Bruton's Tyrosine Kinase Deficiency Research Articles

Loss of BTK ameliorates the pathological cardiac fibrosis and dysfunction

Cardiac fibrosis is a common irreversible pathological feature of diverse heart disorders. Uncontrolled cardiac fibrosis contributes to maladaptive cardiac remodeling and eventually heart failure. However, the molecular determinants of ischemic and non-ischemic pathological cardiac fibrosis remain largely unknown. Here, we investigated the role of Bruton's tyrosine kinase (BTK) in cardiac fibrosis and remodeling of mice under various pathological conditions. BTK expression was increased in myocardium of mice after pressure overload or myocardial infarction (MI). BTK was mainly located in cardiac fibroblasts of myocardium, and its expression in isolated cardiac fibroblasts was also upregulated following TGF-β treatment. The deficiency or pharmacological inhibition of BTK with the second-generation inhibitor Acalabrutinib attenuated cardiac fibrosis, preserved cardiac function and prevented adverse cardiac remodeling, which protected against heart failure in mice following pressure overload or MI. BTK deficiency or inhibitor treatment significantly decreased the expression of pro-fibrotic molecules in isolated cardiac fibroblasts and inhibited the transition of fibroblasts to myofibroblasts in response to diverse pathological stresses. BTK directly bound and phosphorylated TGF-β receptor Ⅰ (TβRⅠ) at tyrosine 182, and then promoted the activation of downstream SMAD-dependent or -independent TGF-β signaling, leading to the enhanced transition of fibroblasts to pro-fibrotic myofibroblasts and the excessive extracellular matrix gene expression. Our finding uncovers a driving role of BTK in cardiac fibrosis and dysfunction following pressure overload and MI stress, and highlights novel pathogenic mechanisms in ischemic and non-ischemic maladaptive cardiac remodeling, which presents as a promising target for the development of anti-fibrotic therapy.

Campylobacter infection in 4 patients treated with ibrutinib.

Ibrutinib is a Bruton tyrosine kinase (BTK) inhibitor used in B-cell lymphoproliferative disorders. Patients with genetic BTK deficiency are susceptible to recurrent and severe Campylobacter infections. We report 4 patients treated with ibrutinib who developed chronic or extra-digestive campylobacteriosis resembling ibrutinib-related adverse events including diarrhea (n = 4), panniculitis (n = 2), and arthritis (n = 1). Microbiological explorations identified Campylobacter jejuni (n = 3) or Campylobacter coli (n = 1). All the patients completely recovered after a short course of oral antibiotic therapy. In patients treated with ibrutinib presenting with chronic diarrhea, dermatological, or rheumatological manifestations, campylobacteriosis should be ruled out before attributing the symptoms to ibrutinib and discuss its discontinuation.

Deficient Expression of Bruton's Tyrosine Kinase in Monocytes From X-Linked Agammaglobulinemia as Evaluated by a Flow Cytometric Analysis and Its Clinical Application to Carrier Detection

AbstractThe B-cell defect in X-linked agammaglobulinemia (XLA) is caused by mutations in the gene for Bruton's tyrosine kinase (BTK). Using the anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of intracytoplasmic BTK protein expressed in monocytes was successfully performed. To examine the possible identification of XLA patients and female carriers by this assay, we studied 41 unrelated XLA families with (35) or without (6) known BTK mutations. A flow cytometric assay showed deficient expression of the BTK protein in 40 of 41 patients, complete BTK deficiency in 35, and partial BTK deficiency in 5. One patient exhibited a normal level of BTK expression. All 6 patients with partial BTK deficiency or normal BTK expression had missense BTK mutations. The cellular mosaicism of BTK expression in monocytes from obligate carriers was clearly shown in 35 of 41 families. The results suggested that most BTK mutations in XLA might result in deficient expression of the BTK protein. We conclude that deficient expression of BTK protein can be evaluated by a flow cytometric assay, and the clinical usefulness and limitations in diagnosis of XLA patients and carriers are discussed.

