Contributions
Abstract: P392
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Inflammation and tissue damage
Objective: We previously reported extensive loss of astrocytic connexins (Cxs) in multiple sclerosis (MS) lesions. Because it is widely accepted that autoimmune T cells cause MS lesions, we hypothesized that inflammatory T cells would affect Cxs expression in astrocytes and contribute to MS lesion formation. We assessed whether interferon (IFN)γ, interleukin (IL)-4, or IL-17, which is produced by Th1, Th2, or Th17 cells, respectively, reduced Cx43 expression in astrocytes.
Methods: Primary mixed glial cell cultures mainly composed of astrocytes and microglia were prepared from the brains of newborn C57BL/6 mice. Microglia were separated from these cultures by anti-CD11b antibody-conjugated magnetic beads. Then, astrocyte-rich cultures and astrocyte and microglia-mixed cultures were reconstituted. These cultures were treated with IFNγ, IL-4, or IL-17 for 24 h. Cx43 expression was evaluated by Western blotting, immunocytochemistry, and quantitative real-time PCR.
Results: Treatment of primary mixed glial cell cultures with IFNγ, IL-4, or IL-17 showed that only INFγ-treatment reduced Cx43 expression in a concentration-dependent manner. Treatment of astrocyte-rich cultures or astrocyte and microglia-mixed cultures with IFNγ exhibited that Cx43 expression was downregulated only in astrocyte and microglia-mixed cultures. IFNγ-treated microglia conditioned medium induced significant downregulation of Cx43 expression in astrocyte-rich cultures. We found that IFNγ concentration-dependently increased IL-1β and tumor necrosis factor (TNF)α in microglia conditioned medium. Furthermore, treatment with IL-1β and TNFα reduced Cx43 expression in astrocyte-rich cultures. Finally, we treated primary mixed glial cell cultures with conditioned media of Th1, Th17 and regulatory T cells differentiated from naïve T cells in vitro. Only Th1 cell conditioned medium significantly reduced Cx43 expression in astrocytes.
Conclusions: These findings suggest that Th1 cell-derived IFNγ activates microglia to release IL-1β and TNFα, which reduce Cx43 expression in astrocytes. Thus, Th1-dominant inflammatory states can disrupt astrocytic intercellular communication and may exacerbate MS.
Disclosure:
Mitsuru Watanabe is funded by a research grant from Japan Society for the Promotion of Science, Japan;
Katsuhisa Masaki: nothing to disclose;
Ryo Yamasaki: nothing to disclose;
Jun Kawanokuchi: nothing to disclose;
Hideyuki Takeuchi: nothing to disclose;
Akio Suzumura: nothing to disclose;
Jun-ichi Kira is a consultant for Biogen Idec Japan and Medical Review. He has received honoraria from Bayer Healthcare, Mitsubishi Tanabe Pharma, Nobelpharma, Otsuka Pharmaceutical and Medical Review. He is funded by a research grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan and grants from the Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology, Japan. Source of funding: This study was supported by “Glial assembly” Grant-in-Aid for Scientific Research on Innovative Areas (Grant Number 25117012) from the Ministry of Education, Culture, Sports, Science and Technology, and Grant-in-Aid for Young Scientists (Grant Number 15K19489) from Japan Society for the Promotion of Science, Japan.
Abstract: P392
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Inflammation and tissue damage
Objective: We previously reported extensive loss of astrocytic connexins (Cxs) in multiple sclerosis (MS) lesions. Because it is widely accepted that autoimmune T cells cause MS lesions, we hypothesized that inflammatory T cells would affect Cxs expression in astrocytes and contribute to MS lesion formation. We assessed whether interferon (IFN)γ, interleukin (IL)-4, or IL-17, which is produced by Th1, Th2, or Th17 cells, respectively, reduced Cx43 expression in astrocytes.
Methods: Primary mixed glial cell cultures mainly composed of astrocytes and microglia were prepared from the brains of newborn C57BL/6 mice. Microglia were separated from these cultures by anti-CD11b antibody-conjugated magnetic beads. Then, astrocyte-rich cultures and astrocyte and microglia-mixed cultures were reconstituted. These cultures were treated with IFNγ, IL-4, or IL-17 for 24 h. Cx43 expression was evaluated by Western blotting, immunocytochemistry, and quantitative real-time PCR.
Results: Treatment of primary mixed glial cell cultures with IFNγ, IL-4, or IL-17 showed that only INFγ-treatment reduced Cx43 expression in a concentration-dependent manner. Treatment of astrocyte-rich cultures or astrocyte and microglia-mixed cultures with IFNγ exhibited that Cx43 expression was downregulated only in astrocyte and microglia-mixed cultures. IFNγ-treated microglia conditioned medium induced significant downregulation of Cx43 expression in astrocyte-rich cultures. We found that IFNγ concentration-dependently increased IL-1β and tumor necrosis factor (TNF)α in microglia conditioned medium. Furthermore, treatment with IL-1β and TNFα reduced Cx43 expression in astrocyte-rich cultures. Finally, we treated primary mixed glial cell cultures with conditioned media of Th1, Th17 and regulatory T cells differentiated from naïve T cells in vitro. Only Th1 cell conditioned medium significantly reduced Cx43 expression in astrocytes.
Conclusions: These findings suggest that Th1 cell-derived IFNγ activates microglia to release IL-1β and TNFα, which reduce Cx43 expression in astrocytes. Thus, Th1-dominant inflammatory states can disrupt astrocytic intercellular communication and may exacerbate MS.
Disclosure:
Mitsuru Watanabe is funded by a research grant from Japan Society for the Promotion of Science, Japan;
Katsuhisa Masaki: nothing to disclose;
Ryo Yamasaki: nothing to disclose;
Jun Kawanokuchi: nothing to disclose;
Hideyuki Takeuchi: nothing to disclose;
Akio Suzumura: nothing to disclose;
Jun-ichi Kira is a consultant for Biogen Idec Japan and Medical Review. He has received honoraria from Bayer Healthcare, Mitsubishi Tanabe Pharma, Nobelpharma, Otsuka Pharmaceutical and Medical Review. He is funded by a research grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan and grants from the Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology, Japan. Source of funding: This study was supported by “Glial assembly” Grant-in-Aid for Scientific Research on Innovative Areas (Grant Number 25117012) from the Ministry of Education, Culture, Sports, Science and Technology, and Grant-in-Aid for Young Scientists (Grant Number 15K19489) from Japan Society for the Promotion of Science, Japan.