Abstract

Bruton's tyrosine kinase (Btk) is a signaling molecule involved in development and activation of B cells through B-cell receptor (BCR) and Toll-like receptor (TLR) signaling. We have previously shown that transgenic mice that overexpress human Btk under the control of the CD19 promoter (CD19-hBtk) display spontaneous germinal center formation, increased cytokine production, anti-nuclear autoantibodies (ANAs), and systemic autoimsmune disease upon aging. As TLR and BCR signaling are both implicated in autoimmunity, we studied their impact on splenic B cells. Using phosphoflow cytometry, we observed that phosphorylation of ribosomal protein S6, a downstream Akt target, was increased in CD19-hBtk B cells following BCR stimulation or combined BCR/TLR stimulation, when compared with wild-type (WT) B cells. The CD19-hBtk transgene enhanced BCR-induced B cell survival and proliferation, but had an opposite effect following TLR9 or combined BCR/TLR9 stimulation. Although the expression of TLR9 was reduced in CD19-hBtk B cells compared to WT B cells, a synergistic effect of TLR9 and BCR stimulation on the induction of CD25 and CD80 was observed in CD19-hBtk B cells. In splenic follicular (Fol) and marginal zone (MZ) B cells from aging CD19-hBtk mice BCR signaling stimulated in vitro IL-10 production in synergy with TLR4 and particularly TLR9 stimulation, but not with TLR3 and TLR7. The enhanced capacity of CD19-hBtk Fol B cells to produce the pro-inflammatory cytokines IFNγ and IL-6 compared with WT B cells was however not further increased following in vitro BCR or TLR9 stimulation. Finally, we used crosses with mice deficient for the TLR-associated molecule myeloid differentiation primary response 88 (MyD88) to show that TLR signaling was crucial for spontaneous formation of germinal centers, increased IFNγ, and IL-6 production by B cells and anti-nuclear autoantibody induction in CD19-hBtk mice. Taken together, we conclude that high Btk expression does not only increase B cell survival following BCR stimulation, but also renders B cells more sensitive to TLR stimulation, resulting in increased expression of CD80, and IL-10 in activated B cells. Although BCR-TLR interplay is complex, our findings show that both signaling pathways are crucial for the development of pathology in a Btk-dependent model for systemic autoimmune disease.

Highlights

  • B cells are crucial players in autoimmunity, as B cell depletion therapy was proven effective in patients with several systemic autoimmune diseases including Sjögren’s syndrome (SjS) and rheumatoid arthritis (RA) [1, 2]

  • Bruton’s tyrosine kinase (Btk) is a critical kinase in the B cell receptor (BCR) signaling pathway and is known to interact with various proteins that are downstream of Toll-like receptor (TLR)

  • Our findings provide evidence that TLR signaling is critical in systemic autoimmunity driven by overexpression of Btk in transgenic mice, which is characterized by the induction of TLR7/9-associated auto-antibody specificities, including dsDNA, histone, RNP-A, RNP-C, and SmB

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Summary

Introduction

B cells are crucial players in autoimmunity, as B cell depletion therapy was proven effective in patients with several systemic autoimmune diseases including Sjögren’s syndrome (SjS) and rheumatoid arthritis (RA) [1, 2]. These diseases are marked by altered B cell selection leading to the production of autoreactive antibodies, an important hallmark in the pathology of systemic autoimmune diseases. BCR signaling is crucial for the selection of B cells. Evidence is accumulating that additional signals derived from CD40, Toll-like receptors (TLRs) and BAFFR affect selection of B cells [4]

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