The Role of Bruton’s Tyrosine Kinase in Immune Cell Signaling and Systemic Autoimmunity

Abstract

Bruton’s tyrosine kinase (BTK) is an intracellular signaling molecule first identified as the molecule affected in X-linked agammaglobulinemia (XLA) patients, who almost completely lack peripheral B cells and serum immunoglobulins. BTK is crucial for B cell development and various B cell functions, including cytokine and natural antibody production. Importantly, it is also expressed in numerous other cells, including monocytes, macrophages, granulocytes, dendritic cells, and osteoclasts. A few rare cases of autoimmune disease in XLA patients have been described. Interestingly, increased BTK protein expression in patients with systemic autoimmune disease appears to be correlated with autoantibody production. In addition, BTK may promote autoimmunity as an important driver of an imbalance in B–T cell interaction. Because of this overwhelming evidence of a pathogenic role of BTK in autoimmunity, several clinical trials in rheumatoid arthritis and systemic lupus erythematosus patients with BTK inhibitors are currently running. Here, we review BTK function in different signaling pathways and in different cell lineages, focusing on the growing body of literature indicating a critical role for BTK in autoimmunity. We also discuss BTK and the promising results of BTK inhibition in animal models of autoimmune disease.

Introduction

Systemic autoimmunity is a condition characterized by an aberrant immune response against self-antigens. Rheumatoid arthritis (RA), Sjögren’s syndrome (SjS), inflammatory bowel disease (IBD), and diabetes mellitus type I (T1D) are among the most common autoimmune diseases. Approximately 23.5 million people in the United States have an autoimmune disorder, and the incidence is slowly rising, with an estimated 7.6–9.4% of people suffering from autoimmunity worldwide. Autoimmune diseases are chronic and, although symptoms can be suppressed by medication, they cannot be cured, placing a heavy burden on society. Understanding the pathogenesis of these diseases is essential to improving current treatment strategies and to identify new therapeutic targets.

A breakdown of B cell tolerance, resulting in autoantibody production, is a major pathogenic event in systemic autoimmunity, and B cell depletion therapy was shown to be effective in several autoimmune diseases. The importance of B cell receptor (BCR) signaling in this process is underlined by genetic studies showing that B cell intrinsic effects are central to autoimmune disease development. Among the risk loci identified by genetic studies are genes that encode intracellular signaling molecules downstream of the BCR or molecules that regulate BCR signaling, including BLK, Lyn, BANK1, and PTPN22.

A crucial signaling molecule downstream of the BCR is Bruton’s tyrosine kinase (BTK). Located on the X chromosome, BTK was first identified as the gene mutated in the primary immunodeficiency X-linked agammaglobulinemia (XLA). In XLA patients, the absence of functional BTK protein in B cells leads to an almost complete arrest in early B cell development in the bone marrow at the pre-B cell stage. As a consequence, almost no circulating B cells are present and antibody levels are very low in the serum of these patients, who suffer from recurring infections from an early age. BTK also plays an important role in BCR signaling in mature peripheral B cells. Mouse studies have shown that BTK is crucial for peripheral B cell differentiation and survival, as well as for their activation upon BCR ligation.

Furthermore, BTK inhibition completely blocks BCR signaling in human primary B cells. Increases in BTK expression or activation have been shown in chronic lymphocytic leukemia (CLL) patients, and treatment with BTK inhibitors is very effective in this disease. Increasing evidence shows that dysregulation of BTK may also be involved in systemic autoimmunity. Increased expression of BTK in B cells in transgenic mice induces a spontaneous autoimmune phenotype, and in RA and SjS patients, BTK levels in peripheral B cells are increased. These findings make BTK an interesting new target in the treatment of autoimmunity.

In this review, we discuss BTK function as a critical tyrosine kinase downstream of the BCR, as well as its involvement in other signaling pathways in various cell lineages of the immune system. We highlight the evidence for involvement of BTK in autoimmune disorders and discuss opportunities for BTK targeting as a therapeutic strategy.

