Distinctive Roles for α7*- and α9*-Nicotinic Acetylcholine Receptors in Inflammatory and Autoimmune Responses in the Murine Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

Qiang Liu,Paul Whiteaker,Fu-Dong Shi,Barbara J Morley,Ronald J Lukas

doi:10.3389/fncel.2017.00287

Abstract

Previous studies have demonstrated immunosuppressive and anti-inflammatory effects of nicotine, including in the experimental autoimmune encephalomyelitis (EAE) model in mice of some forms of multiple sclerosis (MS). Other studies using knock-out (KO) mice have implicated nicotinic acetylcholine (ACh) receptors containing α7, α9, or β2 subunits (α7*-, α9*- or β2*-nAChR) in different, disease-exacerbating or disease-ameliorating processes. These outcomes are in harmony with gene expression analyses showing nAChR subunit mRNA in many classes of immune system cell types. Consistent with influences on disease status, predictable effects of nAChR subunit (and subtype) KO, or of nicotine exposure, are seen on immune cell numbers and distribution and on cytokine levels or other markers of immunity, inflammation, demyelination, and axonal degradation. Providing support for our hypotheses about distinctive roles for nAChR subtypes in EAE, here we have used direct and adoptive EAE induction and a nAChR subunit gene double knock-out (DKO) strategy. Immune cell expression of nAChR α9 subunits as protein is demonstrated by immunostaining of isolated CD4+, CD8+, CD11b+ and CD11c+ cells from wild-type (WT) mice, but not in cells from nAChR α9 subunit KO animals. Nicotine exposure is protective against directly-induced EAE in WT or α7/α9 DKO animals relative to effects seen in WT/vehicle-treated mice, but, remarkably, EAE is exacerbated in vehicle-treated α7/α9 DKO mice. Brain lesion volume and intra-cranial inflammatory activity similarly are higher in DKO/vehicle than in WT/vehicle-treated animals, although nicotine’s protective effects are seen in each instance. By contrast, in adoptive transfer studies, disease severity is attenuated and disease onset is delayed in recipients of splenocytes from WT animals treated with nicotine rather than with vehicle. Moreover, protection as seen in nicotine-treated WT animals is the same in recipients of splenocytes from nAChR α7/α9 DKO mice irrespective of their exposure to nicotine or vehicle. When combined with previous observations, these findings are consistent with disease exacerbation (or even induction) being mediated at least in part via α9*-nAChR in peripheral immune cells. They also suggest protective roles of central nervous system (CNS) α7*-nAChR. The results suggest that both α7*- and α9*-nAChR are potential targets of therapeutic ligands to modulate inflammation and autoimmunity.

Highlights

It is widely recognized and undisputed that controlled inflammatory and immune responses play critical roles in maintenance of normal health by protecting against insults, in part by clearing damaged cells, compromised tissue areas and/or foreign substances (Weiner and Selkoe, 2002; Nikoopour et al, 2008; Viganò et al, 2012)
Our subsequent studies have confirmed those protective effects in the EAE model and have illuminated roles for different nicotinic acetylcholine receptor subtypes in specific features of the disease process or recovery and in protective effects of nicotine exposure (Piao et al, 2009; Shi et al, 2009; Hao et al, 2011, 2013; Simard et al, 2013). nAChR exist as pentamers of nAChR subunits that are encoded from a mammalian family of 16 different genes (Jensen et al, 2005; Lukas and Bencherif, 2006; Taly et al, 2009). nAChR exist as a group of subtypes, each defined by their distinctive subunit composition
Similar assessment in immune cells from nAChR α9 subunit KO mice were negative for subunit immunoreactivity

Summary

INTRODUCTION

It is widely recognized and undisputed that controlled inflammatory and immune responses play critical roles in maintenance of normal health by protecting against insults, in part by clearing damaged cells, compromised tissue areas and/or foreign substances (Weiner and Selkoe, 2002; Nikoopour et al, 2008; Viganò et al, 2012). Studies by us and others at first seemed consistent with a dominant role for α7∗-nAChR in protective effects of nicotine in EAE (Nizri et al, 2009; Hao et al, 2011) In part, this is because the integrated disease response, assessed based on signs of limb and tail weakness in the EAE model, was attenuated by nicotine treatment in wild-type (WT) animals, but not in nicotine-treated nAChR α7 subunit knock-out (α7 KO) mice. Deeper characterization involved determinations of immune cell levels in the periphery and in the central nervous system (CNS) and of levels of expression of markers of inflammation and hyper-immunity These levels were not statistically different for vehicle-treated α7 KO or WT animals, again suggesting lack of involvement of α7∗-nAChR in natural, disease-related, cholinergic processes, which must involve other nAChR subtypes.

MATERIALS AND METHODS

RESULTS

DISCUSSION