WO2024215861A2

WO2024215861A2 - Nisin-based formulations for oral and systemic conditions

Info

Publication number: WO2024215861A2
Application number: PCT/US2024/024033
Authority: WO
Inventors: Yvonne L. KAPILA; Allan RADAIC; Pachiyappan KAMARAJAN
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-04-12
Filing date: 2024-04-11
Publication date: 2024-10-17
Anticipated expiration: 2025-10-12
Also published as: WO2024215861A3

Abstract

Dysbiosis of the oral microbiome mediates chronic periodontal disease, gut microbial dysbiosis, and mucosal barrier disfunction that leads to both brain inflammation as well as steatohepatitis via the enterohepatic circulation. An improvement of this microbial dysbiosis towards health may improve brain, gut and liver disease. Treatment with antibiotics and probiotics have been used to modulate the microbial, immunological, and clinical landscape of periodontal disease with some success. The instant invention describes the use of nisin, an antimicrobial peptide and bacteriocin produced by Lactococcus lactis, to counteract the periodontitis-associated gut dysbiosis and/or to modulate the glycolipid-metabolism and inflammation in the brain and liver.

Description

NISIN-BASED FORMULATIONS FOR ORAL AND SYSTEMIC CONDITIONS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63/495,692, filed April 12, 2023, entitled “NISIN-BASED FORMULATIONS FOR ORAL AND SYSTEMIC CONDITIONS”, and U.S. Provisional Patent Application No. 63/511.360, filed June 30. 2023, entitled “NISIN LANTIBIOTIC MITIGATES BRAIN MICROBIOME DYSBIOSIS AND ALZHEIMER’S DISEASE-LIKE NEUROINFLAMMATION TRIGGERED BY PERIODONTAL DISEASE” the contents of each of which is incorporated by reference herein.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under DE025225 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

Embodiments of the disclosure concern at least the fields of microbiology and medicine.

BACKGROUND OF THE INVENTION

Periodontal disease, a common chronic inflammatory disease of the oral cavity, is caused by the host immune response to an oral polymicrobial dysbiosis present within oral biofilms^1,2. These dysbiotic biofilms, which are predominantly compnsed of anaerobic Gram-negative bacteria, namely periodontopathic bacteria, are continually releasing a lipopolysaccharide (LPS) challenge and other microbial molecules that trigger an altered host immune response and the release of tissue destructive enzymes in the periodontal tissues, thereby leading to periodontal tissue destruction and tooth loss ^2,3. These local microbial and inflammatory products from inflamed periodontal tissues travel into the systemic circulation ⁴'⁷, and thereby are thought to be associated with systemic diseases via several different mechanisms ³. In fact, periodontal disease has been known to exacerbate various metabolic disorders, such as obesity, diabetes, dyslipidemia, and cardiovascular disease ^8-11.

In recent years, growing evidence from basic ^12-16. clinical ^17,18. and epidemiological ^18-23 studies suggest that periodontitis is associated with both brain and liver disease. For example, non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, has been closely associated with periodontal disease ²⁴ NAFLD is characterized by hepatic lipid deposition in the absence of a secondary predisposing factor, such as a habitual drinking history, viral infections or autoimmune diseases ^23-28. A portion of NAFLDs can develop into more severe and progressive forms, namely nonalcoholic steatohepatitis (NASH) ²⁷ ', further leading to cirrhosis and hepatocellular carcinoma, which are end-stage liver diseases ^29,3°.

Enteral translocation of oral bacteria and inflammatory mediators, and gut microbial dysbiosis have been proposed as potential mechanisms that mediate this pathogenesis of NAFLD ^31,32 Based on the unique anatomical characteristics of the liver, all blood from the gut travels through the portal vein to gather into the liver before the blood reaches the systemic circulation ^33,34 Since gut dysbiosis increases the amount of hepatotoxins, such as LPS, ethanol, and volatile organic compounds^35- ³⁸, and further enhances intestinal permeability by impairing intercellular tight junctions in the gut wall, it thereby promotes the translocation of hepatotoxins and enterobacteria and their byproducts to the liver ^39,4°.

Disease in the oral cavity, that is, periodontal disease impacts the gut, liver and brain. Periodontal disease is a chronic disease that impacts more than 1/2 of the US population and many more people globally. Its prevalence increases with age and with our population aging, there will be a large spike in the prevalence of periodontal disease as baby boomers age. There are no therapeutics on the market that specifically treat periodontal disease. There is a need in the art for new therapeutic regimens useful for the treatment of periodontal disease associated pathologies such as non-alcoholic fatty' liver disease and Alzheimer's disease.

SUMMARY OF THE INVENTION

As discussed in detail below, we have discovered that the administration of nisin can significantly shift the in vivo microbiome towards a healthy state while preventing the harmful inflammatory and structural alterations in the brain triggered by polymicrobial infection. We also show that the administration of nisin can prevent other pathologies triggered by polymicrobial infection including fatty liver lipid deposition. Building upon these discoveries, the disclosure presented herein provides methods for administering nisin in therapeutic regimens directed towards pathologies such as Alzheimer’s disease and steatohepatitis.

As discussed below, oral polymicrobial infection and dysbiosis is observed to trigger a significant shift in microbial diversity⁷ and composition in the, gut and liver of infected mice, an elevated cytokine immune response, a decreased expression of tight junction-related genes in the brain and gut. an increase in inflammation in the small intestine concomitant with decreased villi structural integrity, heightened hepatic exposure to bacteria, and lipid and malondialdehyde accumulation in the liver. In contrast, in the context of disease, we demonstrate that nisin treatment of mice significantly shifts the microbiome back towards the healthy control state while preventing the harmful inflammatory and structural alterations in the brain and GI tissues and fatty liver lipid deposition triggered by the polymicrobial infection. Validation with RNA Seq analyses confirmed the significant infection-related alteration of several genes involved in mitochondrial dysregulation. oxidative phosphorylation, and metal/iron binding and their restitution following nisin treatment. In addition, in support of these in vivo findings in an animal model indicating that periodontopathogens induce gastrointestinal and liver distant organ lesions, human autopsy specimens further demonstrated a correlation between tooth loss and severity of liver disease. The studies disclosed herein demonstrate and confirm a nexus between the administration of nisin and improvements in liver and brain health.

In illustrative studies that led to the invention disclosed herein, mice were divided into four groups: control, infection (oral polymicrobial inoculum), nisin (300 pg/ml nisin), and infection + nisin. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum, were administrated topically onto the oral cavity to establish polymicrobial periodontal disease. Microbial composition as assessed by 16S ribosomal RNA sequencing, global RNA Seq gene profiling, expression of inflammatory genes as determined RT-PCR, and histological findings were evaluated in oral, gut and liver tissues following bacterial challenge and/or nisin treatment. Human autopsy specimens and their corresponding dental radiographs were also examined to further evaluate correlations between liver and oral pathological findings.

The studies disclosed below further show that nisin can inhibit or ameliorate brain inflammation and its administration can, for example be used to abrogate the deposition of AP42, Tau, and phosphorylated Tau in the brain following oral polymicrobial infection. The invention disclosed herein harnesses these discoveries in order to provide new therapeutic regimens for the treatment of pathologies associated with brain inflammation as well as hepatopathologies such as steatohepatitis. The studies disclosed herein underscore the ability of nisin to mitigate and correct periodontal disease by correcting inflammation, promoting tissue repair/regeneration, and re-aligning the microbiome including the virome back towards health.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include methods of inhibiting development of and/or ameliorating liver steatosis in a subject, the method comprising administering to the subject a pharmaceutical composition comprising nisin in amounts sufficient to inhibit the development of liver steatosis in the subject. While nisin can be administered via a number of routes, in certain embodiments of the invention, the pharmaceutical composition is administered orally. In some embodiments of the invention, the subject is selected to be a patient diagnosed with a periodontal disease. In certain embodiments of the invention, the subject is selected to be a patient diagnosed with a fatty liver disease such as steatohepatitis. In certain embodiments, amounts of nisin in the composition are sufficient to decrease the number of lipid vesicles observable via histology in a mouse by at least 10% as compared to a control mouse in a murine model of polymicrobial liver steatosis. In certain embodiments of the invention, amounts of nisin in the composition are from about 1 nanogram to about 100 grams (e.g. from about 100 nanograms to about 20 grams or about 50 grams).

Embodiments of the invention also include methods of inhibiting development of and/or ameliorating brain neuroinflammation in a subject, the methods comprising administering to the subject a pharmaceutical composition comprising nisin in amounts selected to be sufficient to inhibit the development and/or progression of brain neuro inflammation in the subject. In certain embodiment of the invention, the pharmaceutical composition further comprises at least one of: a lipid, a nanoparticle, an anti-inflammatory agent, and a polymeric scaffold. Typically in such embodiments, amounts of nisin administered are selected to be sufficient to decrease expression of TNF-a mRNA in the subject. In illustrative embodiments of the invention, amounts of nisin administered are selected to be sufficient to reduce phosphorylated Tau deposition in the subject. In certain embodiments of the invention, the subject is selected to be a patient diagnosed with a neuroinflammatory disease such as Alzheimer’s disease.

Embodiments of the invention include methods of administering nisin alone, or in nisin formulations comprising agents such as delivery vehicles, scaffolds, in nano-sized delivery systems, and/or complexed with other anti-inflammatory molecules, for the treatment of non-alcoholic fatty liver disease (NAFLD), via oral, sublingual, buccal, local and systemic administration routes. Illustrative nisin formulations comprise water- and oil-based vehicles containing agents such as pol oxamers (including, but not limited to poloxamer 188, pol oxamer 407 and/or poloxamer 238), alcohol, glycerol, propylene glycol, vegetable oils, mineral oils, cacao butter, poly(ethylene glycol) (PEG) and/or gelatin. In certain embodiments of the invention, the formulation comprises one or more probiotics such as the Nisin- producing Probiotic organism Lactococcus Lactis. Anti-inflammatory molecules that can be included in such formulations include, but not limited to curcumin, alpha lipoic acid (ALA), and niacin. Scaffolds useful in such formulations include hydroxyapatite, calcium phosphate, ceramic, coral-based bone scaffolds, human bone scaffolds, hydrogels, polymeric scaffolds, such as, but not limited to poly(e- caprolactone) (PCL), poly(lactic acid) (PLA), poly(lactide-co-glycolide acid) (PLGA), poly(ethylene glycol) (PEG), collagen, seaweed cellulose, chitosan, microspheres, printed-based polymer scaffolds and the like. Embodiments of such formulations include nano-sized delivery systems such as lipid-based nanoparticles (such as, but not limited to solid lipid nanoparticles, liposomes, micelles, nanostructured lipid carriers), polymer-based nanoparticles (such as, but not limited to polymeric micelles, nanospheres and nanocapsules) and metal nanoparticles (such as, but not limited to palladium, silver, titanium, gold nanoparticles).

Embodiments of the invention include compositions of matter comprising nisin and a pharmaceutically acceptable carrier. Typically, in these embodiments, nisin is present in the composition in such that amounts of nisin in an individual administered the composition are sufficient to inhibit or ameliorate the harmful inflammatory and structural alterations in the brain and/or liver that are triggered by polymicrobial infection.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Nisin promotes a shift from a disease-associated microbiome toward a healthy state through whole body. Bar graphs show relative abundance of each bacteria taxa at the phylum level (A-C) and genus level (D-F) in oral cavity , small intestine, and liver.

Figure 2. Bar plots show bacterial taxa that exhibited significant differences in relative abundances at species level from oral cavity (A), small intestine (B) and liver (C). *p < 0.05 between groups with Tukey test, fp < 0.05 with t-test.

Figure 3. Nisin prevents the alterations in microbial diversity (A-C) and community structure (D-F) by polymicrobial infection at oral cavity, small intestine, and liver. *p < 0.05 and **p < 0.01 between groups with Tukey test, fp < 0.05 with t- test.

