IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

Abstract

Background Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cells infiltration and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE. Methods In vitro, shRNA-recombinant lentivirus with Glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR and Cytometric bead array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or student’s t-test, as appropriate. Results Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9 activated astrocytes. In which, Gm13568 associates with CBP/P300 is enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process. Conclusions These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.

The LncRNA AK018453 regulates TRAP1/Smad signaling in IL‐17‐activated astrocytes: A potential role in EAE pathogenesis

“…ChIP assay was used as described previously (Liu et al, 2021; Tsai et al, 2010). Briefly, primary mouse astrocytes were stimulated with or without IL‐17 for 6 h, then cells were treated with 1% formaldehyde, and stopped by 1.25 mM of glycine.…”

“…Primary mouse astrocytes were cultured as described previously (Liu et al, 2014, 2019, 2021). Astrocytes were synchronized with non‐serum culture media for 12–16 h prior to IL‐17 treatment.…”

“…The lentivirus vector system contains the basic element of HIV, 5′ LTR and 3′ LTR and other auxiliary components, such as WPRE (woodchuck hepatitis virus post transcriptional regulatory element). Recombinant lentiviral vectors carrying with EGFP and with astrocyte‐specific promoter of GFAP (Liu et al, 2019, 2021) were constructed by Gene Chem (Shanghai, China), which can cross the blood–brain barrier. To silence the mouse TRAP1 and lncRNA AK018453, shRNA sequences against them were designed.…”

“…RIP assay was performed as previously described (Liu et al, 2021; Tsai et al, 2010). Briefly, after stimulating with IL‐17 (50 ng/ml), primary mouse astrocytes were treated with 0.3% formaldehyde in DMEM/F12 medium for 10 min at 37°C.…”