Effects of BTK signalling in pathogenic microorganism infections

Abstract As a cytoplasmic protein tyrosine kinase, Bruton's tyrosine kinase (Btk) is widely considered as a vital kinase in many aspects of different physiologic processes. It is engaged in many important signalling pathways related to the immune response, such as the B cell receptor pathway, pattern‐recognition receptor pathway, and triggering receptor expressed on myeloid cell pathway. Recent studies have increasingly focused on the important role of Btk in various inflammatory diseases, which are related to Btk expression in myeloid innate immune cells, such as macrophages, dendritic cells and neutrophils. Although some investigations have explored the role of Btk in microbial infections, many aspects remain elusive, and some of the results are opposite and controversial. Considering the complicated and multiple roles of Btk in the immune system, we summarized the engagement of Btk signalling in various pathogenic microorganism infections, the possible mechanisms involved and its therapeutic potential in the control of infectious diseases.


| INTRODUC TI ON
Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase belonging to the tyrosine kinase expressed in hepatocellular carcinoma (Tec) family of non-receptor tyrosine kinases (TFKs), 1 which also include Tec, interleukin (IL)-2-inducible T cell kinase (Itk), resting lymphocyte kinase (Rlk) (also called T cell-expressed kinase), and bone marrow-expressed kinase (Bmx). In 1952, the phenotype of Btk deficiency was first described by Dr Bruton in a boy who presented with recurrent bacterial infections due to the deficiency in humoural immunity. 2 This severe primary immunodeficiency is named X-linked agammaglobulinaemia (XLA).
In 1993, the causative gene of XLA, Btk, was first identified and isolated. 3 As the unambiguous causative gene of XLA, many extensive and deep studies on the function of Btk have been performed, focusing on B cells. Recently, many researchers have demonstrated or reviewed that Btk also plays important roles in innate immunity 4 and is closely related to inflammatory diseases, such as autoimmune and allergy diseases. 5,6 For example, in rheumatoid arthritis, Btk overexpression occurs, 5 and inhibition of Btk signalling is an effective approach for its treatment. 7 Similarly, Btk is required for FcεRI-mediated activation and histamine release in mast cells and basophils, and the application of Btk inhibitors greatly improves the outcome of allergy diseases. 6,8 Although there are some studies on the roles of Btk signalling in microbial infections, they are still lacking a comprehensible and in-depth summary, and the results are uncertain and even conflicting. Therefore, we present the current knowledge on the effects of Btk signalling in pathogenic microorganism infections, including mainly those caused by viruses, bacteria, fungi and parasites. Furthermore, the mechanism and disputes involved and therapeutic implications are also discussed.

| BTK E XPRE SS I ON AND MUTATI ON S
In humans, the Btk gene is located in the region Xq21.3-22.1, which contains 19 exons and encodes a 76 kD protein with 659 amino acid residues. The Btk protein comprises five different domains, which are the pleckstrin homology (PH), Tec homology, Src homology (SH) 3, SH2 and kinase domain (SH1) from the N terminus to the C terminus. Among them, SH1 is the catalytic domain. 1 Btk is generally expressed in all haematopoietic lineages except for T cells and plasma cells, including B cells and all innate immune cells. 9 Notably, Btk expression in the B cell lineage occurs in a developmental fashion, which shows inconformity during the different stages of B cell development from marrow-derived haematopoietic stem cells to resting mature cells. 10 In addition, some evidence indicates that Btk may also be expressed in solid tumours. For example, data based on cDNA sequencing and gene silencing showed that Btk is also expressed in the colorectal adenocarcinoma cell line HT-29, 11 and a novel isoform of Btk, Btk-C, is considerably overexpressed in tumorigenic breast cells rather than in normal breast cells. 12 In humans, over 800 mutations have been identified to be responsible for the XLA phenotype, including missense, deletion/ insertion, nonsense and splice site mutations. 13 Similarly, the dysfunction of mouse Btk also results in severe X-linked immunodeficiency (Xid) in mice. The mutation in Xid mice, which is described as R28C and obtained from an arginine to a cysteine in the PH domain, resulted in arrested B cell development. 14 Poor induction of inflammatory responses also exists in Xid mice because of its significant roles in many myeloid cells.