Figure 4. Periodontal inflammation following polymicrobial oral infection is reduced by nisin. A) Total bacteria amount, B) Gene expression of pro-inflammatory cytokine, and C) Histopathological evaluation in periodontal tissue. *p < 0.05 and **p < 0.01 between groups with Tukey tests.

Figure 5. Inflammation of small intestine following polymicrobial oral infection is prevented with nisin treatment. A) Gene expression of immune cytokine profiles from the ileum tissue, B) Histopathological examination to evaluate seventy of inflammation of small intestine, C) Histological score, D) Gene expression of tight junction proteins associated with gut burner function. *p < 0.05 and **p < 0.01 between groups with Tukey test.

Figure 6. Nisin treatment attenuates the burden of total bacterial and periodontal pathogens into the small intestine and liver. In order to further assess the bacterial load on gut-liver-axis, the number of total bacteria and periodontal pathogens was measured in the small bowel feces (A-C) and liver samples (D-F) using RT-PCR in the manner of absolute quantification. *p < 0.05 and **p < 0.01 between groups with Tukey test.

Figure 7. Hepatic lipid deposition by polymicrobial infection is significantly abrogated in mice treated with nisin. Histopathological analysis of liver tissue was conducted to evaluate the ability of nisin to modulate the diseased changes in lipid deposition and inflammatory reaction in the histological images stained by hematoxylin (A) and Oil red (B). Four different fields (100x magnification) were randomly selected on the images of three tissue sections per mouse specimen (n = 3 per group), and number of vesicles (C) and area of orange-stained fatty deposition (D) was measured using ImageJ analysis software. *p < 0.05 between groups and **p < 0.01 between groups with Dunn’s test.

Figure 8. Gene profile in liver tissue analyzed by RNA sequencing (n=6). A) RNA-seq detected 2.949 genes were categorized by hierarchical clustering approach. B) Subcluster analysis were further performed to determine the trends in gene expressions among groups. C) Volcano plot revealed 2,084 DEGs (1185 up-regulated and 899 down-regulated) between the control group and the nisin group (left panel) and 560 DEGs (408 up-regulated and 152 down-regulated) between the infection group and the inf. + nisin group (right panel).

Figure 9. Functional analysis expressed genes in liver tissue for KEGG pathways (n = 6). A) Scatter Plot shows for top 20 significantly enriched KEGG terms, which were determined between the infection group and the inf. + nisin group by ClusterProfiler (version 3.8.1). B) The enriched pathway of Non-alcoholic fatty liver disease (NAFLD, term 04932) in KEGG database. C) The bar graph revealed differential genes among four groups for the NAFLD-related pathway (* adjusted p- value <0.05).

Figure 10. Functional analysis expressed genes in liver tissue for GO pathways (n = 6). A) Bar plot shows for top 20 significantly enriched GO terms at molecular function category, which were determined between the infection group and the inf. + nisin group by ClusterProfiler (version 3.8.1). B) Related genes of iron binding and metal cluster binding in GO database. C) The bar graph reveals differential genes among four groups for the iron binding-, metal cluster binding-, and cellular response to metal ion- related genes (*: adjusted p-value <0.05).

Figure 11. Hepatic malondialdehyde (MDA) deposition following the polymicrobial infection is significantly reduced in mice treated with nisin. MDA in liver tissue was quantified to evaluate the ability of nisin to modulate lipid peroxidation due to oxidative stress in the histological sections stained by immunohistochemistry (A). Four different fields (100* magnification) were randomly selected on the images of three tissue sections per mouse specimen (n = 3 per group), and area of brown-stained MDA deposition (B) was measured using ImageJ analysis software. *p<0.05 between groups and **p < 0.01 between groups with Dunn’s test.

Figure 12. The number of remaining teeth correlated with the severity of liver disease at human autopsy study. The control and periodontitis groups were defined based on the severity and extent of periodontal disease assessed on panoramic radiographs and cone-beam CT images, respectively. Panel A shows representative findings of oral (upper panel) and liver (lower panel) in each group. Blue arrows indicate small fat droplets, red arrows indicate scarring fibrosis, and arrowheads indicate ballooning hepatocytes with cell injury, respectively. The NAFLD activity score (NAS) measured from histological findings was compared between the control group and the periodontitis group using an unpaired t-test (B). The correlation coefficient between the NAS and the number of remaining teeth was analyzed using the Pearson correlation coefficient (C).

Figure 13. A schematic providing a description of the experimental groups and infection and treatment protocols.

Figure 14. Analysis of the microbial abundance by 16s rRNA sequencing shows that nisin reverses the changes in brain microbiome composition induced by oral polymicrobial infection. The groups included Control, Infection, Nisin, Infection+Nisin. Differential abundance analysis for bacteria at phylum (A) and genus level (B). *, the difference between the Control and Infection group was significant (p<0.05). #, the difference between the Infection and Infection+Nisin group was significant (p<0.05).

Figure 15. Analysis of microbial community composition and diversity shows that nisin alters microbial diversity and community structure in brain following oral polymicrobial infection. The groups included Control, Infection, Nisin, Infection+Nisin. A-C. Chaol estimator, Shannon index and Simpson index are analyzed based on the numbers of OTUs from brain tissues. There is no significant difference in Chaol among the four groups. As for Shannon index and Simpson index, the bacterial diversity score of the Infection+Nisin group is significantly lower than that of the Control, Infection and Nisin group. D. PCoA based on Weighted Unifrac distance is shown for different groups. The microbial compositions of Infection and Infection+Nisin group are shifting to different states, while the microbial compositions of Control and Nisin group are in the middle state. E. Analysis of Similarity (Anosim) among different groups are shown. The microbiome compositions of the Control, Nisin and Infection+Nisin group are significantly different from that of the Infection group.

Figure 16. Nisin attenuates the burden of periodontal pathogens in the brain following oral polymicrobial infection. DNA was isolated and purified from the brain samples of four groups (Control, Infection, Nisin and Infection + nisin). The bacteria were quantified by standard real-time PCR using primers corresponding to 16S ribosomal RNA. A. The table demonstrates the detection frequency (%) of periodontal pathogens in all collected brain samples. The copy numbers of each pathogen B. P. gingivalis, C. T. forsythia, and D. F. nucleatum) were detected in every' lOOng DNA. *, the difference between the two groups was significant (p<0.05), ns, the difference between the two groups was non-significant. E. The copy number of each pathogen shown in aggregate for comparisons of relative levels.

Figure 17. Nisin inhibits the expression of proinflammatory cytokines in the brain following oral polymicrobial infection. To evaluate the immune cytokine profiles in brain tissues, mRNA expression of IL-ip (A), IL-6 (B) and TNF-a (C) were measured by real-time PCR. The amount of mRNA in each reaction was normalized to GAPDH. Data are shown as means ± standard deviation from 6 mice per group. *, the difference between the two groups was significant (p<0.05). **, the difference between the two groups was significant (p<0.01). ***, the difference between the two groups was significant (p<0.001), ns, the difference between the two groups was non-significant.

Figure 18. Nisin abrogates the deposition of A 42, Tau, and phosphorylated Tau in the brain following oral polymicrobial infection. To evaluate the effect of nisin on modulating brain pathological changes, ELISA analysis was conducted to determine the levels of Af>42 (A), total Tau (B) and phosphorylated Tau (C) in brain homogenates. Data are shown as means ± standard deviation from 6 mice per group. *, the difference between the two groups was significant (p<0.05). **, the difference between the two groups was significant (p<0.01). ***. the difference between the two groups was significant (p<0.001), ns, the difference between the two groups was non-significant.

Figure 19. Analysis of the microbial abundance by 16s rRNA sequencing show that nisin shifts the oral microbiome back toward healthy control levels following infection. The groups included Control, Infection, Nisin, Infection+Nisin. Differential abundance analysis for bacteria at the phylum level. Data previously highlighted in Kuraj et al., 2023 (33).

DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

As discussed below, the disclosure presented herein provides methods for administering nisin in therapeutic regimens directed towards liver pathologies including non-alcoholic fatty liver diseases. The disclosure presented herein further provides methods for administering nisin in therapeutic regimens directed towards neuropathologies such as Alzheimer’s disease. Nisin is an antimicrobial bacteriocin and lantibiotic produced by a group of Gram-positive bacteria that belong to the Lactococcus and Streptococcus species. The peptide is generally regarded as safe (GRAS) by the FDA since 1988 and it has been included in FDA’s food additive ingredient list (Title 21 CFR) as an antimicrobial agent to inhibit the outgrowth of Clostridium botulinum spores and toxin formation in pasteurized cheese spreads (§184.1538). Pharmaceutical dosage forms of nisin are known in the art and can include sterile aqueous solutions or dispersions or sterile powders comprising compounds which are adapted for the extemporaneous preparation of sterile solutions or dispersions, optionally encapsulated in liposomes. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.

Lantibiotics, overall, have been shown to exert their antimicrobial action by forming a complex w ith lipid II, an essential precursor of the bacterial cell wall, either by inhibiting cell w all synthesis through sequestration of lipid II and/or by disruption of membrane integrity and pore formation. This mechanism is associated with a low likelihood of developing bacterial resistance as has been demonstrated in Methicillin- resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). The studies disclosed herein underscore the ability of nisin to mitigate and correct periodontal disease by correcting inflammation, promoting tissue repair/regeneration, and re-aligning the microbiome including the virome back towards health (see, e.g., Gao et al., npj Biofdms and Microbiomes (2022) 8:45).

Periodontal disease is characterized by microbial infections/dysbiosis and inflammation of the gums and bone that surround and support the teeth, which can lead to tooth loss. Briefly, the disease starts as inflammation of the gums, known as gingivitis. Then, the disease progresses to Periodontitis, an advanced and chronic stage of the disease, in which oral inflammation progresses to critical levels, where the gums start to pull away from the teeth and the bone loss ensues, resulting in loose tooth or tooth loss. A recent CDC report shows that 47.2% of adults aged 30 years and older in the US have some form of periodontitis, reaching up to 70.1% of adults 65 years and older. This condition is more common in men than women (56.4% vs 38.4%), those living below the federal poverty level (65.4%), those with less than a high school education (66.9%), and current smokers (64.2%). Periodontal disease is currently managed by scaling, root planning and the use of local and systemic antibiotics. Periodontal infections are characterized by a broad diversity of periopathogens, including anaerobic, facultative, and aerobic bacteria, both Gram negative and Gram positive. Hence, it is recommended to use more than one antibiotic with different antibacterial spectra, such as metronidazole-amoxicillin or metronidazole plus amoxicillin-clavulanate potassium. However, the rise of multidrug and extensively drug-resistant bacterial strains together with to the steady decline in the discovery of new- and effective antibiotics pose a threat to the management of the disease.

We have previously demonstrated that nisin inhibits the planktonic grow th of oral bacteria at low concentrations (2.5-50 Lig/ml). Nisin also abrogated the development of multi-species biofilms at concentrations >1 pg/ml. Specifically, under biofilm model conditions, nisin interfered with biofilm development and reduced biofilm biomass and thickness in a dose-dependent manner. The treatment of pre- formed biofilms with nisin resulted in dose- and time-dependent disruption of the biofilm architecture along with decreased bacterial viability and no cytotoxicity⁷ towards oral keratinocytes at those concentrations. Further, our in vitro and in vivo studies demonstrate that nisin significantly shifts the oral microbiome composition towards the healthy control state. Our polymicrobial in vivo periodontal disease model demonstrated that health is associated with Proteobacteria, whereas 3 retroviruses were associated with disease. Disease-associated microbial species were correlated with higher levels of the inflammatory cytokines IL-6 and IL- 1 p, compared to control. Nisin significantly reduced these inflammation markers levels back to control (noninfected) levels and demonstrated the ability to shift microbiome back towards control levels. Moreover, nisin also abrogated alveolar bone loss, and the oral and systemic inflammatory host response. Surprisingly, nisin enhanced the population of reparative oral cells; the periodontal fibroblasts and osteoblasts despite the polymicrobial infection, thereby mediating human periodontal ligament cell proliferation dose- dependently by increasing the proliferation marker, Ki-67. In this context, embodiments of the invention are designed to harness nisin's ability to address Per- implantitis/ Peri-implant disease, an oral disease like periodontal disease which is a global "mushrooming" problem around the world (see, e.g., Radaic et al., Microorganisms 2022, 10, 1336).

