Noradrenaline Synergistically Enhances Porphyromonas gingivalis LPS and OMV-Induced Interleukin-1 β Production in BV-2 Microglia Through Differential Mechanisms

Abstract

Infection with Porphyromonas gingivalis (Pg), which is a major periodontal pathogen, causes a large number of systemic diseases based on chronic inflammation such as diabetes and Alzheimer's disease (AD). However, it is not yet fully understood how Pg can augment local systemic immune and inflammatory responses during progression of AD. There is a strong association between depression and elevated levels of inflammation. Noradrenaline (NA) is a key neurotransmitter that modulates microglial activation during stress conditions. In this study, we have thus investigated the regulatory mechanisms of NA on the production of interleukin-1β (IL-1β) by microglia following stimulation with Pg virulence factors, lipopolysaccharide (LPS), and outer membrane vesicles (OMVs). NA (30-1000 nM) significantly enhanced the mRNA level, promoter activity, and protein level of IL-1β up to 20-fold in BV-2 microglia following treatment with Pg LPS (10 μg/mL) and OMVs (150 μg of protein/mL) in a dose-dependent manner. Pharmacological studies have suggested that NA synergistically augments the responses induced by Pg LPS and OMVs through different mechanisms. AP-1 is activated by the β₂ adrenergic receptor (Aβ₂R)-mediated pathway. NF-κB, which is activated by the Pg LPS/toll-like receptor 2-mediated pathway, is required for the synergistic effect of NA on the Pg LPS-induced IL-1β production by BV-2 microglia. Co-immunoprecipitation combined with Western blotting and the structural models generated by AlphaFold2 suggested that cross-coupling of NF-κB p65 and AP-1 c-Fos transcription factors enhances the binding of NF-κB p65 to the IκB site, resulting in the synergistic augmentation of the IL-1β promoter activity. In contrast, OMVs were phagocytosed by BV-2 microglia and then activated the TLR9/p52/RelB-mediated pathway. The Aβ₂R/Epac-mediated pathway, which promotes phagosome maturation, may be responsible for the synergistic effect of NA on the OMV-induced production of IL-1β in BV-2 microglia. Our study provides the first evidence that NA synergistically enhances the production of IL-1β in response to Pg LPS and OMVs through distinct mechanisms.

Keywords: BV2 microglia; Porphyromonas gingivalis; activator protein 1; interleukin-1β; lipopolysaccharide; noradrenaline; nuclear factor-κB; outer membrane vesicles; synergistic augmentation.

Figure 1

Synergistic effects of NA on the Pg virulence factor induced the expression of IL-1β by BV-2 microglia. (A–C) The mean relative level of IL-1β mRNA following treatment with NA, NA + Pg LPS, and NA + OMVs for 1 h. (A) The mean relative level of IL-1β mRNA induced by NA with various concentrations (30–1000 nM). (B,C) The mean relative level of IL-1β mRNA induced by Pg LPS (10 μg/mL) (B) or OMVs (150 μg of protein/mL) (C) in the absence or presence of NA (30–1000 nM). (D–F) The mean relative luciferase activity of the IL-1β probe following treatment with NA, NA + Pg LPS, and NA + OMVs for 1 h. (D) The mean relative luciferase activity of the IL-1β probe induced by NA with various concentrations (30–1000 nM). (E,F) The mean relative luciferase activity of the IL-1β probe induced by Pg LPS (10 μg/mL) (E) or OMVs (150 μg of protein/mL) (F) in the absence or presence of NA (30–1000 nM). Data relative to the values are presented as the mean ± standard error (SE) of 4 independent experiments. ns: not statistically significant.

Figure 2

Time course of the mean relative luciferase activity of the IL-1β probe induced by Pg LPS, OMVs, and their combination with NA. (A,B) The mean relative luciferase activity of the IL-1β probe induced by either Pg LPS alone (10 μg/mL) (A) or combination with NA (1 μM) (B). (C,D) The mean relative luciferase activity of the IL-1β probe induced by either OMVs alone (150 μg of protein/mL) (C) or combination with NA (1 μM) (D). Data relative to the values are presented as the mean ± SE of 3 independent experiments. ns: not statistically significant.

Figure 3

Production of IL-1β by BV-2 microglia following treatment with NA, Pg virulence factors, and their combination. (A) The mean concentration IL-1β in the cell lysates of BV-2 microglia following treatment with NA (1 μM), Pg LPS (10 μg/mL), and their combined application. (B) The mean concentration of IL-1β in the cell lysates of BV-2 microglia following treatment with NA (1 μM), OMVs (150 μg of protein/mL), and their combined application. Data are presented as the mean ± SE of 4 independent experiments. (C) Cell lysates from BV-2 microglia following treatment with NA (3 μM), Pg LPS (30 μg/mL), or their combined application for 1 h were subjected to immunoblotting of IL-1β. (D) Cell lysates from BV-2 microglia following treatment with NA (3 μM), OMVs (150 μg of protein/mL), or their combined application for 1 h were subjected to immunoblotting of IL-1β. Immunoblotting of GAPDH was used as a loading control.

