissue.10 Local enrichment of ACPAs in bronchoalveolar lavage fluids in early stages of ACPA-positive RA11 and the identification of germinal-centre-like structures in rheumatoid lungs12 provide further evidence for local immune activation and potential production of ACPAs in lung tissue.
Although a hypothesis exists for how immunity to citrullinated antigens may be triggered in the lungs,13 it is still unclear whether—and, if so, how—the anti-citrulline immunity generated in the lungs contributes to the development of synovial rheumatoid inflammation. One possibility is the presence of shared ACPA targets in lungs and joints, leading to secondary localisation of the inflammation in the joint. Several potential ACPA targets have been previously identified in the inflamed rheumatoid synovial membrane,14–18 with only scarce presence in the healthy non-inflamed synovial tissue.19 Identification and characterisation of these targets is not easy, because citrullination is a post-translational modification causing a mass shift of 0.9840 Da, which is identical with the mass shift caused by deamidation, one of the most common post-translational modifications that can occur in vivo and artificially during sample preparation.20
In the present study, we investigated the presence of shared citrullinated targets in the lungs and joints of patients with RA using mass spectrometry (MS)-based proteomics. We also characterised immunity to some of these new targets in the peripheral blood of healthy individuals, patients with RA, and disease controls.
Materials and methods
Patients and controls
Biopsy specimens from large bronchi were obtained by bronchoscopy, as previously described,11 from patients with newly diagnosed RA (three smokers and three non-smokers; four female and two male; median age 63; 67% anti-CCP2-positive; patient-reported symptom duration <1 year). Synovial biopsy samples were obtained during orthopaedic surgery from patients with longstanding RA (n=7; five female and two male; median age 58; 71% anti-CCP2-positive; smoking status unknown). Two lung biopsy samples from non-RA individuals (one smoker and one non-smoker) were also analysed. All biopsy samples were snap-frozen and stored at −80°C.
Serum samples were collected from untreated patients with early RA (LURA cohort21 and EIRA cohort7), healthy controls and disease controls (psoriasis arthritis and spondyloarthropathies). Demographic characteristics of the patients and healthy controls are given in table 1. The regional ethics review board in Stockholm approved all studies.
Protein extraction and digestion
Biopsy samples were homogenised by shaking with ceramic beads in 50 mM ammonium bicarbonate/0.1% RapiGest/protease inhibitor cocktail (Complete Mini; Roche Diagnostics) and sonicated; this was followed by buffer exchange with 50 mM ammonium bicarbonate using prepacked PD SpinTrap columns (GE Healthcare). After reduction and alkylation with dithiothreitol and iodoacetamide, proteins were digested with sequencing-grade endoproteinase Lys-C (Roche Diagnostics) at a 1:50 protease/protein concentration and filtered through 10 kDa filters (Pall Nanosep centrifugal device with Omega membrane MWCO 10 kDa; Pall Corp). The samples were dried and resuspended in 0.1% formic acid before analysis.
Liquid chromatography (LC)–MS/MS analyses were performed on an Easy-nLC system (Thermo Scientific) online-coupled directly to the mass spectrometer. The analyses were performed on a hybrid LTQ Orbitrap Velos ETD mass spectrometer (Thermo Scientific, Bremen, Germany). First, 1 µg of each sample was injected from a cooled autosampler on to the LC column. The peptide separation was performed on a 10 cm-long fused silica tip column (SilicaTips New Objective) packed in-house with 3 µm C18-AQ ReproSil-Pur (Dr Maisch, Germany). Chromatographic separation in 90 min was achieved using an acetonitrile/water solvent system containing 0.2% formic acid and a gradient of 5–35% acetonitrile. Mass spectra were acquired at a resolution of 60 000, and the top five (for electron transfer dissociation (ETD) and collision-induced dissociation (CID)) or top four precursors were selected for fragmentation by CID, ETD and heated capillary dissociation (HCD). Other than using shorter dynamic exclusion and a single fragmentation mode, standard settings were used in the analysis.22 For the Q Exactive, the MS acquisition mass spectra were acquired with a resolution of R=70 000, followed by 10 (HCD) consecutive data-dependent MS/MS scans.
