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ROS inhibition increases KDM6A-mediated NOX2 transcription and promotes macrophages oxidative stress and M1 polarization

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Cell Stress and Chaperones Aims and scope

Abstract

Reactive oxygen species (ROS) play an essential role in macrophage polarization. However, the adverse effects of ROS reduction by influencing epigenetics are often ignored. In this study, lipopolysaccharide (LPS) was used to stimulate macrophages to increase the ROS in cells, and N-acetylcysteine (NAC) was used to reduce ROS. Inflammatory factors such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) were used to evaluate the M1 polarization level of macrophages. Chip was used to detect the tri-methylation at lysine 27 of histone H3 (H3K27me3) level at the promoter site. It was found that the decrease of ROS in macrophages would also cause the increase of the H3K27me3 demethylase KDM6A and lead to the reduction of H3K27me3 in the NOX2 promoter, which would increase the transcription level of NOX2 and the production of ROS and ultimately promote the production of inflammatory factors. Knockout of KDM6A can reduce the transcription of NOX2 and the production of ROS of macrophages, thus preventing the M1 polarization of macrophages. The elimination of ROS in macrophages will affect macrophages by increasing KDM6A and making them produce more ROS, thus inducing oxidative stress. In comparison, direct inhibition of KDM6A can reduce ROS production and inhibit macrophage M1 polarization more effectively.

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Funding

This work was supported by grants from the National Key Research and Development Program of China (No.2019YFA0210100)

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Author notes

  1. Yunxi Zhao, Luyang Wang and Mingwei Liu contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiology, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, 210029, Jiangsu, China

    Yunxi Zhao, Luyang Wang, Mingwei Liu, Anning Du, Ming Qiu, Huanyu Shu, Lu Li, Xiangqing Kong & Wei Sun

Authors
  1. Yunxi Zhao
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  2. Luyang Wang
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  3. Mingwei Liu
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  4. Anning Du
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  7. Lu Li
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Contributions

Wei Sun and Xiangqing Kong conceived the project and designed the experiments. Yunxi Zhao and Wei Sun wrote the manuscript. Yunxi Zhao, Luyang Wang, and Mingwei Liu performed most experiments. Ming Qiu, Anning Du, Huanyu Shu, and Lu Li assisted Yunxi Zhao with some experiments. All authors contributed to the article and approved the submitted version.

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Correspondence to Wei Sun.

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Supplementary Information

Fig. 1

: NAC transiently inhibits LPS-induced increase of ROS level in RAW. (A-C) Typical and statistical diagrams of DCFH staining of RAWs treated with 1mg/ml LPS or 3mM NAC for 8h, and RAWs treated with 1mg/ml LPS and 3mM NAC for 8h or 16h, scale bar 100μm. (D) The mRNA levels of NOX2 was detected by qRT PCR after RAWs were treated with 1mg/ml LPS and 3mM NAC for 8h. The above data represent three independent repeated experiments, and the data are presented in the form of mean value ± standard deviation (S.D) *P<0.05, **<0.01, ***<0.001, ****<0.0001, ns, no significance. (PNG 127 kb)

High resolution image (TIF 135 kb)

Fig. 2

: NAC temporarily inhibits LPS induced M1 polarization of macrophages (A-E) The mRNA levels of IL1-β, IL-6, TNF-α, CD80, and CD86 were detected by RT qPCR after RAWs treated with 1mg/ml LPS or 3mM NAC for 8h, and RAWs treated with 1mg/ml LPS and 3mM NAC for 8h or 16h. The above data represent three independent repeated experiments, and the data are presented in the form of mean value ± standard deviation (S.D) *P<0.05, **<0.01, ***<0.001, ****<0.0001, ns, no significance. (PNG 81 kb)

High resolution image (TIF 126 kb)

Fig. 3

: NAC cannot reduce KDM6A transcription in RAW (A-B) The mRNA levels of KDM6A and KDM6B were detected by RT qPCR after RAWs were treated with 1mg/ml LPS and 3mM NAC for 8h (C)The mRNA levels of KDM6B were detected by RT qPCR after THP-1-derived macrophages treated with 1mg/ml LPS and 3mM NAC for 8h. The above data represent three independent repeated experiments, and the data are presented in the form of mean value ± standard deviation (S.D) *P<0.05, **<0.01, ***<0.001, ****<0.0001, ns, no significance. (PNG 1724 kb)

High resolution image (TIF 81.1 kb)

Fig. 4

: Macrophages with different H3K27 methylation levels exist in atherosclerotic plaque (A) Typical diagrams of CD68, H3K27me2, and H3K27me3 level of the aortas of Apoe-/- or C57BL/6 mice fed by western dite for 16 weeks detected by immunofluorescence staining, scale bar 50μm. (High H3K27me2 and high H3K27me3 marked by yellow arrows, low H3K27me2 and low H3K27me3 marked by white arrows.) (PNG 136 kb)

High resolution image (TIF 1.68 mb)

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Zhao, Y., Wang, L., Liu, M. et al. ROS inhibition increases KDM6A-mediated NOX2 transcription and promotes macrophages oxidative stress and M1 polarization. Cell Stress and Chaperones 28, 375–384 (2023). https://doi.org/10.1007/s12192-023-01347-8

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