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

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.


P.g.-odontogenic infection exacerbates pathological progression of NASH through Gal-3 and TGF-β1/Smad pathway.
In HFD group, microvesicular lipid deposition was prominent, but inflammation was slight. Whereas increasing macrovesicular lipid accumulation and hepatic crown-like structures (hCLS), which was aggregation of macrophages and positively correlated with the extent of liver fibrosis [ Supplementary Fig. 2], were seen in HP group. Immunoexpression of Gal-3 and pSmad2 (a key signaling molecule of TGF-β1), which were critical molecules for HSC activation, was examined. Among hepatocytes in HFD, Gal-3 positive spindle cells were scattered. While in addition to increasing Gal-3 positive spindle cells, Gal-3 positive hCLS (arrows) and hepatocytes (arrowheads) were observed. Strong pSmad2 nuclear expression, indicating TGF-β1 signaling activation, was detected in HSCs (arrows) and hepatocytes (arrowheads) of HP group. HFD group showed negative or weak reaction for pSmad2. Interestingly, P.g. was clearly detected in liver of HP group [ Fig. 2c]. Morphometrically, the number of Gal-3 positive hCLS was counted. The number of hCLS in HP group was significantly increased (p < 0.01), [Fig. 2d]. To analyze the degree of liver fibrosis, sirius red staining was performed. Sirius red-positively stained collagen fibers are distributed among hepatocytes with lipid deposition. HP group indicated significantly increased sirius red positive fibrosis areas (p < 0.05), [Fig. 2e]. These data suggest that P.g.-odontogenic infection aggravates inflammatory cell infiltration and liver fibrosis, in which TGF-β1/Smad and Gal-3 pathway are involved.

P.g.-infection and LPS-PG (P.g.-LPS/lipoprotein) stimulation induce HSC activation. It is well
accepted that PAR2 is activated by trypsin-like enzymes and contributes to TGF-β1 production which is resulting in liver fibrosis 22 . Gingipain; a major virulence factor of P.g., is known to activate PAR2 13,23,24 . To determine the effects of P.g.-LPS/lipoprotein, LPS-PG, a ligand for both TLR2 and 4, was used in this study. Palmitate (a main FFA upregulated in serum of NASH patient) treatment significantly upregulated PAR2 [ Fig. 3a] and TLR2 levels [ Fig. 3b], but not toll-like receptor 4 (TLR4) level [ Supplementary Fig. 3a] in LX-2 cells, human hepatic stellate cell line.
Gal-3 production caused by P.g.-infection and -LPS stimulation enhanced myofibroblastic differentiation of HSC through upregulation of TGF-β receptor ii expression. To clarify the mechanism in which LPS-PG stimulation induced myofibroblastic differentiation of HSC, we focused on Gal-3, which is one of the most important molecules to stimulate liver fibrosis 16,20 . LX-2 cells markedly induced Gal-3 production by LPS-PG [ Fig. 5a]. Interestingly, Gal-3 expression was also upregulated by P.g. infection [ Fig. 5b]. The direct effect of Gal-3 on LX-2 cells was examined. Gal-3 upregulated α-SMA expression in LX-2 cells through Smad and ERK pathways as well as TGF-β1 [ Fig. 5c]. It is known that signaling starts with TGF-β binding to TGF-β receptor II and phosphorylates TGF-β receptor I resulting in phosphorylating downstream including Smad2 and 3 26 . Therefore, TGF-β receptor II expression level was examined. Gal-3 upregulated TGF-β receptor II expression of LX-2 with/without palmitate treatment [ Fig. 5d]. Collectively, it is suggested that Gal-3 promoted HSC activation via upregulation of TGF-β receptor II expression resulting in increasing the sensitivity for TGF-β1.  Expression of TLR2 in Hc3716-hTERT cells, immortalized human fetal hepatocytes, was significantly increased by palmitate treatment for 18 hours, however, neither TLR4 [ Supplementary Fig. 3b] nor PAR2 levels were increased [ Fig. 6a,b]. TGF-β1 from Hc3716-hTERT cells with/without palmitate was also significantly up-regulated by P.g.-infection [ Fig. 6c], but not by LPS-PG [ Fig. 6d]. Further, upregulation of Gal-3 production in Hc3716-hTERT cells was prominently caused by not only P.g.-infection [ Fig. 6e] but also by LPS-PG stimulation [ Fig. 6f]. These data indicate that TGF-β1 and Gal-3 produced from hepatocytes additionally promote myofibroblastic differentiation of HSCs in paracrine manner.

