Role of B cells in the pathogenesis of systemic sclerosis

La Revue de médecine interne 38 (2017) 113–124

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Role of B cells in the pathogenesis of systemic sclerosis S. Sanges a,b,c,d,e , T. Guerrier a,b,f , D. Launay a,b,c,d,e,∗ , G. Lefèvre a,b,c,d,e,f , M. Labalette a,b,e,f , A. Forestier a,b,c,d,e , V. Sobanski a,b,c,d,e , J. Corli a,b,e,g , C. Hauspie a,b,f , M. Jendoubi a,b , I. Yakoub-Agha a,b,e,h , P.-Y. Hatron a,c,d,e , E. Hachulla a,b,c,d,e , S. Dubucquoi a,b,e,f a

Université de Lille, U995, Lille Inflammation Research International Center (LIRIC), 59000 Lille, France Inserm, U995, 59000 Lille, France CHU de Lille, département de médecine interne et immunologie clinique, 59000 Lille, France d Centre national de référence maladies systémiques et auto-immunes rares (sclérodermie systémique), 59000 Lille, France e FHU Immune-Mediated Inflammatory Diseases and Targeted Therapies, 59000 Lille, France f CHU de Lille, Centre de biologie-pathologie-génétique, institut d’Immunologie, 59000 Lille, France g CHU de Lille, département de rhumatologie, 59000 Lille, France h CHU de Lille, département des maladies du sang, 59000 Lille, France b c

a r t i c l e

i n f o

Article history: Available online 25 March 2016 Keywords: Systemic sclerosis B cell CD19 BAFF Rituximab

a b s t r a c t Systemic sclerosis (SSc) is an orphan disease characterized by progressive fibrosis of the skin and internal organs. Aside from vasculopathy and fibrotic processes, its pathogenesis involves an aberrant activation of immune cells, among which B cells seem to play a significant role. Indeed, B cell homeostasis is disturbed during SSc: the memory subset is activated and displays an increased susceptibility to apoptosis, which is responsible for their decreased number. This chronic loss of B cells enhances bone marrow production of the naïve subset that accounts for their increased number in peripheral blood. This permanent activation state can be explained mainly by two mechanisms: a dysregulation of B cell receptor (BCR) signaling, and an overproduction of B cell survival signals, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). These disturbances of B cell homeostasis induce several functional anomalies that participate in the inflammatory and fibrotic events observed during SSc: autoantibody production (some being directly pathogenic); secretion of pro-inflammatory and pro-fibrotic cytokines (interleukin-6); direct cooperation with other SSc-involved cells [fibroblasts, through transforming growth factor-␤ (TGF-␤) signaling, and T cells]. These data justify the evaluation of anti-B cell strategies as therapeutic options for SSc, such as B cell depletion or blockage of B cell survival signaling. ´ e´ Nationale Franc¸aise de Medecine ´ Interne (SNFMI). Published by Elsevier Masson SAS. © 2016 Societ All rights reserved.

1. Introduction Systemic sclerosis (SSc) is a rare condition classified within the connective tissue diseases. It is characterized by the progressive development of fibrosis in the skin [allowing classification of the disease as limited cutaneous (lcSSc) or diffuse cutaneous (dcSSc) subset, depending on its extension] and/or the internal organs (affecting, among others, the lungs, digestive tract and heart) [1]. Eventually, it causes disabling complications (notably pulmonary fibrosis and hypertension), impacting on vital [2,3] and functional prognoses [4].

∗ Corresponding author at: Service de médecine interne, hôpital Claude-Huriez, CHRU de Lille, rue Michel-Polonovski, 59037 Lille cedex, France. E-mail address: [email protected] (D. Launay).

SSc pathogenesis is complex and only partially elucidated. It combines, to different degrees, a fibrotic (excessive synthesis of collagen fibers by activated fibroblasts), vascular (microangiopathy due to endothelial dysfunction) and immunological (dysregulation of cellular and humoral immune systems) components [5]. Among the different immunity actors implicated in SSc, the almost-constant presence of autoantibodies and hypergammaglobulinemia has long suggested a potential role of B cells. More recently, the rapid expansion of anti-B cell biotherapies and the development of animal models of SSc (detailed in Table 1) have shed light on the implication of B lymphocytes in the pathophysiology of this disease. Herein, we review the clinical and experimental arguments supporting a participation of B cells in the inflammatory and fibrotic phenomena observed during SSc, both within damaged organs and at a systemic level.

http://dx.doi.org/10.1016/j.revmed.2016.02.016 ´ e´ Nationale Franc¸aise de Medecine ´ 0248-8663/© 2016 Societ Interne (SNFMI). Published by Elsevier Masson SAS. All rights reserved.

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S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

Table 1 Principal experimental animal models of systemic sclerosis. Animal model

Origin of experimental disease

Fibrosis

Vasculopathy

Inflammation

Autoantibodies

TSK mouse (tight-skin 1)

Spontaneous heterozygous fibrillin-1 gene mutation Spontaneous mutation of unidentified genes Daily subcutaneous bleomycin injection Daily subcutaneous hypochlorous acid injection B10.D2 bone marrow grafted into irradiated Balb/c mouse

+

−

−

+

+

+

+

+

+

−

+

+

+

+

+

+

+

+

+

+

Chicken UCD-200 (University of California at Davis) BLM mouse (bleomycin exposure) HOCL mouse (hypochlorous acid exposure) GVH-Scl mouse (chronic sclerodermiform GVH)

+: present; −: absent; GVH: graft-vs.-host disease.