| ROLE OF BTK IN IMMUNE CELL S AND ITS S I G NALLING PATHWAYS
In XLA patients, B lymphocytes without intact Btk fail to reach the mature state and eventually suffer premature death. Lacking functional circulating B lymphocytes, individuals cannot generate any immunoglobulins in response to antigenic stimulations to develop an effective humoural immune response. 2,15 Btk dramatically and extensively affects all stages of B cell development, including proliferation, maturation, differentiation, apoptosis and cell migration. 10 Recent studies have increasingly focused on the awareness of Btk roles in other innate immune cells, such as macrophages, dendritic cells (DCs) and neutrophils. 4 Btk deficiency decreases the number of monocytes/macrophages. Moreover, defective Btk signalling suppresses FcγR-mediated cytokine production in monocytes/ macrophages but not phagocytosis. In Btk-deficient mice, DCs are normal in number but defective in antigen presentation and maturation. The population of neutrophils increases significantly in the bone marrow of Xid mice. However, in the absence of Btk, neutrophils are immature, and their functions are impaired. Btk is also required for neutrophil migration, and the expression of the lineage-determining transcription factors and granule proteins are Btk dependent. Furthermore, Btk is a critical gatekeeper of neutrophil responses because reactive oxygen species production is increased after engagement of Toll-like receptors (TLRs) or tumour necrosis factor (TNF) receptors in Btk-deficient neutrophils, which is reversed by the transduction of recombinant Btk.
Lying downstream of BCR, Btk becomes activated by interaction with partner molecules through the PH and SH domains upon activation of BCR with all types of signalling molecules, eventually modifying and maintaining the normal functions of B cells. 16 Upon BCR activation, immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasm are phosphorylated by Src-family protein tyrosine kinases (such as Lyn) and spleen tyrosine kinase (Syk). 17 At steady-state, Btk is non-phosphorylated, and after BCR activation, it is phosphorylated at Tyr551 in the SH1 domain by Syk or Lyn, followed by autophosphorylation at Tyr223 in the SH3 domain. 18 Meanwhile, Syk facilitates the recruitment and activation of phosphatidylinositol 3-kinase (PI3K) through phosphorylation of B cell adaptor for PI3K (BCAP), an adaptor protein that interacts with the B cell co-receptor CD19. PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5bisphosphate (PIP3), which recruits Btk to the plasma membrane through linking with the Btk PH domain. 19 In addition, Syk phosphorylates B cell linker protein (BLNK). In connection with the adapter BLNK, Btk triggers the downstream signalling pathway for calcium release by subsequent phospholipase Cγ2 (PLCγ2) phosphorylation. 20 Upon the activation of PLCγ2, PIP2 is hydrolysed to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 activates the transcription of nuclear factor of activated T cells (NFAT) by regulating intracellular calcium levels. DAG mediates the activation of protein kinase Cβ (PKCβ), which eventually induces the activation of many key proteins in cellular physiological processes, such as extracellular signal-regulated kinases 1 and 2 (Erk1/2), Jun N-terminal kinase, p38 and nuclear factor кB (NF-кB) pathway components ( Figure 1A). In addition, evidence has shown that Btk is involved in both activating and inhibitory FcR signalling pathways. Similar to the BCR signalling pathway, the Syk-Btk pathway is also activated following the crosslinking of activated FcRs. However, cross-linking of inhibitory FcR (FcγRIIB) and activating receptors such as BCR inhibits the recruitment of Btk, leading to reduced Btk activation 21 ( Figure 1A). Most TLR signalling pathways except that of TLR3 involve a common and vital protein named myeloid differentiation primary response 88 (MyD88) to maintain recognition function. Upon the activation of those TLRs, MyD88 recruits and transmits the signals to downstream molecules, such as IL-1 receptor-associated kinase 1 (IRAK1) and MyD88 adapter-like (MAL). Btk also cooperates with proteins Toll-IL-1 receptor (TIR), MyD88, IRAK1 and MAL to induce activation of a set of transcription factors, including NF-кB, activator protein-1 (AP-1) and interferon regulatory factor 3 (IRF3), suggesting that Btk plays a central role in both innate and adaptive immunities. 22 However, the TLR3 signalling pathway is MyD88-independent, in which Btk phosphorylates TLR3 directly and eventually induces the activation of many transcription factors, such as TLR signalling 23 ( Figure 1B). In addition, Btk is also involved in the NOD-like receptor (NLR), another major family of PRRs, signalling pathway. Being an NLR, NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) plays a crucial role in inflammation. Btk is required for IL-1β release regulated by the NLRP3 inflammasome in macrophages. 24 In-depth research shows that Btk hinges NLRP3 with adaptor apoptosis-associated speck-like protein containing a CARD (ASC) to form an integral functional complex, leading to the oligomerization of ASC and activation of caspase-1, which produces bioactive IL-1β and IL-18 25 ( Figure 1C).
Bruton's tyrosine kinase is also involved in the TREM-1 signalling pathway. Normally, Btk is phosphorylated after TREM-1 stimulation. However, when the expression of Btk is diminished, Ca 2+ mobilization and phosphorylation of Erk1/2 and PLCγ1 are reduced upon TREM-1 triggering. Meanwhile, the production of TREM-1-induced pro-inflammatory cytokines and chemokines is impaired. Therefore, Btk is a positive and essential regulator in TREM-1 signalling. 26 F I G U R E 1 Schematic outline of major Btk signalling cascades. A, In the BCR pathway, upon the activation of BCR, Syk activates PI3K via phosphorylation of BCAP. Then, PI3K phosphorylates PIP2 to generate PIP3, which recruits Btk to the plasma membrane. Btk is phosphorylated by Syk and regulates downstream signalling pathways, such as those of NFAT, Erk1/2, JNK, p38 MAPK and NF-κB. Similar to the BCR pathway, Btk is also involved in FcR pathways, including activating FcRs and inhibitory FcRs (FcγRIIB). B, In the MyD88dependent TLR pathway, after triggering TLRs, Btk cooperates with many proteins, such as MyD88, MAL and IRAKs, and eventually induces transcription factors, including NF-кB, AP-1 and IRF3. In addition, Btk can also phosphorylate TLR3 directly. C, In the activation of the inflammasome, Btk hinges NLRP3 with its adaptor ASC to form the functional NLRP3 inflammasome, leading to the activation of caspase-1, which produces bioactive IL-1β and IL-18. See text for details

