Beyond B cells

BTK’s roles within cells are both diverse and numerous. Aside from B cells, the kinase is found to be highly expressed in mast cells, macrophages and dendritic cells; immune cells are found to be involved in the elimination of pathogens [25]. In addition to B cells, BTK’s function in T cells has also been explored. Nowadays, BTK is considered a vital protein expressed by immunocompetent cells of both innate and adaptive immunity (Figure 3).

Overview of the various roles of Bruton's tyrosine kinase (BTK) in innate immunity. Chemokine receptor (CXCR4) activation upon chemokine binding leads to the dissociation of G proteins made up of Gα, Gβ and Gy subunits and downstream activation of BTK. ITAM-containing (and also ITIM-containing) Fc receptor crosslinking leads to activation of SYK and in turn BTK. Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns (PAMPs) and-damage associated molecular patterns (DAMPs). Activation of TLRs is followed by recruitment of MYD88. BTK in turn interacts with MYD88 leading to activation of transcription factors such as NF-кB. BTK is a direct regulator in the activation of the NLRP3 inflammasome. Efflux of K+ into the cell leads to phosphorylation of BTK, most likely by SYK, followed by activation. This phosphorylation promotes assembly of the inflammasome and leads to the cleavage and secretion of IL-1β [103] T Lymphocytes

BTK inhibition in vivo has been reported to increase the persistence of activated T cells, decrease the Treg /CD4+ T-cell ratio and diminish the immune-suppressive properties of CLL cells via BTK-dependent and BTK-independent mechanisms [26]. Characterization of T-cell compartment in CLL patients upon ibrutinib therapy showed elevated CD4+ and CD8+ T-cell numbers and T-related cytokine levels upon therapy [27]. Ibrutinib has been shown to enhance the antitumor properties of the T-cell compartment suggesting a rationale for immunotherapeutic combinatory treatments [28]. The immunomodulatory effects of ibrutinib and the therapeutic potential of its combination with immune checkpoint inhibitors were also highlighted in a recent study, in which ibrutinib, together with blocking antibodies targeting PD-1/PD-L1 axis, improved CD8+ T-cell effector function and control of CLL [29].

Cells of myeloid origin

Macrophages, originating from monocytes, function in pathogen detection and phagocytosis and together with dendritic cells play a special role linking the innate and adaptive immune systems through antigen presentation [30]. Ren et al. showed that although BTK inhibition with ibrutinib did not affect monocyte FcγR-mediated phagocytosis, it did suppress FcγR-mediated cytokine production. This effect could be rescued by IFN priming when monocytes were co-cultured in vitro with NK cells, suggesting that combining ibrutinib with monoclonal antibody therapy could enhance tumour killing without affecting macrophage effector function [31]. Interestingly, an immunomodulatory action of BTK inhibition on monocyte/macrophage population has been reported in CLL. Specifically, Ibrutinib targets BTK in nurse-like cells (NLCs), which leads to reduced phagocytic ability and enhanced immunosuppression related to NLCs’ expression of M2 markers [32]. Additional evidence for the role of BTK in cells of myeloid origin arose from transcriptomic analysis of XLA-derived monocytes, which revealed downregulation of several innate immunity genes in parallel with upregulation of oxidative phosphorylation and apoptotic pathways. These findings suggest that BTK mutations may significantly impair the innate axis of immunity and indicate a vital role of BTK in innate immune responses [33]. However, another study reported no impact of BTK mutation on monocytes and PMN functions in XLA [34]. Further investigation of the role of BTK in monocytes is needed to provide a better understanding of the events occurring in these studies.

Neutrophils are yet another immune cell type in which BTK has been shown to play a key role [35]. For instance, a neutrophil-BTK-signalosome was reported to selectively activate Mac-1 and thereby enhance neutrophil recruitment during inflammation [35]. In CLL, BTK inhibition suppresses FcγR-mediated neutrophil functions during the early phase of treatment and potentially in a clinically relevant way. The reported short-term neutrophil impairment upon ibrutinib treatment could translate to additional infection risk for CLL patients under ibrutinib [36]. Furthermore, ibrutinib has been reported to inhibit γδT-cell activation and CD107a degranulation and affect neutrophils by reducing NET formation, ROS production and bacteria-killing capacity [37].

Mast cells have been investigated in association with BTK. Zorn et al. [38] reported that BTK plays a role in FcεRI-mediated signal transduction and effector functions in SHIP1-deficient mast cells and that reduced activation can be tackled with BTK inhibitors.

