Extracellular vesicles from periodontal pathogens regulate hepatic steatosis via Toll-like receptor 2 and plasminogen activator inhibitor-1

Abstract

Plasminogen activator inhibitor-1 (PAI-1) is associated with nonalcoholic fatty liver disease (NAFLD) by lipid accumulation in the liver. In this study, we showed that extracellular vesicles (EVs) from the periodontal pathogens Filifactor alocis and Porphyromonas gingivalis induced steatosis by inducing PAI-1 in the liver and serum of mice fed a low-fat diet. PAI-1 induction was not observed in TLR2^-/- mice. When tested using HEK-Blue hTLR2 cells, human TLR2 reporter cells, the TLR2-activating ability of serum from NAFLD patients (n = 100) was significantly higher than that of serum from healthy subjects (n = 100). Correlation analysis confirmed that PAI-1 levels were positively correlated with the TLR2-activating ability of serum from NAFLD patients and healthy subjects. Amphiphilic molecules in EVs were involved in PAI-1 induction. Our data demonstrate that the TLR2/PAI-1 axis is important for hepatic steatosis by EVs of periodontal pathogens.

Keywords: Toll-like receptor 2; extracellular vesicle; nonalcoholic fatty liver disease; periodontal pathogen; plasminogen activator inhibitor-1.

FIGURE 1

PAI‐1 induction by F.alocis EVs in mouse livers. (a) Mice (n = 4) were administered Fa EVs (50 μg) by intraperitoneal injection. After the indicated time periods, the liver was extracted, homogenized with RIPA buffer, and subjected to direct ELISA using anti‐F. alocis rabbit serum. (b, c) Mice (n = 3) were administered Fa EVs (50 μg) by intraperitoneal injection. After 1 h, mRNA was extracted from 50 mg of mouse liver, kidney, spleen, and intestine. The mRNA expression levels of 90 liver disease‐related genes were analysed by qPCR array (b). PAI‐1 mRNA expression levels were analysed by real‐time PCR (c). (d) Mice (n = 3) were administered Fa EVs (50 μg) by intraperitoneal injection. After 3 h, proteins were extracted from mouse livers (100 mg), and liver lysates (500 μg) were subjected to an adipokine array. A total of 38 obesity‐related molecules were analysed by dot blot analysis (upper panel), and the fold increase (lower panel) was determined by densitometry using ImageJ software. (e) Mice (n = 3) were intraperitoneally administered Fa EVs (0.5, 5, and 50 μg) for 3 h. (f) Mice (n = 3) were administered Fa EVs (10 μg/5 μL; 5 μg each in the palate) by palatal injection for 3 h. PAI‐1 expression levels in serum and liver lysates were measured using ELISA. The graphs are shown as the mean values ± standard deviations. Representative data from three biological replicates are shown. Statistical significance was determined by two‐tailed Student’s t test (f), one‐way ANOVA with Bonferroni’s multiple comparison test (a, e) or two‐way ANOVA with Bonferroni’s multiple comparison test (b, c, d). *P 〈 0.05, **P 〈 0.01, ***P 〈 0.001, ****P 〈 0.0001 compared to the nontreatment (PBS) group. ns denotes not significant.

FIGURE 2

Hepatic steatosis by F. alocis EVs in low‐fat diet‐fed mice. (a) Schematic overview of experimental schemes. (b) Body weight of low‐fat diet (LFD, left)‐fed or high‐fat diet (HFD, right)‐fed mice administered PBS‐ or Fa EVs (twice a week for 8 weeks, n = 5). Mice were sacrificed 1 h after the last injection. (c) Liver weight (n = 5). (d) Typical pictures of liver (upper), representative images of Oil Red O stained liver sections (middle, n = 5, Scale bar, 100 μm), and representative images of trichrome stained liver sections (lower, n = 5, Scale bar, 100 μm). (e) Steatosis score, lobular inflammation score, and fibrosis stage of liver sections (n = 5). (f) Triglyceride levels in liver tissue and serum (n = 5). (g) Insulin and glucose concentrations in mouse serum (n = 5). (h) PAI‐1 concentration in liver lysates and serum (n = 5). (i) Serum AST and ALT levels (n = 5). The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two‐way ANOVA with Bonferroni’s multiple comparison test. *P 〈 0.05, **P 〈 0.01, ***P 〈 0.001, ****P 〈 0.0001 compared to the indicated group. ns denotes not significant.

FIGURE 3

Effects of pharmacological inhibition of PAI‐1 on EV‐induced steatosis. (a) Schematic overview of experimental schemes. (b) Body weight of LFD‐fed mice administered PBS, Fa EVs, or Pg EVs (twice a week for 8 weeks) in the presence or absence of TM5441 injection (20 mg/kg, n = 5). (c) Serum active PAI‐1 levels were measured using an active PAI‐1 ELISA kit. (d) Typical pictures of liver and representative images of Oil Red O stained liver sections (n = 5, Scale bar, 100 μm). (e) Liver weight (n = 5) and (f) steatosis score of liver sections (n = 5). (g) Triglyceride levels in liver tissue (n = 5). (h) Gene expression levels of SREBP‐1c, FAS, and ACC in the liver (n = 5). (i) Serum AST and ALT levels (n = 5). The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two‐way ANOVA with Bonferroni’s multiple comparison test. *P 〈 0.05, **P 〈 0.01, ***P 〈 0.001, ****P 〈 0.0001 compared to each PBS group. ^## P 〈 0.01, ^### P 〈 0.001, ^#### P 〈 0.0001 compared to the indicated group. ns denotes not significant.

