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Objective To investigate the role and the possible molecular mechanism of RNA methylase, Wilms' tumor 1-associating protein (WTAP), in microglial activation. Methods HMC3 cells was induced to M1 polarization by treatment of 100 ng/mL IFNγ combined with 1 μg/mL LPS and then served as experimental group, and the untreated cells served as control group. The expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and N6-methyladenosine (m6A) at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting, respectively. After HMC3 cells were transfected with RNAi lentivirus to knock down WTAP, the knockdown efficiency was detected by real-time quantitative PCR and Western blotting. Western blot assay was employed to measure the expression levels of pro-inflammatory cytokines IL-6 IL-1β and TNF-α in WTAP knockdown HMC3 cells. Western blotting was also adopted to detect the protein levels of silent mating-type information regulation 2 homolog 1 (SIRT1), COP1, USP18, EP4, pro-inflammatory transcription factors and some inflammatory pathway receptors in the knockdown cells. SIRT1 agonist SRT1720 and SIRT1 inhibitor EX527 were employed to treat HMC3 cells in the blank-virus group and the knockdown WTAP group, respectively, and the expression level of C/EBPβ was detected by Western blotting. Results Treatment of 100 ng/mL IFNγ combined with 1 μg/mL LPS induced significant up-regulation of IL-6, IL-1β and TNF-α expression in HMC3 cells as compared with the untreated cells (P < 0.05). WTAP was highly expressed in M1 polarized HMC3 microglia (P < 0.05). WTAP knockdown resulted in obviously decreased expression levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α (P < 0.05), increased level of SIRT1 (P < 0.05), and reduced pro-inflammatory transcription factor C/EBPβ (P < 0.05). SIRT1 agonist SRT1720 reduced the relatively high expression of C/EBPβ in the blank-virus group (P < 0.01). While, the inhibitor EX527 increased the relative low expression of C/EBPβ in HMC3 cells with low WTAP knockdown (P < 0.05). Conclusion WTAP promotes M1 polarization in HMC3 cells, possibly through regulating the expression of SIRT1 and thus affecting the C/EBPβ signaling pathway.

Autoimmune uveitis (AU) is a kind of immune‐mediated disease resulting in irreversible ocular damage and even permanent vision loss. However, the precise mechanism underlying dynamic immune changes contributing to disease initiation and progression of AU remains unclear. Here, we induced an experimental AU (EAU) model with IRBP651‐670 and found that day[D]14 was the inflammatory summit with remarking clinical and histopathological manifestations and the activation of retinal microglia exhibited a time‐dependent pattern in the EAU course. We conducted single‐cell RNA sequencing of retinal immune cells in EAU mice at four time points and found microglia constituting the largest proportion, especially on D14. A novel inflammatory subtype (Cd74high Ccl5high) of retinal microglia was identified at the disease peak that was closely associated with modulating immune responses. In vitro experiments indicated that inflammatory stimuli induced proinflammatory microglia with the upregulation of CD74 and CCL5, and CD74 overexpression in microglia elicited their proinflammatory phenotype via nuclear factor‐kappa B signaling that could be attenuated by the treatment of neutralizing CCL5 antibody to a certain extent. In‐vivo blockade of Cd74 and Ccl5 effectively alleviated retinal microglial activation and disease phenotype of EAU. Therefore, we propose targeting CD74 and CCL5 of retinal microglia as promising strategies for AU treatment. [ABSTRACT FROM AUTHOR]