Gudrun Reynisdottir

Structural Changes and Antibody Enrichment in the Lungs Are Early Features of Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

It has been suggested that immunologic events in the lungs may be involved in triggering immunity, in particular production of anti–citrullinated protein antibodies (ACPAs) during early phases of rheumatoid arthritis (RA). The aim of this study was to investigate the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation

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.

Signs of immune activation and local inflammation are present in the bronchial tissue of patients with untreated early rheumatoid arthritis

Objectives Events in the lungs might contribute to generation of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). We investigated if signs of immune activation are present in bronchial biopsies and bronchoalveolar lavage (BAL) of patients with early-untreated RA without clinical signs of lung involvement. Methods Twenty-four patients with RA with symptom duration <1 year and naïve to disease-modifying antirheumatic drugs were subjected to bronchoscopy where BAL and mucosal bronchial biopsies were retrieved. For comparison, 15 bronchial biopsies and 79 BAL samples from healthy volunteers were available. Histological examination was performed to evaluate lymphocyte infiltration, presence of immune cells (T and B cells, plasma cells, dendritic cells and macrophages) and immune activation markers. Cell composition of BAL samples was analysed by differential counting and T cell subsets by flow cytometry. Results Lymphocyte infiltration was more frequently found in ACPA-positive patients (50%) as compared with ACPA-negative patients (17%) and controls (13%). Germinal centres, B cells and plasma cells were only found in ACPA-positive patients. The frequency of T cells in bronchial biopsies of patients with ACPA-positive RA was positively associated with expression of immune activation markers. BAL samples of patients with ACPA-positive, but not ACPA-negative, RA had significantly higher relative numbers of lymphocytes and expressed higher levels of activation markers compared with controls. Conclusions Signs of immune cell accumulation and activation are present both in the bronchial tissue and in BAL of untreated patients with early RA without concomitant lung disease, strengthening the role of the lung compartment as an important player in ACPA-positive RA.

Identification of shared citrullinated immunological targets in the lungs and joints of patients with rheumatoid arthritis

Background The authors have previously demonstrated that smoking induces citrullination in the lungs of healthy smokers and they know that anticitrullinated protein antibodies (ACPA) develop in rheumatoid arthritis (RA) patients many years before disease onset. It was hypothesised that shared citrullinated targets are present in the lungs and joints of RA affected individuals and sought to investigate this by full-proteome analysis of synovial and lung biopsies of RA patients. Material and methods Proteins were extracted from synovial (n=7, five females and two males, median age 58, 66.7% ACPA positive) and lung (n=6, four females and two males, median age 63, 66.7% ACPA positive) biopsies of RA patients. Synovial biopsies were obtained at the time of open surgery from patients with long-standing RA (mean disease duration 24 years). Large bronchi biopsies were obtained by bronchoscopy from patients with newly diagnosed RA (three smokers and three non-smokers) with symptom duration less than 1 year. The proteins were reduced, alkylated and digested with Lys-C, separated by reverse-phase nanoflow-chromatography and analysed by LTQ-Velos-Orbitrap using multiple fragmentation methods. The data were searched against the human International Protein Index database using the Mascot search engine and all citrullinated peptides were manually verified. The degree of modification was quantified manually. The final results were expressed as ratios of citrullinated versus non-modified peptides. Results Over 3300 peptides and 500 proteins were identified in the different samples. The overall protein profiles varied between patients. Five of the identified proteins in the synovium (in total eight sites) and four in the lungs (in total four sites) contained citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in smokers compared to non-smokers (median ratio of 0.03 in smokers and 0.02 in non-smokers for one of the peptides and a median ratio of 4.5 in the smokers and 0.04 in the non-smokers for the second vimentin peptide). While non-modified and citrullinated fibrinogen α-chain derived peptides were present in various amounts in the synovium, only the unmodified sites could be detected in the lungs of a subset of the patients (three out of six). Conclusions The authors demonstrate the presence of shared in vivo citrullinated proteins in the joints and lungs of RA individuals, providing further support for the important pathogenic link between joints and lungs in development of RA.