2. B cell anomalies at the tissue level

3.1. Perturbations of B cell homeostasis

B cells have been found in several tissues of SSc patients. In the skin, B cell infiltrates are not homogeneous and often represent a minority compared to other cell populations [6–13]. When observed, B cells are concentrated in lymphoid aggregates surrounding dermal vessels [6], both in SSc patients’ lesional and clinically healthy skin [7]. The heterogeneity of these infiltrates is probably responsible for the broad variability of immunohistological findings (different numbers of B cells from one slice to another, and from one biopsy to another from the same patient) and contribute to explain the discordant results reported concerning the extent of B cell infiltration into the skin [6–13]. Interestingly, using DNA microarray screening for more than 12,000 genes on skin biopsies [9,12,13], Whitfiled et al. identified, among almost 3000 genes differentially expressed between SSc patients and healthy controls, a cluster of B cell-associated genes [mostly immunoglobulin (Ig) genes] highly upregulated in a subset of dcSSc skin [9,12]. These data suggest that B cell involvement in SSc skin could also differ between patients and be more pro-eminent in certain SSc subgroups. In the lungs, more consistent and extensive B lymphocyte infiltration has been reported. B cells were found within the parenchyma, where they also are organized in aggregates (but sometimes in a more diffuse pattern) [14], and in the bronchoalveolar fluid [15,16]. In the digestive tract, the gastric lamina propria is markedly infiltrated by B cells, also organized in lymphoid aggregates [17]. The origin of these B cell infiltrates and the mechanisms by which they penetrate into tissues are difficult to establish. A possible clue is the documentation in SSc patients of elevated serum levels of certain chemokines known to attract B cells to inflammatory sites [chemokine (C–X–C motif) ligand (CXCL)-13 and chemokine (C–C motif) ligand (CCL)-19]. Interestingly, these chemokines are inversely correlated with the proportion of circulating memory B cells in rheumatoid arthritis [18–20]. The exact role of infiltrating B cells is uncertain but, since their local presence seems only poorly preponderant, some authors suggested that the role of B cells in SSc pathogenesis might be mainly systemic [7].

3.1.1. Modifications of the absolute number of B cells In SSc patients, data regarding variations of the numbers of peripheral B cells during disease course are contradictory. Indeed, even in the absence of any potentially-confounding treatment (corticosteroids or immunosuppressants), the absolute and relative numbers of circulating B cells in SSc patients have been reported to be increased [21], similar [22] or diminished [23] compared to healthy subjects. The disease subset (lcSSc or dcSSc) seems to have little influence on B cell counts [21] and probably does not explain these discordant findings. In contrast, this heterogeneity may be explained by differences between studies in SSc activity and severity, which appear associated with B lymphopenia: indeed, the circulating B cell levels are lower when the modified Rodnan skin score (mRSS) is elevated [23], and in the presence of interstitial lung disease (ILD) or pulmonary arterial hypertension (PAH) [24]. In animal models of SSc, available data mainly concern the splenic B cell compartment and differ markedly from one experimental model to another. Hence, the absolute number of B cells in the spleen is increased in hypochlorous acid-treated (HOCL) mice [25], stable in bleomycin-exposed (BLM) [25] and tight-skin (TSK) mice [26,27], and decreased in sclerodermiform graft-versus-host disease (GVH-Scl) mice [28]. The variations of B cell counts were also studied in lymph nodes, bone marrow and peripheral blood of TSK mice, and revealed no difference compared to controls [27].

3. B cell anomalies at the systemic level Several studies suggest the existence of perturbations of B cell homeostasis (distribution anomalies and activation of certain subsets). These disturbances are caused by dysregulation of the B cell receptor (BCR) signaling, abnormal production of B cell survival signals [B cell activating factor of the tumor-necrosis factor (TNF) family (BAFF), a proliferation-inducing ligand (APRIL)], and stimulation of B cell Toll-like receptors (TLR). They are responsible for several functional anomalies that contribute to the inflammatory and fibrotic events observed during the course of the disease.

3.1.2. Distribution anomalies of B cell subsets In SSc patients, several studies observed the following modifications of the distribution of peripheral B cell subsets, when compared to healthy controls [21,22,29–31]: • memory B cell counts are constantly decreased (both in absolute and relative values) [21,29–31]; • naïve B cell counts are constantly increased (both in absolute and relative values) [21,30,31], with a higher naïve-to-memory B cell ratio [31]; • transitional B cell counts have been described as either increased [30] or decreased [31] (in absolute and relative values); • plasmablast counts are decreased (both in absolute and relative values) [21]; • CD5+ B cell counts are similar between SSc patients and healthy controls [22]. Interestingly, memory B cell levels tend to be lower, and naïve B cell levels to be higher, in SSc patients with short disease duration, diffuse cutaneous subset and pulmonary fibrosis [31]. Moreover, memory B cell counts seem to increase after treatment initiation [29]. Overall, these data suggest that distribution anomalies in B cell subsets could be related to disease activity and severity.

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

In animal models of SSc, the distribution of the main B cell subsets in the spleen (transitional, follicular, marginal zone) and bone marrow (pre-/pro-B cells, immature, mature) does not differ between TSK mice and controls [32]. In contrast, TSK mice have fewer peritoneal B1 cells (CD5+ B220lo ) [26]. 3.1.3. B cell activation status and susceptibility to apoptosis In SSc patients, several teams studied the membrane expression of activation markers [CD25, CD54, CD80, CD86, major histocompatibility complex (MHC) II] and apoptosis markers (CD95, annexin V) on B cells. Overall, the activation status of the peripheral total B cells is comparable [22,24,30] and the percentage of apoptotic cells is diminished [24] compared to healthy controls. When considering B cell subsets, discrepancies exist between studies: some authors reported an overexpression of activation markers exclusively in memory B cells [21], while others observed a preferential activation of naïve and transitional subsets [30]. In addition, the potential influence of SSc characteristics on B cell activation has never been considered. Although the cutaneous subtype (lcSSc or dcSSc) does not seem to modify the expression of activation and apoptosis markers, CD25 is overexpressed at the surface of B cells from patients with PAH or disease duration over 10 years [24]. Moreover, levels of several epigenetic modifications (histone acetylation and methylation, responsible for increased gene transcription) in B cells from SSc patients were found positively correlated with disease activity and mRSS [33]. Overall, these data suggest a possible interaction between SSc activity and severity, and B cell activation state. In animal models of SSc, circulating and splenic B lymphocytes of TSK mice are also activated, as suggested by several experimental findings (MHC II and CD23 overexpression, membrane IgM underexpression, excessive proliferation after BCR stimulation, overproduction of serum Ig) [26,34]. 3.1.4. Abnormal BCR signal regulation In SSc patients, the autoreactivity features observed seem to be explained, at least in part, by a defective regulation of the BCR signal, whose activity is no longer inhibited when interacting with a selfantigen. This regulatory defect is the consequence of hyperactivity of positive regulators of the BCR signal (CD19, CD21) on the one hand, and functional deficit of negative regulators (CD22, CD35), on the other. Analyses of SSc patients’ peripheral blood in several successive studies demonstrated the following findings: • membrane overexpression of CD19 on circulating B cells (especially in the memory subset), correlated with serum Ig levels [21,22,30,31,34]. This anomaly, which seems to be specific to SSc [35], can be explained by elevated circulating concentrations of BAFF (a cytokine known to enhance CD19 expression) [36] and/or by the existence of genetic polymorphisms of the CD19 gene (whose impact seems to differ between ethnic groups) [37,38]; • membrane overexpression of CD21 on circulating B cells [22], associated with lower serum levels of its soluble form (sCD21) [39]; • membrane underexpression and defective activity of CD22, a physiological inhibitor of CD19, on peripheral B cells [30,40,41]. This phenomenon can be explained by the presence of anti-CD22 neutralizing antibodies (whose concentrations are associated with the degree of skin and lung involvement) [40] and/or the existence of CD22 gene polymorphisms [38,41]; • membrane underexpression of CD35, a inhibitory complement receptor that competes with CD21, on peripheral B cells [30]; • possible functional anomalies of intracellular mediators of the BCR signal, notably B-lymphoid tyrosine kinase (BLK) and B cell scaffold protein with ankyrin repeats (BANK), at least partly of