Inflammasomes

The NLRP3 inflammasome is an essential inflammatory complex for various human disorders linked to the activity of IL-1 cytokine. The identification of BTK as a positive regulator, directly acting on the NLRP3 inflammasome, has further shown the significant role this kinase plays in immunity [39]. BTK binds to the caspase recruitment domain (ASC) of NLRP3, ASC oligomerization is induced, and caspase-1 is thereby activated. Pharmacologic ablation and genetic BTK ablation severely diminish NLRP3 activation suggesting a therapeutic opportunity for inflammatory conditions [40]. Additionally, BTK is required for NLRP3 tyrosine phosphorylation and IL-1β release. The BTK-mediated phosphorylation of multiple NLRP3 tyrosine residues can serve as a molecular switch, which could be therapeutically exploited [41].

BTK in infections

The multifaceted role of BTK in immunity, as summarized above, underscores its importance for a wide variety of immune functions, including responses against pathogens. A large body of evidence offers insights into the role of BTK signalling in fungal, bacterial and viral infections, including the SARS-CoV-2 virus and the COVID-19 pandemic.

Fungal infections

Activation of calcineurin–NFAT in macrophages occurs via a phagocytic TLR9-dependent and BTK-dependent pathway in the context of Aspergillus fumigatus infection. Calcineurin inhibition leads to impaired pathogen clearance in the airway due to diminished macrophage inflammatory responses and neutrophil recruitment. In line with that, defecting BTK signalling in macrophages has been associated with susceptibility to pulmonary aspergillosis [42], whereas BTK depletion significantly impaired human macrophage NFAT and NF-κB responses [43]. Furthermore, ibrutinib treatment has been linked to a high incidence of invasive aspergillosis in lymphoma [44].

Apart from macrophages, neutrophils are part of the first line of defence against fungal infections. Patients on ibrutinib due to lymphoid malignancies are characterized by significant functional defects in their neutrophil compartment that impair their response against A. fumigatus [45], whereas data from CLL patients on ibrutinib indicate an increased risk of Pneumocystis jirovecii pneumonia [46].

Bacterial and parasitic infections

Ibrutinib induces changes in gene expression and phenotype in macrophages and severely impairs the macrophage and γδT-cell responses to Mycobacterium tuberculosis [47]. Ibrutinib has been reported to have the potential to inhibit inflammation caused by bacterial infection. Reports have shown that ibrutinib can inhibit the acute lung inflammation associated with pneumococcal pneumonia infections. BTK inhibition through Ibrutinib diminishes myeloid cell activation and migration during lung inflammation and has been identified as a possible therapy for resolving acute lung inflammation during pneumococcal pneumonia [48].

By employing a murine platelet-specific BTK-deficient pneumosepsis model (PF4creBtkfl/Y), Streptococcus pneumoniae and Klebsiella pneumoniae infections were investigated, revealing a role of BTK in maintaining vascular integrity in the lung. However, this mechanism is pathogen-dependent and the platelet BTK is not crucial for antibacterial defence in pneumosepsis [49]. Recently, Nguyen et al reported on another aspect of K. pneumoniae infection using in vivo and in vitro models to show that SKAP2-dependent signalling in neutrophils is important for ROS activation and promotion of bacterial clearance during infection. Interestingly, among the key molecules that were identified for K. pneumoniae-induced neutrophil ROS response was BTK [50].

BTK inhibition has been proposed to confer protection against Leishmania infection via promoting host immunity. In a mouse model for visceral leishmaniasis, ibrutinib treatment was shown to have a protective effect via increasing cytokines’ production, NK T cells’ number in the liver and spleen and granuloma formation [51].

Viral infections

Studies have described in-depth the crucial role of BTK expressed by innate cells in viral infections [25]. In macrophages, TLRs recognize single-stranded RNA from viruses and initiate signalling through BTK-dependent activation of NF-κB, triggering the production of multiple inflammatory cytokines and chemokines, as well as phagocytosis [52]. The latest experimental evidence indicates that BTK is involved in Influenza A virus (IAV) infection. Specifically, it was found that BTK expressed in neutrophils plays a substantial role in regulating inflammation in the respiratory region during acute lung injury in mice. Inhibition of the kinase activity reduced weight loss, increased survival and minimized morphological changes in IAV infection showing a protective effect in the lung during influenza-induced inflammation [53].

COVID-19 pandemic

With the recent emergence of the novel coronavirus, COVID-19, the need for new therapeutic targets has surged as the severity of the pandemic increases. In severe cases of COVID-19, high levels of activation of macrophages have been identified as a cause of the hyperinflammatory immune response seen in these patients. As previously mentioned, BTK regulates the activity of macrophages, prompting the concept that the inhibition of BTK could be used as a therapeutic option for COVID-19 patients. So far, preliminary data have shown promising results. Based on this knowledge of the role of BTK in innate immune cells, COVID-19 patients exhibiting increasing oxygen requirements and hyperinflammation were treated with the second-generation BTKi acalabrutinib. The majority of patients’ conditions rapidly improved upon treatment with increased oxygenation and reduced inflammation. The patients from this study exhibited significantly elevated BTK phosphorylation in monocytes indicating the improved conditions were an on-target effect of BTK inhibition [54].