Additionally, we have designed a nisin-based nanoparticle formulation, wherein nisin is loaded into a solid lipid nanoparticles (SLN) formulation, named SLN-Nisin. Briefly, this formulation was synthesized by heating stearic acid (7 mM) to 75°C. Then, solutions containing DOTAP (final concentration 2.5 mM) and Pluronic F68 (final concentrations 1 mM) were added to the molten lipid. Next, a volume equivalent to 3.25mg of nisin was added to the heated mixture, vortexed and extruded through a heated (at 75°C) polycarbonate membrane (pore size: 0.1 pm) for 15 cycles. Finally, the solution containing the nanoparticles was placed in an ice bath until cooled and stored at 4°C for future testing. In particular, we demonstrated that SLN-nisin surpasses the properties of nisin alone; specifically SLN-nisin compared to nisin alone significantly inhibits to a greater degree the growth of the oral pathogen Treponema denticola, and disrupts significantly more oral biofilms. Further, empirical evidence indicates that the SLN-Nisin formulation increases nisin stability in water by 8-fold. Improved penetration and activity of SLN-Nisin is novel and non-obvious. Such nisin formulations are useful in the methods disclosed herein such as methods of treating pathologies such as non-alcoholic fatty⁷ liver disease (NAFLD).

Non-alcoholic fatty liver disease is defined by macrovesicular steatosis in >5% of hepatocytes, in the absence of a secondary cause such as alcohol or drugs. It encompasses a spectrum of disease from non-alcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis. NAFLD is now a leading cause of chronic liver disease worldwide, with a global prevalence of about 25%. This incidence is, however, increasing with rising levels of obesity, lype-2 diabetes and metabolic syndrome, and NAFLD is predicted to become the leading cause of cirrhosis requiring liver transplantation in the next decade. Lifestyle modification and weight loss remains the cornerstone of management, as no FDA approved pharmacological treatment is available to date. In May 2022, the FDA fast tracked Pfizer’s Ervogastat/Clesacostat Combination for the Treatment of Non-Alcoholic Steatohepatitis (NASH), which is in a randomized placebo-controlled Phase 2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT04321031). Malondialdehyde (MDA) is a common marker for oxidative stress in the liver, which could indicate liver damage. Pending publications from our group have demonstrated that nisin is able to treat NAFLD by significantly reducing the levels of MDA in the liver as well as the number and size of lipid vesicles in the liver in vivo. Additionally, the disclosure presented herein demonstrates that nisin is able to shift the liver microbiome back towards healthy control levels in vivo, supporting that nisin is an appropriate treatment for NAFLD.

Dysbiosis of the oral microbiome mediates chronic periodontal disease, gut microbial dysbiosis, and mucosal barrier disfunction that leads to steatohepatitis via the enterohepatic circulation. The studies disclosed herein demonstrate the use of nisin, an antimicrobial peptide and bacteriocin produced by Lactococcus lactis, to counteract the periodontitis-associated gut dysbiosis and to modulate the glycolipid- metabolism and inflammation in the liver.

Prior studies have reported that compared to individuals with periodontal health, the gut microbiome of patients with severe periodontitis is characterized by differences in microbial composition, low diversity, and changes in ratios of Firmicutes/Bacteroidetes ⁴¹'⁴³. Similarly, in animal models, oral administration of periodontopathic bacteria caused alterations in gut microbiome composition as well as in glucose and lipid metabolism, leading to insulin resistance and hepatic lipid deposition ^32,44'⁴⁶. p. gingivalis-induced gut dysbiosis further downregulated the expression of tight junction proteins, which play a role in gut barrier function, and increased serum LPS levels ^32,47 Therefore, potential liver damage derived from periodontitis-regulated gut dysbiosis may be mediated in the liver via the enterohepatic circulation and it may promote the progression of liver disease.

These facts support the premise that a realignment of the oral and gut dysbiosis towards a healthy state may prevent liver disease ^24,48. Therefore, a microbiome-targeted therapy using probiotics and bacteriocins have been proposed as novel strategies for manipulating the gut microbiome in the management of NAFLD ⁴⁹'⁵¹. Probiotics are defined as live cultured microorganisms that provide health benefits in humans and animals ⁵², and bacteriocins are a generic term for antimicrobial peptides produced by the probiotic bacteria. A recent meta-analysis by Sharpton et al ⁴⁹ revealed that the application of probiotics significantly improved liver-specific markers of hepatic function, liver stiffness, and steatosis in NAFLD patients. Preclinical animal studies have also shown that probiotics suppress the development of hepatic inflammation, insulin resistance, and fatty deposition by regulating the gut microbiota ⁵³‘⁵⁶.

However, little is known about the significance of probiotics and their bacteriocins in the management of liver pathology in patients with periodontal disease. Recently, studies demonstrated that an antimicrobial peptide, nisin, which is produced primarily by Lactococcus lactis species, has effectiveness in the context of periodontal disease ⁵⁷'⁶⁰. Nisin has received a lot of attention in the food industry and the medical field because of its potent and broad-spectrum activity even at trace concentrations, low host cell cytotoxicity at antibacterial concentrations, and low likelihood of promoting the development of bacterial resistance ⁶⁰'⁶⁴. Nisin, also classified as a Class I bacteriocin, is known as a lanthionine-based (lanthionine- containing peptides) antimicrobial based on its chemical structure, because it has unique amino acids that are caused by translational modifications ⁶²-⁶⁵-⁶⁶. Furthermore, studies revealed that nisin and a nisin-producing probiotic Lactococcus lactis decreased the number of pathogenic bacteria while retaining oral commensal bacteria, such as Neisseria species, within salivary-derived biofilms in vitro ⁵⁹. In this context, nisin also significantly inhibited the formation, structure, and viability of biofilms spiked with penodontopathic bacteria and shifted the microbiome composition back toward the healthy control state. We further found that in a polymicrobial infection mouse model of periodontal disease, oral administration of the probiotic L. lactis or its bacteriocin nisin also promoted a shift toward a healthy oral microbiome while preventing gingival inflammation and alveolar bone loss ^57,58. In the present study, the same polymicrobial mouse model ⁶⁷ was induced by oral infection with P. gingivalis, Fusobacterium nucleatum, Treponema denticola, and Tannerella forsythia, and employed to evaluate the effects of nisin in modulating the dysbiosis in the oral, gut and liver microbiome and associated gastrointestinal and liver pathophysiology.

Further aspects and embodiments of the invention are provided in the examples below.

EXAMPLES

EXAMPLE 1: NISIN LANTIBIOTIC PREVENTS LIVER STEATOSIS AND MITOCHONDRIAL OXIDATIVE STRESS FOLLOWING POLYMICROBIAL-PERIODONTAL DISEASE BY ABROGATING ORAL, GUT, AND LIVER DYSBIOSIS AND HEPATIC EXPOSURE TO BACTERIAL LOAD MATERIAL AND METHODS

Infection and treatment of mice

A total of 24 eight-week old BALB/cByJ female mice (The Jackson Laboratories, Bar Harbor, ME) were housed in microisolator plastic cages and randomly distributed into 4 groups (6 mice per group). The description of the experimental groups and infection and treatment protocols are shown in Figure 13. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (IACUC approval number: AN171564-0 IB). Please note that due to the need to reduce the number of control animals to uphold best animal use practices, the oral specimens for the animals used in this study were shared with our previous study ⁵⁷ To start with, all mice were given trimethoprim (0.17 mg per ml) and sulfamethoxazole (0.87 mg per ml) daily for 7 days in the drinking water and their oral cavity was rinsed with 0.12% chlorhexidine gluconate (Peridex) mouth rinse to inhibit the native oral microbiota as described previously ⁶⁷. The polymicrobial inoculum (5x 10⁹ combined bacteria per ml; l *10⁹ cells in 0.2 ml per mouse; 2.5x l0⁸ P. gingivalis, 2.5x l0⁸ T. denticola, 2.5x l0⁸ T. forsythia and 2.5x 10⁸ F. nucleatum) was then administered topically in the morning for 4 consecutive days every week for a total of 8 weeks. Nisin (300 pg/ml, 0.2 ml per mouse) was administered every⁷ day in the evening every week for a total of 8 weeks. A sterile 2% (w/v) carboxymethyl cellulose (CMC; Sigma-Aldrich, St. Louis, MO) solution was administered as the control treatment.

At 8 weeks following the polymicrobial infection, oral swab samples were collected from the oral cavity⁷ of the mice for assessing the status of the oral microbiome. Changes in the oral cavity due to the poly microbial infection have been previously reported ⁵⁷ The teeth and surrounding gingival tissue were wiped with a sterile cotton swab, and the cotton tip was immersed in 10: 1 Trisethylenediaminetetraacetic acid (EDTA) buffer immediately and stored at -80°C until further processing for DNA isolation. Then mice were euthanized, and the maxilla and mandibles were resected from each mouse for immunologic and histologic analysis. In addition, the small-intestinal tissue and it’s bowel feces, and liver tissue were collected for histological observation, microbiologic and immunologic assessment by RT-PCR, and sequencing analysis (microbiome and RNA- Sequencing).

Periodontal bacteria and polymicrobial inoculum

The following periodontal pathogens, namely P. gingivalis FDC 381. T. denticola ATCC 35405, T. forsythia ATCC 43037, and A. nucleatum ATCC 10953, were cultured anaerobically (85% N2, 10% H2, 5% CO2) at 37°C under anaerobic conditions according to methods described in our previous study ⁶⁷. P. gingivalis and F. nucleatum were grown for 3 days in Tryptic Soy Broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 5 mg/ml yeast extract, 0.5mg/ml L-cysteine hydrochloride, 5 pg/ml hemin, I qg/ml menadione and 5% FBS (Gibco Thermo Fisher Scientific, Waltham, MA). T. denticola was cultured in Oral Treponeme Enrichment Broth medium (Anaerobe systems, Morgan Hill, CA) for 5 days. T. forsythia was grown for 7 days in Tryptic Soy Broth containing 5 mg/ml yeast extract. 0.5mg/ml L- cysteine hydrochloride, 5 pg/ml hemin, Ipg/ml menadione, 10 pg/ml N- acetylmuramic acid (Sigma-Aldrich, St. Louis, MO), and 5% FBS. Bacterial concentration was determined quantitatively using a spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA) and each organism was resuspended in phosphate-buffered saline (PBS) at 1 x io¹⁰ bacteria per ml for experiments.

For the oral polymicrobial infection, P. gingivalis was mixed with an equal volume of T. denticola for 5 min. Subsequently, T. forsythia was added to the culture tubes containing P. gingivalis and T. denticola, and the bacteria were mixed gently for 1 min and allowed to interact for an additional 5 min. Finally, F. nucleatum was added and mixed well with P. gingivalis, T. denticola, and T. forsythia. After 5 min, the four bacterial consortium was mixed thoroughly with an equal volume of sterile 4% (w/v) carboxymethylcellulose in PBS, and this mixture was used as the polymicrobial oral inoculum.

Nisin preparation

An ultra-pure (>95%) food grade form of nisin Z (NisinZ® P) was purchased from Handary (S.A., Brussels, Belgium), a primary' manufacturer of nisin in the food industry'. The nisin stock solution was prepared at a concentration of 600 pg/ml in sterile Mili-Q water, filtered using a 0.22 pm syringe filter, and stored at 4°C for a maximum of 5 days for use in experiments ^59,67 For oral treatment of mice, the nisin solution was then mixed with an equal volume of sterile 4% CMC and adjusted to the final concentration of 300 pg/ml.

