Odontogenic infection by Porphyromonas gingivalis exacerbates fibrosis in NASH via hepatic stellate cell activation

Abstract

Odontogenic infection of Porphyromonas gingivalis (P.g.), a major periodontal pathogen, exacerbates pathological progression of non-alcoholic steatohepatitis (NASH). In this study, we aimed to clarify the detailed mechanism in which P.g. induced hepatic stellate cells (HSCs; key effector cells in liver fibrosis) activation. In the liver of high fat diet-induced NASH mouse model with P.g. odontogenic infection, immunolocalization of P.g. was detected. The number of hepatic crown-like structure, which was macrophage aggregation and related to liver fibrosis, was drastically increased and fibrosis area was also increased through upregulating immunoexpression of Phosphorylated Smad2 (key signaling molecule of TGF-β1) and Galectin-3. P.g.-secreted trypsin-like enzyme [gingipain; an activator of protease-activated receptor 2 (PAR2)] stimulated HSC proliferation and differentiation through Smad and ERK signaling induced by TGF-β1 produced from HSCs with P.g.-infection. Further, Galectin-3 produced from HSCs with P.g. infection and P.g.-derived LPS/lipoprotein stimulation stabilized TGFβ-receptor II resulting in increasing sensitivity for TGF-β1, finally leading to HSC differentiation via activating Smad and ERK signaling. In addition to them, hepatocytes (main component cells of liver) contributed to HSC activation through TGF-β1 and Galectin-3 production in paracrine manner. Collectively, P.g.-odontogenic infection exacerbates fibrosis of NASH by HSC activation through TGF-β1 and Gal-3 production from HSCs and hepatocytes.

Figure 1

P.g.-odontogenic infection induces inflammation in jaw. (a) Histological findings of root apex area at 9 weeks after P.g.-odontogenic infection. After 9 weeks of P.g.-odontogenic infection, periapical granuloma was seen at the root apex area of infected tooth (H&E staining). Magnification: X40. (b) Immunohistochemistry of neutrophils in the dental granuloma. Numerous Ly-6B.2 positive neutrophils (brown granules) are observed in HP. Magnification: X100. (c) Immunohistochemically P.g. was detected in necrotic pulp and viable cells in periapical granuloma. Magnification: X1000. Arrow: P.g.

Figure 2

P.g.-odontogenic infection promotes liver fibrosis in model mice. The liver in HFD group (a) and HP group (b) were histologically analyzed including H&E staining (Magnification x100) and immunohistochemistry. Expression of Gal-3 and pSmad2 were analyzed immunohistochemically. Gal-3; Arrow: hCLS. Arrowhead: Gal-3 expressing hepatocyte. pSmad2; Arrow: HSC. Arrowhead: hepatocyte. Magnification: X200. (c) P.g. was detected in hepatocytes with P.g.-odontogenic infection. Arrow: P.g. Magnification: X1000. (d) hCLS was immunohistochemically detected as accumulation of Gal-3-positive macrophages. Magnification: X200. The number of hCLS was counted and number/unit area was calculated. (e) The red-stained fibrosis area was measured, and area/unit area was calculated. Magnification: X200. HFD = High Fat Diet (N = 5), HP = HFD + P.g.-infection (N = 5). Results were shown as mean ± SD. *p < 0.05, **p < 0.01. Gal-3: Galectin-3.

Figure 3

P.g.-infection and LPS-PG induce myofibroblastic activation of HSCs. PAR2 (a) and TLR2 (b) expressions in LX-2 cells with/without palmitate treatment were analyzed with RT-PCR and western blotting, respectively. (c) LX-2 cells with/without palmitate treatment were cultured with P.g. (MOI-100) for 5 days and counted by a coulter counter. Cont/Pal/P.g./Pal, P.g.: N = 5. (d) LX-2 cells with/without palmitate treatment were cultured with 1 µg/ml of LPS-PG for 5 days and counted by coulter counter. Cont/Pal, LPS-PG: N = 4, Pal/LPS-PG: N = 3. (e) LX-2 cells with/without palmitate treatment were cultured with P.g. (MOI-100) for 5 days. α-SMA and type I collagen were detected by western blotting. (f) LX-2 cells with/without palmitate treatment were cultured with LPS-PG (1 μg/ml) for 6 days. α-SMA and type I collagen were detected by western blotting. 18S was used as internal control for RT-PCR and β-actin was used as internal control for western blotting. Results were shown as mean ± SD. **p < 0.01. Pal: palmitate, P.g.: P.g.-infection.

