title: A BAFF/APRIL-dependent TLR3-stimulated pathway enhances the capacity of rheumatoid synovial fibroblasts to induce AID expression and Ig class-switching in B cells.
abstract: OBJECTIVE
  To dissect the role of toll-like receptor (TLR) signalling and B cell survival/proliferating factors in the crosstalk between rheumatoid arthritis synovial fibroblasts (RASF) and B cells.
  
  
  METHODS
  RASF, rheumatoid arthritis dermal fibroblasts (RADF) and osteoarthritis synovial fibroblasts (OASF) were analysed for the expression of B cell survival/proliferating factors BAFF and APRIL in resting conditions and upon stimulation with TLR2/TLR3/TLR4 ligands. Unswitched IgD+ B cells were co-cultured with RASF/OASF/RADF in the presence/absence of TLR ligands and with/without BAFF/APRIL blocking antibodies. Activation-induced cytidine deaminase (AID) mRNA expression, Iγ-Cμ and Iα-Cμ circular transcripts (CTs; markers of ongoing class-switching to IgG and IgA) and IgM/A/G production were measured to assess functional activation of B cells.
  
  
  RESULTS
  TLR3 and to a lesser extent TLR4, but not TLR2 stimulation, induced up to ∼1000-fold BAFF mRNA and increased soluble BAFF release. APRIL was less significantly regulated by TLR3. Resting and TLR3-stimulated RASF released higher levels of BAFF/APRIL compared with RADF. TLR3 stimulation of RASF but not RADF in co-culture with B cells strongly enhanced AID expression, Iγ-Cμ and Iα-Cμ CTs and class-switching to IgG/IgA. Blockade of BAFF/APRIL signalling completely inhibited TLR3-induced, RASF-dependent expression of AID, CTs and the secretion of IgG/IgA.
  
  
  CONCLUSIONS
  RASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, class-switch recombination and IgG/IgA production in IgD+ B cells. Overall, this work highlights a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the rheumatoid arthritis synovium.
authors.affiliation: Centre for Experimental Medicine and Rheumatology, 2nd Floor, John Vane Science Centre, William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. m.bombardieri@qmul.ac.uk
authors.forename: Michele
authors.initials: M
authors.lastname: Bombardieri
authors.forename: Ngar-Woon
authors.initials: NW
authors.lastname: Kam
authors.forename: Fabia
authors.initials: F
authors.lastname: Brentano
authors.forename: Ken
authors.initials: K
authors.lastname: Choi
authors.forename: Andrew
authors.initials: A
authors.lastname: Filer
authors.forename: Diego
authors.initials: D
authors.lastname: Kyburz
authors.forename: Iain B
authors.initials: IB
authors.lastname: McInnes
authors.forename: Steffen
authors.initials: S
authors.lastname: Gay
authors.forename: Christopher
authors.initials: C
authors.lastname: Buckley
authors.forename: Costantino
authors.initials: C
authors.lastname: Pitzalis
mesh_terms: D000368:Aged; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D053264:B-Cell Activating Factor / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D018920:Coculture Techniques; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000276:immunology; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis; D017578:Immunoglobulin Class Switching / Q000276:immunology; D007072:Immunoglobulin D / Q000032:analysis; D007074:Immunoglobulin G / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000276:immunology; D012867:Skin / Q000276:immunology; D013583:Synovial Membrane / Q000276:immunology*; D051196:Toll-Like Receptor 3 / Q000276:immunology*; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D053264:B-Cell Activating Factor; D007070:Immunoglobulin A; D007072:Immunoglobulin D; D007074:Immunoglobulin G; D012333:RNA, Messenger; C495344:TLR3 protein, human; C114631:TNFSF13 protein, human; C505719:TNFSF13B protein, human; D051196:Toll-Like Receptor 3; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase