IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

Nadeem Akhter; Shihab Kochumon; Amal Hasan; Ajit Wilson; Rasheeba Nizam; Ashraf Al Madhoun; Fatema Al-Rashed; Hossein Arefanian; Fawaz Alzaid; Sardar Sindhu; Fahd Al-Mulla; Rasheed Ahmad

doi:10.2147/JIR.S368352

IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

J Inflamm Res. 2022 Jul 27:15:4291-4302. doi: 10.2147/JIR.S368352. eCollection 2022.

Authors

Nadeem Akhter¹, Shihab Kochumon¹, Amal Hasan¹, Ajit Wilson¹, Rasheeba Nizam², Ashraf Al Madhoun^{2

3}, Fatema Al-Rashed¹, Hossein Arefanian¹, Fawaz Alzaid^{2

4}, Sardar Sindhu^{1

3}, Fahd Al-Mulla¹, Rasheed Ahmad¹

Affiliations

¹ Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City, Kuwait.
² Genetics & Bioinformatics, Dasman Diabetes Institute, Kuwait City, Kuwait.
³ Animal and Imaging Core Facility, Dasman Diabetes Institute, Kuwait City, Kuwait.
⁴ Institut Necker Enfants Malades (INEM), French Institute of Health and Medical Research (INSERM), Immunity & Metabolism of Diabetes (IMMEDIAB), Université de Paris Cité, Paris, France.

Abstract

Background: Overexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity.

Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR.

Results: Our results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy.

Conclusion: Our findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Keywords: CCL2; H3K27 ac; IFN-γ; LPS; inflammation; monocytes/macrophages.

Grants and funding

This research was funded by Kuwait Foundation for the Advancement of Sciences, grant number RA AM 2016-007.