DNA isolation from oral swabs, small bowel feces, and liver

DNA from the oral swabs, small bowel feces, and liver was extracted using specific methods for each sample to evaluate microbiological alterations following bacterial challenge and/or nisin treatment by real-time polymerase chain reaction (RT- PCR) and 16S rRNA sequencing. For the oral swabs and liver tissue, the DNA was isolated and purified using the QIAamp®¹ DNA Mini kit (Qiagen, Hilden, Germany) as in our previous reports ^16,67 Ethanol precipitation of DNA from the oral swabs was further performed to prepare the samples for subsequent analysis. In addition, DNA from the small bowel feces was extracted using the QIAamp® Fast DNA Stool Mini kit (Qiagen) following manufacturer’s protocols. All isolated DNA were stored at - 20°C until further processing for real-time PCR and 1 S rRNA sequencing analysis.

RNA isolation from gingival tissue, small intestine, and liver

For RNA isolation, the gingival tissue, small intestine, and liver were treated overnight at 4 °C with RNAlater solution (Invitrogen) immediately after sample collection. Samples were powdered with a mortar and pestle under continuous liquid nitrogen, and total RNA was then isolated from each sample using the RNeasy mini Kit (QIAGEN). The purity and quantity of the RNA were evaluated using the NanoVue Plus spectrophotometer (Biochrom Ltd.). Subsequently, total RNA was synthesized into cDNA with the SuperScript VILO Master Mix (11755050; Invitrogen) following the manufacturer’s protocol.

Microbiome analysis of oral, small intestine, and liver specimens by 16S rRNA sequencing

The purity and quantity of respective DNA isolated from oral swabs, small bowel feces, and liver were deemed suitable and met quality control measures for 16S rRNA sequencing performed by Novogene, Inc. (en.novogene.com). For the sequencing library preparation, the V4 variable region (515F-806R) of the samples was amplified using specific primers with a barcode. All PCR reactions were carried out with Phusion® High-Fidehty PCR Master Mix (New England Biolabs) and the PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries were generated with NEBNext® UltraTM DNA Library Prep Kit for Illumina and were then sequenced by Illumina NovaS eq 6000 System.

Paired-end reads were assigned to samples based on their unique barcodes, truncated by cutting off the barcode and primer sequences, and merged using FLASH (vl.2.7) ⁶⁸. Next, quality filtering on the raw tags was performed under specific filtering conditions to obtain the high-quality clean tags according to the analysis pipeline of QIIME (vl.7.0) ⁶⁹. Subsequently, the tags were compared with the reference database using UCHIME algorithm ⁷⁰ to detect chimera sequences, and the chimera sequences were then removed to obtain the effective Tags. Sequences analysis was performed by Uparse software (v7.0.1001) using all the effective tags. Finally, sequences with >97% similarity were assigned to the same operational taxonomic units (OTUs). and species annotation at each taxonomic rank was performed based on comparison to the SSUrRNA database of SILVA Database using Mothur software ⁷¹. Alpha diversity and the Simpson diversity index were calculated from the number of observed OTUs with QIIME software to evaluate species richness and evenness. Beta diversity analysis was also used to evaluate differences in samples in terms of species complexity. Principal Coordinate Analysis (PCoA) using weighted UniFrac distance based on OTU distribution across samples was performed by QIIME to visualize multidimensional data and provide an overview of microbial dynamics in response to polymicrobial infection and nisin treatment. An Analysis of Similarity (ANOSIM) was further performed to determine whether the difference in microbial community structure among groups was significant. When suggested by a positive R- value that variation among groups was larger than the variation within groups, the difference among groups was considered significant if the P value was less than 0.05. In addition, for the differential abundance analysis of each bacterial taxa, we performed a two-sample t-test to compute the p-values by R software, assuming equal variance in the two groups. The Benjamini and Hochberg procedure was used to correct for multiple comparisons, and the corresponding False Discovery Rate (FDR) was further calculated for conducting pair-wise comparisons (e.g., infection versus control). An FDR adjusted p value (q value) < 0.05 was considered significant.

Quantification of total bacteria and periodontal pathogens by real-time PCR

Absolute quantification by standard real-time PCR was used to evaluate the abundance of the periodontal pathogens in the oral cavity, small intestine, and liver. Total bacteria and four periodontal pathogens used for the polymicrobial infection were measured by PCR using TaqMan primers and probes (Invitrogen) corresponding to the 16S rRNA gene. Tenfold serial dilutions of DNA of known concentration were used to construct standard curves for quantification of total bacteria and periodontal pathogens. The amplification was conducted using a QuantStudio 3 Real Time PCR system (Thermo Fisher Scientific) with a final reaction volume of 20 pL that included TaqMan Fast Advanced Master Mix (Applied Biosystems), DNA (15 ng/ pL), primers, and probes. The optimized thermal cycling conditions were as follows: 95 °C for 10 min followed by 50 cycles of denaturing at 95 °C for 15 s, annealing and extension at 60 °C for 1 min. Data were analyzed using QuantStudioTM Design & Analysis Software vl.4.3 (Thermo).

PCR evaluation of gene expression of gingival tissues and the small intestine

To evaluate immune cytokine profiles from gingival tissues and small intestine, relative gene expression was measured by real-time PCR using the following TaqMan primers and probes (TaqMan Gene Expression Assays; Applied Biosystems): interleukin 1 [3 (Illfi; Mm00434228_ml), IL-4 (114 Mm00445259_ml), IL-6 (116; Mm00446190_ml), tumor necrosis factor-a (Tnf; Mm00443258_ml), C-C Motif Chemokine Ligand 2 (Ccl2; Mm00441242_ml), C-X-C Motif Chemokine Ligand 2 (Cxcl2; Mm00436450_ml), interferon gamma (Ifng; Mm01168134_ml) and transforming growth factor beta 1 (Tgfbl; Mm01178820_ml). Tight junction proteins, which play an important role in gut barrier function, were also analyzed for the small intestine using the following TaqMan primers and probes: Occludin (Ocln; Mm00500912_ml), Tight junction protein-1 (Tjpl; Mm00493699_ml), and Claudin- 1 (Cldnl; Mm00516701 ml). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Mm99999915_g 1 ) was used as a housekeeping gene to normalize the amount of mRNA present in each reaction. PCR was performed in 20 pl reaction mixtures containing the TaqMan Fast Advanced Master Mix, cDNA template (20 ng/pl well), primers, and probes. The optimized thermal cycling conditions were as follows: 20 min at 95°C, followed by 40 cycles per 1 min at 95°C, and 20 min at 60°C. To compare the expression levels among different samples, the relative expression level of the genes was calculated by the comparative CT (AACT) method using QuantStudioTM Design & Analysis Software.

Histopathological evaluation of the maxilla and small intestine

The right maxilla and small intestines were resected from each mouse and immediately fixed in 4% paraformaldehyde for 24h. The maxilla was then decalcified with diethyl pyrocarbonate-treated 0.5M EDTA (pH 8) for 28 days at room temperature. The specimens were dehydrated and embedded in paraffin using a fully- enclosed tissue processor (ASP300S, Leica Biosystems. Buffalo Grove, IL).

The maxilla tissue blocks were cut into serial sections (4 pm) parallel to the mesiodistal plane from the palatal view using a microtome (RM2145; Leica, Hessen), then sections were stained with hematoxylin and eosin (HE; Sigma- Aldrich, St. Louis, MO, USA) for assessment of inflammation. The sections were examined with a stereomicroscope. The number of inflammatory cells within a square field (200 x 200 pm) in the connective tissue adjacent to the gingival epithelium between the first and second molars were counted in three tissue sections per mouse specimen (n = 3 per group) by a skilled examiner in a blinded manner, and all counts were averaged for each group. Data were expressed as the mean number of cells per 1.0 mm² of connective tissue in the maxillary specimens.

Serial sections (6 pm) of the small intestine were similarly prepared and stained with HE. Histological scoring of the tissues was performed on three tissue sections per mouse specimen (n = 3 per group). Histological inflammatory findings was categorized into five distinct groups based on a previous report ^72,73, and each was graded as follows: grade 0, no sign of inflammation; grade 1, very low-level leukocyte infiltration; grade 2, low- level leukocyte infiltration; grade 3, high-level leukocyte infiltration, high vascular density, and thickened colonic wall; and grade 4, transmural leukocyte infiltration, goblet cell loss, high vascular density, and thickened colonic wall. Data was represented as the mean score value.

Histopathological analysis of liver tissue by HE and Oil-red staining

The liver tissues were fixed in 4% paraformaldehyde for 24 hr then embedded in paraffin or frozen. Paraffin sections (6 pm) were stained with HE as mentioned above. To evaluate fatty depositions in the liver, frozen sections (8 pm) were prepared via a cryostat (CM1950; Leica, Hessen, Germany) and analyzed using Oil red staining. The sections were fixed with 4% paraformaldehyde for 5 min, washed with running tap water for 10 min, and incubated with 60% isopropanol for 5 min. Sections were then stained in the Oil red solution (Sigma-Aldrich, St. Louis, MO) at room temperature for approximately 15 minutes until the appearance of bright red staining was observed. After washing with 60% isopropanol and distilled water, tissues were counterstained with hematoxylin, washed with distilled water again, and subsequently mounted with ImmunoHistoMount (Sigma). Four different fields (100* magnification) w ere randomly selected in Oil red-stained images of three tissue sections per mouse specimen (n = 3 per group), and the area of orange-stained fatty deposits was measured using ImageJ analysis software ⁷⁴ Data were expressed as the percent of fatty area per 100 pm² in the liver specimens.

Gene expression profile of liver tissue by RNA sequencing

A total amount of 1 pg RNA extracted from liver tissue per sample was used as the input material for the RNA sample preparations. After purification of mRNA from total RNA using poly-T oligo-attached magnetic beads, sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for (Illumina®, NEB. USA) following manufacturer’s instructions and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina platform and 2 x 101-bp paired-end reads were generated. Raw sequencing read data of FASTQ format were firstly processed through fastp. In this step, clean data were obtained from the raw data by removing reads containing adapter and poly- N sequences and reads with low' quality. Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Paired-end clean reads were aligned to the mmlO reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. FeatureCounts w as used to count the read numbers mapped of each gene, and then FPKM of each gene w as calculated based on the length of the gene and reads count mapped to this gene. DESeq2 R package was used to detect differentially expressed genes (DEGs) between two groups. The resulting P values were adjusted by the FDR with the Benjamini and Hochberg’s correction. DEGs were defined when the adjusted P value < 0.05 and fold change of FPKM was >1.3. The statistical enrichment of DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG, pathways was tested using R package clusterProfiler.

Furthermore, Gene Ontology (GO, http://www.geneontology.org/) enrichment analysis of DEGs was implemented with the annotation dataset for GO biological process and molecular function. KEGG and GO terms with adjusted P value less than 0.05 were considered significantly enrichment.

