Lipopolysaccharide and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts

Abstract

Both lipopolysaccharide (LPS) and interleukin (IL)-1β activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1β interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1β on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1β cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1β exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1β on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1β of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1β, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1β. Taken together, this study showed that LPS and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.

Keywords: Inflammation; Interleukin-1 beta; Lipopolysaccharide; MyD88.

Figure 1

The synergistic effect of IL-1β and Pg LPS on proinflammatory cytokine expression. Human gingival fibroblasts were treated with 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 24 h and the secreted IL-6 (A), granulocyte-macrophage colony-stimulating factor (GM-CSF) (B) and IL-1β (C) in culture medium were quantified using ELISA. The data (mean ± SD) presented are from one of three independent experiments with similar results.

Figure 2

Time course of IL-6 secretion from human gingival fibroblasts stimulated with IL-1β, Pg LPS or both. Human gingival fibroblasts were treated with 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 24 h and the secreted IL-6 in culture medium was quantified using ELISA at 4, 8, 24 h during the treatment. The data (mean ± SD) presented are from one of three independent experiments with similar results.

Figure 3

The synergistic effect of IL-1β and Pg LPS on MyD88 mRNA and protein expression. A. Human gingival fibroblasts were treated with 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 24 h and MyD88 mRNA was quantified at 4, 8, 24 h using real-time PCR during the treatment. The data (mean ± SD) presented are from one of three independent experiments with similar results. B. Human gingival fibroblasts were treated with 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 12 h and MyD88 was detected using immunoblotting.

Figure 4

The effect of MyD88 knockdown on IL-6 secretion induced by Pg LPS, IL-1β or both. Human gingival fibroblasts were transfected with 200 nM of MyD88 siRNA or scrambled siRNA (control siRNA) for 24 h. After the transfection, MyD88 knockdown by siRNA was confirmed using real-time PCR (A). The transfected cells were then treated with or without 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 24 h and IL-6 in culture medium was quantified using ELISA (B). The data (mean ± SD) presented are from one of three independent experiments with similar results.

Figure 5

The cooperative effect of Pg LPS and IL-1β on phosphorylation of ERK, JNK and p38 MAPK. Human gingival fibroblasts were treated with or without 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 3, 10, 20, and 30 min. At each time point, cells were harvested for immunoblotting of phosphorylated and total ERK (A), JNK (B) and p38 MAPK (C). The intensity of phosphorylated ERK, JNK and p38 MAPK was quantified and normalized to that of total ERK, JNK and p38 MAPK, respectively. The data (mean ± SD) presented are from one of three independent experiments with similar results.

Figure 6

The cooperative effect of Pg LPS and IL-1β on NFκB transcriptional activity. Human gingival fibroblasts were transfected with NFκB promoter-containing luciferase reporter vectors for 24 h and then treated with or without 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both for 24 h. After the treatment, cells were harvested for luciferase activity assays. The data are mean ± SD of three independent experiments.

Figure 7

Inhibition by pharmacological inhibitors of IL-6 secretion stimulated by Pg LPS, IL-1β or both. Human gingival fibroblasts were treated with or without 100 pg/ml of IL-1β, 1 µg/ml of Pg LPS or both in the absence or presence of 5 or 10 µM of PD98059 (PD), inhibitor for ERK pathway (A); 5 or 10 µM of SP600126 (SP) (A), inhibitor for JNK pathway; 5 or 10 µM of SB203580 (SB) (B), inhibitor for p38 MAPK pathway; or 1 or 5 µM of Bay11-7082 (Bay) (B), inhibitor for NFκB pathway, for 24 h. After the treatment, IL-6 in culture medium was quantified using ELISA. The data (mean ± SD) presented are from one of three independent experiments with similar results.