Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice

Abstract

On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca(2+) influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.

Selective Inhibition of BTK Prevents Murine Lupus and Antibody-Mediated Glomerulonephritis

“…Interestingly, transgenic expression of a BTK hypomorph in an xid background that exhibits 25% of normal expression levels results in the generation of normal numbers of mature B cells but impaired B cell Ab production after immunization (29). Conversely, transgenic overexpression of BTK results in spontaneous germinal center formation, autoantibody production, and SLE-like disease (30). Thus, these results collectively highlight the critical importance of BTK in regulating B cell activation and tolerance.…”

B-cell subsets, signaling and their roles in secretion of autoantibodies

“…Btk mutations in humans cause X-linked agammaglobulinemia (XLA), a severe inherited disease associated with deficiency of B cells in peripheral blood and severely low Ig production. 65 On the other hand, Kil et al 66 demonstrated that transgenic mice that overexpress Btk exhibit spontaneous GCs and large number of plasma B cells, with ANA production and lupus-like autoimmune diseases. Others showed that Btk deficiency was associated with arrest of B-cell development at the pre-B-cell stage.…”

Enhanced Expression of Bruton’s Tyrosine Kinase in B Cells Drives Systemic Autoimmunity by Disrupting T Cell Homeostasis

“…Fluorescent labeling of cell membrane molecules was performed as described previously (21). For isotype-specific staining of intracellular Ig in plasma cells, cells were fixed and permeabilized using Cytofix/Cytoperm Buffer and Perm/Wash Buffer, respectively (BD Biosciences, San Jose, CA), according to the manufacturer's instructions.…”

“…We recently observed that overexpression of the BCR signaling molecule Bruton's tyrosine kinase (BTK) restricted to B cells in CD19-hBtk transgenic mice is sufficient to induce an SLE/Sjögren-like disease phenotype (21). Btk protein expression in mouse B cells is normally tightly controlled and upregulated upon BCR stimulation by complex regulatory mechanisms involving micro-RNAs and feedforward NF-kB activation (22)(23)(24).…”

“…Btk protein expression in mouse B cells is normally tightly controlled and upregulated upon BCR stimulation by complex regulatory mechanisms involving micro-RNAs and feedforward NF-kB activation (22)(23)(24). B cells overexpressing BTK are selectively hyperresponsive to BCR stimulation, showing enhanced Ca 2+ influx and NF-kB activation and resistance to Fas-mediated apoptosis in vitro, and are not effectively eliminated in vivo when autoreactive (21). A pathogenic role for BTK in rheumatic diseases is further emerging from mouse studies demonstrating that smallmolecule BTK inhibitors prevent or ameliorate lupus nephritis and experimental arthritis (25)(26)(27)(28)(29)(30)(31).…”