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genetic origin (as attested by the existence of polymorphisms associated with SSc) [42–46]. In animal models of SSc, similar observations were made in TSK mice. Indeed, CD19-pathway overactivation (characterized by constitutive hyperphosphorylation of CD19 and downstream signaling molecules) without membrane overexpression [26], and CD22 activity defect (due to anti-CD22 antibodies) are also found in splenic B cells from this model [40]. These abnormalities account for at least part of the experimental disease phenotype: knockout of the CD19 gene (CD19–/– ) improves skin fibrosis and reduces serum Ig levels, anti-topoisomerase I antibodies levels and cytokines [mainly interleukin (IL)-6] concentrations. Conversely, CD19 membrane overexpression is associated with increased Ig and cytokine levels but does not affect the degree of fibrosis [34]. 3.1.5. Abnormal production of B cell survival signals In SSc patients, serum concentrations of BAFF and APRIL, two cytokines implicated in the regulation of B cell survival, activation, proliferation and maturation, are elevated when compared to healthy controls. Indeed, several teams have documented excessive levels of BAFF in the serum (possibly due to an increased monocyte production), heightened synthesis of its mRNA in the skin, and overexpression of the BAFF membrane receptor (BAFF-R) on peripheral B cells [18,47–49]. Although not found in all studies [18], circulating BAFF levels appear associated with the severity of skin involvement, severe digestive complications (pseudo-obstruction, malabsorption) and serum Ig concentrations [47,48,50]. Serum BAFF level variations during follow-up also seem to reflect SSc activity: their decline is associated with mRSS decrease; and their rise predicts the onset of major organ involvement (ILD, renal crisis, PAH) [47]. Similarly, SSc patients have elevated serum APRIL levels, also due to an increased synthesis by peripheral blood mononuclear cells [51–53]. Circulating APRIL concentrations are associated with the severity of lung involvement, severe vascular complications (digital ulcers, pitting scars, active pattern on nailfold capillaroscopy) and serum Ig levels [51–53]. It is noteworthy that the increase of serum BAFF and APRIL levels seems to be mutually exclusive, allowing delineation of two patient clusters: an “elevated-BAFF” profile at risk of skin fibrosis, and an “elevated-APRIL” profile at risk of lung fibrosis [51]. In animal models of SSc, experimental data also indicate BAFF involvement in SSc pathogenesis. TSK mice also have elevated serum BAFF levels, without modification of splenic B cell surface expression of its receptors [transmembrane activator, calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation antigen (BCMA) and BAFF-R]. Blocking BAFF leads to a diminution of cutaneous fibrosis, and reduces serum Ig levels and skin cytokines [transforming growth factor-␤ (TGF-␤), IL-6, IL-10] [32]. 3.1.6. Abnormal stimulation of B cell TLR In SSc patients, several observations suggest a participation of TLR in the activation of B cells. Indeed, circulating B cells display an overexpression of TLR-9, and an increased proliferation and Ig production when cultured with CpG or nucleosomes (TLR-9 ligands) [54]. This phenomenon is no longer observed when a TLR-9 antagonist is added in culture medium [54]. Remarkably, SSc patients have elevated serum levels of nucleosomes [54], core particles composed of histones and helical DNA that originate from apoptotic or necrotic cells, and can interact with TLR-9. It has thus been speculated that tissue damages could be involved in B cell activation by releasing endogenous ligands for TLR [54]. In animal models of SSc, TLR also appear involved in B cell activation. Indeed, B cells isolated from lymph nodes of BLM mice display

