Oral and Gut Microbial Dysbiosis and Non-alcoholic Fatty Liver Disease: The Central Role of Porphyromonas gingivalis

A potential link betweenand NAFLD has been revealed.and its DNA were detected in the oral cavities or livers of NAFLD patients at higher frequency than those of controls (,). NASH patients withinfection had more severe fibrosis than those without infection (). Furthermore, supportive data from variousstudies showed thatinfection can stimulate fat accumulation, increase the immune response, and result in insulin resistance, indicating the impact ofin NAFLD/NASH procession (). P. gingivalis P. gingivalis P. gingivalis in-vivo P. gingivalis P. gingivalis ^{99 100 100 97}

The spread ofto distant organs, such as the liver, occurs through two possible pathways. One is direct release into the blood circulation. During daily procedures such as brushing, or dental treatment,bacteria-associated factors such as LPS and cytokines spread into the bloodthe microulceration in the periodontal pocket, and are transported to the liver through the hepatic artery. The indirect pathway involves swallowing, which can translocate to the gastrointestinal tract and induce alteration of gut microbiota; this in turn negatively affects the liver through the portal vein system (,). Therefore, the pathogenic action of the bacteria is related to both these pathways: directly through the oral-liver axis, and indirectly through the oral-gut-liver axis (). P. gingivalis P. gingivalis and via P. gingivalis ^{93 101} Figure 2

is a gram-negative, obligate anaerobic bacteria. It is a common component of subgingival microbiomes that can colonize oral epithelial cells ().has unique structural components, known as virulence factors. These include some bacteria's own structural components such as fimbriae and LPS, and secretory components such as gingipains, and play critical roles in the survival and spread ofthrough cellular colonization, along with its pathogenic functions, such as inducing host immune response and inflammatory reactions (,). P. gingivalis P. gingivalis P. gingivalis ^{102 98 103}

fimbriae are filamentous structures on the surface of the bacterium that enable bacterial binding to host cells and tissues, enhance bacterial motility and invasiveness, and contribute to biofilm formation (). There are two forms offimbriae: FimA, and the less important minor fimbriae. Studies of NAFLD patients showed that the invasive type II FimA constituted half of the total fimbriae detected (). Type IV FimA, however, is closely associated with advanced liver fibrosis (). P. gingivalis P. gingivalis ^{104 99 105}

The mechanism underlying the impact of FimA on host cells is primarily related to the activation of adhesion and immuno-inflammatory pathwaysinteractions with various host receptors, which facilitates bacterial colonization and leads to host cell inflammation (,,). In addition, FimA can also help the bacteria survive in the host cell over extended periods by triggering the complement system to protect the bacteria from host immune clearance (). via ^{104 106 107 104}

FimA acts through three major types of receptor. Regarding its inflammatory effects, FimA binds to Toll-like receptor (TLR) 2, which activates the nuclear factor kappa B (NF-κB) system to induce the production of various pro-inflammatory cytokines (). TLR2 has been shown to be associated with the pathogenesis of NAFLD (). FimA also triggers the innate immune system by binding to complement receptor (CR) 3 and stimulating macrophages/monocytes (). As a result, FimA inhibits the production of interleukin (IL) (), which is related to bacterial clearance (), thus enhancingsurvival. CR3 is expressed in the liver (), and the activation level of the complement system is related to NAFLD severity (), suggesting the involvement offimbriae in the pathogenesis of NAFLDimmune response induction. ^{108 109 10 12 110 111 112} P. gingivalis P. gingivalis via

Furthermore, FimA activates CXC-chemokine receptor 4 (CXCR4) and TLR2 to induce cyclic adenosine monophosphate-dependent protein kinase A signaling, which not only results in disruption of phagocytosis by macrophages (), but also inhibits host immune clearance of(). Dysfunctions of both macrophages and CXCR4 have been reported to be associated with NAFLD (,). ^{113 114 115 116} P. gingivalis

Beyond FimA,minor fimbriae can also induce inflammatory reactions in macrophages (), and are reported to induce phosphorylation of FOXO1 (), which plays a role in both lipid and glucose metabolism (), indicating that both FimA and minor fimbriae inmay be factors leading to liver injury by this species. P. gingivalis P. gingivalis ^{117 118 119}

