Microglia-derived TNF-α mediates Müller cell activation by activating the TNFR1-NF-κB pathway

Microglia and its interaction with Müller cells are responsible to retinal surveillance during retinal neurodegeneration, however, the role and mechanism of microglia-derived tumor necrosis factor (TNF)-α in the activation of retinal Müller cells have not been fully elucidated. In the present study, primary microglia and Müller cells were isolated from newborn Sprague-Dawley (SD) rats with purities of 88.2 ± 6.2% and 92.2 ± 2.2%, respectively. By performing immunofluorescence and Western blot analysis, we found that TNF receptor (TNFR)-1 and TNFR2 were expressed in Müller cells. After co-cultured with microglia-conditioned medium (MCM), the elevated mRNA levels of glial fibrillary acidic protein (GFAP), proinflammatory factors (TNF-α, IL-1β, CXCL-1, CSF-1, NOS2, COX2) and decreased CNTF mRNA levels were found in Müller cells. However, pretreatment with R-7050 (a TNF-α receptor inhibitor) or anti-TNFR1 significantly abrogated the changes. Simultaneously, pretreatment with anti-TNFR2 slightly inhibited the expression of GFAP in MCM-incubated Müller cells. Meanwhile, anti-TNFR1 treatment reversed the increased expression of CSF-1 and IL-1β induced by TNF-α. Compared to the control groups, the phosphorylation of NF-κB P65, MAPK P38 and ERK1/2 in TNF-α-treated Müller cells was significantly increased. Nevertheless, pretreatment with anti-TNFR1 inhibited the phosphorylation of NF-κB P65 and MAPK p38, especially NF-κB P65. Additionally, pretreatment with Bay117082 (an NF-κB inhibitor) also significantly inhibited NF-κB P65 phosphorylation and GFAP expression. Moreover, anti-TNFR1 and Bay117082 treatment reduced NF-κB P65 phosphorylation of Müller cells induced by MCM. These results suggested that microglia-derived TNF-α served as a vital role in regulating Müller cells activation during retinal neurodegeneration.