The B side of rheumatoid arthritis pathogenesis

Journal Pre-proof The B side of Rheumatoid Arthritis pathogenesis Stefano Alivernini, Barbara Tolusso, Anna Laura Fedele, Clara Di Mario, Gianfranco Ferraccioli, Elisa Gremese

PII:

S1043-6618(19)31356-8

DOI:

https://doi.org/10.1016/j.phrs.2019.104465

Reference:

YPHRS 104465

To appear in:

Pharmacological Research

Received Date:

11 July 2019

Revised Date:

19 September 2019

Accepted Date:

20 September 2019

Please cite this article as: Alivernini S, Tolusso B, Fedele AL, Mario CD, Ferraccioli G, Gremese E, The B side of Rheumatoid Arthritis pathogenesis, Pharmacological Research (2019), doi: https://doi.org/10.1016/j.phrs.2019.104465

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The B side of Rheumatoid Arthritis pathogenesis Stefano Alivernini1,2^, Barbara Tolusso1^, Anna Laura Fedele1, Clara Di Mario2, Gianfranco Ferraccioli2 and Elisa Gremese1,2*.

Division of Rheumatology, Fondazione Policlinico Universitario A. Gemelli – IRCCS, Rome, Italy

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Institute of Rheumatology, Università Cattolica del Sacro Cuore, Rome, Italy.

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Words count: 4137 Figures: 1

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^equally contributed to the work; *Corresponding author:

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References: 133

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Elisa Gremese, MD Associate Professor of Rheumatology Division of Rheumatology Fondazione Policlinico Universitario A. Gemelli – IRCCS Università Cattolica del Sacro Cuore Via Giuseppe Moscati 31, 00168 – Rome, Italy Tel: +39 06 30159659 Email address: [email protected]

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Abstract

In the last years, a dramatic amount of research has been performed increasing the knowledge about the biological mechanism underpinning Rheumatoid Arthritis (RA) inflammation, putting B lymphocytes in the center of RA pathogenesis. Nowadays, B cell phenotypes and autoantibodies

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positivity arose as important biomarkers in early and long-standing disease. Moreover, comparative analysis of peripheral blood and synovial tissue compartments enables the identification of novel physiopathological mechanisms promoting inflammation. In this narrative review we will discuss the biological relevance of B cell derived autoimmunity and in RA course, from disease onset to remission achievement.

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1. Introduction Rheumatoid Arthritis (RA) is a chronic disease in which synovial tissue inflammation causes articular pain and progressive joint damage, leading to deformities and loss of function if not treated. In the last years, a dramatic amount of research has been performed increasing the knowledge about disease pathogenesis and course. Nowadays it is known that in RA course, a preclinical phase exists, before the onset of any joint symptoms. Multiple trigger factors have been implicated in the complex biological process of the loss of immunological tolerance during RA. In

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particular, complex mechanisms regulate the cross-talk between innate and adaptive immune cells triggering the onset and maintenance of synovial tissue inflammation in RA [1]. In this narrative review we will dissect the biological relevance of B cell derived autoimmunity in the pathogenesis

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of RA.

The detection of autoantibodies in peripheral blood of asymptomatic subjects up to 10 years before

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the clinical manifestation of the disease strongly suggests that the loss of tolerance to autoantigens

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occurs much time before the clinical disease onset and that, at clinical onset, the pathology is at an already advanced step. In particular, the availability of genetic markers which enable to predict RA onset or severity supports the notion that such mechanisms are effective over the whole life of a

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person [2,3]. Moreover, genetic studies performed on multiple populations showed that the socalled ”shared epitope” [human leukocyte antigen (HLA)-DRB1 locus] is strongly linked with seropositivity for Rheumatoid Factor (RF) or Anti-Citrullinated Peptide Antibodies (ACPA) [4], in

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addition with other risk alleles (i.e. PTPN22 and CTLA4) which have been demonstrated to be associated with T-cell stimulation, activation and functional differentiation [5-8]. Furthermore, the tight synergistic link between genetic and environmental parameters was widely demonstrated for smoking and other forms of bronchial stress able to increase the risk of RA development in genetically susceptible individuals [9,10]. Among other environmental factors, an increasingly consolidated role is attributed to obesity, that seems to influence the outcomes and prognosis of RA, but also contribute to the raising in disease 3

incidence [11,12], suggesting a role of fat tissue also in the disease pathogenesis [13]. In particular, data from three different RA cohorts indicate that obesity is associated with a likelihood of developing a seronegative RA, mostly in female (OR 1.6-3.5) [14-16].