Immunohistochemistry of malondialdehyde in liver tissue

Slides containing formahn-fixed paraffin-embedded (FFPE) tissue sections were stained for malondialdehyde using the rabbit-specific horseradish peroxidase (HRP)/3,3’ -Diaminobenzidine (DAB) together with the Avidin Biotin Complex (ABC) Detection immunohistochemistry Kit (Abeam, USA), according to manufacturer’s instructions. Briefly, the slides were deparaffinized by submersing the slides in 100% Xylene (Sigma- Aldrich, USA) for 5 minutes twice, and rehydrated by submersing the slides in ethanol solutions containing increasing percentages of w ater in solution (100%, 90% and 80%; 5 minutes each). Next, the samples w ere submersed in a hydrogen peroxide blocking solution (supplied with the kit) for 10 minutes and washed 2 times in phosphate-buffered saline with 1% Tween 20 (PBS-T). The slides were then submersed in a protein blocking solution (supplied with the kit) and incubated for 5 minutes at room temperature, followed by a PBS-T w ash. Next, the samples were immersed in a solution containing rabbit anti-malondialdehyde primary antibody (Abeam, USA) and incubated overnight at 4°C. The next day, the slides were washed 3 times in PBS-T and submersed in the biotinylated goat anti-rabbit secondary antibody (supplied with the kit) for 2 hours at room temperature. After washing 3 times in PBS-T, the samples were submersed in a solution containing streptavidin peroxidase (supplied with the kit) and incubated for 10 minutes at room temperature. Then, the samples were rinsed in PBS-T and a solution containing the DAB Chromogen and its Substrate (diluted to lx; supplied with the kit) was applied to the samples for lOmin. After rinsing the slides in PBS-T. hematoxylin was added to the slides for 1 minute and rinsed in tap water. Finally, the slides were mounted and the samples were imaged in a DM 1000LED Microscope (Leica, Germany).

Human autopsy study

Japanese human cadavers (n=19) donated by the Department of Anatomy of the Nippon Dental University were used in this study. Based on pre-registration information, subjects with a history' of liver disease (alcoholic hepatitis, viral hepatitis, cirrhosis, liver cancer, etc.) and other digestive disorders were excluded. The study was approved by the Human Research Committee of Nippon Dental University' (no. NDU-T2021-17). The human cadavers were obtained from a donor-based system using the guidelines included in the Layv Concerning Body Donation for Medical and Dental Education (the Body Donation Law) and the Law Concerning Cadaver Dissection and Preservation (LCCDP).

Cone beam computed tomography (CBCT; AZ 3000CT, Asahi Roentgen Industry, Kyoto, Japan) yvas used to obtain scanned images of the maxillas and mandibles, including all of the teeth and alveolar bone of the cadavers. The scanning parameters were as follows: the tube voltage was 85 kV. the tube current was 4 mA, the scanning time yvas 17 seconds, the field of view (FOV) was 79 mm cp x 80 mm H, and the voxel size was 0.155 x 0.155 x 0.155 mm. NEOPREMIUM software (Asahi Roentgen Industry⁷, Kyoto, Japan) yvas used to generate CBCT images from CBCT data. The number of remaining teeth and the severity of periodontal disease based on alveolar bone resorption were evaluated, and cadavers were then divided into two subgroups: a control non-periodontitis group and a periodontitis group. The periodontitis group had at least one tooth yvith alveolar bone loss exceeding half the root length in each quadrant of the oral cavity, whereas the control group had no teeth with alveolar bone resorption exceeding ! the root length.

The gingival and liver tissues were then collected and evaluated for an association between periodontal pathogens and hepatic abnormalities by real-time PCR. To this end, DNA was extracted from these tissues using specific methods (QIAamp DNA FFPE Tissue Kit). Standard real-time PCR was used to detect the presence of periodontal pathogens in the gingival and liver tissues. The 16S rRNA genes corresponding to total bacteria and four periodontal pathogens were amplified with TaqMan primers and probes (Invitrogen) . The amplification was conducted with a final reaction volume of 20 pL that included TaqMan Fast Advanced Master Mix (Applied Biosystems) , DNA (15 ng/ pL), primers, and probes using a StepOnePlus Real Time PCR system (Applied Biosystems). Thermal cycling conditions were as follows: 95 °C for 10 mm followed by 50 cycles of denaturing at 95 °C for 15 s. annealing and extension at 60 °C for 1 min. Data were analyzed using StepOnePlus software (Applied Biosystems).

The liver tissue was also fixed with a 20% neutral buffered formalin solution, embedded in paraffin, sectioned (4 pm), and stained with HE. NAFLD activity score (NAS) for the liver specimens was evaluated in the HE stained images in accordance with the definition of Kleiner et al. to diagnose liver disease ⁷⁵. Total NAS in individual subjects was calculated as the sum of three scores, including steatosis, inflammation, and cell injury (ballooning).

Statistical analysis

A power analysis was performed to determine the optimal sample size for the present study based on data from our previous study,⁶⁷ relative to the mean difference and SD for the level of IL-6 gene expression using G*Power 3 analysis software (Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany). The required minimum sample size of mice was determined as 5 to obtain a power of 80% with a = .05. All evaluations were carried out by one calibrated and blinded examiner. SPSS 21.0 statistical software (IBM, Chicago, IL, USA) was used for statistical analysis of the non-sequencing data. The analysis of the bacterial number and immune profile- related gene expression, and inflammatory cell infiltrate in the gingival tissue were compared using ANOVA and Tukey’s test for multiple comparison among 4 groups. Student's t test was used to compare values between two groups. The histological score for enteritis and lipid deposition area in the liver were analyzed by a Kruskal- Wallis H-test followed by a Steel-Dwass test or Dunn’s test for nonparametric data. For the human autopsy study, the Pearson correlation coefficient was used to analyze the correlation between the number of remaining teeth and NAS. A p value less than 0.05 was considered to be significant.

Nisin shifts a disease-associated microbiome toward a healthy state in various organs

To determine the extent to which nisin modifies the microbiome of the oral cavity, small intestine, and liver following an oral polymicrobial infection, the microbial composition and abundance at these sites was analyzed by 16s rRNA sequencing at three taxonomic levels, namely at the phylum, genus, and species level.

In the oral cavity, (Fig. 1A), the relative abundance of the phylum Proteobacteria and Fusobacteria were significantly increased in the infected mice compared to the healthy control mice, but the proportions of Bacteroidetes, Firmicutes, Cyanobacteria, and Actinobacteria were decreased. In contrast, nisin treatment significantly prevented these oral microbiome changes and shifted the microbial composition back toward the healthy control state. At the genus level (Fig. IB), the proportions of Lactococcus and Fusobacterium significantly increased in the infection group compared to the control group, whereas the abundance of Lachnospiraceae NK4A 136. Streptococcus. Cutibacterium. Granulicatella. and Veillonella decreased. However, nisin recovered the disease-associated changes in Lactococcus, Fusobacterium, and Lachnospiraceae NK4A136 back toward the control healthy state. The administration of nisin alone increased Lactococcus and Porphyromonas, and reduced the proportions of Lachnospiraceae NK4A136, Streptococcus, Granulicatella, and Veillonella. At the species level (Fig. 2A), there was an increase in Lactococcus lactis and a decrease in Streptococcus pneumoniae and Pseudomonas brenneri in the infection group but a preventive effect was noted in the infection+nisin group, which was consistent with the microbial findings observed at the genus level.

In the gut, analysis of the relative abundance of microbiota in small bowel feces at the phylum level (Fig. IB) revealed a lower amount of Firmicutes and higher amount of Bacteroidetes in the infection group than in both the control and infection+nisin groups. In contrast, Actinobacteria exhibited a higher abundance in all other three groups compared to the control group, and Tenericutes was lower. At the genus level (Fig. ID), 15 bacterial taxa showed significant changes among groups. In particular, nisin significantly increased the acetate- and butyrate-producing beneficial bacterium, such as Lactobacillus, Lachnospiraceae UCG-001 group, Lachnospiraceae UCG-006 group, Lachnoclos tridium and Acetitomaculum. However, the infection group showed a significant increase in the proportion of Turicibacter and Bifidobacterium, which was prevented by nisin treatment. Interestingly, these genera taxa, which showed significant changes, are predominantly classified in the Firmicutes phylum. Moreover, at the species level (Fig. 2B), the proportion of Lactobacillus gasseri was markedly increased in both the nisin and infection+nisin groups. Clostridium sp. ASF502 and an unidentified bacteria belonging to cmclassified-Ruminococcaceae also showed significant alterations among groups.

In the liver tissue, microbiome changes at the phylum level revealed a significantly higher abundance of Firmicutes in the infection group than in the control group, whereas Proteobacteria and Actinobacteria were lower (Fig IE). Bacteroides showed no significant difference among groups. Importantly, nisin treatment consistently maintained the same relative abundance phylum levels as the healthy controls, thus protecting this organ from the disease-related changes in the microbiota (Fig IE). In addition, at the genus level (Fig. IF), the proportion of Lachnospiraceae NK4A136 group and Turicibacter, which are classified in the phylum Firmicutes, tended to be higher in the infection group compared to the control and infection+nisin groups. In contrast, in terms of the phylum Proteobacteria, the abundance of genus Sphingobium dramatically decreased following the infection and/or nisin treatment, and Psuedomonas, Brevundimonas, and Massilia were significantly increased by nisin treatment alone (Fig IF). As shown in Figure 2C for the liver tissue, at the species level, the proportion of Lactobacillus gasseri was markedly higher in both the nisin and infection+nisin groups, revealing a similar tendency as in the gut microbiome. In contrast, a higher abundance of Clostridiales bacterium CIEAF 020, Ruminoclostridium sp. KB 18, and Clostridiales bacterium CIEAF 012 were detected in the infection group compared to the other groups (Fig 2C); although these bacterial taxa showed significant differences at P<0.05, these differences did not reach further significance when adjusted by FDR.

Nisin prevents the oral polymicrobial infection-mediated alterations in microbial diversity and community structure in the oral cavity, small intestine, and liver

In order to assess the changes in bacterial diversify following the polymicrobial infection and nisin treatment, the Simpson diversify' index was analyzed based on the OTU numbers from the oral cavity , small intestine and liver, respectively. As shown in the Figure 3A, the bacterial diversify scores for the oral microbiome were significantly lower in both the infection and nisin groups compared to the control group (P<0.001; n=6; Tukey test). The infection+nisin group had a higher diversify score compared to the infection and nisin groups (P<0.001), but equivalent to the control group (P=0.115), showing nisin's ability to recover the microbiome diversify back toward a state of health like the control. In the small intestine (Fig. 3B), the diversify’ was significantly lower in the nisin group (P<0.003 and P<0.023, respectively) compared to the control and infection groups. However, the diversify of the infection+nisin group was slightly increased (P<0.148) compared to the nisin group, although this was not significant and intermediate between the infection and nisin groups. In contrast to the oral and gut microbiome, the bacterial diversity in the liver tissue (Fig. 3C) tended to increase following the polymicrobial infection, but this change was mitigated by nisin treatment. Although there was no statistical difference in these microbiome changes among groups when using the Tukey test, the t-test showed a significant difference between the infection and inf+nisin group (P<0.038).

To evaluate the overall similarity and dissimilarly in the microbiome changes among different treatment groups, we further performed PCoA using weighted UniFrac distance. For the oral cavity (Fig 3D), the microbiome composition of the infection and nisin groups revealed significant qualitative differences compared to the control group (P=0.002 with R=0.856 and P=0.002 with R=0.907, respectively; n=6; AN0S1M). However, among infected animals, those treated with nisin were more similar to the control, and there was a significant difference betw een the infection and the inf + nisin groups (P<0.01 with R=0.191). On the other hand, in the small intestine (Fig 3E). the microbial composition of all treatment groups (the infection: P=0.011 with R=0.528, the nisin: P=0.003 with R=0.802, and the inf + nisin: P=0.003 with R=0.557, respectively) were significantly different from the control group. Also, the gut microbiome of the nisin group exhibited a shift to a different state from that of the infection group (P=0.031 with R=0.279). Interestingly, nisin treatment of infected mice further induced a change in the gut microbiome toward a middle state between the infection and nisin groups, which was consistent with the previously mentioned findings for the Simpson diversity⁷. Similarly, for the liver (Fig 3F), the microbiome composition of the infection group was significantly different from the control and nisin groups (R=0.527, P=0.011 and 0.279, P=0.031, respectively). We again found that among the infected mice, those treated with nisin were similar to the control group, indicating that nisin also drives the overall hepatic microbiome composition toward the healthy control state as in the oral and GI tissues. Periodontal inflammation following polymicrobial oral infection is reduced by nisin

To evaluate nisin’s ability to alter the host inflammatory response that mediates periodontal disease, we evaluated the immune cytokine profiles within gingival tissues via changes in gene expression and histological inflammatory cell infiltrate in the periodontal tissues, while validating changes in the total bacterial load in the oral cavity (Fig. 4). The number of total oral bacteria were significantly higher in the infection group compared to the control group as assessed by RT-PCR of oral swabs (Fig.4A) (P<0.001; n=5; Tukey test). However, among the infected animals, nisin markedly decreased the total bacterial count similar to the control levels (P<0.001). For the gene expression analyses (Fig. 4B), the levels of IL-6 and CXCL2 were significantly upregulated in the infection group (PO.OOl and P<0.01, respectively; n=5; Tukey test) compared to the control. However, nisin treatment significantly prevented these cytokine changes in the infection group (P<0.001 and P<0.05, respectively). Other cytokine-related genes, including TNF-a, CCL2, IL-10, TGF0, and IFN-y, showed a similar tendency, but these differences did not reach statistical significance.

