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. 2022 Mar 1:13:813979.
doi: 10.3389/fimmu.2022.813979. eCollection 2022.

ALKBH5-Mediated m6A Modification of A20 Regulates Microglia Polarization in Diabetic Retinopathy

Tingting Chen  1 , Wenhui Zhu  1 , Congyao Wang  1 , Xia Dong  1 , Fenfen Yu  1 , Yihua Su  1 , Jingwen Huang  1 , Lijun Huo  1 , Pengxia Wan  1
Affiliations

ALKBH5-Mediated m6A Modification of A20 Regulates Microglia Polarization in Diabetic Retinopathy

Tingting Chen et al. Front Immunol. .

Abstract

Background: To investigate the role of microglia polarization in the pathogenesis of diabetic retinopathy, and study the mechanism of ALKBH5-mediated m6A modification of A20 of retinal microglia polarization.

Methods: Diabetics rats were constructed and the M1/M2 polarization of retinal microglia was determined using immunofluorescence, flow cytometry, and quantitative real-time PCR (qRT-PCR). Glucose at different concentrations was added to treat the microglia, and the polarization rate was detected. RNA sequencing was performed to identify the differentially expressed gene in glucose treated microglia, and A20 expression was confirmed by qRT-PCR and western blotting. Lentiviruses encoding shRNA for A20 or overexpressing A20 were constructed to clarify the role of A20 in microglia polarization in vitro and vivo. N6-methyladenosine (m6A) modification level and degradation rate of A20 were determined and m6A related proteins were detected.

Results: Diabetics rats showed a higher M1 polarization rate but lower M2 polarization rate of retinal microglia. With the increase of glucose concentration, microglia tend to polarize into M1 inflammatory type rather than M2 anti-inflammatory type. Shown by RNA sequencing, glucose treated microglia showed a differentially expressed gene profile, which was enriched in kinds of inflammatory categories and pathways. A20 expression was lower in microglia with glucose treatment, which was demonstrated to negatively regulate the M1 polarization. Moreover, intraocular injection of A20-overexpression lentiviruses (OE-A20) rectified the enhanced M1 retinal microglia polarization of diabetes rats. The higher m6A modification level and faster degradation rate of A20 was observed in glucose treated microglia, which was mediated by m6A demethylase ALKBH5.

Conclusion: Lower expression A20 resulted in the enhanced M1 polarization of retinal microglia in diabetic retinopathy, which was caused by ALKBH5 mediated m6A modification. This study may provide new perspectives on not only the pathogenesis but also the diagnosis and treatment for diabetic retinopathy.

Keywords: A20; ALKBH5; diabetic retinopathy; m6A modification; microglia polarization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WS declared a shared affiliation with the authors to the handling editor at the time of review.