1.1 Clinical phenotypes of RA Due to the wide heterogeneity in clinical presentation, evolution and response to treatments, several efforts have employed in defining subtypes of RA, but to date the main clinical and biological

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distinction is still based on autoantibody status. It is now recognized that the presence or absence of RF and/or ACPA identify two different disease phenotypes, also from a pathogenetic point of view, as evidenced by the finding of autoantibodies years before the clinical onset of the disease. Studies

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mainly based on the 1987 RA classification criteria [17], supported the hypothesis that seropositive RA also underpinned a more severe prognosis of the disease, as regards radiographic progression

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and response to therapies. More recent studies, mainly based on RA cohorts diagnosed according to

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the new 2010 RA classification criteria [18], show that seronegative RA have more inflammation and higher disease activity at baseline than seropositive patients, probably due to the greater weight given to the presence of autoantibodies in the 2010 criteria, having similar results in terms of

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outcomes in both subtypes of RA [19-21].

On the other hand, the long-term extension analysis of the BeST study, that enrolled early RA patients (disease duration <3 years) with high disease activity, diagnosed according to the 1987

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criteria, showed similar course in seronegative than in seropositive patients at 10 years of follow-up [22]. These findings indicate the need to adopt, in clinical practice, comparable approaches in ACPAneg as well as ACPApos RA patients but taking into account differential physio-pathological underlining mechanisms [23].

1.2 Autoimmunity against citrullinated antigens in RA

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The detection of autoantibodies against citrullinated antigens is the main immunological hallmark of RA. These autoantibodies react against citrullinated peptides derived from arginine after posttranslational modification by the enzyme Peptidyl Arginine Deiminases (PADs) [24], being found in nearly two thirds of RA patients [25-29]. It has been demonstrated that the immune response against citrullinated peptides is initially restricted and increases over time as suggested by autoantibodies levels increase during the predating time period closer to the onset of clinical symptoms [30]. Moreover, a time-dependent expansion of ACPA specificity and subtypes suggests

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the production of different autoantibody specificities mirroring the epitope spreading phenomenon [31]. In addition, the loss of tolerance leading to clinical manifestation of arthritis is tightly associated with the aberrant deregulation of cytokines release [32,33], including TNF- IL-6, IL-

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12p70, IL17 and IFN-, already years before RA onset. Recently, it was demonstrated that the inflammatory activity of ACPA IgG is promoted by Fc fragment glycosylation which is increased

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before RA onset [34], in parallel with a galactosylation decrease and core fucosylation increase

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[35].

However, ACPA seropositivity in RA is not a matter only of specificity but their presence is

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associated with a severe and erosive phenotype of the disease [36-39]. In this contest, ACPA may behave as osteoclasts activator [40], providing the biological basis for the detection of bone erosive features in ACPApos asymptomatic individuals at risk of RA development [41], as suggested by the periarticular bone changes in terms of thinning and fenestration of the cortical bone together with

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milder changes in the trabecular bone fund in such population [41]. These findings challenged the concept that bone damage in RA is exclusively led by inflammation. Moreover, considering that ACPA production is not merely limited to joint environment but may occur at different anatomical sites as lung epithelium [42] and gingival mucosa [43], to date it is still unclear where and how the loss of immunological tolerance takes place and where the ACPA production by autoreactive plasmacells firstly occurs [44].

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However, in daily clinical practice only 2/3 of RA are positive for ACPA and/or RF. Sieghart et al. recently investigated the distribution of different isoforms of ACPA and RF in a wide RA cohort finding that the contemporary analysis of multiple antibody isoforms allows to reduce the definition as seronegative to nearly 20%, suggesting that the determination of a wider range of autoantibody isotype is recommended to reduce not only the diagnostic but even the sensitivity gap left by the routine determination limited to IgM-RF and IgG-ACPA only [45]. Moreover, the combination of ACPA and IgM-RF was demonstrated to exert a direct action, in vitro, on the inflammatory status.