BTK Protein and BCR Signaling

BTK Protein Structure and Its Role in B Cell Development

BTK is a protein kinase consisting of 659 amino acids. It belongs to the strongly conserved family of TEC non-receptor kinases, which includes four additional members: tyrosine kinase expressed in hepatocellular carcinoma (TEC), inducible T cell kinase (Itk), bone marrow-expressed kinase (Bmx), and resting lymphocyte kinase (Rlk). BTK is most similar to Itk and TEC, containing five different domains: a plekstrin homology (PH), TEC homology (TH), and Src homology (SH) 2 and 3 and kinase domain. BTK is expressed in all hematopoietic cells except for T cells and is shown to be especially crucial for B lymphocytes.

Signaling via the BCR is vital for B cell survival during B cell development because BCR affinity—and thereby signaling strength—determines B cell selection and survival. As mentioned above, XLA patients with mutations in the BTK gene show a severe deficiency of peripheral B cells and immunoglobulins due to an arrest of B cell development around the large pre-B cell stage. The CBA/N mouse strain harbors a loss-of-function mutation in the Btk gene. These BTK-mutant mice are known as Xid (X-linked immunodeficiency) mice and do not appear to have a detectable defect in bone marrow B cell development. Rather, these mice show an impaired differentiation of mature peripheral B cells characterized by an approximate 50 percent reduction in total splenic B cells. There is a severe decrease in long-lived IgM low IgD high mature B cells in these mice, illustrating that BTK signaling is crucial for maturation and maintenance of mature peripheral B cells.

The transitional block from IgM positive IgD negative immature B cells to IgM low IgD high mature B cells in BTK-deficient mice is in parallel with findings in XLA patients, in whom residual B cells are also IgM high IgD low. In addition, Xid mice lack the population of B-1 cells in the peritoneal and pleural cavities. Because this cell population is important for the production of natural antibodies and is involved in T cell-independent B cell responses, Xid mice have severely reduced IgM and IgG3 levels in the serum. Consistent with the finding that B cells from Xid mice still have the capacity to form germinal centers, T cell dependent immune responses to model antigens appear normal and serum levels of Ig isotypes other than IgM and IgG3 are unaffected.

Next to naturally occurring mutations in BTK as seen in Xid mice, several groups created C57BL/6 mice with a targeted deletion of the Btk gene. These BTK-deficient mice show impaired differentiation from large pre-B to small pre-B cells in the bone marrow and have a reduced capacity to open the Ig lambda (λ) light chain locus for recombination and therefore show reduced λ light chain usage. However, the effect on B cell development is less severe than seen in XLA patients because BTK-deficient mice present with only a partial block at the pre-B cell stage. Like Xid CBA/N mice, also BTK-deficient C57BL/6 mice have peripheral B cells that show severe defects in their differentiation, survival, and activating capacity. In addition, neither Xid nor BTK-deficient B cells are able to induce cyclin D2 expression upon BCR engagement and as a result they do not reach the S-phase of the cell cycle and do not proliferate.

As mentioned earlier, BTK-deficient mice show only a partial block in B cell development at the large to small pre-B cell transition. However, when BTK is knocked out in combination with either TEC kinase or the SH2 domain leukocyte protein of 65 kD (SLP-65, also known as BLNK), this results in an almost complete arrest at the pre-B cell stage. In addition, BTK-deficient B-lineage cells are outcompeted by BTK-sufficient B cells in heterozygous BTK plus/minus female mice because a significant disadvantage was observed for BTK-deficient cells at the transition from pre-B to the IgM high IgD negative immature B cell stage in the bone marrow. This disadvantage was virtually complete in mature splenic or blood B cells in mice, consistent with findings in the peripheral blood B cell compartment in carrier mothers of XLA patients.

In summary, these findings in humans and mice demonstrate that the presence of BTK in the context of BCR-mediated survival signals is crucial for normal B cell development and maturation.