LPS is a component of the outer cell membranes of gram-negative bacteria (,). It critically contributes to the pathogenicity of microbes, mainly through lipid A, which is the biologically active region of LPS (). LPS is structurally different in different bacterial species due to variations of fatty acid acyl chain composition of lipid A ().LPS contains multiple forms of lipid A, and the structural differences in lipid A may explain whyLPS can initiate differential signaling pathways and immune responses (,). In addition to participating in biofilm formation ()LPS plays an important role in triggering host inflammatory responsesTLR activation; the specific type of TLR activation by LPS depends on the variation in the acylation of lipid A (). However, it was recently reported that the activation of TLR2 byisother molecules on the surfaces of intactcells rather than lipid A (). Activation of TLR2 leads to an increase in the production of cytokines such as tumor necrosis factor α (TNF-α) and IL-6 by macrophages (), while activation of TLR4 elevates the levels of IL-1β, IL-6, and IL-8 (). In addition, NF-κB signaling pathway has also been reported to act downstream of TLRs, and to play a critical role in the inflammatory function ofLPS, including the production of cytokines (). Severalandstudies have been performed to clarify the effects ofLPS on NAFLD. Injection ofLPS into the gingiva of animal models resulted in lipid deposition and inflammation in the liver (–), andstudies using HepG2 cells indicated that LPS may play a role in intracellular lipid accumulation and inflammationboth NF-κB and c-Jun-NH2-terminal kinase signaling (). Moreover,LPS can accelerate the progression of mild fatty liver to NASH (), and studies using steatotic hepatocytes revealed that this activity may be related to increased TLR2 expression, inflammasome mRNA levels, and pro-inflammatory cytokines (). Moreover, recent evidence shows thatLPS may contribute to hepatic fibrosis by activating hepatic stellate cells (HSCs); the mechanism involves triggering TLR4 to increase the production of galectin-3, which is critical for HSC activation (). In addition,LPS is possibly implicated in insulin resistance, either by stimulating the activation of pro-inflammatory cytokines such as TNF-α and IL-6, which play important roles in insulin resistance (), or by directly inhibiting glucose incorporation into smooth muscle cells (). ^{120 121 122 10 10 123 124 10 125 126 127 123 128 130 131 132 100 132 133 134} P. gingivalis P. gingivalis , P. gingivalis via P. gingivalis via P. gingivalis P. gingivalis in-vivo in-vitro P. gingivalis P. gingivalis in-vitro via P. gingivalis P. gingivalis P. gingivalis

Kuraji et al. reported thatLPS accumulated predominantly in the liver, over other organs, and persisted in the livers of HFD-induced steatotic mice longer than in normal mouse liver. In addition, the diseased mice showed enhanced sensitivity to LPS and delayed clearance of LPS from the liver, indicating the potent role ofLPS in liver injury and NAFLD. The mechanism is possibly related to increased and activated hepatic macrophages (Kupffer cells) and TLR signaling (). P. gingivalis P. gingivalis ⁹³

As noted elsewhere in this review, gut bacteria-derived LPS also plays critical roles in the pathogenesis of NAFLD. The different functions and mechanisms involved in gut bacteria-derived LPS andLPS have been investigated. For example,LPS can activate both TLR2 and TLR4, whereasLPS can only bind to TLR4 (); although both LPS types activate TLR4,LPS stimulates different pathways from those affected byLPS (). Furthermore,LPS shows a stronger ability to escape recognition by the host innate defense system thanLPS (), andLPS induces more intracellular fat accumulation in HepG2 cells thanLPS (). In contrast to the substantial accumulated evidence that gut bacteria-derived LPS contributes to NAFLD by inducing oxidative stress (), there is as yet little evidence () supporting direct oxidative stress stimulation byLPS in NAFLD, which should be investigated further. P. gingivalis P. gingivalis Escherichia coli P. gingivalis E. coli P. gingivalis E. coli P. gingivalis E. coli P. gingivalis ^{135 136 137 131 138 139}

