Clarithromycin Modulates Neutrophilic Inflammation Induced by Prevotella intermedia in Human Airway Epithelial Cells

Abstract

Objectives: In the present study, we aimed to clarify the mechanisms by which periodontal pathogens, particularly Prevotella intermedia, induce severe neutrophilic inflammation. In addition, we aimed to test the efficacy of macrolides, which has not been resolved in the neutrophilic inflammation induced by P. intermedia. Methods: NCl-H292 human airway epithelial cells were pre-incubated with clarithromycin for 2 h before incubation with P. intermedia supernatants. Then, C-X-C motif chemokine ligand 8 (CXCL8) transcription and interleukin (IL)-8 production were measured. To elucidate the signaling pathway, mitogen-activated protein kinase inhibitors were added to the cell culture, and the cells were subjected to Western blotting. Results:P. intermedia supernatants promoted CXCL8 transcription and IL-8 production, and the reactions were significantly suppressed by clarithromycin pretreatment. Only trametinib, the selective mitogen-activated extracellular signal-regulated kinase inhibitor, downregulated CXCL8 transcription and IL-8 production. Furthermore, Western blotting revealed that stimulation with P. intermedia supernatants specifically induces extracellular signal-regulated kinases (ERK) 1/2 phosphorylation, which is suppressed by clarithromycin pretreatment. Notably, the interference analysis revealed that ERK3 might be dispensable for IL-8 production under the stimulation of P. intermedia supernatants. Conclusions: Our results provide new insight into the mechanism underlying P. intermedia-induced production of IL-8 from human airway epithelial cells. Furthermore, macrolides might have therapeutic potential in regulating periodontal pathogen-induced neutrophilic inflammation in the lungs.

Keywords: Prevotella intermedia; clarithromycin; human airway epithelial cells.

Figure 1

Enhancement of CXCL8 mRNA expression and IL-8 production by P. intermedia supernatants and inhibition of the reactions by clarithromycin pretreatment. (A) CXCL8 mRNA expression in H292 cells was assessed using RT-PCR under co-incubation with P. intermedia supernatants for 4 h. P. intermedia supernatants enhanced CXCL8 mRNA expression in a protein concentration-dependent manner (n = 6). (B,C) 50 μg/mL of clarithromycin pretreatment was administered for 2 h before stimulation with P. intermedia supernatants. CXCL8 mRNA expression post 4 h (B) and IL-8 production post 24 h incubation (C) were inhibited by clarithromycin pretreatment (n = 4−6). Data are presented as mean +/− standard error of the mean (SEM), and each figure is representative from two independent experiments. Significant differences are determined using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; CAM, clarithromycin.

Figure 2

CXCL8 mRNA expression and IL-8 production by P. intermedia supernatants might be induced through transduction via ERK 1/2. (A) Inhibition of CXCL8 mRNA expression with dimethyl sulfoxide, Trametinib, Adezmapimod, SP600125, and caffeic acid phenethyl ester at a concentration of 10 μM was analyzed using real-time RT-PCR post 4 h incubation (n = 4). (B) CXCL8 mRNA expression was inhibited by Trametinib pretreatment in a concentration-dependent manner (n = 4). (C) IL-8 production post 24 h incubation was inhibited by Trametinib pretreatment (n = 4). (D) CXCL8 mRNA expression was not influenced by Adezmapimod or SP600125 pretreatment in a concentration-dependent manner (n = 4). Data are presented as mean +/− SEM, and each figure is representative from two independent experiments. Significant differences are determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; DMSO, dimethyl sulfoxide; Trametinib, MEK 1/2 inhibitor; Adezmapimod, p38 MAPK inhibitor; SP600125, c-JUN N-terminal kinase inhibitor II; CAPE, NF-kβ inhibitor.

Figure 3

P. intermedia supernatants specifically induce the phosphorylation of ERK1/2 rather than other MAPKs and NF-kB. Western blot analyses under reducing conditions with 2.5% 2-mercaptoethanol of each MAPK and NF-kβ in the cell lysates 60 or 120 min post-stimulation with P. intermedia supernatants are shown (representative from two independent experiments).

Figure 4

Clarithromycin inhibited ERK1/2 phosphorylation induced by P. intermedia supernatants in a concentration-dependent manner. Western blot analyses under reducing conditions with 2.5% 2-mercaptoethanol of total or phosphorylated ERK1/2 in the cell lysates 60 min post-stimulation with P. intermedia supernatants under each concentration of clarithromycin treatment are shown (representative from two independent experiments). CAM, clarithromycin.

Figure 5

ERK3 knockdown upregulated CXCL8 mRNA expression induced by P. intermedia supernatants. After being transfected with 50 nmol/L of ERK3/MAPK6 small interfering RNAs (siRNAs) or control siRNAs for 72 h, the H292 cells were harvested to confirm the efficiency using (A) Western blotting and (B) real-time RT-PCR. (C) Then, the cells were exposed to P. intermedia supernatants for 4 h, and CXCL8 mRNA expression was assessed using real-time RT-PCR. (D) The cells were additionally treated with trametinib, and CXCL8 mRNA expression was assessed similarly. Data are presented as mean +/− SEM. Significant differences are determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.