Discussion
It is well accepted that invasive enterobacteria such as Escherichia coli (E. coli) and their derived PAMPs play a critical role in pathogenesis of NASH 12,27,28 . Especially, LPS derived from E. coli in portal blood reaches the liver and enhances tissue necrosis factor α (TNFα) production from Kupffer cells through TLR4 signaling and leads to pathological progression of NASH. On the other hand, it is suggested that non-invasive bacteria such as Lactobacillus salivarius, acidophilus and Pediococcus pentosaceus do not induce liver fibrosis, or rather prevent liber fibrosis 6,10,[29][30][31][32] . Lactobacillus acidophilus has anti-inflammatory and antifibrotic activities through inhibiting NF-kB and down-regulating expression of TGF-β1, α-SMA and collagen. 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. www.nature.com/scientificreports www.nature.com/scientificreports/ a significantly higher fibrosis score 10 . Interestingly, there was a report describing that the serum AST and ALT levels of 10 NAFLD patients with periodontitis were significantly improved with oral hygiene instructions such as scaling and root planning procedures for 3 months 33 . In this study, P.g. infected from dental pulp induced periapical inflammation with neutrophils-infiltration at local site and was detected in the liver as well as our previous study 10 . We demonstrated that P.g.-odontogenic infection induced liver fibrosis and increased the number of hCLS, positively correlated with the extent of liver fibrosis 34 . Further, the immunohistochemical analysis of the liver highlighted that pSmad2 (a downstream of TGF-β1-signaling) and Gal-3 were significantly upregulated with P.g.-odontogenic infection, indicating these are key molecules for liver fibrosis induced by P.g.-odontogenic infection 16,20 .
P.g. has many pathogenic factors including fimbria, bacterial DNA, gingipain and LPS [6][7][8][9][10]13,23,24 . It was reported that gingipain, a trypsin-like cysteine protease, activated PAR2 in oral epithelial cells, gingival fibroblasts and immune cells in the periodontal tissue to produce cytokines including IL-6, 8 and MMP-2 resulting in periodontal breakdown 23,24 . Previous study indicated that secreted gingipains from P.g. induced TGF-β1 production from gingival fibroblasts 13 . P.g.-LPS also plays a key role in inducing inflammation not only at local but also at distant organs through the circulation. However, there is still controversy in receptors for P.g.-LPS [6][7][8][9][10] . Some studies suggested that P.g.-LPS exhibited an activity mediated by TLR2 though other studies for synthetic lipid A of P.g.-LPS have indicated that they are able to activate cells through TLR4 but not TLR2, suggesting that TLR2 activity induced by P.g.-LPS might be attributed to a contaminant lipoprotein [8][9][10][11]14,35 . Recently, Nativel et al. confirmed that P.g.-LPS activity was mediated exclusively through TLR4 and it only weakly induced proinflammatory cytokine secretion in mouse models 36 . Therefore, in this study we used LPS-PG including ligands for TLR2 (P.g.-lipoprotein) and 4 (P.g.-LPS), to focus on pathological significance of TLR2 since TLR2 was significantly upregulated in palmitate treated hepatocytes and in the liver of HFD-feed mouse 10 . Thus, we hypothesized that gingipain and P.g.-LPS/lipoprotein might be potential aggravating factors of pathological progression of NASH. www.nature.com/scientificreports www.nature.com/scientificreports/ Accumulating data have demonstrated that HSCs are effector cells for liver fibrosis. In response to liver injury, HSC is activated to be a myofibroblastic phenotype, which is highly proliferative and produces type I collagen (HSC activation) 14 . α-SMA and type I collagen, which are induced by TGF-β1/Smad and ERK pathways 14,19,37,38 , are common makers for HSC activation. TGF-β1 is known as one of the most important key mediators of fibrosis in several organs such as the lung, kidney, and liver 14,17,[39][40][41] resulting from proliferation and differentiation of myofibroblasts through Smad and ERK signaling pathways 22,37 .
