In Rheumatoid Arthritis Patients, HLA-DRB1*04:01 and Rheumatoid Nodules Are Associated With ACPA to a Particular Fibrin Epitope

Objectives Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5-amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein/peptide antibodies (ACPAs). Citrulline is a neutral amino acid resulting from post-translational modification of arginine involved in peptidic bounds (arginyl residue) by PeptidylArginine Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrin(ogen) (hFib) and can be specifically detected by the AhFibA assay. Five citrullinated peptides derived from hFib together represent almost all of the epitopes recognized by patients with ACPA-positive RA, namely: α36–50cit, α171–185cit, α501–515cit, α621–635cit, and β60–74cit. The use of antibody fine specificities as markers of clinical phenotypes has become a major challenge. Our objective was to study whether RA clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against the epitopes borne by the five peptides. Methods 184 ACPA-positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patient characteristics including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2 antibodies, AhFibA, and antibodies against the five citrullinated hFib (hFib-cit) peptides were analyzed by ELISA. Results Anti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 – 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were associated with rheumatoid nodules (OR = 2.71 [1.00 – 7.16], p= 0.044). Conclusion Immune complexes containing anti-α501-515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. Apheresis of these epitope-specific antibodies might be a new therapeutic opportunity for patients with rheumatoid nodules.


INTRODUCTION
Rheumatoid arthritis (RA) is the most severe type of chronic autoimmune arthritis. Its prevalence ranges from 0.5% to 1.1% in North America and northern Europe, and between 0.3 and 0.7% in southern Europe (1).
RA features symmetrical bilateral polyarthritis of the small joints. Extra-articular manifestations such as rheumatoid nodules, lung damage, or vasculitis can also be present (2).
RA is usually preceded by the emergence of anti-citrullinated protein/peptide antibodies (ACPAs) and rheumatoid factors of various isotypes. Citrullyl is a neutral residue resulting from post-translational modification of an arginyl residue in the peptidic sequence by PeptidylArginine Deiminases (PADs). The deiminated protein/peptide is said citrullinated.
In ACPA-positive RA, the genetic risk is mostly carried by shared epitope (SE)-positive HLA-DR molecules. The SE (a fiveamino acid motif encompassing positions 70 to 74 of the HLA-DRB1 chain) encoded in the major histocompatibility complex (MHC) is present in approximately 70% of patients with ACPApositive RA (3). Different combinations of HLA-DR alleles (genotypes) confer different relative risks of developing ACPApositive RA, with highest risks for genotypes encoding two copies of the SE (4).
ACPA present in patients with RA recognize citrullinated epitopes on various proteins (5). A major citrullinated autoantigen expressed in the rheumatoid joint is fibrin, both its alpha and beta chain being recognized by ACPA (6). ACPA are likely to play a role in the pathophysiology of the disease. Indeed, ACPAs have been shown to predict progression of undifferentiated arthritis to RA and are associated with severe disease (7). However, given the heterogeneity of the disease's clinical features, more reliable prognostic and phenotypic markers are missing.
The discovery of ACPA led to the development of diagnostic tests based on a first synthetic cyclic citrullinated peptides (CCP) (8). Since then, several generations of anti-CCP tests have been commercialized (9). ACPAs have become one of the 2010 American College of Rheumatology (ACR)/EULAR RA classification criteria (10).
Besides anti-CCP tests, a test for autoantibodies to human citrullinated fibrinogen (AhFibA) can be used for the serological diagnosis of early RA (11).
Previous studies analyzed the recognition by various samples of patients of only 3 (a36-50cit, b60-74cit and FibCit a621-635) out of the 5 major peptides, and only studied early RA defined by the 1987 ACR criteria (14) and not the 2010 ACR/ EULAR criteria.
The primary objective of this work was to study whether, in a cohort of 184 patients with ACPA-positive RA fulfilling the 2010 ACR/EULAR criteria, a particular HLA-DR background or original clinical patterns, were associated with antibodies to the epitopes from the 5 major hFib-cit peptides a36-50cit, a171-185cit, a501-515cit, a621-635cit, and b60-74cit.