BTK Activation and the IgM-BCR Signaling Cascade

BTK is located in the cytoplasm in resting B cells but can be recruited to the cell membrane after BCR stimulation. Upon triggering of the surface IgM BCR, the immunoreceptor tyrosine-based activation motifs (ITAMs) of the BCR co-receptors CD79a and CD79b are phosphorylated by Lyn, resulting in the recruitment and activation of Syk. Simultaneously with BCR activation, Lyn will phosphorylate the cytoplasmic tail of the co-receptor CD19, which results in recruitment and activation of phosphoinositide 3-kinase (PI3K). PI3K can also be activated by B cell adaptor for PI3K (BCAP), illustrating that BCR signaling can target PI3K activity independent of CD19. PI3K generates PIP3, which is essential in recruiting BTK to the cell membrane by interacting with the BTK PH domain. Upon recruitment, activated Syk and Lyn can fully activate SLP-65. This results in phosphorylation of BTK in the kinase domain at tyrosine 551, which subsequently leads to full activation of BTK and auto-phosphorylation at tyrosine 223 in the SH2 domain. Upon full activation and with SLP-65 as a linker protein, BTK can activate phospholipase C gamma 2 (PLCγ2), leading to the activation of several downstream signaling pathways.

Class-Switched BCR Signaling

In contrast to IgM BCR signaling, the kinetics and signaling cascade of class-switched BCRs are slightly different. In IgG and IgE signaling, the heavy chain domains contain longer cytoplasmic tails compared to IgM and IgD BCRs. These cytoplasmic tails contain phosphorylation motifs called immunoglobulin tail tyrosine (ITT) motifs, which can recruit adaptor protein Growth factor receptor-bound protein 2 (Grb2) via their SH2 domains after ITT phosphorylation by Syk. ITT-bound Grb2 binds BTK, thereby amplifying the response triggered by IgG engagement. Using ITT-mutant IgG1 B cells, it was shown that the ITT domain is crucial for a competitive advantage of IgG-switched B cells in vivo, providing an explanation for the requirement of enhanced signaling specifically in switched-memory B cells. In contrast to the signaling domains of IgG and IgE, no studies have been reported on IgA-mediated BCR signaling, so the intracellular signaling mechanism remains to be elucidated.

Calcium Mobilization

Upon BCR-mediated activation of PLCγ2, PIP2 can be cleaved into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 can bind to the IP3 receptor (IP3R), facilitating the influx of calcium into the cytoplasm. This activates signaling via calmodulin, which subsequently binds and activates the phosphatase calcineurin (CaN). Activated CaN dephosphorylates nuclear factor of activated T cells (NFAT), leading to a conformational change that reveals NFAT’s nuclear localization signal and allows its translocation into the nucleus. In the nucleus, NFAT functions as a transcription factor that regulates gene expression required for immune responses.

Calcium Mobilization

Upon BCR-mediated activation of PLCγ2, phosphatidylinositol bisphosphate (PIP2) is cleaved into inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 can bind to the IP3 receptor (IP3R), which facilitates the influx of calcium into the cytoplasm. This rise in cytoplasmic calcium activates signaling through calmodulin (CaM), which subsequently binds and activates the phosphatase calcineurin (CaN). Activated calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT), resulting in a conformational change that exposes the nuclear localization signal and allows NFAT to translocate into the nucleus. In the nucleus, NFAT acts as a transcription factor, regulating the expression of genes required for immune responses.

Cytoskeletal Rearrangements

Besides the activation of transcription factors, BCR triggering leads to dynamic cytoskeletal rearrangements that are critical for B cell spreading, immune synapse formation, and antigen internalization. BCR-induced signals promote the nucleation of new actin filaments through downstream activation of signaling proteins such as WASp and actin-regulatory complexes. The proper organization of the cytoskeleton is essential for efficient antigen processing and presentation by B cells.

AKT-Mediated Signaling

One of the important downstream events following BCR ligation and BTK activation is the recruitment and phosphorylation of AKT, also known as protein kinase B (PKB). Upon activation, AKT promotes cell survival and growth by phosphorylating several substrates that inhibit apoptotic pathways and enhance glucose metabolism. Activation of AKT depends on the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) by PI3K upon BCR engagement. BTK is recruited to PIP3 at the plasma membrane, contributing to the full activation of AKT signaling.

MAPK Pathway Activation

In addition to the above pathways, BTK plays a role in the activation of mitogen-activated protein kinases (MAPKs). Following BCR engagement, a cascade of phosphorylations leads to the activation of ERK, JNK, and p38 MAP kinases. This provides an additional layer of regulation for gene expression, cell proliferation, and differentiation necessary for proper immune responses.

