Interleukin‐21 enhances Toll‐like receptor 2/4‐mediated cytokine production via phosphorylation in the STAT3, Akt and p38 MAPK signalling pathways in human monocytic THP‐1 cells

Abstract

Interleukin-21 (IL-21) is a type I cytokine produced by activated T cells that promotes cytokine production in monocytes. Monocytes are activated by Toll-like receptors (TLRs) to produce pro-inflammatory mediators. However, little is known about the regulatory effect of IL-21 on TLR-mediated inflammation in human monocytes. This study investigated the potential association between IL-21 and TLR2/4mediated inflammation in human monocytic THP-1 cells. First, the expression of the IL-21 receptor (IL-21R) in human monocytic THP-1 cells was examined by flow cytometry. Then, THP-1 cells were treated with either the TLR2 ligand peptidoglycan (PGN) or the TLR4 ligand lipopolysaccharide (LPS) with or without IL-21.Then, the production of several cytokines (IL-6, IL-8, TNF-α, IFN-γ and IL-10), expression of TLR2/4, and activation of the downstream signaling pathways of mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and nuclear factor-kappa B (NF-κB) were determined. We found that IL-21R was highly expressed in human monocytic THP-1 cells and that IL-21 induced TLR2 and TLR4 expression and further enhanced PGN/LPS-mediated TLR2/4 expression. In addition, IL-21 also upregulated the expression of IL-6, IL-8, TNF-α, IFN-γ and IL-10 and enhanced TLR2/4-mediated cytokine production in THP-1 cells via phosphorylation of the STAT3, Akt and p38 MAPK signalling pathways. Our study suggests, for the first time that IL-21 enhances TLR2/4-mediated cytokine production in human monocytic THP-1 cells by activating the STAT3, PI3K/Akt and p38 MAPK signalling pathways.

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“…Specifically, IL-1β is a pro-inflammatory cytokine produced in a variety of cells, including monocytes, tissue macrophages, keratinocytes, and other epithelial cells [51]. IL-6 functions in inflammation and the maturation of ** [52] B cells through activation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT1 and STAT3) (54). IL-8 is a pro-inflammatory and growth-promoting factor [50,53].…”

“…IL-10 is a type II cytokine with numerous immunosuppressive and antiinflammatory capacities, playing a role in the inhibition of antigen-presenting cells, including dendritic cells, monocytes, and macrophages [50]. IL-21 is a type I cytokine produced by activated T cells that promotes cytokine production in monocytes [54]. TNF-α is a potent pro-inflammatory cytokine exerting pleiotropic effects on various cell types [55].…”

IL‐21 regulates macrophage activation in human monocytic THP‐1‐derived macrophages

“…To explore the macrophage polarization, macrophages were harvested, washed, and stained with fluorescein Percp/Cy5.5‐conjugated anti‐human CD86, PE/Cy7‐conjugated anti‐human CD80, PE‐conjugated anti‐CD206, BV421‐conjugated anti‐human CD163. Finally, the above cells were washed three times with PBS, then resuspended in fluorescence‐activated cell sorting (FACS) buffer, finally analyzed using a FACS Calibur Flow Cytometer (BD Biosciences) 9 …”

“…To study the regulatory function of IL‐21, THP‐1‐derived macrophages were cultured for 24 h in the presence or absence of IL‐21 (50 ng/ml) and subsequently stimulated with LPS (100 ng/ml) for 24 h. To determine the regulatory function of IL‐21 in macrophage polarization in vitro, M1/M2 macrophages were incubated with or without IL‐21 for 24 h. After incubation, the culture supernatants were collected, followed by the determination of TNF‐α, IL‐6, IL‐8, and IL‐10 secretion using respective Enzyme‐linked Immunosorbent Assay (ELISA) Kits (eBioscience), according to the manufacturer's protocol 9 …”

“…As mentioned in our previous article, 9 total RNA was extracted, quantified, and reverse‐transcribed to complementary DNA. The primers for each gene were synthesized by Sangon Biotech listed in Table 1.…”