From another point of view, we must be wary of the fact that BTK inhibition impairs various functions of the innate immunity and increases the susceptibility to infections or impaired humoral immunity in patients on BTKi. Awareness of these issues during the current COVID-19 pandemic is essential as weakened immune states increase susceptibility to infection. However, following the rationale stated by Chong et al., the risk of depriving cancer patients of treatment and dulling the hyperactive macrophage outweighs the potential dampened immune response. Furthermore, long-term BTKi therapy may allow for meaningful recovery of humoral immune function, ultimately leading to decreased infection rates [55] and potentially protecting against lung injury in COVID-19 patients [56].

Taking into account the full effect of BTK inhibition in the setting of treating COVID-19-infected B-cell lymphoma patients, a recent controversial debate in the discontinuation of BTK inhibitors to those patients has been brought to attention [57]. Two pilot studies published the clinical characteristics and progress of 6 CLL and 8 WM patients, respectively, with COVID-19 infection who continued or held the BTKi therapy. The authors of both studies suggested that BTK inhibition may indeed have protective effects against SARS-CoV-2 virulence. The small cohort of patients evaluated is a limitation to support the therapeutic approach of BTKi, but ideally, two clinical trials assessing the effect of second-generation BTK inhibitors in hospitalized COVID-19 patients will shed light on that setting [58, 59].

BTK in autoimmune diseases

Since its discovery in XLA, the link of BTK with autoimmune phenomena is strengthened by numerous studies in various autoimmune diseases and mechanistic insights concerning BTK’s contribution in driving autoimmune pathogenesis. For instance, a driving factor for autoimmunity is that increased levels of BTK support autoantibody production [60].

Systemic lupus erythematous

Systemic lupus erythematosus (SLE) is characterized by the secretion of autoantibodies. These autoreactive B cells exhibit increased levels of BTK expression [61]. Murine studies suggest that inhibition of BTK can control the autoreactive B cells with potential therapeutic implications [62]. In line with that, the covalent BTK inhibitor, evobrutinib, has shown efficacy in murine models for SLE, rheumatoid arthritis and cutaneous anaphylaxis. In vivo studies have demonstrated evobrutinib to be potent and highly selective for BTK, showing high BTK occupancy. Current clinical trials are ongoing for its efficacy in the treatment of SLE, among other autoimmune diseases [63, 64].

Rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by autoantibodies causing chronic inflammation and joint pain [65]. Interest in BCR-targeted therapies has been increasing recently, especially BTK due to its role in controlling the Fcγ receptor downstream pathway among others.

Jansson et al. reported a gene on the murine X chromosome linked to susceptibility for developing arthritis. XID mouse models harbouring BTK mutations showed that in the absence of BTK, there are lower chances of developing arthritis [66]. From this development, many BTK inhibitors have been produced and investigated for their use in RA. For instance, the reversible BTK inhibitor 7H-pyrrolo[2,3-d] pyrimidine-4-amine derivative has been shown to have an anti-arthritic effect. Despite initial promising in vivo results, a select few have seen successful developments and been moved onto clinical trials [67].

Currently in phase II clinical trials, branebrutinib is an irreversible, covalent BTK inhibitor that has been reported to be highly selective and efficient even in low doses. Branebrutinib has demonstrated a high BTK occupancy, and it has been suggested this inhibitor would be effective for the treatment of autoimmune diseases, in particular RA and SLE [68, 69]. Fenebrutinib, a reversible and non-covalent BTK inhibitor, has previously been shown to be effective in patients with CLL and is now under investigation for its efficacy in RA and SLE [70].

Pemphigus vulgaris

The rare chronic autoimmune disease pemphigus vulgaris (PV) is identified by characteristic blistering on the skin and mucous membranes. The pathogenesis of PV involves the IgG autoantibodies attacking the desmoglein 3 glycoprotein within the desmosome [71]. T-follicular helper cells have been reported to have a role in PV pathogenesis. Increased BTK expression has been shown to induce the differentiation of these T cells [72]. Studies have shown the use of BTK inhibitors in cases of PV patients who also presented with B-cell lymphomas showed promising results for both diseases. From here, clinical trials of the BTK inhibitor PRN1008 are underway for PV therapy [73]. Studies have been looking to target the BCR-identified BTK as a potential target for PV therapies [74].