Bruton's Tyrosine Kinase Deficiency Alters Gut B Lymphocyte Populations and IgA Recognition of Commensal Bacteria in Autoimmune Disease

Bruton's Tyrosine Kinase Deficiency Alters Gut B Lymphocyte Populations and IgA Recognition of Commensal Bacteria in Autoimmune Disease

Bruton's Tyrosine Kinase (BTK) Is a Novel KMT2A Partner Gene

Histone-lysine N-methyltransferase 2A gene (KMT2A) rearrangements are common genetic events in acute leukemia. They more frequent in infants and are found in 30-45% of acute myeloid leukemias (AML). They demonstrate great molecular heterogeneity with more than 90 partner genes and multiple KMT2A breakpoints involved (Meyer et al., 2018). These characteristics challenge both initial molecular diagnostics and MRD monitoring in ALs with KMT2A rearranged. As the accurate detection of all KMT2A-r types is crucial in order to correct patients' therapy and risk groups' definition, we aim to fully characterize KMT2A rearrangements within our cohort of patients using various techniques. Our recent study demonstrates a novel KMT2A partner gene - Bruton's tyrosine kinase (BTK), in a case of childhood AML. The patient is 9 m.o. girl who presented with WBC 69,3*109/L and hepatosplenomegaly. Morphological, immunochemical and immunological examination revealed acute monocytic leukemia. Bone marrow aspirates were analyzed by G-banded karyotyping, FISH with KMT2A breakapart probe. The karyotype was 45,X,der(X?)t(X;11)(q22.1;q23.3),del(11)(q14),-20[7] with 95% KMT2A-rearranged nuclei. Real-time RT-PCR for 8 most common KMT2A rearrangements screening was negative. Targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA) followed by high-throughput sequencing identified novel fusion transcript KMT2A-BTK with exon 9-exon 2 breakpoint junction. Sanger sequencing was used for validation. The achieved data on KMT2A translocation partner and breakpoint location was used for subsequent patient-specific MRD monitoring. The patient was treated according to AML-MRD-2018 local protocol and achieved complete MRD-negative remission after induction course. Then, a severe myelodepression developed after consolidation therapy, and the patient died due to infectious complications. BTK gene is located at Xq22.1 and encodes for Bruton's tyrosine kinase protein (Hashimoto et al., 1996), which is a cytoplasmic non-receptor tyrosine kinase essential for B-cell maturation and development (Shinners et al., 2007). BTK deficiency is strongly associated with X-linked agammaglobulinemia, where 80-90% of patients express either truncated or misfolded or mutated BTK protein (Valiaho, Smith, & Vihinen, 2006). BTK is also implemented in B-cell acute lymphoblastic leukemia, where it often has a deleted or a truncated kinase domain (Feldhahn et al., 2005). The opposite situation was reported for AML in several studies showing increased BTK activity (Gu et al., 2011; Tomasson et al., 2008). The presented t(X;11)(q22.1;q23.3) childhood AML is to our knowledge the first case of BTK translocations in hematologic malignancies, and its value for leukemogenesis is to be further investigated. However, as the breakpoint junction is located in BTK intron 1, upstream of its coding region (the translation starts in exon 2), we can speculate that BTK's structure itself is not committed and its activity is increased due to upregulated expression together with KMT2A. Therefore, the novel fusion KMT2A-BTK was for the first time revealed during the molecular profiling of KMT2A-rearranged AML in Russian Federation. The work was supported by RFBR grant №17-29-06052. Disclosures No relevant conflicts of interest to declare.

Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization

BackgroundIbrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses. Materials and methodsA mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry. ResultsControl mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p=0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230±60 MFI) and reached peak levels at day 21 (426±61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109±59 MFI, p=0.004) and day 21 (241±86 MFI, p=0.003). FACS analysis found a reduction of B220+ or CD19+ B cell population (p<0.05). In addition, ibrutinib attenuated recall DSA IgG responses to re-sensitization (p<0.05) and reduced CD38+CD138+ plasma cells (p<0.05) in the spleens. ConclusionsIbrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression.

Bruton's Tyrosine Kinase Deficiency Inhibits Autoimmune Arthritis in Mice but Fails to Block Immune Complex–Mediated Inflammatory Arthritis

Bruton's tyrosine kinase (BTK) is a B cell signaling protein that also contributes to innate immunity. BTK inhibitors prevent autoimmune arthritis but have off-target effects, and the mechanisms of protection remain unknown. We undertook these studies using genetic deletion to investigate the role of BTK in adaptive and innate immune responses that drive inflammatory arthritis. BTK-deficient K/BxN mice were generated to study the role of BTK in a spontaneous model that requires both adaptive and innate immunity. The K/BxN serum-transfer model was used to bypass the adaptive system and elucidate the role of BTK in innate immune contributions to arthritis. BTK deficiency conferred disease protection to K/BxN mice, confirming outcomes of BTK inhibitors. B lymphocytes were profoundly reduced, more than in other models of BTK deficiency. Subset analysis revealed loss of B cells at all developmental stages. Germinal center B cells were also decreased, with downstream effects on numbers of follicular helper T cells and greatly reduced autoantibodies. In contrast, total IgG was only mildly decreased. Strikingly, and in contrast to small molecule inhibitors, BTK deficiency had no effect in the serum-transfer model of arthritis. BTK contributes to autoimmune arthritis primarily through its role in B cell signaling and not through innate immune components.

