The mechanisms of Porphyromonas gingivalis-derived outer membrane vesicles-induced neurotoxicity and microglia activation

Abstract

Background/purpose: Periodontitis is associated with various systemic diseases, potentially facilitated by the passage of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs). Several recent studies have suggested a connection between Pg-OMVs and neuroinflammation and neurodegeneration, but the precise causal relationship remains unclear. This study aimed to investigate the mechanisms underlying these associations using in vitro models.

Materials and methods: Isolated Pg-OMVs were characterized by morphology, size, and gingipain activity. We exposed SH-SY5Y neuroblastoma cells and BV-2 microglial cells to various concentrations of Pg-OMVs. Cell morphology, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, an enzyme-linked immunosorbent assay, and Western blot analysis were used to evaluate the cellular mechanism underlying Pg-OMV-induced neurotoxicity in neuronal cells and inflammatory responses in microglial cells.

Results: Exposure to Pg-OMVs induced neurotoxicity in SH-SY5Y cells, as evidenced by cellular shrinkage, reduced viability, activation of apoptotic pathways, and diminished neuronal differentiation markers. Gingipain inhibition mitigated these effects, suggesting that gingipain mediates Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. Our research on neuroinflammation suggests that upon endocytosis of Pg-OMVs by BV-2 cells, lipopolysaccharide (LPS) can modulate the production of inducible nitric oxide synthase and tumor necrosis factor-alpha by activating pathways that involve phosphorylated AKT and the phosphorylated JNK pathway.

Conclusion: Our study demonstrated that following the endocytosis of Pg-OMVs, gingipain can induce neurotoxicity in SH-SY5Y cells. Furthermore, the Pg-OMVs-associated LPS can trigger neuroinflammation via AKT and JNK signaling pathways in BV-2 cells.

Keywords: Gingipain; Lipopolysaccharide; Microglial cell; Neuron; Outer membrane vesicles; Porphyromonas gingivalis.

Figure 1

Characterization of Pg-OMVs. (A) The morphology of Pg-OMVs was evaluated using transmission electron microscope (TEM). (B) Analysis of Pg-OMVs particle diameters and concentration using nanoparticle tracking analysis. (C) Analysis of Pg-OMVs gingipain activity and its inhibition with KYT-1 and KYT-36. The bar represents the mean ± SEM of three different experiments (∗P < 0.05).

Figure 2

Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. (A) Bright-field morphology of undifferentiated SH-SY5Y treated with different concentrations of Pg-OMVs for 24 h. Scale bar, 100 μm. (B) MTT assay evaluation of the effect of Pg-OMVs on neuronal cell viability. For dose-dependence, Pg-OMVs were given at concentrations of 5, 10, and 20 μg/mL for 24 h. (C) Western blot analysis of cleaved PARP (cPARP) in SH-SY5Y cells upon exposure to different concentrations of Pg-OMVs for 24 h. (D) The relative quantification of cPARP expression normalized to β-actin. The bar represents the mean ± SEM of three different experiments (∗P < 0.05). (E) Bright-field morphology of differentiated SH-SY5Y cells treated with different concentrations of Pg-OMVs for 24 h. Scale bar, 100 μm. (F) Western blot analysis of Syn and GAP43 in differentiated SH-SY5Y cells upon exposure to different concentrations of Pg-OMVs for 24 h.

Figure 3

Pg-OMVs-induced neurotoxicity via clathrin-dependent endocytosis and gingipain. (A) The undifferentiated SH-SY5Y cells were pretreated with Pitstop (2 μM) for 30 min and then incubated with DiO₃-labeled Pg-OMVs (green) for another 24 h. Nuclei were stained with DAPI (blue). (B) MTT assay evaluation of the effect of Pitstop on Pg-OMVs-induced neurotoxicity. The bar represents the mean ± SEM of three different experiments (∗P < 0.05). (C) The undifferentiated SH-SY5Y cells were pretreated with KYT-1 (1 μM) or KYT-36 (1 μM) for 30 min followed by treatment with Pg-OMVs (10 μg/mL) for 24 h. The morphology was observed using bright-field microscopy. Scale bar, 100 μm. (D) MTT assay evaluation of the effect of KYT-1 and KYT-36 on Pg-OMVs-induced neurotoxicity. (E) Bright-field morphology of differentiated SH-SY5Y cells pretreated with KYT-1 and KYT-36 followed by treatment with Pg-OMVs for 24 h. Scale bar, 100 μm.

Figure 4

Pg-OMVs activate microglial cells via LPS. (A) Western blot analysis of iNOS expression in BV-2 cells exposed to different concentrations of Pg-OMVs for 24 h. The relative quantification of iNOS expression normalized to GAPDH. The bar represents the mean ±SEM of three different experiments (∗P < 0.05). (B) ELISA analysis of the levels of TNF-α in BV-2 cells exposed to different concentrations of Pg-OMVs for 24 h. The levels of TNF-α were normalized to total protein. The bar represents the mean ± SEM of three different experiments (∗P < 0.05). (C) Western blot analysis of the levels of iNOS in BV-2 cells pretreated with KYT-1 (1 μM) or KYT-36 (1 μM) for 30 min and then incubated with Pg-OMVs (1 μg/mL) for 24 h. (D) ELISA analysis of the levels of TNF-α in BV-2 cells pretreated with KYT-1 (1 μM) or KYT-36 (1 μM) for 30 min and then incubated with Pg-OMVs (1 μg/mL) for 24 h. The levels of TNF-α were normalized to total protein. The bar represents the mean ± SEM of three different experiments (∗P < 0.05). (E) Western blot analysis of iNOS production in BV-2 cells exposed to Pg-OMVs and LPS inhibitor polymyxin B. (F) ELISA analysis of TNF-α production in BV-2 cells exposed to Pg-OMVs and LPS inhibitor polymyxin B. The levels of TNF-α were normalized to total protein. The bar represents the mean ± SEM of three different experiments (∗P < 0.05).

Figure 5

Pg-OMVs-mediated microglial activation is dependent on endocytosis and AKT and JNK signaling pathways. (A) The BV-2 cells were pretreated with Pitstop (2 μM) for 30 min and then incubated with DiO₃-labeled Pg-OMVs (green) for another 24 h. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (B) Western blot analysis of the levels of inducible nitric oxide synthase (iNOS) in BV-2 cells pretreated with Pitstop (2 μM) for 30 min and then incubated with Pg-OMVs (1 μg/mL) for 24 h. (C) ELISA analysis of TNF-α production in BV-2 cells exposed to Pg-OMVs and Pitstop. The levels of TNF-α were normalized to total protein. The bar represents the mean ± SEM of three different experiments (∗P < 0.05). (D) Western blot analysis of pAKT and pJNK production in BV-2 cells exposed to Pg-OMVs (1 μg/mL) for the indicated time. (E) Western blot analysis of the levels of iNOS in BV-2 cells pretreated with different concentrations of AKT inhibitor LY294002 and JNK inhibitor SP600125 for 30 min and then incubated with OMVs (1 μg/mL) for 4 h. (F) ELISA analysis of TNF-α production in BV-2 cells exposed to Pg-OMVs and AKT and JNK inhibitors. The levels of TNF-α were normalized to total protein. The bar represents the mean ± SEM of three different experiments (∗P < 0.05).