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In particular, ACPA/IgM-RF/citrullinated fibrinogen immune complexes were found to increase, in vitro, cytokines release by macrophages (i.e. TNF-, IL-1, IL-6, IL-17), influencing cellular polarization towards pro-inflammatory (M1) phenotype and suggesting a synergistic biological

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effect [46]. Finally, the determination of multiple autoantibody isoforms plays a prognostic role in the earliest phase of RA since the combined seropositivity of IgM-RF and IgG-ACPA characterizes

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early RA patients with high likelihood chance of radiographic progression during the “treat-to-

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target” follow-up [47].

1.3 Additional autoantibody positivities in RA

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The aberrant autoimmune response in RA, may be directed against modified antigens others than the ones daily used in clinical practice. Vimentin is an intermediate filament expressed in mesenchymal cells and macrophages of synovial tissues involved in adhesion, migration and

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survival as well as cells signaling during stress and autoantibodies against modified vimentin (MCV) have higher sensitivity (69%) but less specificity (94%) compared to ACPA (62% of sensitivity and 97% of specificity respectively) in RA [48,49]. However, despite limitations of the available studies investigating this issue, the specificity of anti-MCV decreases to 86% if other rheumatic diseases are used as comparison group, being detectable in other conditions [49]. In particular, the combined analysis of anti-MCV, IgA/IgM-RF and ACPA in a wide early-RA cohort (n=370) showed the importance of anti-MCV detection being able to reduce the gap of 6

“seronegative” patients of nearly 10% (personal data). The utility of anti-MCV detection in RA is supported by the evidence that anti-MCV positive RA patients show a higher prevalence of bone erosions regardless to ACPA positivity not only at disease onset but even after one year follow-up [50], maybe due to the induction of osteoclastogenesis and bone loss promoted by anti-MCV presence leading to cell activation and TNF release [39,51]. Interestingly, anti-MCV autoimmunity was demonstrated being associated with periarticular bone loss, synovitis and bone erosions in animal model leading to the decrease of trabecular bone and to the increase of osteoclasts [52].

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These findings support the concept that the presence of autoantibodies against modified antigens before the clinical disease onset, may play an active role on bone biology. However, despite the data presented in multiple studies suggest that autoantibodies can mediate pathological events in

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RA through multiple mechanisms, as immune-complexes formation and direct agonistic activity,

extensive experimental validation [53].

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the activation role of ACPA on cells involved in the disease pathogenesis is going to require more

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To date, other mechanisms of antigen modification have been identified not directly enzymatically mediated. Carbamilation is a chemical modification mediated by cyanide in which a lysine residue is converted into homocitrulline, resulting in a self-reacting antigen. Similarly to ACPA, anti-CarP

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antibodies alone are not sufficient to break immune tolerance and induce autoimmunity [54]. However, anti-CarP antibodies are detectable during the pre-symptomatic phase of RA and even in unaffected first-degree relatives, despite their lower sensitivity (42%) and similar specificity (96%)

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compared to ACPA, increase the diagnostic accuracy even in case of RF/ACPA negativity [55-58]. Moreover, the concomitant presence of anti-CarP, RF and ACPA increases the diagnostic power for RA of three folds than the routinely used RF/ACPA combination (OR from 36.7 to 112.2) [59]. Interestingly, despite their strict association with RF/ACPA positivity, anti-CarP antibodies seem not to be dependent to known RA risk factors (i.e. share epitope and smoking) suggesting the need to be investigated to uncover possible novel pathogenetic mechanisms involved in RA [60]. Recently, Truchetet et al. showed that anti-CarP positivity is associated with a more severe clinical 7

and radiographic disease in a cohort of early arthritis patients (ESPOIR cohort). In particular, antiCarPpos arthritis patients had a higher chance of ACR/EULAR 2010 criteria fulfillment among eight years and were more frequently treated with at least one biological DMARD compared to antiCarPneg arthritis patients despite RF/ACPA status [61]. Finally, a new class of autoantibodies against PAD enzymes has been discovered in RA patients. Five different isoforms belonging to PAD enzymes class (PAD1-4 and 6) are known and anti-PAD4 was the first to be discovered [62]. It is well known that PAD4 plays an important role in RA

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pathogenesis contributing to autoantigen citrullination, even if only recently it has been shown to be tightly related to the inflammatory process. In particular, the association between PAD4 and its substrates may result with the loss of tolerance since dying cells or netting neutrophils in RA joint

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are able to release enzymatically active PAD4 within the extracellular space where the latter can modify arginine containing proteins in the nearby as collagen or fibrinogen [63]. Anti-PAD4

