FTY720 Modulates Microglia Toward Anti-inflammatory Phenotype by Suppressing Autophagy via STAT1 Pathway

Abstract

Since microglia-associated neuroinflammation plays a pivotal role in the progression of white matter diseases, modulating microglial activation has been suggested as a potential therapeutic strategy. Here, we investigated the anti-inflammatory effects of fingolimod (FTY720) on microglia and analyzed the crosstalk between microglia autophagy and neuroinflammation. Lipopolysaccharide (LPS)-induced primary cultured microglia model was established. Microglial phenotypes were assessed by Western blot, quantitative real-time polymerase chain reaction (RT-PCR) and flow cytometry. Autophagy was evaluated by immunofluorescence, MDC staining and Western blot. Rapamycin was used to investigate the role of autophagic process in regulating microglial phenotypes. The signaling markers were screened by RT-PCR and Western blot. FTY720 shifted microglial phenotype from pro-inflammatory state to anti-inflammatory state and inhibited microglial autophagy under lipopolysaccharide (LPS) treatment. Rapamycin reversed the effect of FTY720 on phenotype transformation of microglia. The results of mechanism studies have shown that FTY720 notably repressed LPS-induced STAT1 activity, which was reactivated by rapamycin. Our research suggested that FTY720 could significantly transform pro-inflammatory microglia into anti-inflammatory microglia by suppressing autophagy via STAT1.

Keywords: Autophagy; FTY720; Microglia polarization; STAT1.

Fig. 1

FTY720 skewed LPS-induced pro-inflammatory state to anti-inflammatory state in primary microglia. Primary microglial cells were exposed to vehicle as Control, LPS stimulation, LPS stimulation plus FTY720 treatment, and IL-4 stimulation. a The protein expression of pro-inflammatory phenotype markers (iNOS, CD16), anti-inflammatory phenotype (CD206, Arg-1) in microglia were detected by western blot. n = 8 per group. b Percentage of CD86- or CD206-positive cells in total CD11b-positive microglia was detected by flow cytometry. n = 6 per group. c The expression of pro-inflammatory microglia markers (IL-1b, TNF-a, iNOS, CD86) and anti-inflammatory microglia markers (FIZZ1, CD163, IL-10, YM-1) was detected by RT-PCR. n = 6 per group. Quantitative analysis was performed. One-way ANOVA with Dunnett’s post-hoc test, **p < 0.01 versus Control, ^##p < 0.01 versus LPS group, ^△△p < 0.01 versus LPS + FTY720 group

Fig. 2

FTY720 repressed autophagy in microglia. a Autophagic Flux markers (Beclin1, P62, LC3II/LC3I) were detected by western blot. b Autophagy was measured by MDC. Autophagic Flux markers (P62, LC3B) were detected by immunofluorescent staining. Quantitative analysis was performed. One-way ANOVA with Dunnett’s post-hoc test, **p < 0.01 versus Control, ^##p < 0.01 versus LPS group. n = 8 per group

Fig. 3

FTY720 altered microglia phenotype toward anti-inflammatory phenotype via inhibiting autophagy. With the treatment of autophagy stimulator RAPA, the protein expression of pro-inflammatory phenotype markers (iNOS, CD16) and anti-inflammatory phenotype (CD206, Arg-1) in microglia was reversed compared with LPS plus FTY720 group; the results were detected by western blot. Quantitative analysis was performed. One-way ANOVA with Dunnett’s post-hoc test, *p < 0.05 and **p < 0.01 versus LPS group, ^##p < 0.01 versus LPS + FTY720 group. n = 8 per group

Fig. 4

FTY720 facilitated pro-inflammatory microglia to anti-inflammatory microglia by suppressing autophagy via STAT1 signaling markers. a The mRNA expression of JAK2, STAT1, NLRP3, STAT5α, JAK1, TRAF6, PPARγ, IRF3, IRF4, JAK3, STAT3, NF-κB, STAT6, IRF8, IRF5 and MSX3 were detected by RT-PCR. b The protein expression of STAT1 in primary microglia was detected by western blot. c Quantitative analysis was performed. One-way ANOVA with Dunnett’s post-hoc test, **p < 0.01 versus Control, ^##p < 0.01 versus LPS group, ^△△p < 0.01 versus LPS + FTY720 group. n = 8 per group

Fig. 5

Summary figure. FTY720 could alleviate microglial inflammatory state through suppressing autophagy via STAT1