TRAF6 upregulation in spinal astrocytes maintains neuropathic pain by integrating TNF-α and IL-1β signaling

The proinflammatory cytokines tumor necrosis factor (TNF) alpha and interleukin (IL) 1 beta have been strongly implicated in the pathogenesis of neuropathic pain, but the intracellular signaling of these cytokines in glial cells is not fully understood. TNF receptor-associated factor 6 (TRAF6) plays a key role in signal transduction in the TNF receptor superfamily and the IL-1 receptor superfamily. In this study, we investigated the role of TRAF6 in neuropathic pain in mice after spinal nerve ligation (SNL). SNL induced persistent TRAF6 upregulation in the spinal cord. Interestingly, TRAF6 was mainly colocalized with the astrocytic marker glial fibrillary acidic protein on SNL day 10 and partially expressed in microglia on SNL day 3. In cultured astrocytes, TRAF6 was upregulated after exposure to TNF-alpha or IL-1 beta. TNF-alpha or IL-1 beta also increased CCL2 expression, which was suppressed by both siRNA and shRNA targeting TRAF6. TRAF6 siRNA treatment also inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) in astrocytes induced by TNF-alpha or IL-1 beta. JNK inhibitor D-NKI-1 dose-dependently decreased IL-1 beta-induced CCL2 expression. Moreover, spinal injection of TRAF6 siRNA decreased intrathecal TNF-alpha- or IL-1 beta-induced allodynia and hyperalgesia. Spinal TRAF6 inhibition via TRAF6 siRNA, shRNA lentivirus, or antisense oligodeoxynucleotides partially reversed SNL-induced neuropathic pain and spinal CCL2 expression. Finally, intrathecal injection of TNF-alpha-activated astrocytes induced mechanical allodynia, which was attenuated by pretreatment of astrocytes with TRAF6 siRNA. Taken together, the results suggest that TRAF6, upregulated in spinal cord astrocytes in the late phase after nerve injury, maintains neuropathic pain by integrating TNF-alpha and IL-1 beta signaling and activating the JNK/CCL2 pathway in astrocytes. (c) 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.