Spleen tyrosine kinase inhibition in the treatment of autoimmune, allergic and autoinflammatory diseases

Spleen tyrosine kinase (Syk) is involved in the development of the adaptive immune system and has been recognized as being important in the function of additional cell types, including platelets, phagocytes, fibroblasts, and osteoclasts, and in the generation of the inflammasome. Preclinical studies presented compelling evidence that Syk inhibition may have therapeutic value in the treatment of rheumatoid arthritis and other forms of arthritis, systemic lupus erythematosus, autoimmune cytopenias, and allergic and autoinflammatory diseases. In addition, Syk inhibition may have a place in limiting tissue injury associated with organ transplant and revascularization procedures. Clinical trials have documented exciting success in the treatment of patients with rheumatoid arthritis, autoimmune cytopenias, and allergic rhinitis. While the extent and severity of side effects appear to be limited so far, larger studies will unravel the risk involved with the clinical benefit.


Introduction
Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase of 72 kDa and a member of the ZAP70 (ζ-chainassociated protein kinase of 70 kDa)/Syk family of the non-receptor-type protein tyrosine kinases (PTKs) [1,2] and contains two SRC homology 2 (SH2) domains and a kinase domain [3]. Syk is expressed in most hematopoietic cells, including B cells, immature T cells, mast cells, neutrophils, macrophages, and platelets [1,3,4], and is important in signal transduction in these cells [2,5].
Syk plays an important role in signal transduction initiated by the classic immunoreceptors, including B-cell receptors (BCRs), Fc receptors, and the activating natural killer receptors [3,6,7]. Syk is associated mainly with ITAM (immunoreceptor tyrosine-based activation motif)dependent pathways and aff ects early development and activation of B cells, mast cell degranulation, neutrophil and macrophage phagocytosis, and platelet activation [1,3,4]. Functional abnormalities of these cells are invariably associated with both autoimmune and allergic diseases. Although there have been many exciting develop ments in the treatment of these diseases, there are still serious limitations of the effi cacy of the used drugs as they are associated with the development of serious side eff ects. Because of the central role of Syk in signaling processes not only in cells of the adaptive immune response but also in additional cell types known to be involved in the expression of tissue pathology in autoimmune, autoinfl ammatory, and allergic diseases, Syk inhibition has attracted considerable interest for further development. In this review, we will provide a brief account of the role of Syk signaling in various cell types and will summarize preclinical and clinical studies, which point to the therapeutic usefulness of Syk inhibition.

Syk and lymphocytes
Th e function of Src-family kinases and Syk kinases in immunoreceptor signaling pathways is well known (Figure 1) [6]. After receptor engagement, Src-family kinases phosphorylate the ITAMs of immunoreceptors and this results in the recruitment and activation of Syk [6,7]. BCR-and FcR-defi ned dual-phosphorylated ITAMs recruit Syk through interaction with their tandem SH2 domains, and this triggers kinase activation and downstream signaling [4,8].
Because the development of B and T cells requires intact antigen receptor-mediated signal transduction, Syk defi ciency leads to a complete absence of mature B cells, and ZAP70 defi ciency results in severe T-cell defects [9,10]. Syk plays an important role in the transition of pro-B cells into pre-B cells [9]. Although it was previously thought that BCR signaling was mediated via Syk and T-cell receptor (TCR) signaling via ZAP70, recent data have shown that ZAP70 has a role in B-cell development and Syk is important in pre-T cell signaling ( Figure 2) [11,12]. It appears that Syk and ZAP70 have overlapping roles in early lymphocyte development [11,12].
For the transmission of BCR-mediated cell signaling events, subsequent activation of diff erent types of PTKs, including Syk, is required [13]. BCR aggregation can directly stimulate activation of pre-associated Syk, resulting in tyrosine phosphorylation of Igα-Igβ ITAMs [6,14,15]. Th is phosphorylation leads to recruitment of additional Syk. Subsequently, recruited Syk is activated by Src-PTK-dependent transphosphorylation and by autophosphorylation [6,14]. Th erefore, Syk is necessary for BCR-mediated tyrosine phosphorylation and signal transduction [6,15].

