Proteomic analysis of P. gingivalis-Lipopolysaccharide induced neuroinflammation in SH-SY5Y and HMC3 cells

Abstract

Chronic periodontitis and its keystone pathogen, Porphyromonas gingivalis, have increasingly been linked with Alzheimer's disease (AD). However, P.gingivalis-lipopolysaccharide (LPS) mediated release of neuroinflammatory proteins contributes to AD remains underexplored. In this study, we utilized data-independent acquisition mass spectrometry to characterize P.gingivalis-LPS induced profile of differentially expressed proteins associated with the neuroinflammatory response in human neuroblastoma (SH-SY5Y) and human microglial (HMC3) cells. We reported a set of 124 proteins in SH-SY5Y cells and 96 proteins in HMC3 cells whose levels were significantly upregulated or downregulated by exposure to P. gingivalis-LPS. Our findings demonstrate that P. gingivalis-LPS contributed to the elevated expressions of dementia biomarkers and pro-inflammatory cytokines that include APP, Aβ_1-42, Aβ_1-40, T-Tau, p-Tau, VEGF, TGF-β, IL-1β, IL-6 and TNF-α through 2 distinct pathways of extracellular sensing by cell surface receptors and intracellular cytosolic receptors. Interestingly, intracellular signaling proteins activated with P. gingivalis-LPS transfection using Lipofectamine™ 2000 had significantly higher fold change protein expression compared to the extracellular signaling with P. gingivalis-LPS treatment. Additionally, we also explored P. gingivalis-LPS mediated activation of caspase-4 dependent non canonical inflammasome pathway in both SH-SY5Y and HMC3 cells. In summary, P. gingivalis-LPS induced neuroinflammatory protein expression in SH-SY5Y and HMC3 cells, provided insights into the specific inflammatory pathways underlying the potential link between P. gingivalis-LPS infection and the pathogenesis of Alzheimer's disease and related dementias.

Keywords: Porphyromonas gingivalis; Amyloid precursor protein; Data-independent acquisition mass spectrometry; Lipopolysaccharide; Neuroinflammation; Tau protein.

Fig. 1

Effect of P. gingivalis-LPS on SH-SY5Y and HMC3 cells viability with MTS assay (A, C) and cytotoxicity with LDH assay (B, D). SH-SY5Y and HMC3 cells were treated or transfected with a 10.0 μg/mL concentration of P. gingivalis- LPS for 4, 12, 24, and 48 h. Untreated cells were used as controls for P. gingivalis- LPS treated cells, and 1.0 μg/mL lipofectamine 2000 treated cells were used as controls for P. gingivalis- LPS transfected cells. Data are mean ± SD of 4 independent experiments (n = 4)

Fig. 2

P. gingivalis-LPS promotes Aβ_1–42 in SH-SY5Y cells, and IL-1β in both SH-SY5Y and HMC3 cells. A Aβ_1–42 ELISA levels were significantly increased at 24 h in SH-SY5Y cells treated or transfected with 10.0 μg/mL P. gingivalis- LPS (n = 4). C In HMC3 cells, neither P. gingivalis- LPS treatment nor transfection affected the expression of Aβ_1–42 at any time point as compared to controls (n = 4). B, D The expression of IL-1β was increased with both P. gingivalis- LPS treatment and transfection in SH-SY5Y at 24 h and HMC3 cells at 4 h (n = 4). Statistically significant differences *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: p > 0.05 by one-way ANOVA with Tukey's post hoc test

Fig. 3

Proteomic analysis of differentially expressed proteins in SH-SY5Y and HMC3 cells treated and transfected with P. gingivalis- LPS cells with adjusted p-values < 0.05 and a log fold change > 2. Volcano plots showed the differential protein expression in (A) the P. gingivalis- LPS treated SH-SY5Y cells and (B) the P. gingivalis- LPS transfected SH-SY5Y cells, (C) the P. gingivalis- LPS treated HMC3 cells and (D) the P. gingivalis- LPS transfected HMC3 cells. Red dots on the right-hand side indicate upregulated proteins, blue dots on the left-hand side indicate downregulated proteins and grey dots indicate no significant change in protein expression levels. Data are based on 3 independent experiments (n = 3)

Fig. 4

Comparative expression of differentially expressed proteins in SH-SY5Y and HMC3 cells treated and transfected with P. gingivalis- LPS. Heat map plot showed the higher log2 fold change protein expression in (A) SH-SY5Y cells transfected with P. gingivalis- LPS compared to that found in treated SH-SY5Y cells (B) HMC3 cells transfected with P. gingivalis- LPS compared to that found in treated HMC3 cells. Orange and blue colors represent the log2 fold change values of upregulated and downregulated proteins, respectively, compared to controls. Data are based on 3 independent experiments (n = 3)

Fig. 5

Protein–protein interactions using the STRING database among all the differentially expressed proteins extracted from log2 fold change expression among P. gingivalis- LPS treated or transfected (A) SH-SY5Y and (B) HMC3 cells. Data are based on 3 independent experiments (n = 3)

Fig. 6

Pie Charts showing the functional classification of differentially expressed proteins among P. gingivalis- LPS treated or transfected (A) SH-SY5Y and (B) HMC3 cells, based on the biological pathway analysis using Gene Ontology (GO) enrichment analysis through PANTHER-classification system. Data are based on 3 independent experiments (n = 3)

Fig. 7

P. gingivalis-LPS upregulated neuroinflammatory markers in SH-SY5Y and HMC3 cells. The relative mRNA expression of T-Tau (Total-Tau), VEGF, TGF-β, IL-6 and TNF-α was increased with both P. gingivalis- LPS treatment and transfection in (A) SH-SY5Y and (C) HMC3 cells. (n = 4). ELISA of p-Tau level at T181 and T217 with both P. gingivalis- LPS treatment and transfection in (B) SH-SY5Y and (D) HMC3 cells. (n = 4). Statistically significant differences *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: p > 0.05 by one-way ANOVA with Tukey's post hoc test

Fig. 8

P. gingivalis-LPS increased amyloidogenic processing of APP in SH-SY5Y and HMC3 cells. (A) Representative western blots show the protein expression of APP after P. gingivalis- LPS treatment or transfection in SH-SY5Y and HMC3 cells. (n = 4); GAPDH was used as a loading control. Quantification of relative protein levels normalized against GAPDH was shown in the graphs (B) SH-SY5Y and (C) HMC3 cells. (n = 4). ELISA of Aβ_1-40 and Aβ_1-42 peptides after P. gingivalis- LPS treatment or transfection in (D, F) SH-SY5Y cells (E, G) in HMC3 cells. Statistically significant differences *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: p > 0.05 by one-way ANOVA with Tukey's post hoc test

Fig. 9

P. gingivalis- LPS activated Caspase-4 dependent non canonical inflammasome pathway in SH-SY5Y and HMC3 cells. Representative western blots show the upregulated protein expression of caspase-4, NLRP3, caspase-1, gasdermin D, and IL-1β, in response to P. gingivalis- LPS transfection using lipofectamine in (A) SH-SY5Y and (C) HMC3 cells. (n = 3); GAPDH was used as a loading control. Quantification of relative protein levels normalized against GAPDH was shown in the graphs (B) SH-SY5Y and (D) HMC3 cells. (n = 3)