Patients
We undertook a prospective study on 184 patients followed at the rheumatology department of Sainte Marguerite Hospital in Marseille. Patients included were considered ACPA-positive RA based on previous results of anti-CCP2 antibodies obtained with various commercial assays, and fulfilled the 2010 ACR/EULAR criteria. Patients treated with Rituximab were excluded due to its potential effect on ACPA levels.
Patient characteristics were collected from their medical files: presence of rheumatoid nodules, smoking habits, age at diagnosis, HLA-DR genotype, IgM rheumatoid factor (RF), activity and erosive characteristics of the disease, treatment response, dry eye syndrome, cardiovascular event, osteoporosis.
-In order to state the presence or absence of rheumatoid nodules, patients medical files and all available chest CT scans were analyzed. -Disease activity was measured using the Disease Activity Score calculated with the level of C-reactive protein (DAS28-CRP) on the day of the sample. -Treatment response was evaluated according to the EULAR response criteria. Patients were considered to have shown a good response if their DAS28 was ≤3.2 or has decreased by >1.2 (15) according to the 2019 update of EULAR recommendations for the management of RA, the time needed to achieve treatment target has been set at 6 months before concluding to treatment failure (16). -Patient personal history of a cerebrovascular accident, myocardial infarction or heart failure was defined as a major cardiovascular event. -Patients were categorized as smokers (active or previous smokers) or non-smokers. -Erosive RA was defined according to the EULAR definition of erosive RA (17). -Dry eye syndrome was defined as a Schirmer's test value ≤ 5 mm/5 min, or an Ocular Staining Score ≥ 5. Dry mouth was defined as an unstimulated salivary flow rate of ≤ 0,1 mL/min. -Osteoporosis was defined either by personal history of osteoporosis fracture, or by bone mineral density with a Tscore below -2,5 for at least one of the testing sites.
specificities were assayed and anti-CCP2 were re-assayed with a same test in all patients.

Ethics
All patients gave informed written consent for this study. Patient data was pseudo-anonymized. Sample collection was approved by the ethics committee under the number DC-2008-327. This study was declared to the Assistance Publique -Hopitaux de Marseille (AP-HM) under the number PADS19-332.

ACPA Testing
For all the ACPA tests, we used the positivity threshold (N) corresponding to 95% diagnostic specificity. Plasma anti-CCP2 assays were performed using the ELISA CCPlus ® IMMUNOSCAN kit (Euro Diagnostica, Arnhem, The Netherlands) according to the manufacturer's recommendations. Antibody titers expressed in arbitrary units (AU/mL) were considered positive above 25 AU/mL.

Rheumatoid Factor Testing
IgM Rheumatoid Factor (RF) was assayed with the RF ELISA from Orgentec and considered positive if >20 UI/mL.

Antibodies to Human Fibrin(ogen) Citrullinated Peptides
Peptides with the following sequences were synthetized with either arginines (R) in control peptides or citrullines (R) in target peptides, and used as immunosorbents: -a36-50: GPRVVERHQSACKDS -a171-185: biotin-ahx*-VDIDIKIRSCRGSCS -a501-515: biotin-ahx*-SGIGTLDGFRHRHPD -a621-635: RGHAKSRPVRGIHTS -b60-74: RPAPPPISGGGYRAR *: ahx: aminohexanoic acid IgG autoantibodies to the fibrinogen-derived citrullinated peptides a36-50cit, a621-635cit and b60-74cit were tested by ELISA according to a previously described method in which peptides are passively adsorbed on polystyrene microtitration plates (6,18). IgG autoantibodies to the peptides a171-185cit and a501-515cit were tested by ELISA with biotinylated peptides linked to previously adsorbed Avidin. Briefly, plates (Maxisorp; NUNC, VWR International, Fontenay-sous-Bois, France) were incubated overnight with Avidin at 5 µg/mL. After washing with PBS 0.1% Tween-20 (PBS-T) biotinylated peptides were incubated for 1 hour at 1µg/mL in PBS 2% BSA (PBS-BSA). After 3 washings the plates were incubated for 1 hour with patient plasma diluted to 1/100 in PBS-BSA then washed 3 times in PBS-T. A peroxidase-conjugated goat anti-human IgG antibody (Southern Biotechnology) diluted to 1/2500 in PBS-BSA was incubated for 1h and, after 3 washings, reactivity was revealed by ortho-phenylenediamine dihydrochloride and hydrogen peroxide in a pH5 citrate buffer. The reaction was stopped after 5 min with H 2 SO 4 and optical density (OD) read at 492 nm. All the steps were performed at room temperature around 20°C (6). The specific antibody reactivity was defined as the OD difference between the citrullinated and the noncitrullinated related control peptide. Interassay variations were corrected by linear regression analysis using reference peptide and plasma, tested on each plate. DOD below the cut-off value of 0.25 were considered negative. Patients with DOD over the cutoff were divided into 3 equal groups (tertiles) with weak (T1), medium (T2) or high titers (T3). Patients from the medium and high-titer groups (T2-T3 combined) were considered together to have a "high level" reactivity.