Syk and phagocytes
FcγR, one of the classic immunoreceptors, typically engages Syk [3,7,16,17], and Syk-defi cient murine macrophages display defective phagocytosis [7,16]. After FcγR engagement, ITAMs in the receptor are phosphorylated by Src-family kinases, resulting in the recruitment and activation of Syk. As a result, Syk-mediated phosphorylation of several adaptor proteins causes activation of downstream pathways, which execute phagocytosis. Syk is also important in complement-mediated phagocytosis resulting from the binding of C3bi-coated particles to complement receptor 3 [3,17]. Downstream of Syk, the signal involves Vav and RhoA to generate contractile forces, which result in the engulfment of the phagocytosed particles [3,17,18].

Syk and mast cells
FcεRI, the high-affi nity surface receptor for IgE, is expressed on the surface membrane of mast cells, and crosslinking of receptor-bound IgE by multivalent antigen starts the activation of mast cells by promoting the aggregation of FcεRI [19,20]. Degranulation and cytokine release occur after the activation signal starts the cascade [20]. Th ese events contribute to the development and continuation of allergic infl ammation. Syk plays an important role in the development of signal transduction events initiated after FcεRI aggregation [2,21], mast cell activation, degranulation, and cytokine production ( Figure 3) [22,23]. All of these facts point to the conclusion that Syk inhibition might be an attractive target for preventing allergen-induced diseases.

Syk in vascular development
Syk is required for the separation of lymphatic vessels in the general circulation [9,29]. Syk-defi cient mice die because blood fi lls the lymphatic vessels [29]. Probably, Syk activation together with platelet activation and aggregation play a role in lymphatic vessel development and their separation from blood vessels [4,9].

Syk and osteoclasts
Osteoclasts are multinucleated cells that degrade bone by releasing proper enzymes. Syk has been claimed to have a role in osteoclast diff erentiation and osteoclast function [3,4]. Although FcγR is associated with osteoblast-osteo clast interactions, DAP12 (DNAXactivating protein of molecular mass 12 kDa) is the responsible protein for relaying an osteoblastindependent signal [30,31]. Syk, which is downstream of DAP12 and FcγR, is required for osteoclast development    and function ( Figure 5) [30,32]. DAP12 phosphorylation recruits Syk through its SH2 domain, leading to autophosphorylation. Phosphorylated Syk associates with cytoskeleton network and actin ring formation [3]. In addition, it was reported that Syk plays a role in the process of osteolysis. Syk, therefore, repre sents an attractive therapeutic target to mitigate increased osteoclastic activity in arthritis.

Syk and fi broblasts
Fibroblast-like synoviocytes (FLSs) represent a signifi cant component of the synovial lining and contribute to the lubrication and preservation of the joint. In rheumatoid arthritis (RA), FLSs expand in numbers, acquire immune cell features, produce proinfl ammatory cytokines and enzymes, and contribute to the infl ammatory process and the eventual destruction of the joint. A number of studies have claimed a role for Syk in the function of FLSs [33,34]. Syk activation is important in tumor necrosis factor-alpha (TNFα)-induced cytokine and metalloprotein ase (MMP) production by RA FLSs [33]. Syk also plays an important role in TNFα-induced c-Jun Nterminal kinase (JNK) activation in FLSs [33]. Th is is an important event as in the future Syk inhibition may be used to supplement the therapeutic eff ect of TNF inhibition in patients who do not display suffi cient response to TNF blockade. Activation of Syk by TNFα causes the activation of the protein kinase Cδ/JNK/c-Jun signaling pathway and this is important for the secretion of a critical cytokine, interleukin-32 (IL-32), by RA FLSs [34].

Syk inhibition therapy in autoimmune and allergic infl ammatory diseases
Although the exact mechanisms of action remain unclear, Syk inhibitors have claimed encouraging therapeutic results in the treatment of patients with allergy, auto immune diseases, or B-cell lineage malignancies [23,35,36]. R406, an orally available active metabolite of the prodrug R788 (fostamatinib), is a competitive Syk inhibitor [37,38]. Th e selectivity for R406 in inhibiting Syk is limited as it may inhibit additional kinases and non-kinase targets. Among those targets are FMS-related tyrosine kinases 3 (FLT3), Lck, and Janus kinase 1 (JAK1) and JAK3, which may also be involved in the expression of autoimmune pathology [4]. Th ese non-Syk targets may enhance the clinical value of R406 in the treatment of autoimmune diseases as JAK inhibitors have been con sidered for the treatment of arthritis. R112 is another Syk inhibitor formulated for intranasal use [39] and has a rapid eff ect and quickly inhibits mast cell activation. Additional Syk inhibitors with less specifi city include piceatannol and BAY 61-3606 [40,41].