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an enhanced expression of TLR-4, and an increased secretion of proinflammatory (IL-6) and pro-fibrotic (IL-4, TGF-␤) cytokines when stimulated by bacterial lipopolysaccharide (a TLR-4 ligand) [55,56]. Knockout of the TLR-4 gene (TLR-4–/– ) in this model decreases the in vitro B cell cytokine overproduction, and lowers the in vivo serum levels of Ig (total IgG, anti-topoisomerase I) [55]. Interestingly, studies have shown that breakdown products of the extracellular matrix (such as hyaluronan, heparan sulfate and chondroitin sulfate) can act as TLR-4 ligands [56]. Levels of these molecules were found elevated in the skin and lungs of BLM mice when compared to controls [56]. Thus, it has been hypothesized that B cells might be activated through the interaction of TLR-4 with extracellular matrix degradation products induced by BLM exposure, providing a singular link between fibrosis and immunity in this model [56]. 3.1.7. Summary: a model of B cell homeostasis perturbations in SSc by Yoshizaki and Sato et al. Mainly based on the results of the studies conducted over the past 15 years, Yoshizaki and Sato proposed a pathophysiological model describing the perturbations of B cell homeostasis in SSc [57]. According to this model, dysregulation of the BCR signal (along with overproduction of survival signals and TLR stimulation) induces a permanent activation of memory B cells, associated with an increased susceptibility to apoptosis. This continual loss of B lymphocytes, by cell death (but also perhaps by their recruitment into tissues), causes a reduction of their absolute number in the peripheral blood. To maintain a constant B cell count, a regulatory feedback increases their bone marrow production, leading to an expansion of the naïve subset. This chronic activation contributes to the occurrence of the functional perturbations observed in the B cells of SSc patients. 3.2. Perturbations of B cell functional properties Several experimental findings suggest that B cells participate in the inflammatory and fibrosing phenomena of SSc through different functional anomalies: production of autoantibodies (some of which are directly pathogenic), secretion of pro-/anti-inflammatory and/or pro-/anti-fibrotic cytokines, and direct intercellular contact (notably with fibroblasts and T cells). 3.2.1. Production of autoantibodies In SSc patients, polyclonal hypergammaglobulinemia and antinuclear antibodies (especially directed against topoisomerase I, centromere proteins and RNA polymerase III [58]) are quasiconstant findings. Although these antibodies (directed against intracellular targets) probably do not have a direct pathogenic effect, recently identified autoantibodies with new specificities (membrane or extracellular antigens) have more convincing pathophysiological roles: • anti-endothelial cell antibodies induce endothelial activation and apoptosis, and are associated with digital ulcers, severe Raynaud phenomenon, pulmonary hypertension and fibrosis and nailfold capillaroscopic anomalies [59–65]; • anti-endothelin-1 type A receptor and anti-angiotensin-II type 1 receptor provoke endothelial activation and secretion of pro-inflammatory and pro-fibrotic cytokines, and are strong predictors of digital ulcers recurrence, PAH occurrence and death [66–70]; • antifibroblast antibodies trigger pro-adhesive and proinflammatory phenotypic changes in fibroblasts, and are more frequent in dcSSc patients (especially those with PAH) [71–77]; • anti-matrix metalloproteinase-1 and -3 antibodies prevent the degradation of excess collagen in the extracellular matrix, are

more prevalent among dcSSc patients and correlate with the extent of skin and lung fibroses [78,79]; • anti-fibrillin 1 antibodies, whose prevalence differs among ethnic groups, are thought to induce fibroblast activation and release of sequestered TGF-␤1 from fibrillin-1-containing microfibrils in the extracellular matrix [80–84]; • anti-platelet–derived growth factor receptor (PDGFR) agonistic antibodies were initially considered as specific to SSc and involved in fibroblast activation [85], but these results have later been challenged [86–89]. In animal models of SSc, high serum Ig levels, antinuclear antibodies and SSc-specific autoantibodies (especially anti-topoisomerase I) are also commonly found [25,26,32,56,90]. 3.2.2. Production of pro-inflammatory and pro-fibrotic cytokines In SSc patients, studies plead for a participation of B cells in the circulating pool of pro-inflammatory and pro-fibrotic cytokines. Indeed, B cells isolated from SSc peripheral blood secrete more IL-6 than those of healthy controls after stimulation by BAFF and Staphylococcus aureus Cowan strain [47]. In contrast, rituximab depletion of B cells leads to a decrease in circulating pro-inflammatory cytokines (IL-6, IL-15, IL-17, IL-23) levels and is associated with less severe skin fibrosis [7,91]. In animal models of SSc, similar observations were made. Splenic B cells from TSK mice secrete more IL-6 than controls after B cell-specific stimulation (BAFF + S. aureus Cowan, anti-CD40 + antiIgM) [26,32]. The abolition of IL-6 oversecretion in CD19–/– mice or after BAFF-blocking treatment confirmed the importance of the BCR signaling and survival signal dysregulations in the occurrence of the B cell functional abnormalities observed in this model. B cells isolated from spleen and lymph nodes of BLM mice also exhibit an increased production of pro-inflammatory (IL-6, IFN-␥, TNF-␣) and pro-fibrotic [IL-4, macrophage inflammatory protein-2 (MIP-2), TGF-␤] cytokines after stimulation by bacterial lipopolysaccharide [55,56]. This cytokine oversecretion is mediated by a BCR-dependent signal (because it decreases in CD19–/– mice) and a TLR-4 stimulation (as it is reduced in TLR-4–/– mice). 3.2.3. Production of anti-inflammatory cytokines: role of regulatory B cells Recently, a new B cell subset, endowed with anti-inflammatory properties, was identified and designed as regulatory B cells (Bregs) [92]. These particular B cells are able to suppress inflammatory responses, notably through IL-10 production (“B10 cells”). Human Bregs usually express high membrane levels of CD24, CD27, CD38, CD48, CD148 and are enriched within the memory and transitional subsets [92]. However, as no specific transcription factor or cell surface antigen has been discovered so far, the most reliable way to identify them remains intracellular IL-10 staining (with Bregs defined as CD19+ IL-10+ cells). The discovery of Bregs introduced the concept of a potentially ambivalent role of B cells in systemic diseases: in experimental autoimmune encephalomyelitis (a murine model for multiple sclerosis), some B cell subsets (notably marginal zone B cells) are harmful through their synthesis of pro-inflammatory cytokines, while others (Bregs) are beneficial through their secretion of IL-10 [92]. In SSc patients, IL-10 secretion by peripheral B cells is impaired compared to healthy control [30]. The absolute and relative numbers of Bregs (CD19+ IL-10+ cells) in the peripheral blood are also lower [29–31]; and most of them are contained in the transitional and/or memory subset [30,31]. These anomalies could be related to defective BCR and TLR-9 intracellular signaling pathways [31]. Of note, quite similar observations were made in chronic GVH disease [93–95].

