Inhibition of effector B cells by ibrutinib in systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease that affects the skin, blood vessels, and internal organs. Within a complex and incompletely understood pathogenesis, the defining pathophysiological features include immune dysregulation with the production of autoantibodies, vasculopathy, and chronic activation of fibroblasts. In particular, the involvement of internal organs critically reduces survival of patients [1]. A stratification of patients in risk-associated groups in the recently developed SCleroderma mOrtality p Eustar (SCOpE) score allows for precise prediction of 3-year mortality, estimating the survival of high-risk patients (SCOpE ≥ 15) at 53% [2]. More than half of the patients die from SSc itself, mostly due to cardiopulmonary complications [3]. Thus, currently available treatment options summarized in the updated EULAR recommendations remain insufficient for controlling disease progression in a clinically satisfactory way [4].

The pathogenesis of SSc remains poorly understood. Accumulating evidence suggests that B cells are involved in SSc beyond the mere production of autoantibodies. Alterations to the B cell compartment maintain the hyperreactivity and chronic activation of large portions of the immune system. The sensitive equilibrium between effector B cells and regulatory B cells (Bregs) is disrupted, and immunoregulatory Bregs prove to be numerically and functionally impaired [5]. Furthermore, evidence for an important pathogenetic role for effector B cell-derived profibrotic IL-6 and TNF-α, as well as for protective effects of anti-inflammatory IL-10, has been published recently [6].

Consequently, different approaches have been tested to address the hyperactivation of B cells in SSc. In a phase III study, the IL-6-receptor-α inhibitor tocilizumab failed to reduce skin thickening, but a trend toward improvement of the modified Rodnan Skin Score and pulmonary function was observed [7]. Complete B cell depletion with rituximab showed better efficacy with regard to the reduction of skin fibrosis and respiratory restriction in a case-control study [8]. The clinical improvement in both trials might have been limited by the lack of specificity, as depletion of all B cell subsets eliminates the protective effects conveyed by Bregs in the context of autoimmunity.

The specific inhibition of autoreactive, profibrotic, and chronically activated B cell subsets represents a more promising approach to achieve effective treatment of SSc. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib could inhibit hyperactivated B cell subpopulations to counteract underlying pathogenetic mechanisms. First FDA-approved for mantle cell lymphoma in 2013 and chronic lymphocytic leukemia in 2014, a potential application for ibrutinib has been suggested for any autoimmune disease in which B cells play an important role [9, 10]. Here, we report on the potential of this small molecule inhibitor to alter B cell pathology in primary patient samples in order to pave the avenue for a clinical application of ibrutinib in patients with SSc.

Ibrutinib (IC₅₀ = 0.5 nM for BTK inhibition) is a first-in-class, irreversible inhibitor of the BTK that can effectively inhibit BCR signaling by selective active-side binding [14]. The BCR pathway plays an important role in B cell development and survival, as it regulates proliferation, differentiation, and apoptosis [15]. Targeting BCR signaling holds the prospect of significantly altering diseases with pathological B cell activation and proliferation. Consequently, ibrutinib was first tested in the context of B cell malignancies and proved effective in patients with relapsed or refractory non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, or Waldenström macroglobulinemia [16]. Importantly, ibrutinib as a single agent showed proficient tolerability and a low side-effect profile [9, 10, 17]. On the other hand, while a beneficial impact of ibrutinib in autoimmune diseases like rheumatoid arthritis or SSc has been proposed from the very beginning, the transition to a clinical application in this field is incomplete [18].

In SSc, B cells are assumed to be important players in disease onset and progression [19, 20]. B cells of patients with SSc show elevated expression levels of the regulatory surface molecule CD19, which reduces the threshold of BCR signaling, thereby importantly influencing B cell activation and survival [21]. This overexpression of CD19 has been linked to the production of SSc-specific autoantibodies, as well as increased levels of profibrotic cytokines, and contributes to a chronically hyperactivated B cell population [22, 23]. We hypothesize that inhibition of the BTK could be the key to restoring B cell physiology and might, therefore, provide a substantial improvement to SSc treatment.