Syk inhibition in arthritis
Despite enormous advances in the treatment of RA, a signifi cant number of patients either fail to respond to treatment or develop signifi cant side eff ects. Based on a number of laboratory fi ndings and preclinical studies, including the fact that RA synovium displays increased amounts of phosphorylated Syk compared with osteoarthritis synovium [33], signifi cant eff ort is currently being devoted to determine whether Syk inhibition can be used to treat patients with RA (Table 1).

Animal arthritis models and Syk inhibition
Strong preclinical studies point to the therapeutic potential of Syk inhibition. Syk-defi cient bone marrow murine chimeras do not allow the development of arthritis following the injection of arthritogenic K/BxN serum [42], suggesting the importance of hematopoietic cell Syk-dependent signaling in the development of arthritis. Administration of R406 reduced clinical arthritis in two antibody-induced arthritis models (K/BXN serum and collagen antibody). In addition, R406 suppressed bone erosions detected by radiography, pannus formation, and synovitis in these animal models [37]. It was also observed that the expression of Syk in synovial tissues corre lated with the levels of infl ammatory cell infi ltrates in the joints and was virtually undetectable in R406treated mice subjected to collagen-induced arthritis in rats [38]. In addition, Syk inhibition reduced synovial fl uid cytokine levels and cartilage oligomeric matrix protein in serum in these animals [38]. R406 was also found to limit an Arthus reaction in mice [37] and rats [38] and reverse passive Arthus reaction in murine chimeras with Syk-defi cient hematopoietic cells [43,44]. Th is eff ect is probably due to the suppression of immunecomplex-mediated infl ammation by inhibiting the Fc receptor signaling.

Human studies
After a small phase I study [45] in which clinical effi cacy of the R788 Syk inhibitor in patients with RA was not associated with serious side eff ects, a 12-week, randomized, placebo-controlled trial in which active RA patients who were also receiving methotrexate (MTX) were enrolled was carried out [46]. Twice-daily oral doses of 100 and 150 mg of R788 were demonstrated to be signi ficantly superior to placebo and 50 mg twice a day of R788. Interestingly, the clinical eff ect was noted as early as 1 week after the initiation of treatment. Patients receiv ing 100 and 150 mg R788 achieved excellent ACR20 (American College of Rheumatology 20% improvement criteria) (65, 72%), ACR50 (49, 57%), and ACR70 (33, 40%) responses. Also, signifi cant reductions in serum IL-6 and MMP-3 levels were noted within the fi rst week of treatment. Diarrhea and other gastrointestinal adverse eff ects such as nausea and gastritis, neutropenia, and elevation in transaminase level were the reported major side eff ects.
In the follow-up study, 100 and 150 mg (twice daily) of R788 were compared with placebo at 6 months in 457 active RA patients who were MTX incomplete responders [47]. Th e ACR20 response was achieved in 66% and 57% of patients in the 100 and 150 mg groups, respectively, compared with 35% in the placebo group. Both R788 dosing regimens achieved statistical signifi cance compared with placebo at the sixth month. In this study, the onset of clinical eff ect was again rapid with maximum improvement achieved by week 6 and maintained through out the study. Th e most common side eff ect was reversible and dose-dependent diarrhea. Transient neutro penia, hypertension, and elevation of liver function tests were also recorded.
Another randomized placebo-controlled phase II study was conducted in 219 RA patients who had failed treatment with at least one biologic agent [48]. Patients received either 100 mg (twice daily) of R788 or placebo. ACR20 response and magnetic resonance imaging (MRI) images of the hands and wrists were evaluated 3 months later. Th ere was no statistical diff erence in the ACR20 response between the two groups. However, a signifi cant decrease in erythrocyte sedimentation rate and C-reactive protein and improvement in synovitis and osteitis scores on MRI were observed in the R788 group compared with the placebo group.