Peptide identification and validation
Mass lists were extracted from the raw data using Raw2MGF (an in-house written programme) and Mascot Distiller V.2.4.0 (Matrix Science, London, UK). The mgf files were searched against a concatenated version of the human IPI database V.3.86 using the Mascot search engine (Matrix Science). The following parameters were used: Lys-C digestion with a maximum of two missed cleavages; carbamidomethylation (C) was used as a fixed modification, while pyroglutamate (Q), oxidation (M), deamidation (NQ) and citrullination (R) were used as variable modifications; a precursor tolerance of 5 ppm and a fragment tolerance of 0.25 Da (CID and ETD) or 0.1 Da (HCD). All peptides identified as citrullinated with a Mascot score of at least 20 were investigated. A score of 20 is a commonly used threshold23 and is defined as −10 log(P), where P is the probability of a random match between the theoretical MS/MS spectra of a peptide and the measured MS/MS spectrum (http://www.matrixscience.com). For some short peptides, even lower scores were considered. CID, ETD and HCD spectra of unmodified, deamidated (NQ) and citrullinated spectra of each peptide were compared to find evidence of citrullination. Special attention was given to the shift in retention time and the mass shifts of specific fragment ions.
Quantification of citrullination
To quantify the peptides, the spectra across the LC elution peak of each peptide were combined, and the abundances of the monoisotopic masses of unmodified and citrullinated forms of the validated peptides were compared. To simplify the quantification, deamidated peptides were ignored. The average charge state and the proportion of the peptide ion present at each charge state were determined using synthesised peptides. The degree of modification was quantified manually by comparing the abundance of the monoisotopic peaks, with a correction for the difference in charge state distribution between the unmodified and modified species. Since the citrullinated peptides were only identified by MS/MS in a few of the samples, in other samples these peptides were identified by the accurate mass and retention time. Unmodified versions of the peptides were present in almost all samples, and were used to correct for variation in peptide retention times between the samples. Strict mass filters were used to ensure the validity of the identifications. To accurately determine the molar amount in each sample for one of the citrullinated peptides present in multiple samples, TVETRDGQVINETSQHHDDLE and its citrullinated analogue were synthesised with heavy leucine (+7 Da) (Chinapeptide, Shanghai, China) and used as internal standards, as in the so-called AQUA methodology.24 The peptide was spiked into the samples in both unmodified and citrullinated forms at 50 fmol/µg protein. Two technical replicates were analysed for each sample.
Several biotinlyated cit-vim peptides and their native counterparts were designed and tested in an in-house ELISA, with cit-vim 435–455 being the most appropriate. Briefly, streptavidin-coated high-binding capacity ELISA plates were coated with 2.5 μg/mL biotinylated vimentin 435–455 (vim 435–455) peptide, in its native and citrullinated forms (DTHSK-cit-TLLIKTVET-cit-DGQVI; Genecust, Luxembourg). For detection of the antibodies, serum samples were diluted 1:100 in phosphate-buffered saline/0.05% Tween 20 and incubated at room temperature. The bound antibodies were detected with horseradish peroxidase-conjugated goat anti-human IgG F(ab)2 (Jackson ImmunoResearch Europe). Detection was performed using the chromogenic substrate, 3,3,5,5-tetramethylbenzidine (Sigma-Aldrich, Stockholm, Sweden), and absorbance was measured at 450 nm with reference at 650 nm. A standard was included in each plate to convert absorbance into arbitrary units (AUs). The cut-off value in AUs/mL was set to 98% specificity and was determined using reactivity in the healthy subjects. To test for cross-reactivity between the new cit-vim 435–455 peptide and the previously described cit-vim peptide 60–75, serial dilutions of an RA serum sample reactive with both peptides were first incubated with 50 μg/mL cit-vim 435–455 peptide and then tested by ELISA for remaining reactivity against either cit-vim 435–455 or cit-vim 60–75.
HLA typing was performed by sequence-specific primer PCR assay (DR low-resolution kit; Olerup SSP, Saltsjöbaden, Sweden) as previously described25 in 103 patients from the LURA cohort (31 missing data) and 241 patients from the EIRA cohort (18 missing data). Shared epitope (SE) alleles were defined as DRB1*01, DRB1*04 or DRB1*10.
Comparisons between groups of patients were analysed by analysis of variance follow by Dunnett's and Sidak's multiple comparison tests. Univariate analysis of categorical variables was performed using the χ2 test.