NF-κB Pathway

A central role of BTK following BCR signaling is the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. BTK, through the phosphorylation and activation of downstream substrates like PLCγ2, ultimately leads to the activation of the IκB kinase (IKK) complex. The IKK complex phosphorylates inhibitory IκB proteins bound to NF-κB, resulting in their ubiquitination and proteasomal degradation. Consequently, NF-κB translocates into the nucleus, where it regulates the expression of survival, proliferation, and inflammatory genes.

BTK in Other Immune Cells

Although BTK is most critical for B lymphocyte development and activation, it is also expressed in other hematopoietic cells, including myeloid lineage cells such as monocytes, macrophages, dendritic cells, granulocytes, and osteoclasts. In these cells, BTK is involved in a variety of signaling pathways, especially those mediated by Fc receptors and Toll-like receptors (TLRs).

BTK and Myeloid Cell Signaling

In monocytes and macrophages, BTK contributes to signal transduction via immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptors. Upon immune complex binding, Fc receptors become phosphorylated and initiate downstream signaling involving Syk, PLCγ2, and BTK, ultimately leading to cytokine production and phagocytosis. BTK also participates in TLR signaling by modulating the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. These signals play important roles in both defense against pathogens and the pathogenesis of autoimmune inflammation.

BTK in Dendritic Cells

Dendritic cells express BTK and utilize it in the integration of danger signals through TLRs and other pattern recognition receptors. TLR engagement leads to the recruitment of BTK, which in turn can promote the activation of downstream pathways including NF-κB and MAPKs. This enhances the expression of co-stimulatory molecules, the secretion of inflammatory cytokines, and ultimately the ability of dendritic cells to prime T cell responses.

BTK in Osteoclasts

BTK is also expressed in osteoclasts, where it plays a role in receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated differentiation and bone resorption. BTK deficiency impairs osteoclast formation and function, which can impact bone metabolism and may have implications for inflammatory bone loss observed during autoimmune diseases such as rheumatoid arthritis.

Role of BTK in Systemic Autoimmunity

Multiple lines of evidence point to a central role for BTK in the pathogenesis of systemic autoimmunity. Genetic studies in humans have identified BTK as a susceptibility gene for autoimmune conditions. Increased BTK expression and activation have been detected in B cells from patients with autoimmune diseases such as rheumatoid arthritis and Sjögren’s syndrome. In animal models, overexpression of BTK in B cells leads to spontaneous autoantibody production and the development of autoimmune phenotypes.

Breakdown of B Cell Tolerance

The development of autoantibodies is a hallmark of many autoimmune diseases. BTK is key in regulating both central and peripheral B cell tolerance checkpoints. Defective BTK signaling can disrupt the negative selection of autoreactive B cells in the bone marrow and compromise anergy induction in the periphery, leading to the survival and activation of self-reactive clones. These autoreactive B cells contribute to ongoing inflammation and tissue damage upon differentiation into antibody-secreting cells.

Imbalance of B and T Cell Interactions

BTK plays a role in regulating the cross-talk between B and T cells. Dysregulated BTK activity can enhance the capacity of B cells to present antigen and provide co-stimulatory signals to T cells, potentially breaking self-tolerance. The imbalance in B–T cell interactions is thought to be a driver of systemic autoimmunity.

BTK as a Therapeutic Target

Given its central role in multiple aspects of immune cell signaling and autoantibody production, BTK represents an attractive therapeutic target in autoimmune diseases. Small-molecule BTK inhibitors have shown efficacy in preclinical animal models of systemic lupus erythematosus, rheumatoid arthritis, and other conditions. These inhibitors suppress B cell activation and reduce inflammatory cytokine production in myeloid cells. Clinical trials of BTK inhibitors in humans with systemic autoimmune disorders are ongoing, and preliminary results suggest beneficial effects on disease activity.

Concluding Remarks

In summary, Bruton’s tyrosine kinase is a critical intracellular signaling molecule in B cells and other hematopoietic lineages. Defects in BTK lead to profound immunodeficiency, while overactivation or increased expression is associated with the development of systemic autoimmunity. BTK integrates signals from various receptor pathways, orchestrating cell survival, activation, and effector responses. Targeting BTK with selective inhibitors offers a promising approach for the treatment of autoimmune diseases driven by dysregulated B cell and innate immune cell function. Future research will further define the therapeutic potential GSK’963 of BTK inhibition in human autoimmunity.