Syk inhibition in lupus animal models
In systemic lupus erythematosus (SLE), the FcγR-Syk associates with the TCR in lieu of the zeta-chain ZAP70 [49]. Th is rewiring of the TCR has been claimed to account, at least partly, for the overactive T-cell pheno type observed in SLE [35]. In addition, the pathogenesis of SLE has been associated with B-cell activation in which Syk may play an important role. Th erefore, Syk inhibition therapy was used in lupus animal models ( Table 2).
Long-term (24 to 34 weeks) administration of R788 to lupus-prone NZB/NZW mice before and after disease onset [50] resulted in delayed onset of proteinuria and renal dysfunction, decreased kidney infi ltrates, and prolonged survival in these mice. Although antibody titers were minimally aff ected, a dose-dependent reduction in the numbers of CD4 + activated T cells expressing high levels of CD44 or CD69 in spleens from R788treated mice was noted. Arthus responses were also reduced in NZB/NZW mice pretreated with R788. Also, a Syk inhibitor was reported to reduce the severity of established antibody-mediated experimental glomerulonephritis in rats [51].
Treatment of lupus-prone MRL/lpr and BAX/BAK mice with R788 not only prevented the development of skin and renal pathology but also treated established disease [52]. Syk inhibition reduced splenomegaly and lymphadenopathy and other immune parameters. Th e fact that Syk inhibition suppresses SLE in at least three lupus-prone mice suggests that Syk inhibition in patients with SLE may be of clinical value.

Syk inhibition in allergic diseases
Mainstay therapy for allergic diseases remains avoidance of allergens and allergen-specifi c immunotherapy [23]. However, allergen avoidance and immunomodulation therapies are usually impractical, complex, and timeconsuming [23]. Targeting activation of mast cells to prevent release of mediators represents an important treatment alternative [20,39,52,53]. An eff ec tive way to inhibit the production and release of all mast cell mediators should aim at interfering with the action of IgE by blocking the FcRε with biologics [20,23]. Alterna tively, targeting the intracellular signaling cascade may represent an attractive approach. Appropriately, protein tyrosine kinases such as Syk, Lyn, and Btk have been directly implicated in IgE-dependent mast cell activation and have been suggested as targets for therapeutic intervention [39][40][41]. Syk represents the most attractive target because studies with mast cells derived from Sykdefi cient indicated mice showed that Syk is important in the activation of mediators of degranulation, eicosanoid, and cytokine production [23,39]. Syk inhibition therapies in allergic diseases are summarized in Table 3.

Animal allergic disease models and Syk inhibition
Seow and colleagues [40] examined the eff ect of piceatannol, a Syk inhibitor, on ovalbumin-induced anaphylactic contraction of isolated guinea pig bronchi and release of histamine and peptidoleuketrienes in vitro. Piceatannol pretreatment slightly suppressed peak anaphylactic bronchial contraction but facilitated relaxation of the contracted bronchi. Piceatannol did not inhibit direct histamine-, leukotriene D4-, or KCl-induced bronchial contraction or revert an existing anaphylactic bronchial contraction but did signifi cantly prevent ovalbumininduced release of both histamine and peptidoleukotrienes from lung fragments. But piceatannol did not inhibit exogenous arachidonic acid-induced release of peptidoleukotrienes from lung fragments. In an antigeninduced airway infl ammation model in rodents, the Syk inhibitor BAY 61-3606 blocked both degranulation and lipid mediator and cytokine synthesis in mast cells and suppressed antigen-induced passive coetaneous reaction, bronchoconstriction, bronchial edema, and airway infl ammation [41]. R406 inhibited pulmonary eosinophlia, goblet cell meta plasia, and airway hyper-responsiveness (AHR), which developed in BALB/c mice exposed to aerosolized 1% ovalbumin for 10 consecutive days [20]. In addition, treatment with R406 suppressed the presence of eosinophils and lymphocytes and IL-13 in broncho alveolar lavage fl uid. Suppression of Syk in bone marrow-derived dendritic cells was considered important in the suppression of AHR. Th is preclinical information has justifi ed attempts to determine whether Syk inhibition may have clinical value.

