Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

Abstract

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.

Keywords: Chemokine; Cytokine; IKKα, inhibitor kappa B kinase alpha; IKKβ, inhibitor kappa B kinase beta; IκBα/β, inhibitor kappa B alpha and beta; LPS, lipopolysaccharide; Lipopolysaccharide (LPS); Mitogen-activated protein kinase (MAPK); NF-κB, nuclear factor-kappa B; Nuclear factor kappa B (NF-κB); Tetracyclines.

Fig. 1

(A) Time-dependent changes in cytokine and chemokine production in LPS-stimulated THP-1 cells. After LPS (10 μg/ml) treatment for 15, 30, 60, or 120 min without any agents and with minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml), cytokines and chemokines were measured using Multi Plex according to the manufacturer’s protocols. * p<0.05 compared with LPS only at 60 min. ** p<0.05 compared with LPS only at 120 min. Mino: minocycline, Tige: tigecycline, Doxy: doxycycline. (B) The rate of cytokine and chemokine production in the THP-1 cell line compared to the production of cytokines and chemokines by LPS stimulation without tetracyclines. After LPS treatment (10 μg/ml) for 30, 60, 120 or 240 min without any agents and with minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 mg/ml), cytokines and chemokines were measured with Multi Plex.

Fig. 2

Effects of minocycline, doxycycline, and tigecycline on the modulation of NF-κB, phospho-NF-κB, IKKα/β, phospho-IKKα/β, IκBα, and phospho-IκBα in LPS-stimulated THP-1 cells. THP-1 cells were incubated without or with 10 μg/ml LPS, or with LPS plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 30, 60, or 120 min. NF-κB, phospho-NF-κB, IKKα/β, phospho-IKKα/β, IκBα, and phospho-IκBα were assessed with Western blotting.

Fig. 3

Effects of tetracyclines (minocycline, doxycycline, and tigecycline) on the activation of phospho-ERK1/2 and phospho-p38 in LPS-stimulated THP-1 cells. THP-1 cells were incubated without or with 10 μg/ml LPS, or with LPS plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 30 or 60 min. Phospho-p38 and phospho-ERK1/2 were assessed with Western blotting.

Fig. 4

SB203580, U0126 and BAY11-7082 suppressed TNF-α and IL-8 production in LPS-stimulated THP-1 cells on treatment with or without tetracyclines. THP-1 cells were pre-incubated by SB203580 (10 μM), U0126 (5 μM) and BAY11-7082 (5 μM) for 30 min, followed treatment without or with LPS (10 μg/ml), or with LPS (10 μg/ml) plus minocycline (50 μg/ml), doxycycline (50 μg/ml), or tigecycline (50 μg/ml) for 60 min. TNF-α were measured with ELISA. *p<0.05, **p<0.01, ***p<0.001 compared to the measurement without the inhibitor in the same group. Abbreviation; SB: SB203580, U: U0126, BAY: BAY11-7082.