CXCL4, a chemokine upregulated in systemic sclerosis patients, abrogates TLR9 signaling and central tolerance in B cells

Abstract

Abstract Background: Systemic sclerosis (SSc) is an autoimmune connective tissue disease, characterized by defective central B cell tolerance. Deficiency of MYD88, which mediates the function of TLRs, results in a failure to silence autoreactive B cells. CXCL4, a chemokine found elevated in the sera of SSc patients, potentiates IFN-α secretion from TLR9-induced plasmacytoid dendritic cells. The objective is to investigate whether CXCL4 regulates TLR9 signaling and central tolerance in B cells. Methods: B cells from SSc patients or healthy donors were cultured with TLR9 ligand (TLR9-L) alone or with CXCL4. Cytokines gene expression and secretion were analyzed. Delivery of TLR9-L was analyzed by Amnis and confocal microscopy. NOD-scid-common gamma chain (γc) knockout immunodeficient mice were engrafted with CD34 +human fetal hematopoietic stem cells transduced with lentivirus expressing human CXCL4. New emigrant/transitional B cells were sorted. Immunoglobulins chains of these cells were cloned and transfected in fibroblasts. Polyreactivity of the antibodies released by the fibroblasts were assessed. Results: Defective TLR9 signaling was observed in B cells of SSc patients. CXCL4 abrogated TLR9 response in human B cells. TLR9-L when given to B cells alone, was localized in the lysosomes and LAMP-2 +compartments while CXCL4 prevented the delivery of TLR9-L to these vesicles. CXCL4 in vivoexpression led to defective TLR9 responses and increased number of polyreactive B cells. Conclusions: Our data provide evidence for a novel mechanism by which CXCL4 impairs central B cell tolerance by altering the intracellular trafficking of TLR9 ligands, hence inhibiting TLR9 response which is required for the removal of developing autoreactive B cells.