Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1β Release From NK92 Cells

Abstract

Introduction

Although natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1β (IL-1β), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity.

Methods

NK92 cells were examined for the synthesis and release of IL-1β utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)_1, HCA₂ and HCA₃ was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses.

Results

LPS-induced the release of IL-1β from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis.

Discussion

Our results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1β. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients.  

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Keywords:

• pyroptosis
• gasdermin D
• NK cells
• IL-1β
• dimethyl fumarate
• monomethyl fumarate

Abbreviations

ASC, apoptosis-associated speck-like protein containing CARD domain; ATP, adenosine triphosphate; cDNA, complementary deoxyribose nucleic acid; DAPI, 4ʹ,6-diamidino-2-phenylindole; DMF, dimethyl fumarate; DNMTs, DNA methyltransferases; ELISA, enzyme-linked immunosorbent assay; GDNA, genomic deoxyribose nucleic acid; GSDMD, gasdermin-D; HCA, hydroxy-carboxylic acid receptors; IFN-γ, interferon-gamma; IL, interleukin; LPS, lipopolysaccharide; MMF, monomethyl fumarate; MSP-PCR, methylation specific polymerase chain reaction; NK, natural killer; NLRP3, NOD-like receptor pyrin domain containing 3; qPCR, quantitative polymerase chain reaction; TLR, toll-like receptor.

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

AAM is supported by the University of Sharjah grant numbers 1701090222-P and 1701090223-P and by Terry Fox Foundation. JSM is supported by the University of Sharjah grant numbers 1801090131-P and 1801090139, and by Al-Jalila foundation grant number AJF2018036. RAH is supported by Al-Jalila grant number AJF201741 and the Sharjah Research Academy grant number MED001.