Statistical Analysis
Association studies were carried out with contingency tables using the Chi2 test and Fisher's exact test when Chi2 was not applicable.
The Baptista-Pike method was used to obtain the 95% confidence interval for Odds Ratio (OR).
Spearman's rank correlation coefficient rho was used to measure the correlations between anti-CCP2 antibodies, AhFibA, and antibodies to the 5 peptides and tested against the null hypothesis of rho =0.
Statistical analysis was carried out using PRISM software for Windows (GraphPad Software, 169 San Diego, California, USA) and IBM ® SPSS ® Statistics version 20. The significance threshold was set at p < 0.05.

Clinical Description of the Population
The clinical characteristics of the 184 patients with ACPA-positive RA are shown in Table 1 (see details in Supplementary Data File 1). Half of patients were treated with methotrexate, 24% in monotherapy, and 76% in association with a bDMARD. Among

Epitopic Specificity of Anti-Citrullinated Fibrin Peptide Antibodies and Clinical Characteristics
Multiple Peptide Recognition

DISCUSSION
We studied the associations between HLA-DR background, specificity of ACPAs to epitopes borne by citrullinated fibrin

The Clinical and Biological Characteristics
The clinical and biological characteristics of the RA population studied here were classical. In our population of patients with ACPA-positive RA, 17% of patients had rheumatoid nodules which is in accordance with other European populations prevalence of 18%-22.4% and less than in northern European population (30-40%) (19). It was previously reported that cvDMARDs or bDMARDs could accelerate rheumatoid nodulosis, particularly methotrexate (20) and TNF-alpha inhibitors (21). In our cohort we could not find any overrepresentation of rheumatoid nodules in patients treated with DMARDs, specially with methotrexate.

Prevalence of ACPAs to the Citrullinated Fibrin Peptides
The prevalence of antibodies to the peptides a36-50cit (15%), a621-635cit (45%), and b60-74cit (58%), were in close agreement with those previously described with the same ELISAs (11,13,(22)(23)(24)(25)(26) and also those recently described in very large cohorts of patients with a multiplex peptide chip (27)(28)(29)(30)(31)(32). The pilot study (6) which identified the peptides bearing the immunodominant epitopes, only used sera selected for highest titers of ACPA, therefore leading to prevalences which are not representative of patient population. The prevalence of antibodies to the peptides a171-185cit (76%) and a501-515cit (67%) had been previously studied by ELISA only in the pilot study (6) and were 45% for the 2 peptides. However in the present study, the peptides were used biotinylated and linked to Avidin versus immunoadsorbed on solid phase in the pilot study. Such an increased reactivity when using biotinylated peptides, depending on a different presentation of the peptides, was clearly demonstrated with the peptide b60-74cit in a recently published study (33).

