Modulation of TLR3/TLR4 inflammatory signaling by the GABAB receptor agonist baclofen in glia and immune cells: relevance to therapeutic effects in multiple sclerosis.

Tadhg Crowley,John F Cryan,Orna O’Toole,Teun Kuijper,John-Mark Fitzpatrick,Eric J Downer,Olivia F O’Leary

doi:10.3389/fncel.2015.00284

Abstract

The GABAB receptor agonist, baclofen, is used to treat muscle tightness and cramping caused by spasticity in a number of disorders including multiple sclerosis (MS), but its precise mechanism of action is unknown. Neuroinflammation drives the central pathology in MS and is mediated by both immunoreactive glial cells and invading lymphocytes. Furthermore, a body of data indicates that the Toll-like receptor (TLR) family of innate immune receptors is implicated in MS progression. In the present study we investigated whether modulation of GABAB receptors using baclofen can exert anti-inflammatory effects by targeting TLR3 and(or) TLR4-induced inflammatory signaling in murine glial cells and human peripheral blood mononuclear cells (PBMCs) isolated from healthy control individuals and patients with the relapse-remitting (RR) form of MS. TLR3 and TLR4 stimulation promoted the nuclear sequestration of NF-κB and pro-inflammatory cytokine expression in murine glia, while TLR4, but not TLR3, promoted pro-inflammatory cytokine expression in PBMCs isolated from both healthy donors and RR-MS patients. Importantly, this effect was exacerbated in RR-MS patient immune cells. We present further evidence that baclofen dose-dependently attenuated TLR3- and TLR4-induced inflammatory signaling in primary glial cells. Pre-exposure of PBMCs isolated from healthy donors to baclofen attenuated TLR4-induced TNF-α expression, but did not affect TLR4-induced TNF-α expression in RR-MS patient PBMCs. Interestingly, mRNA expression of the GABAB receptor was reduced in PBMCs from RR-MS donors when compared to healthy controls, an effect that might contribute to the differential sensitivity to baclofen seen in healthy and RR-MS patient cells. Overall these findings indicate that baclofen differentially regulates TLR3 and TLR4 signaling in glia and immune cells, and offers insight on the role of baclofen in the treatment of neuroinflammatory disease states including MS.

Highlights

Autoimmunity drives the development of multiple sclerosis (MS), involving central nervous system (CNS) infiltration of immune cells, myelin degradation, reactive changes in glia and axonal loss (Compston and Coles, 2002)
TLR4 Activation Time-Dependently Increases Nuclear nuclear factor (NF)-κB p65 Expression and tumor necrosis factor-α (TNF-α) Release in Mixed Glia TLR4 and TLR3 were initially targeted given their involvement in EAE progression (Touil et al, 2006) and evidence that their expression is dysregulated in MS lesions (Bsibsi et al, 2002)
To initially characterize the impact of TLR4 stimulation on proinflammatory signaling in primary murine mixed glial cells, we temporally assessed the impact of LPS on the distribution of the NF-κB p65 subunit in response to LPS treatment

Summary

Introduction

Autoimmunity drives the development of multiple sclerosis (MS), involving central nervous system (CNS) infiltration of immune cells, myelin degradation, reactive changes in glia and axonal loss (Compston and Coles, 2002). Innate immunity is regulated by complex mechanisms involving pattern-recognition receptors (PRRs) that recognize molecular signatures of microbes. Toll-like receptors (TLRs) belong to the family of signaling PRRs (Jeannin et al, 2008) that initiate innate immune reactions by activating transcription factors such as nuclear factor (NF)-κB, in addition to inducing the expression of interferons (IFNs) and cytokines. TLRs are localized in endosomal compartments (including TLR3, TLR7, TLR8, TLR9), or are cell membranebound (as with all other TLRs; O’Neill, 2004), and couple to specific signaling cascades, inducing gene transcription and controlling immune processes, with this specificity reliant on the TLR adaptor proteins recruited (O’Neill, 2004). TLR3 (and TLR4) induces MyD88-independent signaling to couple to NF-κB via Toll-Interleukin-1 Receptor (TIR)-domaincontaining adaptor-inducing IFN-β (TRIF) protein. TLRs are key players in CNS diseases, and with respect to MS, key roles of TLRs have been shown in murine models of MS (Touil et al, 2006), while the expression of TLRs characterized on immune cells and CNS glia and neurons (Nishimura and Naito, 2005)

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Conclusion