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

Interestingly, circulating levels of Bregs are negatively correlated with the occurrence of ILD, with C-reactive protein (CRP) values, and with anti-topoisomerase I and anti-centromere antibodies titers [29,31]. Moreover, Bregs counts tend to increase after treatment initiation [29]. These data suggest that levels of Bregs could be associated with disease activity in SSc. In animal models of SSc, the role of Bregs has so far been studied in GVH-Scl mice [28]. Unlike other murine models of SSc, GVH-Scl B cells seem to play mostly a protective role, since a functional defect in B cells (obtained by grafting the bone marrow of CD19–/– mice) enhances cutaneous fibrosis. This phenomenon is explained by the expansion of the proportion of Bregs during the course of the experimental disease: thus, engraftment of CD19–/– bone marrow combined with adoptive transfer of Bregs cells corrects the previously observed worsening of skin fibrosis. This beneficial effect is achieved by an anti-inflammatory action on T cells and monocytes-macrophages: indeed, Breg-secreted IL-10 decreases T cell production of interferon-␥ (IFN-␥), as well as IL-6 and TNF-␣ production by monocytes-macrophages [28]. 3.2.4. Cellular cooperation: B cell-fibroblast In SSc patients, different studies have highlighted the importance of B cell-fibroblast cooperation in the development of pulmonary and cutaneous fibroses. In the lungs, Kondo et al. recovered and cloned the non-adherent fraction of primary cultures of lung tissues from SSc patients, and created a cell line that demonstrated phenotypic characteristics of B cell lineage [96]. Coculture of that cell line with SSc pulmonary fibroblasts leads to the acquisition of pro-fibrotic properties (mainly IL-6 overproduction) by fibroblasts, induced by a B cell secretion of cytokines (IL-1, TNF-␣). In the skin, quite similar observations were made. Under the influence of different factors (IFN-␥ stimulation, TLR-3 activation), SSc dermal fibroblasts are able to secrete BAFF [97]. When those fibroblasts are cocultured with healthy control B cells, BAFF induces a B cell secretion of pro-fibrotic cytokines (IL-6, CCL-2, TGF-␤1), that triggers a fibroblast production of fibrosis markers [collagen, ␣-smooth muscle actin (␣-SMA) and tissue inhibitor of metalloprotease-1 (TIMP-1)] [98]. Moreover, this phenomenon is weaker when the cells are cocultured in Transwell© plates (that block direct cell contact by a microporous membrane), indicating a participation of direct intercellular contacts in addition to soluble mediators. Finally, rituximab depletion of B cells in the skin and peripheral blood is associated with a decreased production of reactive oxygen species and fibrosis markers (collagen, ␣-SMA) by skin fibroblasts [10]. This effect could be mediated through a reduced secretion of anti-PDGFR antibodies [10], and an attenuation of PDFGR expression and activation on cutaneous spindle-like cells [99]. 3.2.5. Cellular cooperation: B cell–T cell In SSc patients, B cell–T cell interactions also appear critical for SSc pathogenesis, and require both soluble mediators (such as T cell production of T helper 2 cytokines, that induce B cell activation [100]) and direct intercellular contacts. Hence, B cells retrieved from anti-topoisomerase I-positive SSc patients do not produce anti-topoisomerase I antibodies when cultured alone with pokeweed mitogen and recombinant topoisomerase I [101]. Antitopoisomerase I antibody secretion is only detected when B cells are cocultured with T cells (mostly CD4+ ) and requires direct intercellular contact [101]. These intercellular contacts are mediated by several pairs of membrane receptors, primarily MHC II/T cell receptor (TCR), CD80-CD86/CD28, CD40/CD40 ligand (-L) [30,101] and inducible costimulator (ICOS)/ICOS-L [102,103]. Indeed, peripheral B cells from SSc patients display an increased membrane expression of

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CD40 [30]. Moreover, serum ICOS-L levels are elevated in SSc patients and associated with diffuse skin involvement, extensive ILD and CRP concentrations. Surprisingly, ICOS-L expression on circulating B cells is similar in SSc patients and controls; a phenomenon also seen in systemic lupus erythematosus and attributed to a downregulation induced by ICOS overproduction [102]. In animal models of SSc, the importance of B cell–T cell cooperation in the induction of fibrosis has long been known. Phelps et al. studied the effects of the infusion of TSK splenocytes to irradiated C57BL/6 mice [104]. While separate transfers of T or B cells have little influence, the simultaneous injection of the two lymphocyte populations almost completely reproduces the TSK phenotype: dermis thickening, enhanced collagen gene transcription, inflammatory infiltration of the skin and production of autoantibodies. Several communication pathways between B and T cells have been incriminated in the different mouse models of SSc. The CD40/CD40-L axis seems critical in TSK mice [90]: blockage of that pathway by anti-CD40-L antibodies limits fibrosis development and Ig production; membrane CD40-L expression is increased on T cells; and anti-CD40 antibody stimulation enhances B cell proliferation. Similarly, in BLM mice, the ICOS/ICOS-L axis appears to be particularly important [105]: B cell surface expression of ICOS-L is inversely associated to the severity of skin and lung fibroses. 3.2.6. Cellular cooperation: B cell-endothelial cell In SSc patients, preliminary findings seem to indicate a potential participation of B cells in the vascular damages observed during the course of the disease. Indeed, rituximab depletion of B cells is associated with a decrease of arterial stiffness (suggesting modifications in the elastic properties of large vessels walls) [106], an improvement of Raynaud phenomenon and digital ulcers, and a decrease of nailfold capillaroscopic anomalies [107,108]. The modalities of interactions between B cells and endothelial cells are poorly elucidated but could include endothelin-1, an endothelium-derived peptide involved in both angiogenesis (by an autocrine loop) and inflammation. Since B cell surface expression of endothelin receptors in SSc patients is identical to healthy controls, the endothelin pathway is probably not a major activation mechanism of B cells in this disease [68,109]. Conversely, B cells can activate endothelial cells by secreting anti-endothelin-1 type A receptor stimulatory antibodies [66,67,69]. Other autoantibodies also mediate the effects of B cells on endothelium, such as antiendothelial cell [59–65] and anti-angiotensin-II type 1 receptor antibodies [66,67,69]. 4. Anti-B cell targeted therapies In light of these results suggesting a significant role of B cells in the pathogenesis of SSc, it seems relevant to include anti-B cell biotherapies in the therapeutic armamentarium against this disease [110]. Several modalities of B cell regulation are currently being evaluated in SSc: B cell depletion by anti-CD20 antibody, inhibition of survival signals by BAFF-blocking molecules, proteasome inhibition by bortezomib, and B cell-specific protein kinase inhibition by fostamatinib and ibrutinib. 4.1. B cell depletion: anti-CD20 antibody Initially validated for the treatment of lymphoproliferative malignancies, the use of rituximab (RTX), a chimeric monoclonal antibody directed against the CD20 antigen, has since been successfully extended to the field of autoimmune diseases (rheumatoid arthritis, antineutrophil cytoplasm antibody-associated vasculitides, systemic lupus erythematosus).