Next, we evaluated the effects of the infection and nisin treatment on the morphologic features and inflammatory cell infiltrate of the periodontal tissues using HE-stained histological sections (Fig. 4C). In the control group, very few inflammatory cells were present in the gingival connective tissue just below the thin junctional epithelium. In contrast, the gingival tissues from the infection group exhibited epithelial hyperplasia with rete ridge elongation and an infiltration of numerous inflammatory cells (p < 0.001; Fig. 4D). Treatment with nisin significantly reduced the inflammatory cell infiltrate (p < 0.001) and the rete ridge elongation in the infection group, as previously reported ⁵⁷.

Inflammation in small intestine following polymicrobial oral infection is prevented with nisin treatment To evaluate the effect of the oral polymicrobial infection and nisin treatment on the small intestine, we performed gene expression assays to evaluate the immune cytokine levels in the ileum (Fig. 5). The gene expression levels of the pro- inflammatory cytokines, including IL-6, TNF-a, and CCL2, were significantly elevated in the infection group compared to the control group (Fig. 5A). However, this cytokine upregulation in the infection group was suppressed by nisin treatment (Fig. 5A). The gene expression levels of the anti-inflammatory cytokines, IL-4 and TGF-p. showed similar significant changes like the pro-inflammatory cytokines, which may reflect a resolution of inflammation and pro-resolving response.

The severity of the inflammatory changes in the small intestine were further evaluated by histopathological examination. In the infected animals, compared to the control group, the ileum showed marked wall thickening with an exfoliation of the mucosal epithelium, decreased villi height, and a severe inflammatory cellular infiltrate into the lamina propria (Fig. 5B). Conversely, the intestinal tissues from the nisin-treated infected animals had a mild inflammatory cell infiltration of the mucosa. Furthermore, nisin treatment significantly improved the villi height and histological scores in terms of the inflammatory cell infiltrate, vascular density, loss of goblet cells, and thickening of the intestinal wall (p < 0.05; n=3; Steel-Dwass test; Fig. 5C).

Moreover, we used RT-PCR to evaluate the gene expression of tight junction proteins, which are responsible for regulating intestinal barrier function and intestinal permeability (Fig. 5D).

The expressions of Ocln and Tjpl were significantly downregulated in the small intestine following the polymicrobial oral infection. However, among the infected mice, this downregulation was also present in the infection+nisin group, and was not prevented by treatment with nisin, although some positive trends were noted for TjpliZO-\ . For Cldnl gene, there were no significant changes among groups.

Nisin treatment attenuates the total bacterial burden and periodontal pathogens that enter the small intestine and liver In order to further assess the bacterial load on the oral-gut-liver-axis, the number of total bacteria and periodontal pathogens were measured in the small bowel feces and liver samples using RT-PCR to determine their absolute quantification (Fig. 6). For the small bowel feces, the copy number of the universal 16S rRNA gene (total bacteria) was significantly reduced following nisin treatment both in the presence and absence of the polymicrobial infection (P<0.05; n=5; Tukey test; Fig. 6A). Interestingly, in the small bowel feces, P. gingivalis and T. forsythia exhibited significantly higher levels in the infection group than the control group (P<0.05 and P<0.001, respectively; Fig. 6B and 6C), whereas nisin markedly decreased the levels of these periodontal pathogens among the infected animals (P<0.05 and PO.OOl, respectively). Moreover, in the liver tissue, the number of total bacteria was significantly elevated in the infection group compared to the control group, and this increase was prevented by nisin treatment in the infected animals (P<0.05; Fig 6D). Similar to the small bowel feces, T. forsythia in the liver of the infected group (P<0.05) was significantly higher than in all the other groups, and nisin abrogated its increase in the infected mice (Fig 6F). P. gingivalis also tended to decrease in the infected group when treated with nisin, although this decrease did not reach statistical significance (Fig 6E). However, the other oral periodontal pathogens that were part of the oral polymicrobial infection, F. nucleatum and T. denticola, were not detected in the feces or liver of the infected mice (Data not shown).

Hepatic lipid deposition by polymicrobial infection is significantly abrogated in mice treated with nisin

Histopathological analysis of the liver tissue w as conducted to further evaluate the effects of the oral polymicrobial infection and nisin treatment on the liver. As shown in the histological images of the representative liver tissues stained with H-E (Fig. 7A) and Oil Red O (Fig. 7B), limited lipid deposition and infiltrating inflammatory cells were observed in the healthy control animals. In contrast, the infection group showed an obvious and severe lipid deposition, a number of perivenous small lipid droplets with vesicles, and detachment of endothelial cells in the central vein. However, in the inf + nisin group, only limited lipid deposition and vesicles were observed similar to the control and nisin groups, indicating that nisin improved the lipid metabolic changes in the liver following the polymicrobial infection.

Hematoxylin stained liver sections were analyzed using image analysis software in order to quantify the number of vesicles. In agreement with the other histological findings, the polymicrobial infection significantly increased the number of vesicles by 103-fold (from 3.8+4.7 vesicles in the control group to 393.4+146.1 vesicles in the infection group; P < 0.0001; n=3; Dunn's test; Fig 7C). However, nisin significantly decreased the number of vesicles in the infection group (P < 0.01). with no significant difference compared to the control group (p=0.5674; 3.8±4.7 vesicles in the control group versus 11.30+11.36 in infection + nisin group). To further quantify the degree of lipid deposition, oil red O-stained liver sections were analyzed. Similar to the findings for the increased vesicle numbers, the area of hepatic lipid deposition significantly increased by 15% in the infection group (P < 0.05; n=3; Dunn' s test; Fig 7D). Whereas, among the infected animals, nisin markedly decreased the lipid deposition (P < 0.05), although there was still a significant difference between the infection+nisin group and the control group (P < 0.05).

Nisin restores changes in gene expression related to hepatic mitochondrial function and oxidative stress following polymicrobial infection

RNA sequencing of mouse liver tissue was performed to explore the underlying mechanisms of the regulatory action of Nisin in fatty liver disease induced by polymicrobial infection. A total of 18,614,688-30,662,424 (mean 22,562,974) paired-end clean reads per sample (n = 6) were generated by the Illumina MiSeq system. The distribution of fragments per kilobase of exon per million mapped fragments (FPKM) for all genes (54532 genes) was consistent across all samples, but the majority of genes had low expression (FPKM < 1.0). After removing the low- expressed genes between groups, a final set of 2,949 genes was used in this study (Fig. SA). Among these genes, two gene subclusters were detected that showed different expression patterns (Fig. 8B). The expression level of 1678 genes in subcluster 1 tended to be upregulated in the infection group and downregulated in the nisin and the inf + nisin groups compared to the control group. On the other hand, the expression of 1267 genes in subcluster 2 tended to be increased in the nisin and the inf + nisin groups compared to the control and the infection groups. Therefore, to detect genes that may be important regulators of the effect of nisin in the liver, we performed a differentially expressed genes (DEGs) analysis (fold change > 1.3, q-value < 0.05) between the control and the nisin group or between the infection and the inf + nisin group, resulting in 2,084 and 560 genes, respectively (Fig. 8C).

To examine the global function of genes whose expression was altered by nisin treatment in the polymicrobial infected mice, a gene functional analysis for Kyoto encyclopedia of genes and genomes (KEGG) pathways was performed. The top 20 significantly enriched KEGG terms (q-value < 0.05) were identified for the DEGs between the infection group and the inf + nisin group. KEGG terms related to energy metabolism, such as Ahermogenesis", "oxidative phosphorylation’; "PPAR signaling pathway”, and "metabolism of xenobiotics of cytochrome P450" were detected (Fig. 9A). Interestingly, the "non-alcoholic fatty' liver disease (NAFLD)" term was detected with the fourth highest count and contained 28 genes classified as energy metabolism-related terms (Fig. 9B). Among these, genes related to electron transfer complexes in mitochondria (Cxi: Ndufb6, Ndufa8, Ndufs7, Ndufb7, Ndufal2, Ndufs4, Ndufs5, Ndufa9, Ndufal3, Ndufs8, Ndufvl, Ndufa4; CxII: Sdhb; CxIII: Uqcrc2, Uqcrq, UqcrlO, Uqcrh; CxIV: Cox6al, Cox4il, Cox5a, Cox7a2) were more common (Fig. 9C). In addition, for other cell organelles, peroxisome-related genes (Adiporl, Adipor2. Ppara). Cytochrome P450-related genes (Cyp2el) and Cytochrome c-related genes (Cycs) were significantly lower in the inf + nisin group than the infection group. Next, gene ontology (GO) enrichment analysis was performed between the infection and the inf + nisin groups to assess cellular structure and molecular function in more detail, and the top 20 significantly enriched GO terms (q value < 0.05) were identified (Fig. 10A). For GO terms at the cellular component level, genes related to mitochondrial protein complexes, membrane structure, and respiratory chain were more abundant in the infection group, consistent with KEGG pathway analysis. Furthermore, at the molecular function (MF) level, we detected GO terms related to the binding capacity of metal ions, such as iron and copper, in addition to NADH dehydrogenase, electron transfer and antioxidant activities. These metal ions play an important role in oxidative phosphorylation via electron transfer complexes in mitochondria, and the list in Figure 10B shows the role of genes related to metal ion binding that have significant differences between groups in the GO database. Indeed, the expression of many genes in the iron and copper ion binding and iron-sulfur cluster binding term was enhanced in the infection group, while it was significantly attenuated in the inf + nisin group (Fig. 10C). In addition, the MT1 gene, which is involved in cellular response and homeostasis of metal ions, such as copper and zinc, was significantly downregulated in the infection group compared to the control group.

Lipid peroxide deposition in the liver enhanced by polymicrobial infection is significantly inhibited in nisin-treated mice

Given the increased numbers of lipid vesicles, lipid deposition, and gene clusters related to NAFLD in the infection group, malondialdehyde (MDA) expression in liver tissue was further evaluated as an indicator of lipid peroxide and oxidative stress, especially because of the importance of genes related to mitochondrial oxidative phosphorylation. Brown-staining MDA was rarely observed in the inf + nisin group as well as the control and nisin groups (Fig. 11A). In the infection group, on the other hand, MDA deposition was more abundant within the cytoplasm, vesicles, and vascular sinusoids. Using semi-quantitative analysis, it was determined that the MDA deposition significantly increased by 3-fold in the polymicrobial infection group compared to control (from 15.08±6.37% in control to 46.65±9.90%; p < 0.01; n=3; Dunn's test; Fig 11B). Reiterating previous results, MDA was significantly reduced in the inf + nisin group (6.76±3.30%, p< 0.0001).