Human allergic diseases and Syk inhibition
Syk inhibition has tried in patients suff ering of allergic disorders to determine whether it mitigates clinical manifestations. A nasal allergen challenge study in volunteers with allergic rhinitis showed that one intranasal dose of R112 is clinically safe and signifi cantly reduces the level of prostaglandin D2, a key mediator of allergic nasal congestion, but not histamine and tryptamine levels [54]. In this 2-day, multiple-dose, double-blind, placebo-controlled clinical study with seasonal allergic rhinitis patients, R112 signifi cantly decreased the global clinical symptom score compared with placebo. Each individual symptom, like sneezing, stuffi ness, itching, runny nose, cough, postnasal drip, facial pain, and headache, was also signifi cantly improved in the R112 group compared with control treatment. Th e most important feature of R112 was noted to be the rapid onset of action. Within 45 minutes, rhinitis symptoms were relieved by using R112, and the duration of action extended to 4 hours. It appears that larger studies to validate the effi cacy of Syk inhibition in the treatment of allergy are in order.

Syk inhibition in immune thrombocytopenic purpura
In patients with immune thrombocytopenic purpura (ITP), there is an accelerated clearance of circulating IgGcoated platelets through Fcγ receptor-bearing macrophages in the spleen and the liver [55]. Syk inhibition should limit platelet destruction in patients with ITP, probably by blocking FcγR signaling. Injection of mice with an antibody directed to integrin αIIb leads to profound thrombocytopenia, which is prevented in mice pretreated with R788 [56]. Also, pretreatment with Syk inhibitors prevented anemia in a mouse model of autoimmune hemolytic anemia (AHA) [56]. At the clinical level, treatment of a small number of patients (n = 12) suff ering from ITP with an R406 led to therapeutic success. Specifi cally, in 8 patients, the clinical response was sustained, whereas in the remaining 4, the response was of limited duration. Obviously, larger studies are needed to determine clinical effi cacy.

Syk inhibitors in intestinal ischemia reperfusion injury
Because hematopoietic cells are involved in the expression of intestinal ischemia-reperfusion injury (IRI), we investigated the ability of R788 to protect mice against IRI [57]. Mice were fed with Syk inhibitor (3 or 5 g/kg day) for 6 days before intestinal IRI was performed. We observed that R788 signifi cantly suppressed both local intestinal and remote lung injury. Th e benefi cial eff ect was associated with reduced IgM and complement 3 deposition to the aff ected tissues and signifi cant reduction of polymorphonuclear cell infi l tra tion. Th e value of this study is that it extends the clinical range of the therapeutic value of Syk inhibitors to conditions involving IRI, such as organ transplant and coronary and carotid revascularization.

Syk signaling in autoinfl ammatory disorders
Recent studies have revealed essential roles for Syk in the infl ammasome production of cytosolic Nlrp3 (NLR family pyrin domain-containing 3) [58,59]. Syk signaling is important for the production of reactive oxygen species and gene transcription factors important in the expression of pro infl am ma tory factors like IL-1β. Pro-IL-1β synthesis is regulated by the Syk-caspase recruitment domain 9 (Syk-Card9) pathway ( Figure 6) [58]. Nlrp3 infl ammasome has been shown to be involved in monosodium urate (MSU)-mediated activation of monocytes [60]. It was reported that the MSU-triggered infl ammatory response requires Nlrp3 and adaptor protein apoptosis-associated speck-like protein contain ing Card [61]. MSU causes strong Syk tyrosine phos phorylation in human neutrophils, which can be suppressed in the presence of piceatannol [62]. Apparently, Syk is required for MSU-mediated activated protein kinase activation and IL-1β production, and Syk recruitment leads to Card9 activation, which controls pro-IL-1β synthesis ( Figure 6) [58,60,61]. Card9 has been known to mediate events downstream of Syk in ITAM-mediated activation [63]. Th ese studies have generated a rationale for the use of Syk inhibitors in the treatment of crystal-induced arthritis and other autoinfl ammatory diseases.

