The antibiotics doxycycline and minocycline inhibit the inflammatory responses to the Lyme disease spirochete Borrelia burgdorferi - PubMed (original) (raw)

The antibiotics doxycycline and minocycline inhibit the inflammatory responses to the Lyme disease spirochete Borrelia burgdorferi

Andrea L F Bernardino et al. J Infect Dis. 2009.

Abstract

Tetracyclines moderate inflammatory responses of various etiologies. We hypothesized that tetracyclines, in addition to their antimicrobial function, could exert control over the inflammation elicited by Borrelia burgdorferi. To model systemic effects, we used the human monocytic cell line THP-1; to model effects in the central nervous system, we used rhesus monkey brain astrocytes and microglia. Cells were stimulated with live or sonicated B. burgdorferi or with the lipoprotein outer surface protein A in the presence of increasing concentrations of doxycycline or minocycline. Both antibiotics significantly reduced the production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8 in a dose-dependent manner in all cell types. Microarray analyses of the effect of doxycycline on gene transcription in spirochete-stimulated monocytes revealed that the NFKB and CHUK (alias, IKKA) genes were down-regulated. Functionally, phosphorylation of IkappaBalpha and binding of NF-kappaB to target DNA were both reduced in these cells. Our results suggest that tetracyclines may have a dual therapeutic effect in Lyme disease.

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Figures

Figure 1

Figure 1. Doxycycline and minocycline affect the secretion of pro-inflammatory mediators by stimulated THP-1 cells

Concentrations of IL-6 (A, D), TNF-alpha (B, E), and IL-8 (C, F) in THP-1 cells were determined after incubation in medium alone or with 0.005 mM, or 0.01 mM of doxycycline (1) or minocycline (2) for 24 h, followed by incubations with fresh doxycycline at the above concentrations, and with 0.25 μg/ml L-OspA, live, sonicated B. burgdorferi (both at 10 MOI), or no stimulant. Shown are the mean values ± SD obtained from triplicate specimens. * (P<0.05) and ** (P<0.005) indicates significant differences between untreated and doxycycline/minocycline-treated cells in a dose-dependent manner. Similar results were obtained with supernatants from cells of 3 additional independent experiments.

Figure 2

Figure 2. Doxycycline markedly reduces the secretion of pro-inflammatory mediators in stimulated primary glial cells

IL-6 (A, C) and IL-8 (B, D) production by microglia (1) and astrocytes (2) was measured after pre-incubation of the cells in medium alone or pre-treatment with 0.0025 mM, or 0.005 mM of doxycycline for 24 h, followed by 24 h incubation with fresh doxycycline at the above concentrations and 0.25 μg/ml L-OspA, live, sonicated B. burgdorferi (both at 10 MOI), or not stimulant. Shown are the mean values ± SD obtained from triplicate specimens. * (P<0.05) and ** (P<0.005) indicates significant differences between untreated and doxycycline-treated cells in a dose-dependent manner. Similar results were obtained with supernatants obtained from cells purified from 3 additional rhesus macaques.

Figure 3

Figure 3. Microarray analysis showing the NFκB signaling pathway affected by doxycycline in stimulated THP-1 cells

Cells were incubated in medium alone or pretreated with 0.01 mM of doxycycline for 24 h, followed by incubation with fresh 0.01 mM doxycycline and sonicated B. burgdorferi (10 MOI-equivalent) for 2 or 12 h. Pathway analysis was performed using Ingenuity Pathways Analysis (IPA) software, version 5.0. Two pathways with high significance of genes regulated by doxycycline in THP-1 cells at 2 h (A) and 12 h (B) were identified and merged. Color indicates the degree of up- (red) or down-regulation (green) of the genes identified by microarray analysis and others are those associated with the regulated genes based on the pathway analysis.

Figure 3

Figure 3. Microarray analysis showing the NFκB signaling pathway affected by doxycycline in stimulated THP-1 cells

Cells were incubated in medium alone or pretreated with 0.01 mM of doxycycline for 24 h, followed by incubation with fresh 0.01 mM doxycycline and sonicated B. burgdorferi (10 MOI-equivalent) for 2 or 12 h. Pathway analysis was performed using Ingenuity Pathways Analysis (IPA) software, version 5.0. Two pathways with high significance of genes regulated by doxycycline in THP-1 cells at 2 h (A) and 12 h (B) were identified and merged. Color indicates the degree of up- (red) or down-regulation (green) of the genes identified by microarray analysis and others are those associated with the regulated genes based on the pathway analysis.

Figure 4

Figure 4. DNA-binding activity of NFκB in untreated and doxycycline/minocycline-treated stimulated THP-1 cells

The activity of NFκB subunit p65 in THP-1 cells was measured using the TransAM ELISA kit. Cells were incubated in medium alone or treated with doxycycline or minocycline at a concentration of 0.005 mM, or 0.01 mM, followed by incubation with sonicated B. burgdorferi (10 MOI-equivalent) for 2h or 4h. Shown are the mean values obtained from triplicate specimens ± SD. *(P<0.05) and **(P<0.005) indicate a significant difference between untreated and doxycycline/minocycline-treated cells in a dose-dependent manner.

Figure 5

Figure 5. Western blot analysis showing the effects of doxycycline on IκBα phosphorylation

THP-1 cells were pre-treated for 24 hours with doxycycline at 0.01 mM, washed, and then incubated with fresh doxycycline followed by stimulation with sonicated B. burgdorferi for 15, 30, or 60 min. IκBα phosphorylation was determined by Western blot (A) using anti-phospho-IκBα antibody; β-tubulin was the control for equal protein loading. Similar results were obtained in three independent experiments.

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