While the BCR is composed of a unique antigen-specific immunoglobulin binding a certain epitope, TLRs recognize a variety of molecular patterns associated with pathogens or cell damage. Signaling downstream of TLR9, a member of the TLR family recognizing unmethylated single-strand DNA, is known to be augmented in SSc, supporting collagen deposition from fibroblasts and synergizing with BCR signaling for B cell activation and immunoglobulin class-switching [11, 24]. It is suggested that circulating fragments of self-DNA acting as endogenous TLR9-ligands could have a role in SSc development and progression [25, 26]. In our in vitro model, the TLR9-agonist CpG (ODN2006) increased survival of cultured B cells and induced the production of various cytokines and anti-Scl-70-antibodies. While no indications of increased B cell apoptosis were observed, ibrutinib treatment showed convincing potential to counteract the production of key inflammatory cytokines, specifically IL-6 and TNF-α. Both cytokines play pivotal roles in the perpetuation of fibrotic signaling leading to skin thickening and organ fibrotic transformation [27, 28]. As a single agent, ibrutinib combines the effects on IL-6 and TNF-α and could alter the production of more cytokines involved in the pathogenesis of SSc as well. A central transcription factor downstream of TLR9 signaling inducing the production of these inflammatory agents is NFκB. Our flow cytometry analysis of phosphorylated NFκB shows increased activation of TLR9 signaling even 24 h after stimulation. Ibrutinib reduced the abundance of pNFκB significantly, supporting previous findings that describe BTK as a key signaling molecule of the TLR9 pathway [29]. The reduction of anti-Scl-70 under ibrutinib treatment is further encouraging and might be true for other autoantibodies. Even though autoantibodies are more of a diagnostic marker than a prognostic factor in SSc, a contribution to disease development via immune activation cannot be ruled out [30]. In vitro findings suggest that anti-Scl-70-antibodies could be of direct pathogenetic relevance by binding to the cell surface of fibroblasts [31].

In our model, ibrutinib was able to reduce the release of proinflammatory IL-6 and TNF-α at doses even below the effective concentrations achieved in vivo (Fig. 2). Importantly, physiologically applicable doses of ibrutinib showed a biased inhibition of fibrogenic cytokines while maintaining IL-10 and IFN-γ levels. Serum IL-10 levels are generally not decreased in patients with SSc, but a significant reduction in IL-10-producing B cells has been described [5, 32, 33]. The preservation of the anti-fibrotic effects of IL-10 under ibrutinib treatment is considered important in the context of aggravated skin fibrosis in a B^IL10−/− mouse model by Matsushita et al. [6] On the other hand, the role of interferon type II (IFN-γ) is controversially discussed in SSc. Some describe a reduction of serum IFN-γ and a pathogenic imbalance of Th1 and Th2 cytokines [33], while others found elevated IFN-γ production without any correlation to clinical outcomes [34]. Functionally, IFN-γ as a Th1-cytokine is categorized to have anti-fibrotic effects and might be reactionarily increased in patients with SSc in an attempt to control fibrotic transformation [35]. Thus, an increase in IFN-γ under ibrutinib treatment could contribute to restore the physiological Th1-Th2-balance.

In a flow cytometric analysis of intracellular cytokines, ibrutinib inhibited the production of IL-6 preferentially in the naïve (CD27⁻) B cell subpopulation. A reduction of IL-6⁺ memory (CD27⁺) B cells was restricted only to high-dose ibrutinib treatment (10 μM). In SSc, the composition of the B cell population is pathologically altered. Memory B cells are reduced and show a high susceptibility for apoptosis-inducing signals, while naïve B cells are numerically increased [20, 36]. Treatment with ibrutinib for 24 h did not change the relative ratio of B cell subsets, but the biased inhibition of IL-6 production represents an indicator for a differential influence of ibrutinib on B cell subpopulations that could translate to meaningful differences between subsets in survival and activation in vivo.

In summary, our in vitro data could pave the avenue for a clinical application of ibrutinib as a novel treatment of the underlying pathogenetic immune imbalance in SSc. Overall, we provide convincing evidence that ibrutinib has the potential to improve B cell pathology contributing to disease onset and progression, encouraging a clinical translation to the benefit of patients with SSc.