Lung changes detected by high resolution tomography are present in ACPA positive RA patients already at disease onset

Background/purpose The authors have previously shown that smoking increases citrullination in the lungs of healthy smokers and hypothesised that this process is an important step in the early development of rheumatoid arthritis (RA). To test this the authors investigated lung function in a cohort of RA patients with very early disease, already at the time of diagnosis and compared it with an aged, smoke and gender matched cohort of individuals without RA. Methods 82 patients, 56 female and 26 male, median age 60 (range 22–84) with symptom duration less than 1 year at the time of diagnosis and naive to disease-modifying antirheumatic drug treatment were included in the RA cohort. A second cohort of age, smoking and gender matched non-RA individuals (n=43) were investigated with an identical protocol. Lung function (x-ray, high-resolution CT (HRCT) and dynamic spirometry) was tested in both RA and controls at baseline and repeated after 6 months in the RA group. In a subgroup of patients bronchoscopy was performed at inclusion (n=18) and 6 months later (n=11) and bronchoalveolar lavage samples as well as mucosal large bronchial biopsies were retrieved. Presence of peptydilamino deiminase (PAD) enzymes were evaluated by immunohistochemistry. Results 63% of the patients were anticitrullinated protein antibodies (ACPA) positive, 26% were current smokers and 22% have reported a previous lung pathology. All patients had active disease with a mean disease activity score of 5.5±0.1 at inclusion that decreased significantly to 3.3±0.2 after 6 months. A majority of the patients (55%) had any type of changes on HRCT either focal or diffuse. Lung changes of any type were more frequent in RA as compared to non-RA individuals (35%) with a higher prevalence of lung fibrosis (15/82 in the RA cohort and 0/43 in the control cohort). In the RA cohort one patient (1.2%) was diagnosed with lung cancer following HRCT screening while none in the control cohort. ACPA and rheumatoid factor but not smoking status associated with the presence of changes on HRCT. Immunohistochemistry demonstrated a significant increase in expression of both PAD2 and PAD4 in the lungs of current smokers independent of the ACPA status with no significant changes following 6 months of treatment. Conclusion Presence of ACPA associates with early lung HRCT changes in RA. The authors suggest that smoking (and other yet unidentified factors) promotes site specific citrullination (such as in lungs) leading to generation of ACPA immunity and lung changes very early in the disease process.

From Citrullination to Specific Immunity and Disease in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease resulting from the complex interaction between genes and environment. In a majority of patients this interaction leads to formation of anti-citrullinated proteins antibodies (ACPA). Recent advances have demonstrated that external factors (such as smoking) are able to induce posttranslational extra-articular modifications of proteins (such as in the lungs). In genetic susceptible hosts the new generated epitopes are presented more efficiently to the immune system leading to break of tolerance and generation of ACPA already before disease onset. A second hit (such as minor trauma or transient viral infection) might promote synovial citrullination with local emergency of same citrullinated epitopes as originally present at the initiation site. In this way ACPAs will localize in the joint and initiate antibody- and immune complex-mediated events leading to chronic inflammation. The current chapter focuses on the role of citrullination in breaking immune tolerance to first generate ACPA and then lead to the onset of chronic joint inflammation.

Characterisation of lung inflammation and identification of shared citrullinated targets in the lungs and joints of early rheumatoid arthritis

Background and Objectives To investigate if changes in the lungs are present in rheumatoid arthritis (RA) patients early in the disease process and to address the contribution of these changes to the initiation of the disease. Materials and Methods 24 RA patients with patient-reported symptom duration less than one year and naive to DMARD treatment and 15 healthy individuals were subjected to bronchoscopy and mucosal bronchial biopsies were retrieved. Histological analysis for identification of inducible bronchia associated lymphoid tissues (iBALT) and lymphocyte infiltration, as well as immunohistochemistry (IHC) for PAD enzymes, CD3, HLA-DQ and HLA-DR were performed. Presence of citrullinated targets were detected by IHC using biotinylated ACPA isolated from synovial fluid of RA patients. Mass spectrometry was used for identification of citrullinated epitopes in 6 lung and 8 synovial biopsies from RA patients. An in-house ELISA was setup to measure reactivity against new identified citrullinated targets in the serum of RA patients in two distinct early RA cohorts (n = 393) and a cohort of non-RA patients (n = 236) as disease controls. Results Bronchial lymphocyte infiltration and iBALT formation was observed in 55% of the ACPA+ RA patients but only 17% of ACPA- patients and 7% of healthy volunteers. Higher expression of CD3, HLA-DQ, HLA-DR and citrullinated targets was observed in bronchial biopsies of ACPA+ as compare to ACPA- RA patients. BAL fluids were enriched in both IgG and IgA ACPA as compared to paired serum samples. Mass spectrometry identified 5 proteins in the synovium (in total 8 sites) and 4 in the lungs (in total 6 sites) containing citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of both synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in the smokers as compared to non-smokers.14.5% of the RA patients tested by ELISA showed antibody reactivity against the new identified citrullinated target compared to 3.4% in the disease controls. Conclusions Signs of inflammation and local ACPA enrichment are present early in bronchial tissues of ACPA+ RA patients. Shared citrullinated targets in the lung and joints as well as systemic reactivity against these targets are present in RA patients. Our findings support the notion that early inflammatory events in the lungs may represent a critical initiating factor in the development of ACPA+ RA.