| Effect of Btk in viral infections
Since Epstein-Barr virus (EBV) was discovered in African Burkitt lymphoma in 1964, it has been remarkably identified as an oncogenic virus of B cell lymphoma because of its ability to transform resting B cells. As EBV immediate-early genes, BZLF1 and BRLF1 transcription is initiated from a proximal promoter named Zp, whose activation requires intact BCR-Syk-Btk pathways. 27 Moreover, the signalling of latent membrane protein 2A

| Effect of Btk in fungal infections
In general, fungal infections are considered opportunistic infections in immunocompromised patients, which are life-threatening even with optimal medical therapy. In response to fungal spores, endogenous reactive oxygen in macrophages is produced, leading to the rapid phosphorylation of Btk. 51 54 Another study also illustrated that Btk activation in response to Aspergillus fumigatus participates in phagocytosis through the TLR9-Btk-NFAT pathway. 55 By contrast, Btk deficiency plays protective roles against intestinal colonization by C albicans because decreased infiltrating macrophage numbers and elevated pro-inflammatory cytokine expression are observed in dextran sodium sulphate (DSS)-induced Xid mice colitis, suggesting that Btk inhibition combined with C albicans colonization can be a possible therapy for the treatment of inflammatory bowel diseases. 56 Thus, we suppose that during the crosstalk between macrophages and fungi, Btk is phosphorylated rapidly to participate in the formation of phagosomes or regulate the secretion of inflammatory mediators.

| Effect of Btk in parasitic infections
Single-agent treatment with ibrutinib increases the risk of atypical Pneumocystis jirovecii pneumonia in CLL patients. 57 Other pub- has also been investigated by scientists using knockout mice and a specific kinase inhibitor to obtain contrary outcome in ConA-induced hepatitis. 65 Every domain of Btk has a site for specific molecules to participate in various signalling pathways related to different physiological activities, which makes Btk function complex and variable.
Meanwhile, Btk, Tec and Itk partially overlap not only in expression patterns but also in functions, 66 and Tec has been reported to partially compensate for Btk functions in mice. 10  HIV HIV-1-infected cells LFM-A13, Ibrutinib Btk was up-regulated in HIV-1-infected cells, and antibody treatment, inhibitors and Btk knockdown by siRNA showed anti-HIV effects.

IAV IAV-infected mice Ibrutinib
The Btk inhibitor has a protective effect in IAVinduced acute pneumonia.

Staphylococcus aureus Staphylococcus aureus infection in vivo and in vitro Ibrutinib
Βtk could be a potential drug-target for the treatment of NLRP3 inflammasome-linked inflammation.

Streptococcus pneumoniae Streptococcus pneumoniaeinfected mice
Ibrutinib Ibrutinib has the potential to protect against pneumonia.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Both Bingjue Ye and Cheng Zhou drafted the manuscript, Huiting Guo supplemented the manuscript. Min Zheng reviewed the manuscript and provided revisions. All authors reviewed the manuscript.