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antibodies are detectable in the pre-clinical phase of RA being strongly associated with ACPA and

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with radiographic damage and progression [64-66]. Despite similar specificity for RA (96%), antiPAD4 autoantibodies show lower sensitivity (35-38%) than ACPA [67,68]. It was proposed that anti-PAD4 are able to interfere with PAD4 unfolding or directly affecting antigen binding due to

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the modification of the number of citrulline residues derived by PAD4. Controversial data have been produced on the action exerted by anti-PAD4 antibodies on the enzymatic activity of PAD4 itself. Darra et al. demonstrated that a subset of anti-PAD4 antibodies cross-reacting with anti-

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PAD3 are able to increase PAD4 catalytic efficacy through a decrease of enzyme’s calcium requirement [69]. Based on the upcoming data, further investigations are needed to better define the pathogenetic role of such autoimmune response products, mainly in ACPA/RFneg RA.

1.4 Aberrant B lymphocyte activation in RA The presence of autoantibodies and the successful effect of B cell targeted therapy clearly place the adaptive immune cells at the center of RA pathogenesis [70,71]. Specifically, RA patients show, at 8

synovial tissue level, abundant myeloid and plasmacytoid dendritic cells that express cytokines and costimulatory molecules which are involved in antigen presentation, loss of immunological tolerance and T cell activation [72]. Moreover, B lymphocytes exert multiple role in the pathogenesis of RA not limited only to autoantibodies production but also modulating both T and dendritic cells function, promoting ectopic lymphoid neogenesis development as well as the release of inflammatory mediators [73]. Moreover, the presence of T cell oligoclonality and B cell hypermutation, in synovial tissue, already detectable in preclinical phases increasing overtime

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closer to disease onset, suggest the aberrant activation of the adaptive immune system as early event promoting disease development [74-77].

Differential synovial tissue composition characterizes RA patients, from a pauci-immune phenotype

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to a follicular well-structured synovitis. Synovial B cells are located in the context of lymphoid aggregates where they undergo to clonal expansion and support the production of autoantibodies

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[75-77].

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Recently, Gerlag and colleagues reported the results of the PRAIRI study, in which, ACPA/RF positive asymptomatic individuals were included in a randomized, double-blind, placebo-controlled study to receive a single infusion of 1000 mg of Rituximab or placebo showing that B cell targeted

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therapy is able to significantly delay the development of arthritis, providing evidence for the pathogenetic role of B cells in the earliest, pre-arthritis stage of autoantibody positive RA [78]. Despite ACPA positivity in the preclinical phase, their presence does not cause apparent pathology,

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since ACPA and/or RF positive individuals do not show proven synovitis, in terms of CD3pos, CD22pos, CD55pos, CD68pos or CD138pos cells [79]. Multiple evidences suggested that alteration of B cells subsets distribution is present at the beginning of RA in peripheral blood compartment [80]. In particular, RA patients with disease duration less than 6 weeks showed lower percentages of memory B cells and pre-switched antigen experienced memory B cells (IgDposCD27pos) than healthy controls [81]. Considering comparative studies enrolling RA patients with different disease durations, higher percentages of switched9

memory B cells and plasmablasts characterized long-standing RA with a strong direct correlation with disease duration [80,82]. It has been proposed that synovial tissue becomes enriched of memory B cells under the influence TNF during the disease course [80,82,83]. Moura RA et al. showed that IgDnegCD27neg memory B cells are suppressed by TNF inhibitor and Tocilizumab treatment in RA supporting the notion that TNF and IL-6 represent crucial stimuli for B cell activation and survival [84]. Recently, a new previously unknown mechanism through which B cells may act on bone damage

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was demonstrated to be mediated by CCL3-TNF signaling that inhibits osteoblast differentiation by the activation of ERK and NF-kB signaling pathways. These findings demonstrated the effect of B lymphocytes on bone formation inhibition through the direct production of multiple osteoblasts

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inhibitors in addition to the direct effect on bone exerted by autoantibodies themselves [85].