In this study, P.g.-infection markedly stimulated HSC differentiation including upregulation of α-SMA and type I collagen production through activating Smad and ERK pathways. Moreover, TGF-β1 production was upregulated by P.g.-infection. Hence, TGF-β1 induced by P.g.-infection is the major molecule for HSC activation. As described the above, accumulating evidence indicated that activation of PAR2 by extracellular serine proteases induced TGF-β1 production from HSCs, moreover, it was demonstrated that TGF-β1 protein expression were decreased in PAR2 knock-out mice resulting in reduced liver fibrosis 25,42 . Therefore, we hypothesized that gingipain-derived from P.g. contributed to production of TGF-β1 from HSCs. As we expected, our study demonstrated that gingipain inhibitors completely inhibited TGF-β1 production from HSCs. Furthermore, palmitate is a major FFA in serum of NASH patients and palmitoylation is critical for efficient PAR2 signaling 43 . In this study, we elucidated that PAR2 was markedly upregulated with palmitate treatment and it significantly increased TGF-β1 production from HSCs with P.g.-infection. Gingipain inhibitors reduced TGF-β1 production. Moreover, TGF-β receptor I inhibitor suppressed HSC activation caused by P.g.-infection. It is suggested that PAR2 activation caused by gingipain results in liver fibrosis through HSC activation. The possibility may remain that infected www.nature.com/scientificreports www.nature.com/scientificreports/ P.g. include quite small amount of LPS, but the amount was supposed to be too small to have effects on cells, which could be ignored 44,45 .
In addition, HSC proliferation with/without palmitate treatment was significantly promoted by P.g.-infection. Proliferation rate of palmitate-treated HSC showing increased activation of gingipain/PAR2/TGF-β1 axis was similar to that of non-treated HSC. It is suggested that other molecules than TGF-β1 induced by P.g.-infection may also contribute to HSC proliferation. To clarify the significance of gingipain induced TGF-β1 in P.g.-odontogenic infection NASH mouse model, in vivo experiments using gingipain inhibitors are needed in near future.
Gal-3 is reported to be another key molecule for liver fibrosis through HSC activation 16,20,46 . Serum Gal-3 level was reported to be higher in advanced cases of liver fibrosis 47 . Gal-3 is produced from HSCs by NF-kB activation or by phagocytosis via integrin 16 . Interestingly, P.g. is well known to be phagocytosed via integrin α5β1 and α5β3 while P.g.-LPS and lipoprotein activate NF-kB pathway through TLR4 and 2, respectively 16,[48][49][50] . Our data showed that Gal-3 was significantly produced from HSCs with P.g.-infection and P.g.-LPS/lipoprotein stimulation. Moreover, Gal-3 upregulated TGF-β receptor II at protein level in HSCs. Gal-3 cross-links N-glycans on TGF-β receptors including receptor II and delays their removal by endocytosis 21,51 . Gal-3 expression caused by P.g.-infection and P.g.-LPS/lipoprotein stimulation may contribute to enhancing the sensitivity of HSCs to TGF-β1 by upregulating TGF-β receptor II, resulting in HSC activation. Moreover, in palmitate-treated HSCs, TLR2 expression is upregulated. Therefore, palmitate-treated HSCs tended to produce higher amount of Gal-3 by P.g.-LPS/lipoprotein stimulation, resulting in exacerbating liver fibrosis. These results suggest that steatosis induces upregulations of TLR2 expression, which contribute to high sensitivity to gingipain and P.g.-LPS/lipoprotein leading to severe inflammation and fibrosis. www.nature.com/scientificreports www.nature.com/scientificreports/ Interestingly, hepatocyte, which is major constitutive parenchymal cell of the liver and exists adjacent to HSC, significantly produced TGF-β1 and Gal-3 with P.g.-infection and/or P.g.-LPS/lipoprotein as well as HSCs. It is indicated that additional production of TGF-β1 and Gal-3 from hepatocytes may induce further activation of HSCs in a paracrine manner. Moreover, palmitate treatment also upregulated TLR2 expression in hepatocyte, which might increase Gal-3 expression in hepatocyte. Thus, it is suggested that the interaction between HSCs and hepatocytes has critical role in HSC activation caused by P.g.-infection and -LPS stimulation, especially in fatty liver.