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Table 2 Therapeutic trials evaluating rituximab efficacy in systemic sclerosis. Reference

Study characteristics

Baseline data

Treatment modalities

Follow-up data

n

Inclusion criteria

Exclusion criteria

Mean SSc duration

Serological profile

Skin (m ± SD)

Lungs (m ± SD)

Previous treatments

Rituximab administration scheme

Associated treatments

Follow-up duration

Skin (m ± SD)

Lungs (m ± SD)

Lafyatis et al., 2009 [6]a

Interventional, uncontrolled, open-label study

15

Early dcSSc (< 18 months)

14.5 months (range 9–18)

ATA (3/15) ARA (5/15) ACA (1/15)

dcSSc (15/15) mRSS: 21 ± 9

ILD (?/15): FVC 89 ± 26%; DLCO 80 ± 16%

N/A

RTX 1 g 2 inf. separated by 15 d

None

12 months

Stable mRSS: 21 ± 10

Stable FVC 93 ± 20% DLCO 78 ± 16%

Bosello et al., 2010 [7]

Interventional, uncontrolled, open-label study

9

dcSSc ↑ mRSS > 10% on CYC therapy

49 months (range 12–240)

ATA (6/9)

dcSSc (9/9) mRSS: 21 ± 9

ILD (?/9): FVC 92 ± 21%; DLCO 58 ± 16%

CYC (9/9) MTX (2/9)

RTX 1 g + MTP 100 mg 2 inf. separated by 15 d (3 received a 2nd cycle at 12–18 m)

None (7/9) MTX (2/9)

36 months

Improved mRSS: 4 ± 1

Stable FVC 97 ± 2% DLCO 58 ± 14%

Bosello et al., 2015 [111]

Interventional, uncontrolled, open-label study

20

dcSSc ↑mRSS > 10%

30.4 months (SD ± 35.6)

ATA (14/20)

dcSSc (20/20) mRSS: 22 ± 10

ILD (14/20): FVC 87 ± 20%; DLCO 55 ± 15%

CYC (16/20) MTX (1/20) None (3/20)

48.5 months (SD ± 20.4)

Improved mRSS: 11 ± 7

Stable FVC 88 ± 20% DLCO 60 ± 21%

Interventional, randomized, controlled, open-label study

6 (CTL)

ATA-pos. dcSSc ILD on CT-scan and/or PFT Treatment stable for > 12 months

8.33 years (range 2–15)

ATA (6/6)

dcSSc (6/6) mRSS: 12 ± 2

CS (5/6) MMF (2/6) CYC (1/6)

Stable mRSS: 10 ± 3

ATA (8/8)

dcSSc (8/8) mRSS: 14 ± 7

CS (5/6) and/or MMF (2/6) and/or CYC (2/6) CS (6/8) and/or MMF (4/8) at stable doses throughout the study

12 months

6.87 years (range 1–15)

ILD (6/6): FVC 86 ± 20%; DLCO 65 ± 21% ILD (8/8): FVC 68 ± 20%; DLCO 52 ± 21%

RTX 1 g + MTP 100 mg 2 inf. separated by 15 d (8 received a 2nd cycle) –

None

Daoussis et al., 2010 and 2012 [8,99]

FVC or DLCO < 50% LVEF< 40% Cardiac arrhythmia IS or CS > 10 mg/d Evolution > 18 months Resting dyspnea Heart failure Infection, tuberculosis Immunodeficiency Cancer Severe comorbidities Resting dyspnea Heart failure Infection, tuberculosis Immunodeficiency Cancer None

12 months

Improved mRSS: 8 ± 7

24 months

Improved mRSS: 5 ± 2

6 months

Improved mRSS: 14 ± 4

24 months

Improved mRSS: 14 ± 6

Stable FVC 82 ± 21% DLCO 60 ± 23% Improved FVC 75 ± 20% DLCO 62 ± 23% Improved FVC 77 ± 20% DLCO 63 ± 22% Stable FVC 88 ± 9% DLCO 73 ± 18% Stable FVC 85 ± 13% DLCO 73 ± 16%

Smith et al., 2010 and 2013 [112,113]

Interventional, uncontrolled, open-label study

8 (RTX)

8

Early dcSSc (< 4 years) mRSS > 14 EUSTAR score > 3

FVC < 50% DLCO < 40% LVEF < 40% Severe comorbidities Infection Cancer

16.5 months (range 8–34)

ATA (3/8) ARA (3/8)

dcSSc (8/8) mRSS: 25 ± 3

ILD (5/8): FVC 93 ± 9%; DLCO 73 ± 23%

Stable doses for > 1 year: CS (6/8) MMF (4/8) Stopped > 3 years: CYC (3/8) D-pen (3/8) MTX (1/8)

RTX 375 mg/m2 1 inf/week for 1 month 4 cycles separated by 6 m

MTX and/or CS (1 patient: D-pen, etanercept, CYC) (1 patient: SLZ)

RTX 1 g + MTP 100 mg 2 inf. separated by 15 d

MTX (5/8) and/or CS (5/8)

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

Type

Table 2 (Continued) Reference

Jordan et al., 2014 [115]a

Study characteristics

Baseline data

Treatment modalities

Inclusion criteria

Exclusion criteria

Mean SSc duration

Serological profile

Skin (m ± SD)

Lungs (m ± SD)

Previous treatments

Rituximab administration scheme

Associated treatments

Follow-up duration

Skin (m ± SD)

Lungs (m ± SD)

Observational, case-control study

63

SSc Treatment by RTX > 1 follow-up visit

Hematopoietic stem cell transplantation during study

6 years (range 3–11)