Shared citrullinated targets are present in the lungs and joints of patients with RA
We identified 5322 unique peptides and 1200 proteins in the RA bronchial and synovial tissues. Only a minority of the MS/MS spectra identifying peptides with a score of at least 20 corresponded to citrullinated peptides. In total, 10 citrullinated peptides from seven proteins were identified by MS/MS and quantified (see online supplementary table S1). Further analysis, using accurate mass and retention time, detected eight citrullinated peptides in synovial and seven in bronchial biopsy samples, with five peptides shared between the two. Of these, two cit-vim peptides were detected in the majority of both synovial and lung tissues: cit-vim 446–466 present in all bronchial and most of the synovial samples (86%, 6/7) and cit-vim 440–445 present in most bronchial (83%, 5/6) and all synovial samples. A higher, but non-significant, average ratio of citrullinated/unmodified cit-vim 446–466 was observed in biopsy samples from the smokers (median 1.260, range 0.025–2.052) than in those from the non-smokers (median 0.039, range 0.012–0.568). In addition, one citrullinated annexin peptide (47–65) was detected in all tested bronchial tissues and 29% (2/7) of the synovial tissues, one citrullinated actin peptide (62–68) in a minority of both bronchial (29%, 2/7) and synovial (33%, 2/6) tissues, and one citrullinated histone peptide in one synovial and two bronchial samples. Furthermore, three citrullinated fibrinogen-α peptides were detected only in synovial samples, and one citrullinated cysteine-rich protein 1 and one citrullinated haemoglobin subunit-α peptide was detected only in bronchial samples. In a separate experiment, none of these 10 citrullinated peptides were identified in the non-RA non-smoker lung, whereas a positive, although weak, signal was identified in the non-RA smoker lung for the cit-vim 446–466 peptide (with a Mascot score of 13). Further quantification confirmed this observation, showing low citrullinated/unmodified ratios for the cit-vim peptides, with the other eight citrullinated peptides being unquantifiable. A summary of the MS results is given in table 2, and detailed information is included in online supplementary table S2.
Identification of antibody reactivity to the shared lung and joint citrullinated targets in patients with RA
In order to investigate the relevance of the newly identified citrullinated vimentin peptides as autoantigens in RA, we examined immune reactivity against both native and citrullinated forms of a peptide containing the two citrullinated sites identified by MS (ie, cit-vim 435–455) in peripheral blood of healthy controls and patients with rheumatic diseases. The cut-off was first set in a cohort of 152 healthy individuals showing 1.8% (3/152) positivity (figure 1B). In the LURA cohort, 14.9% of the sera were positive for cit-vim 435–455 (p<0.05 compared with healthy controls), while, in the second validation cohort derived from the EIRA cohort, 14.3% were positive (p<0.05 compared with healthy controls). Disease controls showed low reactivity (8/236, 3.4%), which was slightly, but no significantly, higher than in healthy controls (figure 1A). A large proportion of the anti-(cit-vim 435–455)-positive RA cases were confined to the anti-CCP2-positive RA group, with 23.4% of anti-CCP2-positive RA patients also being positive for anti-(cit-vim 435–455), while only 2.6% of the anti-CCP2-negative RA patients were positive for anti-(cit-vim 435–455) (p<0.05) (figure 1B). In all, 85% (44/52) of the patients with RA showing reactivity against cit-vim 435–455 were SE positive, as compared with 71% (208/292) of the patients not showing this reactivity (p<0.05), while no association with smoking was observed.
In the cross-reactivity ELISA test performed in a patient showing double reactivity for both cit-vim 435–455 and cit-vim 60–75, serum blocking with the cit-vim 435–455 peptide did not interfere with ELISA reactivity against cit-vim 60–75 (figure 2A), but abolished reactivity against cit-vim 435–455 (figure 2B), suggesting no cross-reactivity.
Recent advances in understanding the longitudinal development of RA suggest that initiation of the disease-specific immune processes might take place at extra-articular rather than articular sites. One such potential extra-articular site is the lung. We demonstrate here that lungs and joints of patients with RA share certain common citrullinated peptides (in particular, cit-vim peptides), which can be identified by unbiased proteomics. Antibodies to these shared citrullinated targets were identified in the peripheral blood of patients with RA.
