Yonsei Med J.  2021 Nov;62(11):1023-1031. 10.3349/ymj.2021.62.11.1023.

Activation of CpG-ODN-Induced TLR9 Signaling Inhibited by Interleukin-37 in U937 Human Macrophages

• Kim SK
• Choe JY
• Park KY

• KMID: 2521453
• DOI: http://doi.org/10.3349/ymj.2021.62.11.1023

Abstract

Purpose
Interleukin-37 (IL-37) is an anti-inflammatory cytokine that inhibits a broad spectrum of inflammatory responses in various human cells, including neutrophils, macrophages, and endothelial cells. The aim of this study was to identify the role of IL-37 in toll-like receptor 9 (TLR9) signaling in human macrophages.
Materials and Methods
Human macrophage U937 cells treated with CpG-oligonucleotides (CpG-ODN), recombinant IL-37, or dexamethasone were used in an in vitro study. IL-37 small interfering RNA (siRNA) and TLR9 siRNA were used to silence endogenous IL-37 and TLR9, respectively. Expression levels of phosphorylated nuclear factor-κB (NF-κB), IκBα, IL-37, IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 protein were assessed by real-time quantitative polymerase chain reaction and Western blotting. CpG-ODN-mediated IL-37 expression stimulated by dexamethasone was detected using immunofluorescent analysis.
Results
U937 cells treated with CpG-ODN induced activation of the NF-κB pathway and increased the expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, but reduced that of IL-37. Recombinant IL-37 attenuated phosphorylation of NF-κB and IκBα and the expression of IL-1β, TNF-α, and IL-6 stimulated by CpG-ODN. Human macrophages transfected with IL-37 siRNA augmented the expression of IL-1β, TNF-α, and IL-6 mRNA and protein in cells treated with CpG-ODN. Dexamethasone markedly inhibited expression of pro-inflammatory cytokines in U937 cells, whereas IL-37 expression was increased with the addition of dexamethasone. Inflammatory responses elicited by CpG-ODN were dependent on an MyD88-TRAF6 pathway. IL-37 inhibited CpG-ODN-induced ubiquitination of TRAF6 in U937 macrophages.
Conclusion
IL-37 inhibits CpG-ODN-mediated inflammatory responses through regulation of a TRAF6- NF-κB pathway in human macrophages.

Keyword

IL-37; TLR9; CpG-ODN; NF-κB; cytokine