Human autopsy study

Additionally, a human autopsy observational study was conducted to provide stronger support for the relationship between periodontal disease and liver disease. In this study, a total of 19 Japanese humans, which included ten male cadavers (82-94 years old; mean, 88.1 years) and nine female cadavers (78-99 years old; mean, 87.1 years old). The control (n=7) and periodontitis (n=5) groups were defined based on the severity of periodontal disease as assessed by the amount of alveolar bone resorption and the number of remaining teeth, respectively. Figure 12A shows representative findings for each group. The NAFLD score of the periodontitis group tended to be higher than that of the control group, but the difference was not significant (p=0.054, unpaired t-test, Fig. 12B). On the other hand, the number of remaining teeth showed a significant negative correlation with the NAFLD score (n=12, r=-0.58, p<0.05, Pearson correlation coefficient, Fig. 12C).

With regard to bacterial detection, F. nucleatum was detected in 58.3% (7/12 cadavers) of the gingival tissues, T. forsythia in 33.3% (4/12 cadavers) and T. denticola in 8.3% (1/12 cadavers), but these detection rates were not significantly different between the control and the periodontitis groups (2x2 chi-square, p>0.05). In liver tissue, only F. nucleatum was detected in 33.3% (4/12 cadavers) of samples, with no significant difference between groups (p>0.05). Similarly, total bacterial counts in gingival tissue (the control group: 14.3 ± 3.3 copies / mg of tissue vs the periodontitis group: 25.8 ± 25.7 copies / mg of tissue) and liver tissue (the control group: 8.2 ± 3.2 copies I mg of tissue vs the periodontitis group: 11.6 ± 3.0 copies / mg of tissue) were measured by unpaired t-tests, but showed no significant differences (p>0.05). A number of studies have recently highlighted that periodontal disease negatively affects glycolipid metabolism and immune responses in the liver via oral and gut dysbiosis ^24-48. In this regard, microbiome-targeted therapy using probiotics and bacteriocins may be an effective approach for shifting not only the periodontal disease-related oral dysbiosis but also the gut dysbiosis toward a healthy state and subsequently preventing the development and progression of liver disease, such as NAFLD and NASH. Therefore, in this study, nisin, a bacteriocin produced by L. lactis, was orally administered in the context of a polymicrobial periodontal disease mouse model, and the effects of nisin on gut dysbiosis and liver disease were evaluated, revealing the significant therapeutic potential for this approach.

Periodontal disease is a chronic polymicrobial infectious and inflammatory disease, characterized by the presence of several hundred bacterial species that inhabit the oral cavity and reside within oral biofilms ⁷⁶'⁷⁸. Various mouse models have been proposed in the literature to study human periodontal disease, including ligature models ⁷⁹‘⁸⁰, injection models ¹⁵, monomicrobial infection models ^81,82, and polymicrobial infection models ⁸‘"⁸⁵. Of these, it has been suggested that in polymicrobial infection models, there are synergistic and reciprocal effects on host immunity and physiological responses, and that the pathogenesis of periodontal disease differs from that in monomicrobial infection models ^85,86. In particular, polymicrobial infection models make use of P. gingivalis, T. deniicola. and T. forsythia as these are classified as periodontopathic microorganisms and categorized as members of the so called “red complex” because they are strongly associated with clinical parameters of severe periodontal disease, including deep periodontal pocket formation and bleeding on probing ⁸⁷'⁹⁰. Also, F. nucleatum, an “orange complex” Gram-negative bacterium that is closely related to the red complex, can aggregate with numerous oral bacteria and can act as an important microbial bridge during biofilm formation ⁹⁰'⁹². These pathogenic bacteria contribute to the pathogenesis of periodontal disease through various mechanisms, including secretion of proteolytic enzymes, host cell invasion, and activation and modulation of host immune responses by LPS and other surface effector molecules ⁹³'⁹⁶. For example, in a previous study that utilized a polymicrobial infection model of periodontal disease with the three red complex species (P. gingivalis, T. deniicola. and T. forsylhia) and F. nucleatum, we found an enhanced serum antibody response to these periodontal pathogens, an altered oral microbiome composition, and a corresponding altered cytokine immune response plus alveolar bone resorption characteristic of periodontal disease ^57,67. Therefore, this well characterized polymicrobial infection mouse model that recapitulates the characteristic features of naturally occurring periodontal disease was also used in this study. As expected, the results showed that in the infection group, the compositional changes and decreased diversity in the oral microbiome characterized by an increase in the Proteobacteria and Fusobacteria phylum were accompanied by a significant increase in inflammatory cytokines and an inflammatory cellular infiltrate into the gingival tissues (Fig. 1-4).

The concept that periodontal pathogens induce gut dysbiosis is supported by the fact that the oral microbiome acts as an endogenous reservoir that supplies novel bacteria to the gut microbiota ⁹⁷. Humans continuously and unconsciously swallow pathogens present in saliva and dental plaque ⁴¹, and some of those oral bacteria can pass through the harsh acidic environment of the stomach and reach the intestinal tract, even in systemically healthy individuals ^43,97. Several clinical studies have reported marked differences in the composition of the gut microbiome between periodontitis patients and healthy subjects ^41-43. Loureityo et al⁴¹ found that the gut microbiome of patients with chronic periodontitis had a higher abundance of the Firmicutes, Proteobacteria, Verrucomicrobia and Euryarchaeota phylum and a lower abundance of the Bacteroidetes phylum, plus the diversity of the microbiota was decreased. Kawamoto et al ⁴² showed that in fecal samples from patients with severe periodontitis, the Bacteroidia and Actinobacteria phylum exhibited a lower abundance and there was a greater enrichment in several families and genera of the Firmicutes phylum. Furthermore, the microbiomes of periodontitis patients contained a greater abundance of the genus Acidaminococcus, Clostridium, Lactobacillus, Bifidobacterium, Megasphaera, and Romboutsiac. Interestingly, Bao et al ⁴³ reported that transplantation of salivary⁷ microbiomes from patients with severe periodontitis into wild-type C57BL6 mice increased the proportion of Porphyromonadaceae and Fusobacterium in the gut and concurrently increased the levels of inflammatory cytokines and decreased the expression of tight junction proteins, which are associated with the intestinal barrier in the intestinal epithelium.

Many animal studies have shown that oral administration of periodontal pathogens induces gut dysbiosis and changes in intestinal metabolites, insulin resistance, and hepatic fat deposition in rats and mice ³²-⁴³-⁴⁴-⁴⁷-⁹³ p_{or exam}p[_e p gingivalis -induced gut dysbiosis suppressed the gene expression of tight junction proteins, causing an increase in serum levels of lipopolysaccharide (LPS)^32,47. Yamazaki et al ⁹⁸ demonstrated that in a mouse model of NAFLD, mice fed a high-fat diet and inoculated with P. gingivalis or P. intermedia exhibited an altered gut microbiome and blood metabolism, and a shift in hepatic transcriptional expression toward an NAFLD phenotype. In contrast, application of Actinomyces naeslundii and Veillonella rogosae, which are endemic oral bacteria, had no effect on NAFLD progression. With regard to a polymicrobial infection model, Blasco-Baque et al ⁴⁵ found that in mice fed a high-fat diet, combined administration of P. gingivalis, F. nucleatum, and P. intermedia induced impaired blood glucose metabolism and insulin resistance with concomitant changes in the gut microbial composition. Consistent with these findings, the polymicrobial infection model used in the present study showed marked changes in the composition and diversity of the gut microbiome, inflammation in the intestinal mucosa, and decreased expression of tight junction proteins (Fig. 1-3 & 5).

Gut dysbiosis increases intestinal permeability due to the disruption of intercellular junctions of the intestinal mucosa, and increases the translocation of enteric bacteria and their metabolites to the liver via enterohepatic circulation ³³ " ¹⁰⁰ Thus, it is likely that in pathological conditions, the liver is constantly exposed to a variety of intestinally-derived substances, including enterobacteria and LPS. Nakajima et al ⁴⁷ reported when the gut microbiota was altered by oral administration of P. gingivalis, the gene expression of tight junction proteins decreased in the gut tissues, LPS levels increased in serum, and larger amounts of bacterial DNA were detected in the liver of mice. Similarly, as shown by the disclosure presented herein, the total bacterial DNA count in the liver of the infected mice was significantly increased compared to the control mice, and the microbiome composition and diversity in the liver was also markedly different (Fig. 1-3 & 6). An increase in Firmicutes and a decrease in Proteobacteria at the phylum level, and an increase in Lachnospiraceae at the genus level were characteristic features of the infection group. This indicates that a periodontal polymicrobial infection may increase the bacterial load on the liver through oral and gut dysbiosis. Importantly, a polymicrobial infection markedly increased liver vacuolar degeneration and fat deposition around the central vein in the liver (Fig. 7), similar to previous findings in monomicrobial periodontal infection-induced gut dysbiosis models ³²-⁴⁶-⁴⁷-⁹⁸

Since gut dysbiosis may cause subsequent fatty liver disease, microbiome- targeted therapeutic approaches using probiotics, prebiotics, and bacteriocins may help prevent the development and progression of NAFLD in patients with periodontal disease. In particular, improving the gut microbiome with probiotics for the treatment of NAFLD has been favorably accepted and supported by many studies ^49-3158. in preclinical animal studies, probiotics have been shown to ameliorate the increased liver adiposity by suppressing the development of insulin resistance and hepatitis signaling through the regulation of the gut microbiota ⁵³’³⁶. Randomized controlled trials in NAFLD patients revealed that administration of polymicrobial probiotics (containing Bifidobacteria, Lactobacillus acidophilus, L. bulgaricus, L. paracasei, L. plantarum, and Streptococcus thermophiliis) significantly reduced the fatty liver phenotype, inflammation, and fibrosis ^{53 101}>¹⁰²

In this study, in the context of a polymicrobial periodontal infection, oral administration of nisin, a bacteriocin produced primarily by L. lactis, shifted the oral, gut, and liver microbiome toward a healthier state. This prevented periodontal disease and enteritis, and subsequently reduced the bacterial exposure in the liver (Fig. 1-6). In addition, nisin mediated a marked protective effect against vacuolar degeneration and fat deposition in the liver (Figure 7). Limited studies have examined the therapeutic effects of nisin and other bacteriocins on NAFLD, and several in vitro and in vivo studies have been conducted on the effects of oral administration of nisin and nisin-producing L. lactis on the digestive tract ¹⁰³'¹⁰⁶, although none of these studies were performed in the context of periodontal disease. Use of nisin and/or nisin- producing L. lactis re-established a balanced gut microbiota and alleviated symptoms compared to conventional antibiotics (vancomycin, metronidazole) and antiinflammatory drugs (sulfasalazine) in various inflammatory bowel disease mouse models ¹⁰³'¹⁰⁵. A study by Jia et al ¹⁰⁶ on the gut-brain axis using a mouse model of Escherichia co/z-induced diarrhea reported that nisin modulates not only the gut microbiota but also important neurochemicals in the brain and central nervous system, such as norepinephrine, 5-hydroxytryptamine and dopamine. Some in vivo experiments have also reported on the therapeutic effect of /., lactis on NAFLD ¹⁰⁷'¹⁰⁹. Lee et al ¹⁰⁸ showed that oral administration of L. lactis strain NZ3900 pre-stimulated with nisin significantly limited the formation of a fatty liver phenotype and the progression of early atherosclerosis in a rabbit model fed a high cholesterol diet. Also, Naudin et al. ¹⁰⁹ reported that in mice fed a high-calorie Western diet, oral administration of L. lactis subsp cremoris improved glucose tolerance and reduced weight gain, obesity, serum cholesterol levels, and hepatic lipid deposition compared to the beneficial bacteria Lactobacillus rhamnosus GGL. In addition, in our study, nisin treatment prevented the increase in Bacteroidetes and decrease in Firmicutes phylum in the polymicrobial infection-induced gut dysbiosis, and improved the bacterial composition to a state similar to that of control mice (Fig. 1). This reduction in the Firmicutes/Bacteroidetes ratio is known to be involved in the disturbance of liver glycolipid metabolism and promotion of fatty liver formation in periodontal disease-infected mice ³²‘⁹⁸. Interestingly, at the genus level, the abundance of Lactobacillus gasseri in the gut and liver microbiomes was increased in the nisin and the infection+nisin groups (Fig. 2). Lactobacillus spp. have been known to play a protective role against NAFLD, which may be one explanation for the liver-friendly effects ofnisin ¹¹⁰-¹¹²