FRI0137 Association between Number and Type of Different ACPA Fine Specificities and Parenchymal Lung Changes in High Resolution Computed Tomography in Patients with Early Rheumatoid Arthritis

Background Anti-CCP2 antibodies are associated with parenchymal lung changes in early RA. Objectives To examine the association between ACPA fine specificities and parenchymal lung changes in early RA. Methods Patients with newly diagnosed RA, glucocorticoids and DMARDs-naive, were included. High-resolution computed tomography (HRCT) was performed to assess parenchymal lung changes (ground glass changes, nodules, infiltrates or fibrosis). ImmunoCAP was used to detect RF IgA, RF IgM, anti-CCP2 IgA, anti-CCP2 IgG and ISAC microarray system was used to detect antibodies against 10 citrullinated (Cit) peptidic antigens: CCP-1 (Filaggrin), CEP-1 (α-enolase), Vim 2–17, Vim 60–75 (Vimentin), Fib β 36–52, Fib α 573, Fib α 591, Fib α 36–50, Fib β 60–74, Fib α 621–635 (Fibrinogen). Logistic regression was performed to examine associations between lung changes and autoantibodies, adjusted for age and sex. Due to risk for effect modification of smoking and co-linearity between smoking and Cit peptides, we stratified the cohort according to ever vs. never smokers. Results A total of 106 patients were included. The mean (SD) age was 57 (14) years. 69% were females; 73% were ever smokers; 70% were RF positive and 69% were positive for anti-CCP2. Parenchymal lung changes were found in 58 patients (54.7%). Higher age [≥65 vs <65, OR (95% CI) =2.5 (1.1–5.9)], pos RF IgA [OR (95% CI) =2.7 (1.2–5.9)], pos CCP2 IgG [OR (95% CI) =2.3 (1.0–5.4)], ever smoking [OR (95% CI) =2.6 (1.1–6.2)] and pack-years above 24 [OR (95% CI) =6.9 (2.0–23.5)], were significant predictors of parenchymal lung changes. Some ACPA fine specificities were associated to parenchymal lung changes in ever smokers (table 1). Having five or more ACPA specificities at the time of diagnosis increased the risk of having lung changes in ever smokers by 6.6 times (table 1).Table 1. Association between ACPA fine specificities and parenchymal lung changes according to smoking status Non-smokers [N=29] Ever smokers [N=77] OR (95% CI) P-value OR (95% CI) P-value Cit Fibrinogen peptides 2.3 (0.4–13.1) 0.35 3.0 (1.0–8.8) 0.05  Fib β 36–52 cit 1.1 (0.2–5.9) 0.87 2.7 (0.9–7.6) 0.07  Fib α 573 cit 2.3 (0.4–13.0) 0.35 3.9 (1.3–12.5) 0.02 Cit Vimentin peptides 3.5 (0.5–24.9) 0.22 2.9 (1.0–8.5) 0.06  Vim 2–17 cit 1.2 (0.2–6.5) 0.80 2.9 (1.0–8.6) 0.05  Vim 60–75 cit 4.9 (0.7–32.7) 0.10 2.2 (0.8–6.3) 0.15 CEP-1 (α-enolase) 0.4 (0.06–2.4) 0.30 2.0 (0.7–6.0) 0.20 N. ACPAs 0 (ref)  1–4 2.6 (1.9–35.9) 0.47 5.2 (1.3–21.2) 0.02  >4 3.6 (0.3–44.7) 0.32 6.6 (1.6–27.3) 0.009 Conclusions RF IgA, anti-CCP2 IgG, antibodies to Cit Fib and Cit Vim peptides were strongly associated to parenchymal lung changes in ever smokers with early RA. The more ACPA fine specificities, the higher the risk of parenchymal lung changes. Disclosure of Interest None declared

OP0171 Characterization of Lung Inflammation and Identification of Shared Citrullinated Targets in the Lungs and Joints of Early RA