Figures

Figure 1
Figure 1
Increased M1 polarization but decreased M2 polarization of retinal microglia in diabetes rats. (A) The percentage of CD16+ M1 microglia increased from 0 to 3 months after STZ induction. The percentage of CD206+ M2 microglia decreased at the 2 and 3 months after STZ induction. (B) The mRNA levels of IL-1β, IL-6 and TNF-α increased from 0 to 3 months after STZ induction. The mRNA levels of IL-4, IL-10 and TGF-β decreased from 0 to 3 months after STZ induction. (C) The 3 months after STZ induction group have more CD16+ M1 microglia but fewer CD206+ M2 microglia compared to the control group. Values are presented as the mean ± SD. ** indicates P < 0.01, and *** indicates P < 0.001.
Figure 2
Figure 2
Glucose promoted M1 polarization but inhibited M2 polarization of microglia. (A) The percentage of CD16/32+ M1 microglia gradually increased with glucose treatment from the concentration of 0 to 100 mM. (B) The percentage of CD206+ M2 microglia gradually decreased with glucose treatment from the concentration of 0 to 100 mM. (C) The secretion levels of IL-1β, IL-6 and TNF-α up-regulated with the increase of glucose concentration, and the secretion levels of IL-4, IL-10 and TGF-β down-regulated with the decrement of glucose concentration. Values are presented as the mean ± SD. ** indicates P < 0.01, and *** indicates P < 0.001.
Figure 3
Figure 3
A20 expression of microglia decreased with glucose stimulation. (A) The heatmap of differentially expression gene profiles. (B) The top 10 enriched categories of molecular function in GO analysis. (C) The top 10 enriched categories of biological processes in GO analysis. (D) The top 10 enriched signal pathways of KEGG analysis. (E) The PPI network of differentially expressed mRNA. (F) The A20 expression at mRNA level decreased from 10 to 100 mM glucose treatment. (G) The A20 expression at protein levels decreased with 50 and 100 mM glucose treatment. Values are presented as the mean ± SD. ** indicates P < 0.01, and *** indicates P < 0.001.
Figure 4
Figure 4
A20 negatively regulated the M1 polarization of BV2 microglia. (A) The percentage of CD16/32+ M1 microglia was higher in the Lv-A20 group than in the Lv-NC group. The percentage of CD206+ M2 microglia was lower in the Lv-A20 group than in the Lv-NC group. (B) The IL-1β, IL-6 and TNF-α expression at mRNA levels were higher in the Lv-A20 group than the Lv-NC group, and the IL-4, IL-10 and TGF-β expression at mRNA levels were lower in the Lv-A20 group than the Lv-NC group. (C) The IL-1β, IL-6 and TNF-α secretion levels were higher in the Lv-A20 group than the Lv-NC group, and the IL-4, IL-10 and TGF-β secretion levels were lower in the Lv-A20 group than the Lv-NC group. (D) The percentage of CD16/32+ M1 microglia was lower in the OE-A20 group than in the OE-NC group. The percentage of CD206+ M2 microglia was higher in the OE-A20 group than in the OE-NC group. (E) The IL-1β, IL-6 and TNF-α expression at mRNA levels were lower in the OE-A20 group than the OE-NC group, and the IL-4, IL-10 and TGF-β expression at mRNA levels were higher in the OE-A20 group than the OE-NC group. (F) The IL-1β, IL-6 and TNF-α secretion levels were lower in the OE-A20 group than the OE-NC group, and the IL-4, IL-10 and TGF-β secretion levels were higher in the OE-A20 group than the OE-NC group. Values are presented as the mean ± SD. *** indicates P < 0.001.
Figure 5
Figure 5
Intravitreal injection of A20-overexpression lentiviruses rectified enhanced M1 polarization of retinal microglia in diabetes rats. (A) The percentage of the CD16+ M1 microglia in the OE-A20 group was lower than that in the OE-NC group. The percentage of the CD206+ M1 microglia in the OE-A20 group was higher than that in the OE-NC group. (B) The mRNA levels of IL-1β, IL-6 and TNF-α were lower in the OE-A20 group compared to the OE-NC group. The mRNA levels of IL-4, IL-10 and TGF-β were higher in the OE-A20 group compared to the OE-NC group. (C) The OE-A20 group has less CD16+ M1 microglia but much more CD206+ M2 microglia compared to the OE-NC group. Values are presented as the mean ± SD. *** indicates P < 0.001.
Figure 6
Figure 6
Decreasing ALKBH5 led to lower A20 expression through m6A modification in microglia with glucose stimulation. (A) The m6A modification level of A20 was higher in the glucose treatment group than in the control group. (B) ALKBH5 expression at protein level decreased with glucose treatment. The expression of METTL3, METTL14 and FTO kept unchanged. (C) Lv-ALKBH5 significantly inhibited the expression of both ALKBH5 and A20. (D) The m6A modification level of A20 was higher in the Lv-ALKBH5 group than in the Lv-NC group. (E) OE-ALKBH5 significantly promoted the expression of both ALKBH5 and A20. (F) The m6A modification level of A20 was lower in the OE-ALKBH5 group than in the OE-NC group. (G) The degradation rate of A20 mRNA shown by t1/2 value was higher in the glucose treatment group than in the control group. (H) Lv-ALKBH5 decreased the t1/2 value, and OE-ALKBH5 increased the t1/2 value of A20 mRNA. Values are presented as the mean ± SD. *** indicates P < 0.001.
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