ATA (42/61) ARA (3/52) ACA (3/62)

dcSSc (46/63) mRSS: 22 ± 10b

ILD (9/63): FVC 61 ± 7%; DLCO 41 ± 8%

N/A

RTX 1 g: 2 inf. in 15 d (47/63) RTX 1 g: 1 inf. (13/63) Other modalities (3/63)

Monotherapy (32/63): MTX (13), AZA (6), CS (5), MMF (4), CYC (1) Bitherapy (8/63): CTC +CYC (2), +AZA (1), +MTX (1) Tritherapy (1/63): CTC + MTX + CSA None (22/63) MTX (3/5) or AZA (2/5) at stable doses during the preceding 12 weeks

7 months (IQR 4–9)

Improved mRSS: 18 ± 10b

Stable FVC (61 ± 4%) Improved DLCO (45 ± 2.7%)

12 months

Improved mRSS: 12 ± 3

24 months

Improved mRSS: N/A

CS (10/10) MMF (3/10) LFL (2/10)

37 months (SD ± 21)

Stable mRSS: 20 ± 7b

Improved FVC 89 ± 3.2% DLCO 66 ± 4% Improved FVC N/A DLCO N/A N/A

CS (2/6) Colchicine (1/6)

6 months

Improved mRSS: 17 ± 5

Interventional, uncontrolled, open-label study

5

dcSSc ILD Failure of CYC therapy

None

None

Giuggioli et al., 2015 [91]

Observational, descriptive study

10

Active SSc Treatment by RTX Failure of previous treatments

Fraticelli et al., 2015 [10]a

Interventional, uncontrolled, open-label study

6

Anti-PDGFRNone pos. SSc ↑ mRSS > 4 in 6 months Visceral involvements stable for > 12 months Failure/contraindication of CYC therapy

3 years (range 2–9)

ATA (5/5)

dcSSc (5/5) mRSS: 27 ± 8

ILD (5/5): FVC 72 ± 5%; DLCO 48 ± 7%

CYC (5/5)

6.3 years (range 2–25)

ATA (4/10) ACA (5/10)

dcSSc (5/10) mRSS: 25 ± 4b

ILD (8/10): N/A

N/A

3 years (range 2–7)

ATA (4/6) ACA (1/6)

dcSSc (5/6) mRSS: 23 ± 10

ILD (4/6): N/A

CYC (2/6) CS (2/6) Colchicine (2/6) D-pen (1/6) HCQ (1/6)

RTX 500 mg + MTP 100 mg 2 inf. separated by 15 d 4 cycles separated by 3m RTX 375 mg/m2 1 inf/week for 1 month (9 received a 2nd cycle at 6 m) (4 received a 3rd cycle at 1 y) (3 received a 4th cycle at 2 y) (1 received a 5th cycle at 3 y) RTX 375 mg/m2 1 inf/week for 1 month 1 cycle only

N/A

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

n

+ MTP 100 mg (31/63)

MoazediFuerst et al., 2014 and 2015 [107,108]

Follow-up data

Type

↑: increase; ACA: anti-centromere antibodies; ARA: anti-RNA polymerase III antibodies; ATA: anti-topoisomerase I antibodies; AZA: azathioprine; CS: corticosteroids; CSA: cyclosporine A; CTL: control; CT-scan: computed tomography; CYC: cyclophosphamide; d: day; D-pen: D-penicillamine; dcSSc: diffuse cutaneous SSc; DLCO: diffusing capacity of carbon monoxide; EUSTAR: EUropean Scleroderma Trial and Research; FVC: forced vital capacity; HCQ: hydroxychloroquine; ILD: interstitial lung disease; inf.: infusion; IQR: interquartile range; IS: immunosuppressants; LFL: leflunomide; LVEF: left ventricular ejection fraction; m ± SD: mean ± standard deviation; m: month; MTP: methylprednisolone; MMF: mycophenolate mofetil; mRSS: modified Rodnan skin score; MTX: methotrexate; n: number of patients included; N/A: not available; PFT: pulmonary function tests; pos.: positive; RTX: rituximab; SLZ: sulfasalazine; SSc: systemic sclerosis; y: year. √ √ a For better inter-study comparison, standard deviations (SD) were calculated from standard errors (SE) [SD = SE × (N)] and from 95% confidence interval (CI) [SD = CI × (N)/1.96]. b Values of mRSS for dcSSc patients only.

119

120

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

In animal models of SSc, the efficacy of anti-CD20 antibody treatment was evaluated in TSK mice [27]. Early B cell depletion (initiated as of day 3 of life) induces a lower cutaneous fibrosis, a correction of hypergammaglobulinemia and autoantibody secretion (especially anti-topoisomerase I), and a normalization of cytokine synthesis (TGF-␤, IL-4, IL-6, IL-10, TNF-␣, IL-2 and IFN-␥) in the skin and spleen. Interestingly, these effects are not observed when anti-CD20 treatment is started later (as of day 56 of life): hence, B cell depletion prevents the evolution of skin fibrosis but does not reverse already established fibrotic lesions. In SSc patients, there are currently eight uncontrolled studies [6,7,10,107,108,111–113], two randomized open trials [8,114], one case-control study [115] and one descriptive cohort [91] that have assessed RTX efficacy and safety (Table 2, Supplemental Table 1). From one study to another, the patients included were fairly heterogeneous in terms of skin extension, severity of lung

involvement, disease duration and prior therapeutic lines. Most often, the indication for RTX use and study inclusion was skin or lung deterioration in dcSSc patients. Despite this heterogeneity, all the authors agreed on RTX efficacy in stabilizing or improving mRSS and pulmonary function parameters (Table 2). This effect appears greater in patients with an early form of the disease. Moreover, RTX seems to be associated with regression of the musculoskeletal involvement, stabilization (or even diminution) of digital ulcers, and lower disease activity and severity scores (Supplemental Table 1). In terms of safety, severe adverse events are rare and mostly infectious. To formally prove the usefulness of RTX in SSc patients, double-blind randomized controlled trials are warranted, and currently ongoing for SSc-associated polyarthritis (RECOVER study: ClinicalTrials.gov Identifier NCT01748084), ILD (RECITAL study: ClinicalTrials.gov Identifier NCT01862926) and PAH (ClinicalTrials.gov Identifier NCT01086540).