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1.5 Synovial tissue features based on the autoimmune profile in RA

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During RA synovial tissue inflammation may display various degrees of synovial lining layer hyperplasia, mononuclear cells infiltrates within the sublining areas, stromal cells hyperplasia and vascularity, leading to an extreme heterogeneity among RA patients from pauci-immune to diffuse

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synovial lymphoid neogenesis (follicular synovitis) [76]. synovitis development caused by leukocytes homing from peripheral blood towards synovial tissue is mediated by endothelial cells activation in synovial vessels, whose number is increased and the expression of adhesion molecules

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(as integrins and selectins) is enhanced [86]. Multiple studies have been conducted to dissect the possible differences in terms of synovial tissue inflammation features among RA patients stratified based on the autoantibody positivity with controversial results. Van Oosterhout and colleagues, found that ACPApos long-standing RA patients showed, at synovial tissue level, higher rate of infiltrating lymphocytes, less extent of fibrosis and a thinner lining layer than ACPAneg RA. Moreover, the authors hypothesized that the attraction of leukocytes within the synovial tissue of ACPApos RA may be mediated through CXCL12 [87]. On the contrary, Gómez-Puerta et al. did not 10

report a significant difference in terms of synovial infiltrates and lymphoneogenesis enrolling RA patients with long-standing disease (disease duration more than 40 months). However, the analysis of cytokines milieu in the synovial fluid of RA patients stratified based on ACPA positivity revealed that the synovial fluid of ACPApos RA was enriched of IL-1, IL-10, CCL20, IL-17 and IL-10 compared to ACPAneg RA [88]. Similar results have been obtained stratifying RA patients based on RF positivity [89]. Investigating the synovial features of early RA patients based on ACPA/RF positivity, a higher rate of follicular synovitis was found in seropositive RA patients than

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seronegative ones [90]. In this contest, mast cells (CD117+ cells) can be detected in the synovial tissue of ACPA/RFneg RA patients as recently found to characterize ACPA/RFpos synovitis

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promoting autoantibodies production [91,92].

1.6 Epigenetic mechanisms underpinning B cell abnormal function in RA

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The genetic concordance between monozygotic twins was estimated to be nearly 15%, suggesting

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the additional contribution exerted by other factors, as epigenetic changes in the regulation of cellular homeostasis [93]. Epigenetic mechanisms, including covalent modification of DNA (Methylation of cytosine at position C5 in CpG pairs mainly in regulatory regions) and histones

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modifications (methylation and acetylation) able to remodel chromatin leading to activation or silencing of gene transcription are potentially heritable and able to regulate gene expression without DNA sequence changes [93]. In particular, Julià and colleagues performed, in a limited cohort of

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RA patients, an epigenome-wide association study (EWAS) to identify disease-specific alterations in DNA methylation in B cells finding that differential degree of methylation was observed in 64 CpG sites leading to the identification of multiple biological pathways differentially expressed in RA B cells [94]. Recently, microRNA (miRNA) acting as post-transcriptional regulators of gene expression through repression of target mRNA [95], arose as fundamental in the regulation of the innate and the adaptive immune system in RA [96,97]. In particular, miR-155 was confirmed as fine tuner of 11

macrophages/dendritic cells as well as T and B lymphocytes biology [96]. Anaparti and colleagues investigated the peripheral blood microRNA expression signature showing that patients with RA and their seropositive first-degree relatives have similar expression of miR-155 significantly higher than healthy unrelated controls suggesting that the abnormal epigenetic regulation of the autoimmune cascade is an early phenomenon within RA course [98]. Moreover, miR-155 knock out mice are resistant to collagen induced arthritis, bone damage and autoantibody production by B cells [95]. These findings are due to the repressive activity exerted by miR-155 on

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Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (SHIP-1) which is an anti-inflammatory molecule in macrophages [99] and on PU.1 that is a transcription factor need for B cell maturation and antibody production [90]. Moreover, at synovial tissue level, RA patients with synovial ectopic

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follicular structures show the highest rate of miR-155 compared to RA patients with diffuse/pauciimmune synovitis suggesting that the miR-155/PU.1 axis can be added to the molecular pathways

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involved in lymphoneogenesis promotion in inflamed tissues in RA. Moreover, considering the B

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cell subpopulations in relation to the epigenetic signature, IgDnegCD27neg memory B cells were shown to have the highest endogenous expression of miR-155 compared to other B cell subsets in

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RA patients [90].