FIGURE 4

Role of TLR2 in periodontal pathogen EV‐induced liver steatosis. (a) Schematic overview of experimental schemes. (b) Body weight of LFD‐fed WT or TLR2^‐/‐ mice administered PBS, Fa EVs, or Pg EVs (twice a week for 8 weeks, n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). Mice were sacrificed 1 h after the last injection. (c) Liver weight (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). (d) Typical pictures of liver (upper) and representative image of Oil Red O stained liver sections (lower, n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups, Scale bar, 100 μm). (e) Steatosis score of liver sections (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). (f) Triglyceride levels in liver tissue (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). (g) PAI‐1 concentration in liver lysates and serum (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). (h) Gene expression levels of SREBP‐1c, FAS, and ACC in the liver (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). (i) Serum AST and ALT levels (n = 5 for PBS groups; n = 6 for Fa EV and Pg EV groups). The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two‐way ANOVA with Bonferroni’s multiple comparison test. *P 〈 0.05, **P 〈 0.01, ***P 〈 0.001, ****P 〈 0.0001 compared to the PBS group. ^# P 〈 0.05, ^## P 〈 0.01, ^### P 〈 0.001, ^#### P 〈 0.0001 compared to the indicated group. ns denotes not significant.

FIGURE 5

PAI‐1 levels, TLR2‐activating ability, and periodontal pathogen proteins of serum from NAFLD patients. (a, b) HEK‐Blue hTLR2 cells (5 × 10⁴ cells/well in 96‐well plates) were pretreated with 5 μg/mL anti‐hTLR2 IgA or isotype human IgA2 for 1 h. Then, the cells were treated with 20 μL of serum from healthy subjects (n = 100) or NAFLD patients (n = 100) for an additional 20 h (a). Pam3CSK4 (100 ng/mL) and ultrapure LPS (100 ng/mL) were used as controls for the TLR2 ligand and TLR4 ligand, respectively (b). The TLR2‐activating ability of serum was measured by SEAP assay using a spectrophotometer. (c) PAI‐1 levels in serum from healthy subjects (n = 100) or NAFLD patients (n = 100) were measured by ELISA. (d) Correlation plot between the PAI‐1 levels in human serum and the TLR2‐activating ability of human serum. (e) Concentration of F. alocis proteins in human serum were measured using an anti‐F. alocis rabbit serum. (f) Correlation plot between the PAI‐1 levels in human serum and the F. alocis protein concentration of human serum. (g) Correlation plot between the TLR2‐activating ability of human serum and the F. alocis protein concentration of human serum. (h) Concentration of P. gingivalis proteins in human serum were measured using an anti‐P. gingivalis monoclonal antibody. (i) Correlation plot between the PAI‐1 levels in human serum and the P. gingivalis protein concentration of human serum. (j) Correlation plot between the TLR2‐activating ability of human serum and the P. gingivalis protein concentration of human serum. Statistical significance was determined by two‐tailed Student's t test (a, b, e, h), two‐way ANOVA with Bonferroni's multiple comparison test (c), or Spearman's rank correlation test (d, f, g, i, j). *P < 0.05, **P < 0.01, ****P < 0.0001 compared to the serum from healthy subjects. ^#### P < 0.0001 compared to the indicated group. ns denotes not significant.

FIGURE 6

PAI‐1 induction by periodontal pathogen EVs in hepatocytes. (a) Hepatocyte cell lines (2 × 10⁵ cells in 48‐well plates for ELISA, 4 × 10⁵ cells in 6‐well plates for RT‒PCR) were stimulated with 10 μg/mL indicated stimuli for 24 h (ELISA) or for 3 h (RT‒PCR). PAI‐1 expression at the protein level (upper panel) and mRNA level (lower panel) was analysed by ELISA and RT‒PCR, respectively. (b) Huh7 cells (4 × 10⁵ cells in 6‐well plates) were stimulated with 10 μg/mL of Fa EVs for 1, 2, or 3 h. Phosphorylation of each molecule was determined by Western blotting. Band intensities in the Western blots were quantified by densitometry using ImageJ software and presented as the relative ratio to the total form of each signalling molecule. (c) Huh7 cells (2 × 10⁵ cells in 48‐well plates) were pretreated with the indicated inhibitors (1 or 10 μM) for 1 h. Then, the cells were stimulated with 10 μg/mL of Fa EVs for 24 h. (d) Huh7 cells (2 × 10⁵ cells in 48‐well plates) were pretreated with TLR2‐blocking antibody or isotype control for 1 h and stimulated with the indicated stimuli (left panel). Huh7 cells (4 × 10⁵ cells in 6‐well plates) were transfected with TLR2 siRNA for 18 h and stimulated with 10 μg/mL of Fa EVs for 3 h (right panel). (e, f, g) Huh7 cells (2 × 10⁵ cells in 48‐well plates) were stimulated with the indicated stimuli (10 μg/mL) for 24 h. PAI‐1 protein levels in the cell culture supernatants were analysed by ELISA (c, d, e, f, g). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the PBS group. ^# P < 0.05, ^#### P < 0.0001 compared to the indicated group. ns denotes not significant.