Fig. 1. Perturbations of B cell homeostasis and functional properties during systemic sclerosis. During SSc, quiescent B cells are activated by four main mechanisms: BCR stimulation (by auto-Ag such as topoisomerase I) and dysregulation (by abnormal expression of regulating coreceptors, auto-Ab such as anti-C22 antibodies, and genetic polymorphisms), survival signal stimulation by BAFF and APRIL (secreted by PBMC and myofibroblasts), TLR stimulation by endogenous ligands (nucleosomes released from apoptotic cells, extracellular matrix degradation products) and Th2-cell stimulation by Th2 cytokines and direct cell-to-cell contact. Activated B cells are mostly contained in the memory subset and display an increased susceptibility to apoptosis, which is responsible for their decreased number. This chronic loss of B cells enhances bone marrow production of the naïve subset that accounts for their increased number. Activated B cells participate in the inflammatory and fibrotic events observed during SSc through four main effector mechanisms: increased production of pro-inflammatory and pro-fibrotic cytokines (that promotes activation of T cells and fibroblasts), decreased production of anti-inflammatory cytokines (responsible for a lesser suppression of inflammatory responses), secretion of pathogenic autoantibodies (targeting fibroblasts, extracellular matrix proteins, or endothelial cells), and direct cell-to-cell contact (notably through T cell costimulatory molecules). Blue: B cell activating mechanism; red: B cell effector mechanism; ↑: increased; ↓: decreased; (+): stimulation; (−): inhibition. Ab: antibodies; Ag: antigens; anti-AT1R: anti-angiotensin-II type 1 receptor antibodies; anti-EC: anti-endothelial cell antibodies; Anti-ETAR: anti-endothelin-1 type A receptor antibodies; anti-MMP1: anti-matrix metalloproteinase-1 antibodies; anti-MMP3: anti-matrix metalloproteinase-3 antibodies; anti-PDGFR: anti-platelet–derived growth factor receptor antibodies; APRIL: a proliferation-inducing ligand; BAFF: B cell activating factor of the TNF family; BAFF-R: BAFF receptor; BCMA: B cell maturation antigen; BCR: B cell receptor; CD: cluster of differentiation; CK: cytokines; ICOS-L: inducible costimulator ligand; IFN: interferon; IL: interleukin; Mem B: memory B cells; MHC II: major histocompatibility complex; PBMC: peripheral blood mononuclear cells; TACI: transmembrane activator, calcium-modulator and cyclophilin ligand interactor; TGF: transforming growth factor; Th2: T helper 2 cells; TLR: Toll-like receptor; TNF: tumor-necrosis factor.

S. Sanges et al. / La Revue de médecine interne 38 (2017) 113–124

4.2. Inhibition of B cell survival signals: BAFF-blocking molecules As BAFF and APRIL play a major role in SSc pathogenesis, it seems relevant to consider inhibition of survival signals as a potential therapeutic strategy. In animal models of SSc, notably TSK mice, treatment with a soluble BAFF receptor limits cutaneous fibrosis, modifies splenic and bone marrow B cell phenotypes (with expansion of the most immature subsets), reduces Ig levels (anti-topoisomerase I, IgM, IgG2a, IgG2b and IgG3) in the serum and cytokine production (TGF␤, IL-6, IL-10) in the skin [32]. In SSc patients, in light of those encouraging results, a therapeutic trial evaluating the efficacy and safety of belimumab, an anti-BAFF monoclonal antibody, is ongoing (ClinicalTrials.gov Identifier NCT01670565).

121

This pivotal role makes anti-B cell biotherapies a promising therapeutic perspective for SSc management. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments The authors acknowledge the Groupe Francophone de Recherche sur la Sclérodermie (GFRS), the Association des Sclérodermiques de France (ASF), the Société Franc¸aise de Greffe de Moelle (SFGM)-Capucine, Novartis and Roche for supporting their work.

4.3. Proteasome inhibition: bortezomib

Appendix A. Supplementary data

Through their inhibitory effect on activated B cells and plasma cells, proteasome inhibitors (namely bortezomib) are a privileged therapeutic class for the management of several Blymphoproliferative disorders: their efficacy was demonstrated in the treatment of multiple myeloma and mantle-cell lymphoma. By extension, their use is now considered for other conditions involving B cells, notably chronic GVH disease and SSc. In animal models of SSc, a recent study demonstrated that early administration of bortezomib to GVH-Scl mice prevents the occurrence of skin lesions and lowers histological and clinical fibrosis scores [116]. However, treatment initiation at a later stage does not induce regression of already constituted lesions. In this model, bortezomib mode of action passes, at least partially, through induction of B cell apoptosis in germinal centers, thereby decreasing their numbers in spleen and skin. In SSc patients, no data is yet available on the efficacy and safety of bortezomib, although preliminary studies in the treatment of chronic GVH disease are encouraging [116,117]. A therapeutic trial comparing and combining bortezomib and mycophenolate mofetil in SSc-ILD is currently ongoing (ClinicalTrials.gov Identifier NCT02370693).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.revmed.2016.02.016.

4.4. Inhibition of B cell-specific protein kinases: fostamatinib and ibrutinib In animal models of SSc, several preliminary studies reported a potential efficacy of small molecules inhibiting B cell-specific intracellular signaling pathways. Fostamatinib, a spleen tyrosine kinase (Syk) inhibitor, seems to improve skin fibrosis and normalize cutaneous production of cytokines (notably TGF-␤) in BLM and GVH-Scl mice [118–120]. Similarly, ibrutinib, a Bruton tyrosine kinase (Btk) inhibitor, appears to decrease clinical and histological scores of skin fibrosis, slow disease progression, and improve survival in GVH-Scl mice [121,122]. In SSc patients, no data is currently available on the efficacy and tolerance of these protein kinase inhibitors. 5. Conclusion Although the intimate mechanisms underlying SSc remain poorly elucidated, several studies agree in implicating B cells in the inflammatory and fibrosing events that characterize the disease. The phenotypic and functional abnormalities displayed by B cells give them a significant place in its pathogenesis, and provide a unique link between autoimmunity and fibrosis (Fig. 1). Furthermore, the involvement of regulatory B cells, whose antiinflammatory properties are beneficial, remains to be specified.

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