NF-κB-Driven STAT2 and CCL2 Expression in Astrocytes in Response to Brain Injury

Abstract

Tissue response to injury includes expression of genes encoding cytokines and chemokines. These regulate entry of immune cells to the injured tissue. The synthesis of many cytokines and chemokines involves NF-κB and signal transducers and activators of transcription (STAT). Injury to the CNS induces glial response. Astrocytes are the major glial population in the CNS. We examined expression of STATs and the chemokine CCL2 and their relationship to astroglial NF-κB signaling in the CNS following axonal transection. Double labeling with Mac-1/CD11b and glial fibrillary acidic protein revealed that STAT2 up-regulation and phosphorylation colocalized exclusively to astrocytes, suggesting the involvement of STAT2 activating signals selectively in astroglial response to injury. STAT1 was also up-regulated and phosphorylated but not exclusively in astrocytes. Both STAT2 up-regulation and phosphorylation were NF-κB -dependent since they did not occur in the lesion-reactive hippocampus of transgenic mice with specific inhibition of NF-κB activation in astrocytes. We further showed that lack of NF-κB signaling significantly reduced injury-induced CCL2 expression as well as leukocyte infiltration. Our results suggest that NF-κB signaling in astrocytes controls expression of both STAT2 and CCL2, and thus regulates infiltration of leukocytes into lesion-reactive hippocampus after axonal injury. Taken together, these findings indicate a central role for astrocytes in directing immune-glial interaction in the CNS injury response.

Injury-Induced Type I IFN Signaling Regulates Inflammatory Responses in the Central Nervous System

“…Adult female mice deficient in STAT1 (STAT1-KO) 129s6/SvEv background (23) were purchased from Taconic Farms (Germantown, NY), IFNAR-KO on 129s6/SvEv background were purchased from B&K Universal Limited (Hull, U.K.), IRF7-KO on C57BL/6 background were purchased from Rikendentate perforant path projection was transected as described previously (21).…”

“…For dual-labeling immunofluorescence, sections were incubated with rabbit anti-IRF7 and with rat anti-mouse Mac-1/CD11b or monoclonal mouse anti-GFAP-cy3-conjugated Abs (C9205; Sigma-Aldrich), followed by incubation with fluorescent secondary Abs, Alexa 488-labeled goat antirabbit (A11034; Invitrogen, Taastrup, Denmark) and Alexa 568-labeled goat anti-rat (A11077; Invitrogen), as described previously (21). Images were acquired using either an Olympus BX51 microscope (Olympus, Ballerup, Denmark) connected to an Olympus DP71 digital camera and a Confocal laser scanning fluorescence microscope (Olympus FV1000; Olympus, Hamburg, Germany).…”

“…Immunohistochemical staining was performed as described previously (21). In brief, after blocking endogenous peroxidases, the sections were incubated overnight with rabbit anti-IRF7 Ab (SC-9083; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse Mac-1/CD11b (MCA711; Serotec, Oxford, U.K.) (to identify microglia/macrophages), or rabbit anti-GFAP (Z0334; DakoCytomation, Glostrup, Denmark) (to identify astrocytes), and in the following day, the sections were incubated with secondary biotinylated Ab (Amersham Biosciences, Little Chalfont, U.K.).…”

“…Involvement of TLR signaling as well as cytokines (TNF-a and IL-1b) and chemokines (CCL2 and CXCL10) in glial response and recruitment of leukocytes in the lesion-reactive hippocampus have been described previously (15,17,21,22). We previously described glial upregulation of STAT1 and STAT2 (21), which suggested the involvement of type I IFN signaling.…”

“…The transection of projections from entorhinal cortex to the hippocampus leads to anterograde axonal degeneration and loss of synapses in the outer molecular layer of the dentate gyrus, followed by glial activation, leukocyte infiltration, and sprouting (13)(14)(15)(16)(17)(18)(19)(20). Involvement of TLR signaling as well as cytokines (TNF-a and IL-1b) and chemokines (CCL2 and CXCL10) in glial response and recruitment of leukocytes in the lesion-reactive hippocampus have been described previously (15,17,21,22). We previously described glial upregulation of STAT1 and STAT2 (21), which suggested the involvement of type I IFN signaling.…”