Conclusions and future directions
Syk, initially recognized as a critical signaling molecule in mast cells and lymphocytes, has been documented to be important in the function of additional cells like platelets, monocytes, macrophages, and osteoclasts. As all of these cells are involved in the instigation and establishment of tissue pathology in autoimmune allergic and auto infl ammatory diseases, Syk inhibition has gained signifi cant interest as an important therapeutic tool.
Preclinical evidence argues convincingly that patients suff ering from diseases such as RA, SLE, ITP, and AHA and allergic rhinitis stand a good chance to benefi t from Syk inhibition. Interestingly, reperfusion injury, which follows ischemia in mice, is greatly suppressed by Syk inhibitors, extending the range of diseases with possible clinical benefi t to organ transplantation and revascu larization procedures. Th e clinical experience is limited to patients with RA and ITP. Yet the rapidity of action and the extent of clinical improvement call for further clinical trials.
Obviously, there are serious questions that need attention. Is Syk involved in the function of additional cells? What other kinases or non-kinase molecules are targeted by the available Syk inhibitors? Can medicinal chemistry enable the development of inhibitors that are more specifi c? Th e RA trial noted several, albeit manageable, side eff ects. Do the noted side eff ects hint at additional unrecognized target molecules aff ected by the used Syk inhibitor? Do the side eff ects point to the presence of Syk in additional cells (for example, intestinal epithelial cells). Th e recorded hypertension in patients treated with the Syk inhibitor needs special consideration.
We believe that now that Syk inhibitors have earned a place in the line of drugs to be further developed for clinical use, eff ort should be invested to further understand the mechanism of inhibition of Syk enzymatic activity in an eff ort to derive compounds with increased
specifi city. Th e need to further study cells and processes controlled by Syk is exemplifi ed by a recent report in which a Syk-positive myeloid population of cells stimulates lymphangiogenesis in vivo and disruption of Syk among others is associated with inappropriate homing of leukocytes [64]. Th e RA clinical trial noted a prompt clinical improvement in patients receiving background treatment. Can Syk inhibitors be used in monotherapy? Does prolonged treatment preserve the clinical benefi t, and if so, for how long? Does discontinuance of treatment result in a prompt rebound of disease? Do existent erosions heal? Th e current trend in RA trials remains the parallel administration of biologics in conjunction with MTX to patients who fail MTX. Th is has led to the development of a number of biologics, many of which belong to the same category. For example, several anti-TNF biologics are already available for the treatment of patients with RA. Should Syk inhibitors attain approval for the treatment of RA, an opportunity may arise (provided that the cost is not too high) to try them in tandem with the biologics or as therapeutic adjuvant to biologics. Should trials in patients with SLE, ITP, AHA, or gout be initiated, a similar and probably longer list of questions should be addressed. Th e report on the benefi cial eff ect of Syk inhibition in IRI begs for additional preclinical studies to determine the role of Syk inhibition in organ transplant and other models of IRI, such as muscle, heart, and liver. Abbreviations ACR20, American College of Rheumatology 20% improvement criteria; ACR50, American College of Rheumatology 50% improvement criteria; ACR70, American College of Rheumatology 70% improvement criteria; AHA, autoimmune hemolytic anemia; AHR, airway hyper-responsiveness; BCR, B-cell receptor; Card9, caspase recruitment domain 9; CLEC2, C-type lectin-like receptor 2; DAP12, DNAX-activating protein of molecular mass 12 kDa; FLS, fi broblast-like synoviocyte; GPVI, glycoprotein VI; IL, interleukin; IRI, ischemia-reperfusion injury; ITAM, immunoreceptor tyrosine-based activation motif; ITP, immune thrombocytopenic purpura; JAK, Janus kinase 1; JNK, c-Jun N-terminal kinase; MMP, metalloproteinase; MRI, magnetic resonance imaging; MSU, monosodium urate; MTX, methotrexate; Nlrp3, NLR family pyrin domain-containing 3; PTK, protein tyrosine kinase; RA, rheumatoid arthritis; SH2, SRC homology 2; SLE, systemic lupus erythematosus; SLP76, SH2 domain-containing leukocyte proteins 76; Syk, spleen tyrosine kinase; TCR, T-cell receptor; TNF, tumor necrosis factor; ZAP70, ζ-chain-associated protein kinase of 70-kDa.