J. Rivard, A. James, J. David, L. Brandenburg, N. Lee, R. Fuerstenberg, K. Brumbaugh, L. Leong, G. Wegner, K. Bradley, K. Reagan
The anti-inflammatory cytokine Interleukin-10 (IL-10) is known to be necessary for down regulating proinflammatory responses toward pathogens. IL-10 inhibits the production of many cytokines downstream of its receptor via the Jak1/Tyk2-STAT3 signaling pathway. The small molecule Tofacitinib, also called CP 690550, is a selective inhibitor of the Jak family of cytoplasmic tyrosine kinases. It has been shown to potently inhibit both Jak3- and Jak1-dependent STAT activation. Mouse microglial BV-2 cells are an established in vitro model for neuronal inflammation and have been reported to respond to IL-10 immunosuppression in a manner similar to primary mouse microglial cells. In this model, IL-10 pretreatment attenuates IL-6 and TNF-alpha secretion in BV-2 cells treated with Interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) cells. In order to better understand the molecular mechanisms involved in IL-10 suppression of proinflammatory cytokine expression, we examined the effects of Tofacitinib on IL-10-induced immunosuppression of proinflammatory cytokine secretion and activation of Jak/STAT signaling. The Proteome Profiler™ Mouse XL Cytokine Array (R&D Systems, Catalog # ARY028) was used to screen cell culture supernates from BV-2 cells that were either treated with IFN-gamma and LPS for 6 hours or remained untreated. We identified increased secretion of several cytokines upon IFN-gamma/LPS treatment including CCL3/4 (MIP-1 alpha/MIP-1 beta), CCL5/RANTES, CCL12/MCP-5, CD40/TNFRSF5, CXCL2/MIP-2, CXCL9/MIG, CXCL10/IP-10/CRG-2, G-CSF, ICAM-1/CD54, IL-1 alpha/IL-1F1, IL-1r alpha/IL-1F3, IL-6, IL-27, Serpin E1/PAI-1, and TNF-alpha. IL-10 pretreatment decreased the secretion of these cytokines. We also observed that Tofacitinib treatment attenuated the immunosuppressive actions of IL-10. Secretion of CCL2/JE/MCP-1, CCL5/RANTES, G-CSF, CCL3/4 (MIP-1 alpha/MIP-1 beta), and CXCL2/MIP-2 was restored to levels comparable to what was observed after treatment with IFN-gamma/LPS. Interestingly, inhibition of the Nuclear Factor-kB (NFkB) signaling pathway by the NEDD8 E1 activating enzyme inhibitor MLN4924 did not appear to abrogate IL-10 immunosuppression. Array results were confirmed using a quantitative Luminex® multiplex assay. Because Tofacitinib is known to inhibit Jak family kinases, we evaluated tyrosine phosphorylation of STAT proteins. Western blot analysis revealed that Tofacitinib inhibited phosphorylation of STAT1 (Y701) and STAT3 (Y705) in BV-2 cells in a time-dependent manner. While it is well established that LPS-induced transcription of proinflammatory cytokines is regulated by mitogen-activated protein kinases (MAPKs) and NFkB, the molecular mechanisms involved in IL-10 suppression of proinflammatory cytokine expression are largely unknown and may be affected indirectly by the action of Tofacitinib.