Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

Abstract

Background: Autoimmune disorders, including Systemic Lupus Erythematosus (SLE), are associated with increased incidence of hematological malignancies. The matricellular protein osteopontin (OPN) has been linked to SLE pathogenesis, as SLE patients show increased serum levels of OPN and often polymorphisms in its gene. Although widely studied for its pro-tumorigenic role in different solid tumours, the role of OPN in autoimmunity-driven lymphomagenesis has not been investigated yet. Methods: To test the role of OPN in the SLE-associated lymphomagenesis, the SLE-like prone Fas(lpr/lpr) mutation was transferred onto an OPN-deficient background. Spleen from Fas(lpr/lpr) and OPN-/-Fas(lpr/lpr) mice, as well as purified B cells, were analysed by histopathology, flow cytometry, Western Blot, immunohistochemistry, immunofluorescence and gene expression profile to define lymphoma characteristics and investigate the molecular mechanisms behind the observed phenotype. OPN cellular localization in primary splenic B cells and mouse and human DLBCL cell lines was assessed by confocal microscopy. Finally, gain of function experiments, by stable over-expression of the secreted (sOPN) and intracellular OPN (iOPN) in OPN-/-Fas(lpr/lpr) -derived DLBCL cell lines, were performed for further validation experiments. Results: Despite reduced autoimmunity signs, OPN-/-Fas(lpr/lpr) mice developed splenic lymphomas with higher incidence than Fas(lpr/lpr) counterparts. In situ and ex vivo analysis featured such tumours as activated type of diffuse large B cell lymphoma (ABC-DLBCL), expressing BCL2 and c-MYC, but not BCL6, with activated STAT3 signaling. OPN-/-Fas(lpr/lpr) B lymphocytes showed an enhanced TLR9-MYD88 signaling pathway, either at baseline or after stimulation with CpG oligonucleotides, which mimic dsDNA circulating in autoimmune conditions. B cells from Fas(lpr/lpr) mice were found to express the intracellular form of OPN. Accordingly, gene transfer-mediated re-expression of iOPN, but not of its secreted isoform, into ABC-DLBCL cell lines established from OPN-/-Fas(lpr/lpr) mice, prevented CpG-mediated activation of STAT3, suggesting that the intracellular form of OPN may represent a brake to TLR9 signaling pathway activation. Conclusion: These data indicate that, in the setting of SLE-like syndrome in which double strand-DNA chronically circulates and activates TLRs, B cell intracellular OPN exerts a protective role in autoimmunity-driven DLBCL development, mainly acting as a brake in the TLR9-MYD88-STAT3 signaling pathway.