1.7 Autoantibodies in clinical remission in RA As stated above, the seropositivity for ACPA is a useful biomarker not only for diagnostic purpose

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but even for prediction of a worse prognosis concerning joint damage [100]. In particular, memory B-cell subset(s) distribution correlates with worse long-term clinical outcomes at disease onset in RA [101]. Importantly, it has been demonstrated that, despite the achievement of clinical remission, through the “treat to target” strategy, ACPA seropositivity generally persists. The seroconversion for IgG-ACPA determining the so called “immunological remission” is a rare phenomenon in clinical practice compared to IgM/IgA isotypes [102]. This phenomenon, over the first year of pharmacological treatment does not translate into a higher chance of drug-free remission in RA 12

[103]. The immune-biologic basis for this persistence is still obscure as ACPA production in ectopic synovial lymphoid tissue is presumably decreased with disease remission. In fact, the analysis of synovial tissue histological composition in RA patients in sustained clinical and ultrasound remission revealed that the rate of synovial lymphoid aggregates is significantly less compared to RA patients at disease onset but not abrogated [104]. Therefore, it can be postulated that ACPA continue to be spontaneously produced by long-lived plasma cells in the synovial tissue, bone marrow or other sites. Pelzek and colleagues have recently investigated the behavior of

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disease-associated autoreactive peripheral B cells in treatment induced clinical remission in RA patients, showing that recirculating ACPA memory B cells generally persist despite DMARD or TNF-inhibition, rationalizing why therapeutic cessation most often results in disease reactivation

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and clinical flare [105]. Haschka and colleagues investigated the predictive power of multiple factors associated with disease flare occurrence in RA patients after treatment discontinuation once

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disease remission is achieved finding that ACPApos RA patients show significant higher change of

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disease flare compared to ACPAneg ones [106]. Moreover, ACPA positivity significantly enhances the disease flare risk of more than 3 times, after treatment discontinuation, in RA patients in disease remission if combined with aberrant Multi-Biomarker Disease Activity (MDBA) panel [107].

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Based on these findings, B cell abnormalities in RA should be considered at the time of remission achievement to foresee the risk of disease flare after treatment modifications.

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1.8 Pharmacological effects on B cells in peripheral blood and synovial compartments of RA Based on their crucial effects on cytokines production and antigen presentation, B cells targeted therapy, as Rituximab, is included in the therapeutic armamentarium to treat RA [108]. Rituximab treatment enables a significant reduction of serum levels of IgM/IgA-RF, ACPA and Anti-MCV autoantibodies [109,110] as well as reduction of BCR clones in the peripheral blood of RA [111]. Considering the synovial tissue environment, Rituximab causes a significant reduction in B cells number [112] but not a complete depletion [109,111]. 13

Moreover, other therapies that are not strictly targeting B-cells, showed a significant effect on B cells in terms of cellular maturation and autoantibody production. Among them, conventional Disease Modifying Anti-Rheumatic Drugs (c-DMARDs) as Methotrexate and Sulphasalazine are able to significantly inhibit clonal growth of B cells in vitro and antibody production [113-115]. Moreover, Methotrexate significantly increases global DNA methylation of B cells in RA [116] and their maturation [117] and Methotrexate/Hydroxychloroquine/Sulfasalazine triple therapy significantly represses genes related to plasmablasts/plasma cells differentiation in synovial tissue

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of RA patients [118]. Moura and co-workers showed that TNF-inhibition enables the reduction of IgDneg/CD27neg B cells in the peripheral blood of RA patients [84] despite no significant change in BCR gene rearrangements [119] whereas Bankò and colleagues demonstrated that Etanercept

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induces significant increase of IL-10 producing B cells (B10) [120]. Considering the synovial tissue compartment, TNF-inhibitor represses memory B cell network despite the persistence of residual B

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lymphocytes in the synovial tissue in RA patients in sustained clinical and ultrasound sustained

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remission [104,121]. The effects of IL-6R inhibition on B cells biology confirmed the crucial role of IL-6 in the maturation and activation of B cells in RA pathogenesis. In particular, a significant reduction of mutational frequency in immunoglobulin receptor and BCR gene rearrangements were

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found in RA patients treated with IL-6R inhibitor together with a significant decrease of peripheral blood IgDneg/CD27neg B cells [84,119,122] together with a significant reduction of mutational frequency in immunoglobulin receptor and ACPA titer [123,124]. Moreover, IL-6R inhibition was