Figure 4

Effects of NF-κB signaling pathway inhibitors on the production of IL-1β by BV-2 microglia following treatment with NA, Pg virulence factors, and their combination. (A,B) The mean relative luciferase activity of the IL-1β probe induced by NA (1 μM), Pg LPS (10 μg/mL), and their combined application in the absence or presence of wedelolactone (30 μM) (A) or NAI (100 nM) (B). (C,D) The mean relative luciferase activity of the IL-1β probe induced by NA (1 μM), OMVs (150 μg/mL) and their combined application in the absence or presence of wedelolactone (30 μM) (C) or NAI (100 nM) (D). Data relative to the values are presented as the mean ± SE of 3–5 independent experiments. ns: not statistically significant.

Figure 5

Effects of AP-1 signaling pathway inhibitors on the production of IL-1β by BV-2 microglia following treatment with NA, Pg virulence factors, and their combination. (A,B) The mean relative luciferase activity of the IL-1β probe induced by NA (1 μM), Pg LPS (10 μg/mL), and their combined application in the absence or presence of U0126 (15 μM) (A) or SR11302 (10 μM) (B). (C,D) The mean relative luciferase activity of the IL-1β probe induced by NA (1 μM), OMVs (150 μg of protein/mL), and their combined application in the absence or presence of U0126 (15 μM) (C) or SR11302 (10 μM) (D). Data relative to the values are presented as the mean ± SE of 3 independent experiments.

Figure 6

Involvement of Aβ₂R in the synergistic effect of NA and Pg virulence factors in the production of IL-1β by BV-2 microglia and effects of signaling inhibitors on the production of IL-1β induced by NA or Pg LPS alone. (A,B) Effects of ICI on the production of IL-1β by BV-2 microglia following stimulation of NA (1 μM) plus Pg LPS (10 μg/mL) or NA (1 μM) plus OMVs (150 μg/mL). (C,D) Effects of β₂ ICI-118551 (0.1 μM), NAI (100 nM), U0126 (15 μM), and SR11302 (10 μM) on the production of IL-1β by BV-2 microglia following treatment with NA (3 μM) or Pg LPS (30 μg/mL). (E,F) Possible involvement of phagocytosis-related pathways on the production of IL-1β by BV-2 microglia following treatment with NA (1 μM) plus OMVs (150 μg/mL) was examined using the TLR9 inhibitor E6446 (10 μM) (E) or the p52 inhibitor SN52 (40 μg/mL) (F). Data relative to the values are presented as the mean ± SE of 3–4 independent experiments.

Figure 7

Possible changes in expression levels and protein complex formation of p65 and c-Fos in BV-2 microglia following combined treatment with NA and Pg LPS. (A) Western blotting to analyze the protein levels of p65 and c-Fos in BV-2 microglia following treatment with NA and Pg LPS. Immunoblotting of GAPDH was used as a loading control. (B,C) Co-immunoprecipitation combined with Western blotting to analyze the interaction between p65 and c-Fos in BV-2 microglia following treatment with NA and Pg LPS. Co-immunoprecipitated samples using normal or anti-p65 IgG were subsequently subjected to Western blotting using anti-p65 IgG (B) or anti-c-Fos IgG (C). Each experiment was repeated at least three times.

Figure 8

Prediction of c-Fos binding to NF-κB p65:p50 heterodimer. (A) The structural model of c-Fos-bound NF-κB p65:p50 heterodimer generated using AlphaFold2, presented as a ribbon colored pLDDT score. c-Fos binds to the molecular surface of p65. The figure to the right shows the complex structure vertically rotated by 90° from the figure to the left. (B) PAE plots of the c-Fos and p65:p50 complex model. (C) The structural model of the p65:p50:c-Fos complex forms a composite surface for DNA recognition. For the model construction, DNA bound to the p65:50 heterodimer (PDB code 2I9T) was bound to the AlphaFold2 model of the p65:p50:c-Fos complex. The long disordered regions in the N-terminus and C-terminus of c-Fos were removed from the figure to facilitate visualization. The NF-κB of p65 and p50 are in green and light blue, respectively. c-Fos is in purple. The two DNA strands are in orange. The figure to the right shows the complex structure vertically rotated by 90° from the figure to the left. The figures were drawn using PyMOL 3.1 [27].