Multiple sclerosis

Recent work has highlighted the potential of blocking BTK activity as a therapeutic option to improve anti-CD20 therapies such as rituximab for multiple sclerosis (MS) patients. Trials using a BTK inhibitor with a brain-penetrating property, tolebrutinib, have shown promising results. These trials have reported that BTK inhibition can halt the engulfing of myelin sheaths by microglia and prevent demyelination. Tolebrutinib has a favourable outcome in MS patients compared with other BTK inhibitors such as ibrutinib. The off-target effects of ibrutinib make it unsuitable for the treatment of diseases outside of malignancies [75].

BTK in lymphoproliferative disorders Chronic lymphocytic leukaemia

BTK has been established as an attractive mark for targeted therapies for the B-cell malignancy chronic lymphocytic leukaemia (CLL) due to its upregulated expression. Through targeting BTK, cell death has been established as a direct consequence due to the blockage of signalling pathways and impeding cell proliferation. Reports of BTK inhibition influencing downstream targets MAPK and NF-κB have only heightened interest in its impact in CLL [76].

The BTK inhibitor ibrutinib has already proved to be highly effective in the treatment of CLL not only through affecting BTK but also through the inhibition of other kinases and growth factors; it has caused a shift in the paradigm of treatment. Despite the success of Ibrutinib, the emerging resistance clones are leading to fewer patients achieving complete remission, whereas discontinuation of therapy due to off-target effect hampers further drug efficiency. New inhibitors with higher levels of specificity and efficacy have been gaining momentum in the hopes of overcoming the rising levels of resistance [77].

Mantle cell lymphoma

Another lymphoproliferative disorder highly dependent on BCR signalling and BTK is mantle cell lymphoma (MCL). BTK has been identified as a possible target for the treatment of this aggressive malignancy. MCL cells overexpress BTK, which seems essential for its pathogenesis. Ibrutinib has been shown to be effective in the treatment of MCL in many studies, and it has been approved for treating relapsed/refractory (R/R) patients [78, 79].

Although the use of BTK inhibitors has made considerable improvements to the overall outcome of MCL, it is still largely an incurable disease; there is a need for novel targeted therapies. Combination therapies are an attractive option for overcoming resistance and improving overall outcomes such as BTK inhibition and venetoclax (BCL2 inhibitor) treatment. Matsumura-Kimoto et al. demonstrated the potential of a combination of the serine/threonine kinase ribosomal protein S6 kinase (RSK2) and the BTK inhibitor ibrutinib. The inhibition of RSK2 was reported to affect downstream proteins involved in the BCR signalling pathway such as BLNK and CD19, as well as proteins from other pathways, blocking the B-cell pathogenesis [80].

Waldenström's macroglobulinaemia

BTK has been reported to be constitutively activated in the less common haematological malignancy Waldenström's macroglobulinaemia (WM). Characterized by the excessive secretion of monoclonal IgM antibodies, WM is defined and diagnosed by a MYD88L265P somatic mutation [81]. BTK is a downstream component that is affected by the mutation MYD88L265P and leads to activation of NF-κB. In WM, a higher level of phosphorylated BTK has been observed than in healthy counterparts with the preferential formation of a complex consisting of phosphorylated BTK and MYD88L265P [82].

Recent studies have identified MYD88 mutations in a complex with the protein kinase SYK, a component of the BCR signalling pathway upstream of BTK. Munshi et al. reported the inhibition of both BTK and SYK had a synergistic effect and caused an increased level of cell death than either treatment alone. This was due to BTK and SYK having different pathways for pro-survival signalling. This combination of Ibrutinib and the SYK inhibitor could be a promising target for future therapies of mutated WM [83].

Other, less common, mutations identified in WM include the C-X-C chemokine receptor type 4 (CXCR4), present in around 40% of patients. CXCR4 mutations have been reported to cause shorter and decreased response rates for WM patients under ibrutinib. This emphasizes the importance of a clear understanding of the genetic landscape when treating a disease, as it has been shown here mutations can effect on BTK and its inhibition in patients with WM [84].

Diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell malignancy divided into distinct molecular subtypes gene expressional profiling. BCR signalling has been identified to be upregulated in DLBCL, and DLBCL tumours are dependent on this signalling, but this differs between subtypes. Due to this BCR dependency, BCR-inhibitory therapies have received a large amount of interest, including BTK and SYK inhibitors. Studies have reported that DLBCL cell lines can be sensitized to venetoclax, through treatment with ibrutinib or the SYK inhibitor, fostamatinib, due to a shift in the binding of BIM and MCL1 [85].