With these experimental datasets including in vivo and in vitro, we suggest the potent mechanisms of HSC activation through TGF-β1 (Fig. 7a) and Gal-3 (Fig. 7b) in steatotic cell. Figure 7(a) PAR2 expression is significantly upregulated with fatty accumulation resulting in excessive TGF-β1 production caused by P.g.-infection through gingipain-PAR-2 axis (1). TGF-β1 up-regulates phosphorylation of Smad and ERK via TGF-β receptor I/II complex, leading to marked HSC activation in autocrine manner (2). TGF-β1 produced from steatotic hepatocyte with P.g-infection also stimulates HSC activation in paracrine manner (3). Figure 7(b) shows Gal-3-related HSC activation. P.g.-lipoprotein induces intense TLR2 signaling via upregulated TLR2-expression caused by fatty accumulation, leading to excessive Gal-3 production together with weak TLR4 signaling by P.g.-LPS (4). Gal-3 was also produced with P.g. endocytosis by infection (5). Secreted Gal-3 promotes HSC activation through Smad and ERK pathways in autocrine manner. In the mechanism, formation of bridges between TGF-β receptor II and Gal-3, which may keep TGF-β receptor II on the cell surface for longer than usual, eventually resulting in enhanced sensitivity to TGF-β1 (6). Steatotic hepatocyte with P.g. infection and/or P.g.-LPS/lipoprotein stimulation also produces Gal-3, which accelerates HSC activation in paracrine manner (7).
Conclusion. The present study indicates that P.g.-odontogenic infection exacerbates pathological progression of NASH by stimulating activation of HSCs through TGF-β1 and Gal-3 production. Moreover, TGF-β1 and Gal-3 production from P.g. infected and/or P.g.-LPS/lipoprotein stimulated hepatocytes contribute to pathological progression of NASH.

Please refer to the Supplementary Materials and Methods for more detailed descriptions.
Animal studies. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Hiroshima University Animal Research Committee and American Veterinary Medical Association (AVMA) Guidelines on Euthanasia. The experimental protocol was approved by the animal care committee of Hiroshima University (A16-58). HFD was fed to 10 mice (HFD-60; Oriental Yeast Co., Ltd., Tokyo, Japan) for 12 weeks to induce fatty liver. Then, the mice were segregated into 2 groups (with or without P.g.-odontogenic infection). P.g. W83 strain was dentally applied to 5 mice following the same procedure as previously described and 5 mice were named as HP 10 . The remaining 5 mice without P.g.-infection were named as HFD. After 9 weeks of P.g.-infection, body weights were measured and tissue samples such as periodontal tissue and liver were taken for histological analysis.
Morphometry. The number of hCLS, which were Mac-2 positive macrophage aggregates, was counted [ Supplementary Fig. 2] at randomly selected 5 different fields in each liver section under 200 magnification (gross area; 3.0 × 10 6 μm 2 ). The sirius red positive fibrosis areas were captured as same method as hCLS and measured with image processing software "Image J" (https://imagej.nih.gov/ij/). Cell culture. A commonly used human hepatic stellate cell line (LX-2), originally provided by Dr. Tomohiro Ogawa (Kindai University, Hiroshima, Japan), and immortalized human fetal hepatocytes (Hc3716-hTERT), established and provided by Professor. Hidetoshi Tahara (Hiroshima University, Hiroshima, Japan), were used in the present study 52 . cell treatment. Please refer to the Supplementary Materials and Methods for more detailed descriptions.
In order to clarify the mechanism of pathological progression of NASH, palmitate, which was FFA to be the major mediators of excessive hepatic lipid accumulation and elevated in circulation of NASH patients, was used in this study, following the previous study 10,53 . In brief, each cell line was cultured in medium containing palmitate (0.2 mM) for 18 hours to induce accumulation of lipids mimicking a fatty liver 10,53 . FFA-free BSA-treated cells were used as control. The cells were incubated in fresh medium without or with P.g. infection at multiplicity of infection (MOI) 100, along with P.g.-LPS/lipoprotein (1 μg/ml), which is a TLR2 and TLR4 ligand, (LPS-PG; InvivoGen, California, USA), human TGF-β1 (10 ng/ml; R&D Systems, Minnesota, USA), and Gal-3 (1 µg/ml; Peprotech, New Jersey, USA) recombinant protein. For gingipain inhibition, KYT-1 and -36 (Peptide Institute, Inc. Osaka, Japan) were used. KYT-1 and -36 are widely used to inhibit gingipain RgpA, RgpB (arginine-specific