Gingipains are a family of secretory cysteine proteinases that are known to be the main virulence factors related to the pathogenicity of. They consist of lysine and arginine gingipains (,). As with fimbriae and LPS, gingipains also play roles in biofilm formation and produce immune-inflammatory responses by activating various immune cells (,). Evidence shows that gingipains protectfrom the host defense system in the following ways: they enhance the production of TNF-α by neutrophils by activating the TLR2/phosphatidylinositol-3 kinase (PI3K) pathway (), thereby facilitating bacterial survival within host cells; they inhibit pathogen clearance by negatively regulating the production of neutrophil-derived molecules () and the macrophage immune receptor CD14 (); and they promote the adaptability ofby triggering the complement system, for example by modulating the C5a receptor (C5aR) and its crosstalk with TLR2 signaling in macrophages (). P. gingivalis P. gingivalis P. gingivalis ^{140 141 10 142 143 144 145 146}

Furthermore, gingipains may play a role in evading the host adaptive immune system by regulating T-cell immunity (). Like LPS (), they can induce the production by Th17 cells of the cell-specific cytokine, IL-17, by directly inducing the expression of CD69 and CD25 on T-cells (). T-cell immunity, especially the Th17/IL-17 signaling pathway, plays a role in protecting pathogens and promoting inflammation, and was recently reported as important in the development of NAFLD (–). Moreover, recent work highlights a remarkable function of gingipains in NAFLD, revealing that they contribute to liver fibrosis by activating HSCsthe proteinase-activated receptor (PAR) 2 and TGF-β pathway (). It should also be noted that gingipains can inactivate PI3K, protein kinase B (Akt), and Akt downstream proteins, including glycogen synthase kinase 3 (GSK3) and mammalian target of rapamycin (mTOR) (). The PI3K/Akt signaling pathway plays multiple roles in various cell functions, including cell survival and glucose metabolism, and anstudy has revealed thatsuppresses glycogen synthesis in HepG2 cells by inhibiting the insulin receptor substrate 1/Akt/GSK3β pathway (). This indicates that gingipains possibly participate in glucose metabolism impairment/insulin resistance, which needs to be investigated further. ^{147 148 149 150 153 132 154 155} via in-vitro P. gingivalis

Biofilms and outer membrane vesicles (OMVs) are two important microorganism-produced structures responsible for the survival, spread, and pathogenicity of microbes. Biofilms are composed of water, bacterial cells, and extracellular polymeric substances, which are responsible for microorganism protection and resistance to clinical treatments, such as antibiotics (–).virulence factors, such as fimbriae, LPS, and gingipains, have been reported to contribute to biofilm formation, as mentioned previously, and the pathogenicity ofis enhanced by biofilm (,). In addition to being a reservoir of pathogenic bacteria, biofilms also contribute to inflammation in many diseases, including inflammatory bowel disease and hepatobiliary carcinomas (); however, the pathogenic effect ofbiofilm, such as direct reactions of biofilm in NAFLD, remains unclear. ^{156 158 159 160 161} P. gingivalis P. gingivalis P. gingivalis

OMVs are small, spherical, bilayered membrane structures that are constantly released from the bacterial surface during growth. Each vesicle is composed of outer membrane proteins, lipoproteins, LPS, and some periplasmic components (,). Like other bacteria such asandcan produce OMVs ().OMVs can concentrate virulence factors such as gingipains and LPS in the form of OMVs and discharge them to the environment to participate in bacteria-associated disorders (,).OMVs play roles in biofilm formation by binding to other periodontopathogens (,), and facilitate bacterial adhesion and invasion in host cells, adaption to stress, and immune defense evasion (). In addition to contributing to the destruction of periodontal tissues,OMVs can migrate to the blood and play an important role in the pathogenesis of various systemic diseases, such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, and carcinoma; the mechanism involved has been recently reviewed by Zhang et al. (). Although the direct impact ofOMVs in NAFLD has not been completely elucidated,OMVs carrying gingipains can transfer to the liver and impair hepatic glycogen synthesis in a mouse model; they can also inhibit insulin-induced Akt/GSK-3β signaling in a gingipain-dependent manner in HepG2 cells (), indicating a potential role of these gingipain-carrying OMVs in the development of NAFLD through negative regulation of glucose metabolism and insulin sensitivity. ^{162 163 164 165 166 167 168 168 169 170} Francisella Pseudomonas putida, P. gingivalis P. gingivalis P. gingivalis P. gingivalis P. gingivalis P. gingivalis

