Th1 cells downregulate connexin 43 gap junctions in astrocytes via microglial activation.

Mitsuru Watanabe,Takuya Matsushita,Ryo Yamasaki,Hideyuki Takeuchi,Katsuhisa Masaki,Jun-Ichi Kira,Jun Kawanokuchi,Akio Suzumura

doi:10.1038/srep38387

Mitsuru Watanabe, Takuya Matsushita + Show 6 more

Open Access

https://doi.org/10.1038/srep38387

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Abstract

We previously reported early and extensive loss of astrocytic connexin 43 (Cx43) in acute demyelinating lesions of multiple sclerosis (MS) patients. Because it is widely accepted that autoimmune T cells initiate MS lesions, we hypothesized that infiltrating T cells affect Cx43 expression in astrocytes, which contributes to MS lesion formation. Primary mixed glial cell cultures were prepared from newborn mouse brains, and microglia were isolated by anti-CD11b antibody-conjugated magnetic beads. Next, we prepared astrocyte-rich cultures and astrocyte/microglia-mixed cultures. Treatment of primary mixed glial cell cultures with interferon (IFN) γ, interleukin (IL)-4, or IL-17 showed that only IFNγ or IL-17 at high concentrations reduced Cx43 protein levels. Upon treatment of astrocyte-rich cultures and astrocyte/microglia-mixed cultures with IFNγ, Cx43 mRNA/protein levels and the function of gap junctions were reduced only in astrocyte/microglia-mixed cultures. IFNγ-treated microglia-conditioned media and IL-1β, which was markedly increased in IFNγ-treated microglia-conditioned media, reduced Cx43 protein levels in astrocyte-rich cultures. Finally, we confirmed that Th1 cell-conditioned medium decreased Cx43 protein levels in mixed glial cell cultures. These findings suggest that Th1 cell-derived IFNγ activates microglia to release IL-1β that reduces Cx43 gap junctions in astrocytes. Thus, Th1-dominant inflammatory states disrupt astrocytic intercellular communication and may exacerbate MS.

Highlights

Connexins (Cxs) are a family of vertebrate proteins that form gap junction (GJ) channels, the major intercellular channel that facilitates direct signalling between cytoplasmic compartments of adjacent cells
We demonstrated that IFNγactivated microglia to release IL-1βthat reduced astrocytic connexin 43 (Cx43) mRNA and protein levels, and functionally inhibited GJs in astrocytes
It was previously reported that functionally coupled astroglial cells determined by a dye injection method are decreased in astrocyte/microglia cocultures from newborn rat brains in the presence of 5% microglia following administration of either TNFα, IL-1β, or IFNγ20

Summary

Introduction

Connexins (Cxs) are a family of vertebrate proteins that form gap junction (GJ) channels, the major intercellular channel that facilitates direct signalling between cytoplasmic compartments of adjacent cells. Astrocytes are functionally coupled to adjacent astrocytes and oligodendrocytes by GJs and form the “glial syncytium” that maintains the homeostasis of glial and neuronal cells[4]. Cx43 expression levels are lower than in grey matter, and Cx43 is present in the foot processes of perivascular astrocytes[7,8,9]. We previously reported early and extensive loss of astrocytic Cx43 in active white matter lesions of MS, neuromyelitis optica (NMO), and Baló’s concentric sclerosis (BCS) pateints[10,11,12]. Perivascular lymphocytic cuffing mainly consisting of T cells has been observed significantly more frequently in active demyelinating lesions with Cx43 loss[11]. Some proinflammatory factors have been reported to reduce astrocytic expression of Cx43 in vitro[15], the mechanisms of Cx43 loss remain to be elucidated in demyelinating diseases. We investigated whether CD4+ T cells, such as T helper (Th) 1, Th17, or regulatory T (Treg) cells, directly or indirectly influence Cx43 protein levels in astrocytes using a primary glial cell culture system

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