Eosinophilic gastroenteritis in a patient with Bruton's tyrosine kinase deficiency

Eosinophilic gastrointestinal diseases (EGID) are relatively rare diseases characterized by eosinophilic infiltration of the gastrointestinal tract resulting in various gastrointestinal symptoms. EGID are often caused by allergic reactions or systemic eosinophilic disorders, but their comorbidity with Bruton's tyrosine kinase (BTK) deficiency has not been previously documented. Here, we report a case of eosinophilic gastroenteritis (EG) in a patient with BTK deficiency. Despite adequate replacement immunoglobulin (Ig) therapy, trough serum IgG was not maintained. To identify the underlying cause of the low trough level and chronic diarrhea, the intestine was investigated on endoscopy. We also screened for the variable number of tandem repeat polymorphism in FCGRT. Genetic analysis could not explain the low trough IgG, but endoscopy indicated eosinophilic enterocolitis. EG may be an important differential diagnosis when primary immunodeficiency patients have chronic diarrhea or continued low serum IgG.

CD19 controls Toll-like receptor 9 responses in human B cells

CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency. We sought to analyze whether CD19 is required for TLR9 function in human B cells. Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA. B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency. CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells.

Toll-like receptor signaling is impaired in dendritic cells from patients with X-linked agammaglobulinemia

Bruton's tyrosine kinase (BTK), which is defective in patients with X-linked agammaglobulinemia (XLA), is expressed not only in B cells but also in monocytes and dendritic cells (DCs). DCs play a crucial role in the innate immune response against infections by sensing pathogens through Toll-like receptors (TLRs). However, it is not known whether BTK deficiency in XLA might impair TLR-mediated signaling in DCs, which are susceptible to various infections. The phenotypic maturation and cytokine production mediated by TLRs were examined in monocyte-derived DC from XLA patients and normal controls. The TLR expression in DCs was analyzed by flow cytometry. TLR-mediated signaling in DCs was evaluated for the phenotypic maturation based on CD83 expression and production of cytokines, such as TNF-α, IL-6 and IL-12p70. TLR levels in DCs were similar between XLA and controls. TLR2, TLR4 and TLR7/8 ligands elicited less phenotypic maturation of DCs from XLA patients than normal controls based on CD83 expression. Stimulation with TLR2, TLR4 and TLR7/8 ligands, as well as TLR3 ligand, resulted in significantly lower production of TNF-α, but neither IL-6 nor IL-12p70, by DCs from XLA patients in comparison to normal controls. These findings suggest that BTK may thus be required for TLR signaling in DCs. The impaired TLR signaling in DCs may therefore be partly responsible for the occurrence of severe infections with bacteria and some viruses in XLA patients.

Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis

Background: X-linked agammaglobulinemia (XLA), caused by mutations in Bruton's tyrosine kinase (BTK ), is the most common form of inherited antibody deficiency. We previously reported that a flow cytometric evaluation of BTK expression in monocytes could easily detect XLA as well as its carrier. Objective: Our purpose was to perform further flow cytometric analysis in additional XLA families in Japan. Methods: In all, 106 hypogammaglobulinemic males (from 91 families) of various ages with a lack of mature B cells (<1%) were investigated. Results: Flow cytometric assessment revealed the deficient BTK expression status in 78 families (93 patients), and mutations in BTK were identified in 76 of 78 families with presumed XLA. Of the patients with normal BTK expression, 2 showed missense mutations in which the normal amount of altered BTK transcript would cause the XLA phenotype. As many as 30% of these patients with XLA were clinically or genetically recognized beyond 5 years of age. Higher concentrations (>300 mg/dL) of serum IgG were evident in the cases diagnosed among adults, seemingly preventing severe infections. Fifty-seven of 70 mothers of patients with BTK deficiency were diagnosed as obligate carriers on the basis of a bimodal BTK expression pattern. Nine of the remaining 13 mothers showing nonmosaic BTK expression had no mutations in 2 alleles; surprisingly, the other 4 mothers had the mutated alleles. Conclusions: A diagnostic approach based on flow cytometric assessment for XLA should be initially considered in genetic investigation of antibody deficiencies, regardless of the patient's age. (J Allergy Clin Immunol 2001;108:1012-20.)