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demonstrated to significantly reduce B cell count and the expression of immunoglobulin genes in the synovial tissue of RA patients [125,126]. Interestingly, CTLA4-Ig therapy causes a significant reduction of post-switch memory B cells and a decrease of Syk phosphorylation in B cells within the peripheral blood compartment of RA patients with a parallel decrease of ACPA-specific B cell clones and IgG/IgA-ACPA and IgM/IgA/IgG-RF serum levels [127-129]. Finally, multiple research groups recently demonstrated that JAK1/JAK3 blockade causes a strong inhibition of plasmablasts development and immunoglobulin secretion in vitro [130] and a significant decrease of synovial 14

tissue expression of CXCL13 suggesting a possible mechanism by which B cells homing within the target tissue may be reduced in RA [131]. In addition, JAK2/JAK3 inhibition was found to significantly suppresses the differentiation of B cells in plasmablasts [132] despite an early increase of peripheral blood B cell count [133]. Therefore, based on these findings, B cells-derived biomarkers may be useful in the assessment of therapeutic response to different conventional and biological therapies in RA management (Table 1).

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2. Conclusions The terrific progress of research on B cells led new knowledge about the multiple functions exerted by B lymphocytes and the molecular pathways underpinning their abnormal function in RA.

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Moreover, the investigation of multiple body compartments (i.e. peripheral blood and synovial tissue) allowed the identification of additional biomarkers associated with the clinical phase and

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disease phenotype (Figure 1). Based on this, we believe that the use of new advanced technologies

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will reveal, in the next future, their utility in a deeper personalized management of RA patients.

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94. Julià A, Absher D, López-Lasanta M et al. Epigenome-wide association study of rheumatoid arthritis identifies differentially methylated loci in B cells. Hum Mol Genet 2017;26:2803-2811. 95. Filipowicz W, Bhattacharyya SN, Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 2008;9:102-114. 96. Alivernini S, Gremese E, McSharry C, et al. MicroRNA-155-at the Critical Interface of Innate and Adaptive Immunity in Arthritis. Front Immunol 2018;8:1932.

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101. Silverman GJ, Pelzek A. Rheumatoid arthritis clinical benefits from abatacept, cytokine blockers, and rituximab are all linked to modulation of memory B cell responses. J Rheumatol. 2014; 41:825-828. 102. Schett G, Emery P, Tanaka Y, et al. Tapering biologic and conventional DMARD therapy in rheumatoid arthritis: current evidence and future directions. Ann Rheum Dis 2016;75:1428–37. 103. de Moel EC, Derksen VFAM, Trouw LA, et al. In RA, becoming seronegative over the first year of treatment does not translate to better chances of drug-free remission. Ann Rheum Dis. 2018 Dec;77(12):1836-1838. 104. Alivernini S, Tolusso B, Petricca L, et al. Synovial features of patients with rheumatoid arthritis and psoriatic arthritis in clinical and ultrasound remission differ under anti-TNF therapy: a clue to interpret different chances of relapse after clinical remission? Ann Rheum Dis 2017;76:1228-1236.

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128. Iwata S, Nakayamada S, Fukuyo S, et al. Activation of Syk in peripheral blood B cells in patients with rheumatoid arthritis: a potential target for abatacept therapy. Arthritis and Rheumatol 2015;67:63-73. 129. Lorenzetti R, janowska I, Smulski CR, et al. Abatacept modulates CD80 and CD86 expression and memory formation in human B-cells. J Autoimmun 2019;101:145-152. 130. Rizzi M, Lorenzetti R, Fischer K, et al. Impact of tofacitinib treatment on human B-cells in vitro and in vivo. J Autoimmunity 2017;77:55-66. 131. Boyle DL, Soma K, Hodge J, et al. The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signaling in rheumatoid arthritis. Ann Rheum Dis 2015;74:1311-6. 132. Kubo S, Nakayamada S, Sakata K, et al. Janus kinase inhibitor baricitinib modulates human innate and adaptive immune system. Front Immunol 2018;9:1510.

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133. Tanaka Y, McInnes IB, Taylor PC, et al. Characterization and changes of lymphocyte subsets in baricitinib-treated patients with rheumatoid arthritis: An integrated analysis. Arthritis Rheumatol 2018;70:1923-1932.

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Figure and figure legend.

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Figure 1. B cell aberrant activation in peripheral blood and synovial compartments in RA.

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Figure 1 legend. The B cell phenotype analysis in the peripheral blood compartment in RA patients revealed the dynamics of B cell subpopulations distribution consistently with disease phase and treatment response. Autoantibodies as ACPA and RF may be detected years before the onset of clinical symptoms and may decrease based on the different isotypes at the time of remission achievement (immunological remission).