In summary, these bacteria-associated structures play roles in bacterial protection, as well as affecting the virulence of, by activating various risk factors for NAFLD, such as fat accumulation, inflammation, insulin resistance/disturbance of glucose metabolism, and fibrosis. The mechanism involves immune cell-related inflammatory reactions and various intracellular signaling pathways (). The scope of this review includes only the reported virulence mechanisms ofin relation to the pathogenesis of NAFLD; the functioning of these mechanisms beyond NAFLD and in other diseases also requires consideration. Moreover, other virulence factors, such as heat shock protein 60 (), have been mentioned in relation to liver diseases (). However, the majority ofstudies have focused on circulating immune cells, while few studies have investigated the role ofin hepatic cells such as Kupffer cells and hepatocytes. Further studies are needed to elucidate the roles of the pathogenic components ofin NAFLD. P. gingivalis P. gingivalis in-vitro P. gingivalis P. gingivalis Figure 2 ^{171 172}

The effects ofon the modulation of gut microbiota have been investigated in many studies. Both intravenous injection and oral administration of the bacteria in a mouse model produced alterations in the gut microbiota (,,). Clinical studies have detected remarkably higher proportions ofin the guts of NAFLD patients than in those of non-NAFLD controls 99; patients with chronic periodontitis tended to have less diversity in their gut microbiomes (); the major changes in the gut microbiota of patients with liver cirrhosis result from invasion by oral bacterial species (). These results indicate a possible relationship between the bacteria, gut, and NAFLD (). Studies involving oral administration ofto mouse models show that, in contrast to the increased blood endotoxin levels that are typical of gut dysbiosis/barrier dysfunction and lead to systemic inflammation contributing to liver injury, nowas detected in the blood system (), or outgrowth of the bacteria in the gut (). This strongly supports the speculation that oral-induced endotoxemia-related liver injury indirectly, by inducing gut dysbiosis and barrier dysfunction. P. gingivalis P. gingivalis P. gingivalis P. gingivalis P. gingivalis ^{50 173 174 175 176} Figure 2 ^{174 80}

However, despite the evidence that oralcan survive in the acidic conditions induced by gastric juice (), enabling it to enter the gastrointestinal tract, and that dead bacteria from the mouth may stimulate several gut pathogens, upregulate bacterial virulence genes, and thereby increase cytotoxicity (), the mechanism underlying bacteria-induced gut dysbiosis remains unclear. For example,must inhabit the host cells and trigger immune-inflammatory reactions to initiate subsequent actions, andstudies have shown that intestinal inflammation can be detected following oral administration of(), but there is as yet no evidence to show whether and howcan localize in the gastrointestinal tract; in addition, the bilateral interactions between oral bacteria and gut microbiota should be considered. Furthermore, the lack of large-scale epidemiologic studies and some inconsistent results in different animal models make it difficult to define a clear mechanism underlying the effects ofin NAFLDmodulation of gut microbiota (). P. gingivalis P. gingivalis in-vitro P. gingivalis P. gingivalis P. gingivalis via ^{49 177 50 93}

On the other hand,is not the only periodontopathogen that can contribute to the pathogenesis of NAFLD.can induce gut dysbiosis and impairing glucose metabolism (); the detection frequency ofin the NAFLD patients was significantly higher than that in the control subjects (); in patients with liver cirrhosis, more than half (54%) of the patient-enriched, taxonomically assigned bacterial species were of oral origin (mostly veillonella and streptococci) (,). Moreover, in comparison to growing evidence that supportsas a cause of gut dysbiosis, the precise mechanism through whichexerts its effects has yet to be determined. Further studies are needed to complete our knowledge of the roles ofin NAFLDthe oral-gut-liver axis. P. gingivalis A. actinomycetemcomitans T. denticola P. gingivalis P. gingivalis P. gingivalis via ^{178 99 176 179}

Given the remarkable impacts of periodontal disease in NAFLD, improving oral hygiene, and other approaches targeting pathogenic oral bacteria, are regarded as effective treatment strategies for bacteria-associated NAFLD. Administration of broad-spectrum antibiotics can effectively protect against various liver diseases, and some specific antibiotic treatments have produced improvement in the clinical symptoms of NAFLD, by means of eliminating harmful microbes, lowering circulating endotoxin and transaminase levels, and preventing lipid accumulation in the liver (). Notably, despite their powerful positive effects, long-term administration of antibiotics may cause toxic side effects, eliciting antibiotic-resistant bacterial strains and itself generating gut dysbiosis (). There is therefore increasing interest in the safety and specificity of therapeutic approaches targeting pathogenic oral bacteria, such as. ^{180 181} P. gingivalis

