In this study, we sought to evaluate the efficacy and characterize the mechanism of action of lanraplenib, a selective oral SYK inhibitor, in human B cells and in vivo in the NZB/W murine model of SLE. Our results demonstrate that lanraplenib-mediated inhibition of SYK can block in vitro B cell activation and maturation of isolated primary human B cells, which are believed to be a driver of disease. Additionally, lanraplenib blocked the progression of LN-like disease in NZB/W mice. Altogether, these data suggest that the effect of lanraplenib in preventing disease progression was at least in part due to inhibition of B cell maturation.
B cells are critical mediators of SLE pathology and have generated research interest. B cells have multiple functions that affect SLE pathogenesis, including cytokine production, antigen presentation to autoreactive T cells, and autoantibody generation and production [1–3]. Antinuclear antibody (ANA) production is a hallmark of SLE; the most commonly found ANA recognizes dsDNA and can be found in the serum of 30–70% of patients with SLE [4, 36, 37]. ANAs can form ICs with nuclear materials released during apoptosis [38, 39], necrosis , and NETosis . In healthy individuals, ICs are bound by complement proteins and are quickly cleared ; however, many SLE patients have defects in one or more complement pathway components , leading to reduced clearance of ICs and resultant type III hypersensitivity .
In the present study, we did not observe a change in serum dsDNA antibody concentrations between 28 and 40 weeks of age with disease progression. It is likely that the effects of lanraplenib or cyclophosphamide on serum dsDNA antibody concentration were negligible due to the initiation of treatment after dsDNA antibody production had already plateaued. Bahjat et al observed a similar lack of dsDNA antibody change in vehicle-treated NZB/W after 28 weeks of age . We have previously tested the effects of a molecule in the same class as lanraplenib in the MRL/lpr murine model of lupus. Between 14 and 20 weeks of age, vehicle-treated MRL/lpr mice showed significantly increased serum dsDNA antibody levels; however, mice treated with either a SYK inhibitor or cyclophosphamide had significantly lower serum dsDNA antibody concentration compared to the vehicle-treated mice at week 20 (see Supplemental Fig. 8, Additional File). These data suggest that SYK inhibition can prevent the development of autoantibodies in vivo. Additionally, by reducing B cell activation and maturation, inhibition of SYK may prevent the intra- and interantigenic epitope spreading observed in SLE patients [44–47].
BCR signaling is necessary for B cell activation and maturation into antibody-producing cells , and SYK is a central player in BCR signal transduction. Upon binding to doubly phosphorylated ITAM of the BCR [49, 50], SYK is auto-  and transphosphorylated [52, 53], and recruits signaling molecules including BLNK [54, 55], PI3K [56, 57], PLC-γ [58, 59], and VAV [60, 61]. Inhibition of SYK phosphorylation results in dissociation of SYK from the ITAM and internalization of the BCR . Conditional SYK knockout mice have defects in B cell development [10, 12] and produce decreased levels of antigen-specific antibodies after exposure to antigen . In this study, we observed an inhibition in maturation of B cells within the spleens of NZB/W mice treated with lanraplenib (0.25%).
SYK has a role in many biological pathways that could contribute to SLE pathogenesis, and its modulation may have a therapeutic benefit beyond affecting B cell function and maturation. Through its dual Src homology 2 domains, SYK has been demonstrated to interact with ITAMs downstream of integrins [63, 64], FcR , and other signaling molecules [66–68]. SYK is expressed in many different cell types that have ITAM-containing signaling receptors, including neutrophils, conventional dendritic cells (DCs), plasmacytoid DCs (pDCs), and others.
In neutrophils, SYK signaling occurs after β2 integrin activation during rolling adhesion [69, 70] and transcellular migration . In addition, proinflammatory cytokines and chemokines, including MCP1, MCP2, and macrophage inflammatory protein (MIP)-1α, are involved in neutrophil recruitment; B cells and myeloid cells can produce MCP1, MCP2, and MIP-1α via TLR7 and TLR9 signaling pathways after endocytosis or phagocytosis of nucleic acid-containing ICs through FcR molecules [72–77]. Furthermore, stimulation of pDCs with serum isolated from SLE patients can induce production of several proinflammatory cytokines, including TNF-α, through the uptake of nucleic acid-containing ICs via FcR molecules [78, 79]. SYK inhibition has been demonstrated to block FcR-dependent IC uptake by classical DCs . In the present study, treatment with lanraplenib reduced the serum concentration of MCP1, MIP-1α, and TNF-α in NZB/W mice (Fig. 4B).
The T cell receptor (TCR) signals through several ITAMs [81, 82], and it has been proposed that SYK plays a role in TCR signaling in SLE patients [83–86]. However, the role of SYK in TCR signaling in NZB/W mice is unknown. The observed effects on splenic T cells with lanraplenib treatment (decreased T cell memory maturation and reduced Tfh abundance) are likely secondary effects and not attributable to direct SYK inhibition of T lymphocyte signaling. We hypothesize that the inhibition of proinflammatory cytokine production observed in this study reduced CD11b+ DC activation and subsequent activation of T cells and differentiation into Tfh [87, 88]. This could further support the role of SYK inhibition in treatment of Sjögren’s syndrome.
SYK has a central role in diverse functional activities across multiple cell types, including BCR signal transduction, neutrophil adhesion, and FcR-mediated endocytosis. In this study, we observed a reduction in splenic B cell maturation with lanraplenib treatment.