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Moreover, the test of additional autoantibody specificities (i.e. anti-MCV, anti-CarP and anti-PAD4), other than ACPA and RF, allows the reduction of the seronegative immunological gap in RA. The new advances in synovial tissue collection, through mini-invasive techniques, allowed the study of synovial tissue inflammation. Different synovitis pathotypes have been found in RA with a higher rate of follicular synovitis in ACPApos compared to ACPAneg RA patients, with a differential rate of response to targeted therapies and a negative impact on bone damage in the former group. In particular, resident synovial B cells, within ectopic lymphoid structures, are enriched of miR-155, whose increase promotes cellular activation, maturation and

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autoantibody release. RA: Rheumatoid Arthritis; ACPA: Anti-Citrullinated Peptide Antibody; miR: microRNA; MCV: Modified Citrullinated Vimentin; CarP: Carbamylated Protein; PAD: Peptidyl Arginine

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Deiminase; RF: Rheumatoid Factor; CD: Cluster Designation.

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Table 1. Pharmacological effects on B cells compartment and autoantibodies of conventional and biological therapies used for RA.

PERIPHERAL BLOOD

SYNOVIAL TISSUE

Conventional-DISEASE MODIFYING ANTI-RHEUMATIC DRUGS (c-DMARDs)

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Sulfasalazine suppresses the production of IgM and IgG, represses B cell hyperactivity [113,114] Methotrexate reduces B cells clonal growth and increases global DNA methylation in B cells [115,116] DMARDs reduce the absolute numbers of total B cells, switched memory B cells, CD21low cells, transitional B cells and plasmablasts [117]

Methotrexate/Hydroxychloroquine/Sulphasalazine triple therapy significantly represses genes related to plasmablasts/plasma cells differentiation [118]

TNF-INHIBITOR

al P

Reduction of IgDneg/CD27neg B cells [84] No effect on mutational frequency of BCR gene rearrangements at week 12, 24 and 52 in RA patients [119] Significant increase of IL-10 productive B cells after 6 months without difference in ACPA serum levels [120]

IL-6R INHIBITOR

Significant reduction of mutational frequency in Immunoglobulin Receptor [123] Significant reduction in mutational frequency of BCR gene rearrangements [119] Significant reduction of IgDnegCD27neg B cells [84,119,122] Significant reduction of ACPA titer in RA after 24 weeks treatment [124]

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Persistence of B cells in the sublining of RA in sustained clinical and ultrasound remission [104] Inhibits memory B lymphocytes via effects on lymphoid germinal centers and follicular cell network [121] Reduction of immunoglobulin genes and CD20 [125,126]

CTLA4-Ig

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Significant reduction of IgG/IgA/IgM and free light chains plasma serum levels in RA. Significant reduction of IgG/IgA-ACPA and IgM/IgA and IgG-RF in RA. Significant reduction of post-switch memory B cells in RA [127] Significant reduction of phosphorylation of Syk in B cells of RA [128] Significant decrease of ACPA-specific switched memory B cells after treatment [129]

RITUXIMAB

Serum levels of IgM- RF and ACPA decreases significantly at 24 and 36 weeks respectively [109] Significant reduction of ACPA, anti-MCV and IgA-RF [110] After 4 weeks, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones [111]

Synovial B cells were significantly decreased, but were not completely depleted in all patients [109,112] No significant changes in the synovial tissue BCR repertoire until week 16, when a reduced clonal overlap with baseline and an increased mutation load is observed [111]

JAK-inhibitor Tofacitinib strongly inhibits plasmablasts development and immunoglobulin secretion in vitro [130] B cells increase after 4 weeks of Baricitinib [133]

Tofacitinib significantly reduced synovial mRNA expression of MMP-1, MMP-3 and chemokines CCL2, CXCL10 and

CXCL13 [131]

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Baricitinib suppresses the differentiation of human B cells into plasmablasts by B cell receptor and type-I IFN stimuli and inhibits the production of IL-6 from B cells [132]

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Table 1 legend. BCR: B cell Receptor; ACPA: Anti-Citrullinated Peptide Antibodies; RF: Rheumatoid Factor; MCV: Mutated Citrullinated Vimentin; MMP:

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metalloproteinases; RA: Rheumatoid Arthritis; JAK: Janus Kinase.

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