MS identification of citrullinated peptides in complex mixtures is challenging due to both the unfavourable dynamic range and the problem of having abundant, frequently occurring modifications by deamidation of the same mass present in the same sample. Chemical modification has been previously used to detect citrullinated peptides independently of the backbone proteins by western blot,26 and similar chemistry has been shown to be compatible with MS.27 Although the methodology seemed promising, and has been used to both increase the specificity28 ,29 and for affinity purification,30 it has not yet been used to analyse the presence of citrullinated proteins/peptides in tissue samples. Our proteomic assay showed low frequency of spectra from citrullinated peptides, indicating that the modification is of low abundance, in agreement with previous results.31 Since the quality of MS/MS spectra correlates with the abundance of the precursor, it appears unlikely that spectra from low abundant components would be of high enough quality to contain the specific peaks that can distinguish the modification states. Thus, identification of additional citrullinated peptides of even lower abundance will probably require additional enrichment steps, such as affinity purification using antibodies from patients with RA, followed by MS studies.
Three of the proteins (vimentin, fibrinogen-α and actin) identified in this study to contain citrullinated peptides have previously been validated as ACPA targets.32 ,33 However, only one peptide, from fibrinogen-α, has also been found in the previous studies.31 Similarly to other fine specificities, most of the anti-(cit-vim 435–455)-positive individuals were also anti-CCP2-positive, although a few anti-CCP2-negative patients also showed reactivity to the newly discovered peptide. Higher levels of the cit-vim 435–455 peptide in lungs of smokers and association of antibodies to (cit-vim 435–455) with SE presence are in line with previous reports,4 ,34 but a larger number of samples to address the specific relation between these antibodies and genetic and environmental risk factors is needed. An interesting result of the present study is the identification of new citrullinated peptides shared between lungs and joints of patients with RA, giving further support to the hypothesis of a direct connection between these two sites.
One limitation of the present study is the lack of paired synovial and lung biopsy specimens from the same patients, which is due to the obvious challenges in collecting these biological samples. However, detection of at least two citrullinated peptides in most of both the lung and synovial unpaired samples further support the validity of our findings. All lung biopsy samples came from patients with newly diagnosed RA, naïve to antirheumatic treatment, therefore minimising potential therapeutic influences. In contrast, synovial biopsy specimens came from cases of longstanding RA. However, we and others have previously shown similar patterns of citrullination in synovial tissue from patients with new-onset and longstanding disease (by means of immunohistochemistry).19 ,35–37 We chose to use in this study biopsy samples from longstanding RA, because a larger amount of material, needed for initial validation of the technique and further development of the project, was available. Another methodological limitation is the restricted access to lung biopsy samples from healthy non-RA individuals. However, the increased frequency of an immune response to the new cit-vim peptide in the blood of patients with RA compared with both healthy and diseases controls confirms the relevance of this peptide as an RA autoantigen in vivo. Our ELISA inhibition assay suggests minimal cross-reactivity between antibodies directed against two distinct parts of the cit-vim protein (435–455 and 60–75). However, some degree of cross-reactivity does exist for different ACPAs,38 and this might also be the case for antibodies to cit-vim 435–455 when tested in other patients.
In summary, we have demonstrated that certain citrullinated proteins are present in both biopsy specimens from lungs of patients with early RA and inflamed joints of patients with RA. Two defined citrullinated peptides are present in both lungs and joints of patients with RA, and specific immunity against one of these peptides is present in the peripheral blood of patients with RA. The data give further support to the hypothesis that immunity to citrullinated proteins may be initiated in the lungs and potentially contribute to inflammation in the joints. Further detailed analysis of targets identified by the present methodology for potential disease-inducing immunity in lungs and joints may inform us about the mechanisms responsible for triggering anti-citrulline immunity and thus also provide clues to future interference with this triggering mechanism.
We thank Monika Hansson and Lena Israelsson for excellent technical support.
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Handling editor Tore K Kvien
Contributors All authors provided substantial contributions to (1) conception and design, or analysis and interpretation of data and (2) drafting the article or revising it critically for important intellectual content and (3) all gave final approval of the version to be published.
Funding This work was supported by research funding from the Swedish Foundation for Strategic Research, Innovative Medicine Initiative BTCure (115142-2), FP7-HEALTH-2012-INNOVATION-1 Euro-TEAM (305549-2), the Initial Training Networks 7th framework program Osteoimmune (289150), the Swedish Research Council, the Swedish Heart Lung Foundation, and through the Regional Agreement on Medical Training and Clinical Research (ALF) between Stockholm County Council and Karolinska Institutet.
Competing interests None.
Patient consent Obtained.
Ethics approval Regional ethics review board in Stockholm.
Provenance and peer review Not commissioned; externally peer reviewed.