One of the treatment modalities is the administration of probiotics. Two lactic acid bacteria,and, have millennia-long histories of use in the fermentation of foods and are generally accepted as safe for human consumption (“Generally Recognized As Safe,” or GRAS, in US Federal Drug Administration terminology) (). It has been reported that, together with one of its derivatives, nisin, was effective in periodontal disease, with potent antibacterial ability but low side effects. Its mechanism involves modulation of the formation, composition, and survival of oral bacterial biofilms (). Moreover,treatment reduced hepatic fat accumulation and showed anti-inflammatory effects in the liver, indicating its potential in the treatment of NAFLD (,). Furthermore, antibacterial effects onand other oral pathogens were seen not only from livecells but also supernatants from cell-free cultures and heat-killed(), indicating a potent yet safe antibacterial activity of this probiotic. Lactococcus lactis Lactobacillus reuteri L. lactis L. lactis P. gingivalis L. reuteri L. reuteri ^{182 82 82 182 183}

With regard to the therapeutic approaches specifically targeting the structure of, further methods for inhibiting biofilm formation by modulating OMVs have been investigated, in addition to the biofilm modulation approach mentioned above. These include RNA interference technology, antimicrobial agent-induced membrane vesicles, and some natural products, which have all been reviewed recently (). Plant-derived products have attracted considerable attention in chronic disease therapy, given their safety. Natural compounds have been demonstrated to inhibit the growth and virulence factor activity of bacteria (–). We have reported that carnosic acid, extracted from rosemary plants, exerts potent effects, inhibiting lipid accumulation in both the adipose tissue and liver, improving glucose metabolism and liver functions in theobesity mouse model (), and inhibiting lipid accumulation in HepG2 cells (). Moreover, our studies have clarified that carnosic acid can protect normal hepatocytes from H2O2-induced oxidative stress (). Interestingly, carnosic acid has been reported to have remarkable antibacterial activities (,). The mechanism is related to its inhibition of biofilm information (); carnosic acid can also protect against LPS-induced liver injury (), and is thus a potential new therapeutic approach for treating NAFLD. In our laboratory, we are currently investigating the roles of carnosic acid in NAFLD associated withinoculation in both mouse and cell models. P. gingivalis ob-ob P. gingivalis ^{120 184 186 187 188 189 190 191 192 193}

Anti-fimbrial Ab has also been reported as effective in inhibiting the adherence ofto its host cells (). Inhibition of or pre-immunity against potent virulence factors ofuch as gingipains, was effective against periodontitis and systemic diseases (,). Moreover, as the CR3 receptor plays an important role in thefimbriae-stimulated adhesion signal, as described above, CR3 attenuation has been considered as an approach for treating periodontitis and-related systemic diseases (). However, little is known about the significance of the above materials for the management of NAFLD associated with infection by oral pathogenic bacteria such as, which should be investigated further. P. gingivalis P. gingivalis, s P. gingivalis P. gingivalis P. gingivalis ^{194 195 196 197}

Since the gut microbiota is closely related to the pathogenesis of NAFLD, various therapeutic approaches targeting gut microbiota have been developed. They include antibiotics, probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, which have recently been reviewed (,). In particular, dietary fiber is cited as useful: it may improve early-stage NAFLD by reducing calorie absorption and correcting the imbalance of gut microbiota ();. reuteri not only improves the dysbiosis of oral microbiota by targeting biofilm, but also shows the ability to correct gut dysbiosis and protect the liver from inflammatory damage (). Moreover, a gut symbiont,, and one of its membrane proteins has produced anti-inflammatory activity and improved gut permeability in animal models (,,). Furthermore, human studies have shown that administration ofleads to decreased body weight, and the markers associated with inflammation and liver dysfunction in obese objects (). However, it should be noted that while microbiome-based therapeutic approaches are usually regarded as safe, they cannot be considered risk-free, as they are still bioactive. Therefore, large-scale studies are required to evaluate their safety. ^{76 180 198 199 30 200 201 202} L Akkermansia muciniphila A. muciniphila