Figure 4

P.g.-infection and LPS-PG induce myofibroblastic activation of HSCs through Smad and ERK signaling pathways. P.g. infection induces TGF-β1 production through PAR2 activation by gingipain. (a) Smad2, Smad3, and ERK1/2 were detected by western blotting at 24 hours after P.g.-infection. (b) LX-2 cells with/without palmitate treatment were cultured with P.g (MOI-100) for 24 hours. The amount of TGF-β1 in each supernatant was measured by ELISA. Cont/Pal: N = 5, P.g./Pal, P.g.: N = 6. (c) LX-2 cells with/without gingipain inhibitors (3 µM) were cultured with P.g. (MOI-100) for 24 hours. Cont/Gingipain Inh: N = 8, P.g.: N = 7. (d) After culturing for 24 hours with/without TGF-β receptor I inhibitor (1 μg/ml), LX-2 cells were cultured with P.g. (MOI-100) for 24 hours. Smad2 and Smad3 were detected by western blotting. (e) Smad2, Smad3, and ERK1/2 were detected by western blotting at 4 days after LPS-PG stimulation. (f) LX-2 cells were cultured with LPS-PG (1 μg/ml) for 24 hours. The amount of TGF-β1 in each supernatant was measured by ELISA. Cont/Pal/LPS-PG/Pal, LPS-PG: N = 5. β-actin was used as internal control. Results were shown as mean ± SD. *p < 0.05, **p < 0.01. Pal: palmitate, P.g.: P.g.-infection, Gingipain Inh: Gingipain inhibitor, β1: TGF-β1, TGF-β R I Inh: TGF-β receptor I inhibitor.

Figure 5

Gal-3 produced from HSCs with P.g.-infection or LPS-PG enhances sensitivity to TGF-β1 via upregulating TGF-β receptor II expression. LX-2 cells with/without palmitate treatment were cultured with (a) P.g. (MOI-100) or (b) LPS-PG (1 μg/ml) for 2 days. Gal-3 expression was analyzed by western blotting. (c) LX-2 cells with/without palmitate treatment were cultured with recombinant human Gal-3 (1 µg/ml) for 3 days. α-SMA, Smad2, Smad3, and ERK1/2 were detected by western blotting. (d) LX-2 cells with/without palmitate treatment were cultured with Gal-3 (1 µg/ml) for 2 days. TGF-β receptor II was detected by western blotting. β-actin was used as internal control. Pal: palmitate, Gal-3: Galectin-3.

Figure 6

Hepatocytes produce TGF-β1 by P.g.-infection and Gal-3 by P.g. infection/LPS-PG stimulation. PAR2 (a) and TLR2 (b) expressions in Hc3716 cells with/without palmitate treatment were detected by RT-PCR and western blotting, respectively. (c) Hc3716-hTERT cells with/without palmitate treatment were cultured with P.g (MOI-100) for 24 hours. Cont/Pal/P.g.: N = 3, Pal, P.g.: N = 4. (d) Hc3716-hTERT cells with/without palmitate treatment were cultured with LPS-PG (1 μg/ml) stimulation for 24 hours. Cont/Pal/LPS-PG/Pal, LPS-PG: N = 5. The amount of TGF-β1 in each supernatant was measured by ELISA. Hc3176-hTERT cells with/without palmitate treatment were cultured with (e) P.g (MOI-100) or (f) LPS-PG (1 μg/ml) for 2 days. Gal-3 expression was analyzed by western blotting. 18 S was used as internal control for RT-PCR and β-actin was used as internal control for western blotting. Results were shown as mean ± SD. **p < 0.01. Pal: palmitate, P.g.: P.g.-infection, Gal-3: Galectin-3.

Figure 7

Schematic representation of the characteristic mechanisms of pathological progression of NASH caused by odontogenic infection of P.g. (a) The mechanisms of HSC activation caused by TGF-β1. (b) The mechanisms of HSC activation caused by Gal-3.