Association Between Multiple Peptide Recognition and Clinical Characteristics
Multiple peptide recognition (>3) was not associated with erosive arthritis, in accordance with previous studies that showed that the diversity of recognized epitopes was not associated with the severity of the disease (34,35). Multiple peptide recognition (>3) was associated with a better response to TNF-alpha inhibitors in our cohort. In the literature, ACPA have been associated either with a reduced response (36), no effect (37) or a better response (38). A better response to TNF-alpha inhibitors may explain the less erosive pattern observed in our patients with multiple peptide recognition. Indeed, TNF-alpha inhibitors limit radiographic progression in patients with RA as already demonstrated (39).
A study of van Beers et al. (40) failed to find association between ACPA fine-specificity profiles in early rheumatoid arthritis patients and clinical features at baseline or with disease progression, however, the citrullinated peptides used in our study were not analyzed, neither rheumatoid nodules.  Recently, Manca et al. studied the clinico-serological association of ACPA detected using VCP1 and VCP2 (EBVderived citrullinated peptides) and HCP1 and HCP2 (histone-H4-derived citrullinated peptides) in 413 established RA. Higher number/levels of ACPA subtypes were associated with lung involvement but not with erosive disease (41).
In Joshua and al. association between ACPA fine specificities and lung abnormalities were studied. Three out of our 5 antibodies (FibCit a36-50, FibCit b60-74, and FibCit a621-635) were used in the study. There was no association between reactivity to any of them and lung abnormalities studied by HR-CT scan but in contrast a significant correlation when reactivity to the various fibrin peptides was considered as a whole (42).
Here, CT scanners were not available for the whole population studied, so we could not confirm any association between number of specificities and lung involvement.
HLA-DRB1*04:01 Was Associated With Anti-a501-515cit Antibodies Associations between fine-specificity antibodies and the presence of the SE have been already analyzed.
The C. Terao et al. study (29) investigated whether subtle differences in ACPA serological profiles might be connected to different driving HLA alleles, by analyzing 18 ACPA finespecificities in RA-affected individuals. A clustering of the peptide reactivities led to divide the patients as having "noncanonical" (including CCP2-negative) or "canonical" serologies (including CCP2-positive). When associations with HLA-A, B, and DR alleles were analyzed, a significant association with aspartic acid residue at Bpos-9 was found between patients with non canonical serologies and an inverse correlation with the canonical ones. In the multiplex peptide array used in that study, 7 peptides from Fibrinogen were present among the 18 tested but only 3 of our 5 peptides, namely a36-50cit, a621-635cit, and b60-74cit. Thus, our study appears as the first correlation analysis of HLA-DR and reactivities to a171-185cit and a501-515cit.
In the development of IgG antibody responses, HLA-DR molecules intervene by presenting antigenic peptides to T follicular helper (Tfh) lymphocytes that provide help to B lymphocytes with surface IgM of a given specificity. Specificity of the antibody response relies on the IgM molecule on the B lymphocyte, not on the antigenic peptide presented by-HLA-DR. Thus, association of HLA-DRB1*04:01 with anti-a501-515cit antibodies does not indicate that this RA-associated allele is capable to choose between different B cell epitopes. Rather it suggests that HLA-DRB1*04:01 can provide help to B lymphocytes specific for the a501-515cit peptide and that others HLA-DR alleles can't. This suggests special efficacy of HLA-DRB1*04:01 as a peptide processor and presentator which in turn allows the development of a more efficient Tfh response. HLA-DR molecules encoded by HLA-DRB1*04:01 have been shown to undergo a shortcut from endoplasmic reticulum to lysosomes which allows association with better processed antigenic peptide (44,45).
"High Level" Anti-a501-515cit Antibodies Were Associated With Rheumatoid Nodules and With IgM Rheumatoid Factor Association between high level anti-a501-515cit antibodies, RF and rheumatoid nodules refers to rheumatoid nodules nature. As previously described, they contain fibrin and IgG but nothing is known about the specificity of those IgG. Necrotic centers were shown to contain citrullinated proteins and RF (46,47). Our data suggests that at least some of the IgG are anti-a501-515cit antibodies. They might form immune complexes with citrullinated fibrin secondarily transformed in macroimmune complexes by rheumatoid factors (48,49) and be at the origin of rheumatoid nodules.
That link of reactivity to a particular fibrinogen peptide and the presence of rheumatoid nodules needs to be further examined and replication studies must be performed. Moreover, while these findings raise new hypothesis about the pathogenesis of rheumatoid nodules, no study of antigen specificity of the IgG present in the nodules has been performed to date. It could be the purpose of further investigations.
In this cohort we analyzed the epitopic specificity of ACPAs using five major citrullinated fibrin peptides which bear the immunodominant ACPA epitopes and checked whether the specificity was associated with particular clinical, biological, or genetic patterns. Our main finding is the association of antibodies to the epitope borne by the citrullinated peptide a501-515 on the alpha chain of fibrin, with HLA-DRB1*04:01 and with rheumatoid nodules. This unexpected epitope specificity of ACPAs in HLA-DRB1*04:01 patients may relate to the original intracellular route of HLA-DRB1*04:01. The association of anti-a501-515cit antibodies with rheumatoid nodules may suggest that the antibodies to the epitope borne by the a501-515cit peptide particularly contribute to the immune complexes in rheumatoid nodules. This finding pave the way of a potential treatment of rheumatoid nodules by purifying patient's serum from this particular ACPA subtype.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Comitéd'ethique Aix Marseille Université-DC-2008-327. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
NB, GS, and GL designed research and analyzed data. MM, NB, and IA collected samples. MP, GO, and CC tested and analyzed fine specificities. VP performed statistical analysis. GL, NB, and PL analyzed patients files. GL, NB, and JR wrote the manuscript. NB, JR, and GS corrected the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by INSERM.