Background RA associated immunity develops years before disease development and might be initiated at extra-articular sites, such as the lungs. Objectives To investigate molecular lung changes in patients with rheumatoid arthritis (RA) at disease onset and to address the contribution of these changes to disease initiation. Methods 24 RA patients with patient-reported symptom duration less than 1 year and naive to DMARD treatment and 9 healthy individuals were subjected to bronchoscopy and mucosal large bronchial biopsies were retrieved. For comparison 16 large bronchial biopsies and 79 BAL samples from healthy volunteers were available. Histological analysis for identification of inducible bronchia associated lymphoid tissues (iBALT) and lymphocyte infiltration were performed. Further immunohistochemical analysis was performed in RA biopsies to detect PAD enzymes, CD3, HLA-DQ and HLA-DR and to identify citrullinated targets. Mass spectrometry was used for identification of citrullinated epitopes in 6 of the lung RA biopsies and additional 8 synovial RA biopsies. One candidate peptide was synthesized and used to investigate presence of antibodies by ELISA in RA patients (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in RA patients. Results Bronchial lymphocyte infiltration and iBALT formation was observed at baseline in half of the ACPA+ RA patients but only 1 out of 6 ACPA- patients (17%) and 1 out of 9 healthy volunteers (10%). Higher expression of HLA-DR and citrullinated targets was observed in bronchial biopsies of ACPA+ as compare to ACPA- RA (p<0.05). CD3 and HLA-DQ expression also showed a tendency to higher expression in the ACPA+ as compared to ACPA- RA patients. ACPA+ RA patients had significantly higher number of lymphocytes and neutrophils as compared to healthy controls (p<0.05). Markers of T cell activation (CD69 and CD103) were expressed by significantly more CD4+ BAL T cells in ACPA+ RA patients as compared to healthy controls. BAL fluids of ACPA+ RA patients were enriched in both IgG and IgA ACPA as compared to paired serum samples. Mass spectometry identified 5 proteins in the synovium (in total 8 sites) and 4 in the lungs (in total 6 sites) containing citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of both synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in the smokers as compared to non-smokers. Antibodies against a synthesized cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 15% of RA patients and 3.4% of disease controls and associated with presence of the SE alleles in RA. Conclusions Signs of inflammation, accumulation of highly activated and differentiated BAL CD4+ T cells and local ACPA enrichment are present early in bronchial tissues of ACPA+ RA patients. Shared citrullinated targets in the lung and joints as well as systemic reactivity against these targets are present in RA patients. Our findings support the notion that early inflammatory events in the lungs may represent a critical initiating factor in the development of ACPA+ RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4449

A1.4 Early Signs of Subclinical Inflammation and Local Antibody Production in Early Rheumatoid Lungs

Objective To investigate if lung changes are present in RA patients early in the disease process and to address the contribution of these changes to disease initiation. Patients and Methods 21 RA patients with symptom duration less than 1 year at the time of diagnosis and naive to DMARD treatment and 8 healthy individuals were subjected to bronchoscopy and mucosal large bronchial biopsies were retrieved. Histological analysis for identification of inducible bronchia associated lymphoid tissues (iBALT), PAD enzymes, CD3, HLA-DQ and HLA-DR expression were performed. Presence of citrullinated targets were detected by immunohistochemistry using biotinylated ACPA isolated from synovial fluid of RA patients. Presence of ACPA and ACPA fine specificities were tested by ELISA in the serum and BAL of RA patients. Mass spectrometry was used for identification of citrullinated epitopes in 6 of the lung biopsies and additional 8 synovial RA biopsies. Results iBALT formation and higher expression of CD3, HLA-DQ, HLA-DR and citrullinated targets was observed in bronchial biopsies of ACPA positive RA. A majority of serum ACPA positive RA patients subjected to lung bronchoscopy had detectable levels of ACPA in the BAL fluids both IgA and IgG. IgG from BAL fluids of ACPA-positive patients showed a higher ACPA reactivity as compared to serum IgG from the same patients. Mass spectometry identified 5 proteins in the synovium (in total 8 sites) and 4 in the lungs (in total 6 sites) containing citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of both synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in the smokers as compared to non-smokers. Conclusions Lung subclinical inflammation is present already at the earliest visit to a rheumatology specialist early after disease onset in ACPA + RA patients. These findings suggest that the lungs might be the primary local initiation sites of the anti-citrulline response in RA.