Mitochondrial dysfunction and oxidative stress may be important mechanistic processes by which nisin attenuates periodontal disease-induced hepatic lipidation and lipid peroxidation. Gene expression analysis in the liver in this study revealed that nisin treatment of polymicrobial-infected mice significantly suppressed the expression of oxidative phosphorylation-related genes in mitochondria and peroxisomes, including cytochrome P450 (Figs. 8 & 9). These genes clustered in pathways involved in the pathogenesis of NAFLD, most of which were associated with the mitochondrial electron transfer complex. In general, oxidative stress is defined as a detrimental condition resulting from an imbalance between excessive production of reactive oxygen species (ROS), such as singlet oxygen, superoxide, and hydrogen peroxide, and a lack of antioxidant capacity ^{113 114} The major intracellular source of ROS is the mitochondria, and superoxide anion radicals are produced via two main subunits when adenosine triphosphate (ATP) is synthesized through oxidative phosphorylation by the electron transport chain: complex I (NADH dehydrogenase) and complex II (ubiquinone-cytochrome C reductase) ¹¹⁵. Oxidative stress is strongly involved in the pathogenesis of NAFLD, especially the accumulation of free fatty acids and ROS production in liver tissue, which have been reported to have mutually adverse effects ^{116 118}. The process by which free radicals pull away electrons from lipids in cell membranes is called lipid peroxidation, resulting in an increase in malondialdehyde (MDA) and 4-hydroxy-2,3-transnonenal (4-HNE), lipid peroxides resulting from cell membranes that cause cell damage and inflammation ¹¹⁹-¹²⁰. Lipid peroxides suppress the function of ATP production in the mitochondrial electron transport chain, reduce mitochondrial function through mitochondrial DNA damage and cytochrome c depletion, and further promote ROS production. Indeed, a decreased number and enlargement of mitochondria, increased lipid peroxidation, and reduced ATP levels have been reported in the livers of NAFLD patients ¹²¹. In the present study, the gene expression of molecules involved in lipid metabolism, PPARa and CYP2E1, was also increased in infected mice, but significantly reduced by nisin treatment. Free fatty acids are known to induce the expression of PPARa, a nuclear receptor-type transcription factor that encodes uncoupling protein-2 (UCP-2), a protein associated with fatty acid oxidation in mitochondria and peroxisomes ¹²² Increased expression of UCP-2, a membrane protein present in the mitochondrial inner membrane, impairs oxidative phosphorylation for ATP production. In addition. PPARa also regulates transcription of Acyl-Co-A oxidase, the rate-limiting enzyme for [3-oxidation in peroxisomes, and PPARa in hepatic peroxidase is activated in conditions of hepatic lipid accumulation ¹²³. In other words, increased expression of PPARa contributes to the enhancement of oxidative stress because hydroxyl radicals, the most toxic being ROS, are readily produced during P-oxidation, the process that degrades free fatty acids. In addition, the expression level of CYP2E1, a type of cytochrome P450 enzyme that plays an important role in the metabolism of fatty acids and cholesterol, is increased by free fatty' acids and is reported to be significantly higher in the liver tissue of NAFLD patients ¹²⁴ CYP2E1 enhances NAD PH oxidase activity- and superoxide production.

Interestingly, genes involved in iron binding and iron-sulfur cluster binding were increased in the livers of infected mice, but suppressed by nisin treatment (Fig. 10). In contrast, gene expression for metallothionein (Mt) 1, which encodes a metal binding protein important as a metal transporter and antioxidative protein that exerts a metal detoxification function, was significantly decreased in the infection group compared to the control group, and this decrease was prevented by nisin-treatment. Iron, a transition metal element with two oxidation states (divalent and trivalent), catalyzes a reaction in the mitochondrial electron transport chain that converts oxygen to a large amount of hydroxyl radicals with strong peroxidative capacity ^l25-^,2G. The liver is the largest iron storage organ in humans, and in normal hepatocytes, the majority⁷ of iron is stored within the shell of ferritin, an intracellular iron storage protein, and is therefore nontoxic ¹²⁶. However, when there is an iron overload in hepatocytes, the increased free iron induces cytotoxicity. An observational study reported by Kowdley et al ¹²⁷ found that intrahepatic iron deposition and hyperferritinemia are frequently observed in patients with NAFLD. Consistent with this result, in the present study, the ferritin gene (Ft) 1 was also significantly increased in the infection group, but this increase was prevented by nisin treatment.

On the other hand, decreases in antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione have been reported to correlate with the severity of NAFLD ^128,129 SOD is known as an important antioxidant enzyme that converts superoxide to hydrogen peroxide and oxygen, and its active center has zinc, copper and manganese ions as cofactors ¹²⁶. Mtl gene expression, which was markedly decreased by infection but corrected by nisin treatment, is known to bind to these heavy metals and thereby act protectively against oxidative stress and lipotoxicity-induced cellular damage in the liver ^130,131. Hence, as a putative therapeutic mechanism for nisin, Mtl may counteract periodontal disease-triggered oxidative stress in the liver by alleviating both ROS overproduction and antioxidant deficiencies in this tissue. This is further supported by the fact that nisin significantly prevented the increase in lipid peroxidation markers (MDA) in the liver tissue of the infected mice (Fig. 11).

The prophylactic application of nisin proposed in this study is based on the premise that periodontal disease exacerbates the development and progression of NAFLD in humans. Akinkugbe et al ²⁰ demonstrated that there is a causal relationship between periodontal disease and NAFLD and that periodontal disease is a risk for NAFLD based on a population-based prospective cohort study of non-NAFLD subjects. At study entry, subjects were divided into three groups based on the cumulative percentage of periodontal disease present (0%, <30%, and >30%), and their liver status was evaluated by ultrasonography and serum ALT after 5 or more years. The results showed that the con founding-adjusted NAFLD incidence rate ratios were statistically significant at 1.28 and 1.60 for the less than 30% and greater than 30% diseased sites, respectively, compared to subjects without periodontal disease. A systematic review by Alakhali et al ²⁰ reported a significant correlation between periodontal or bacteriological parameters and NAFLD in all but one of the 12 selected epidemiological studies. In addition, some studies have also found a relationship between NAFLD and tooth loss, the true endpoint of periodontal disease. In this regard, Qiao et al ¹³² used multivariate logistic regression to analyze the association between patients' self-reported number of missing teeth and NAFLD diagnosed by liver ultrasound in a cross-sectional study of 24.470 Chinese adults, and demonstrated that the number of missing teeth was significantly correlated with the presence of NAFLD in men. Weintraub et al ¹³³ also analyzed the relationship between NAFLD, periodontitis, and tooth loss by logistic regression analysis in a population-based cross-sectional study using data from the National Health and Nutrition Examination Survey III in the United States. In a model adjusted for socioeconomic factors, compared to those with good oral health, adults with fewer than 20 teeth or moderate- severe periodontitis had a higher prevalence of NAFLD depending on the measure, such as ultrasonography, Fibrosis Score, and Fath⁷ Liver Index. Consistent with these results, a human autopsy study revealed a negative correlation between the number of remaining teeth and NAFLD tissue scores (Fig. 12). We did not find a correlation with the extent and severity of periodontal disease possibly because measurements of periodontal clinical parameters were not possible in the formalin-fixed cadavers. Therefore the evaluation of periodontal disease was limited to evaluation of CBCT images. Also, although the periodontopathic bacteria that may have mediated the liver damage could not be detected by PCR, they may have exerted their effects earlier on by activating a chronic immune response and triggering epigenetic effects on the liver toward chronic progression of liver disease.

In summary, we have shown that improving oral and gut dysbiosis with nisin lantibiotic treatment is a potential preventive and therapeutic strategy for NAFLD- associated periodontitis. Nisin is safe for daily human consumption, easy to use, and is environmentally friendly (unlike antibiotics whose widespread use has led to contaminated water sources and soils), and can be used alone or in combination with periodontal therapy ²⁴ In addition, the fact that adjuvant therapy with probiotics for the treatment of periodontal disease is already being tested in clinical applications will help facilitate this new treatment strategy ⁵⁸. However, this study has some limitations. One limitation is that the gut microbiome dysbiosis was induced not by naturally occurring periodontal disease, but by an oral polymicrobial infection with well-known periodontal pathogens, namely red complex bacteria and F. nucleatum. Although we’ve confirmed that periodontal disease develops gradually in this model and it mimics the chronic nature of the disease ^57,67 , the composition of the oral bacterial burden that impacts the intestinal tract may not exactly replicate that in naturally occurring periodontal disease in humans. Also, hematogenous diffusion of inflammatory cytokines and periodontal bacteria have been proposed as another pathway by which periodontal disease induces systemic inflammation ^{l 5}-^{l6J 34}. and our study could not completely rule out this effect of periodontitis on the intestine and liver. Nevertheless, given the high prevalence of periodontal disease and the global increase in NAFLD in recent years, this new approach seems extremely important from both a clinical/medical and basic science perspective. The development of this microbiome-targeted therapy for NAFLD in patients with periodontal disease is still in the early stages. Further studies are warranted to establish the optimal efficacy for host immunomodulatory mechanisms, potential effective combinations of various probiotics and bacteriocins, efficient oral/intestinal delivery methods, and long-term maintenance of microbial composition and functional changes ^24,48. Finally, another notable finding from the sequencing analysis in this study is that polymicrobial oral infections have a significant impact on the liver, far from the site of infection, the periodontal tissue. Consistent and dynamic changes in the liver microbiome, histopathological findings, and gene expression may have critical implications for elucidating the mechanisms by which periodontal disease exacerbates NAFLD.

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EXAMPLE 2: NISIN LANTIBIOTIC MITIGATES BRAIN MICROBIOME DYSBIOSIS AND ALZHEIMER’S DISEASE-LIKE NEUROINFLAMMATION TRIGGERED BY PERIODONTAL DISEASE

Periodontitis, a chronic inflammatory disease triggered by an oral microbial dysbiosis in a susceptible host, is one of the most prevalent diseases affecting nearly 50% of the population worldwide (1). In periodontitis, periodontal tissues are infected by oral microorganisms resulting in destruction of tooth supporting tissues and eventually tooth loss (2). In addition, evidence has accumulated that links periodontal inflammation with many systemic diseases, including diabetes, cardiovascular disease, cancer, adverse pregnancy outcomes, and neurodegenerative diseases (3,4,5). Although the association between periodontal disease and systemic diseases is well known, the underlying mechanisms are not fully understood. Evidence indicates that the inflammatory response induced by periodontal disease is not confined to periodontal tissues (6). Instead, oral microorganisms and inflammatory mediators can affect other organs through their circulation in the bloodstream via bacteremias (7). Levels of IL-10, IL-6, IL-17, TNF-a, and systemic C-reactive protein are elevated in patients with periodontal disease (8-10). Ultimately, the synergistic effects of the bacterial infection and immune response originating from the periodontal tissues may contribute to various systemic diseases. Therefore, control of periodontal infections is considered an important strategy for the prevention and treatment of a series of systemic conditions (11-13). Alzheimer’s disease (AD), the most common form of dementia, is the leading cause of cognitive disorders (14). AD is a complex, multifactorial disease affecting about 50 million individuals globally. The characteristic pathological changes of AD brains are the accumulation of intracellular hyperphosphorylated tau-positive neurofibrillary tangles (NFT) and insoluble amyloid 3 (A ) plaques, which stimulate glial cell activation and elicit local innate immune responses (15). The main etiologic factors of AD include brain hypoperfusion, traumatic brain injury, autoimmune disorders, insulin resistance, and other infectious diseases leading to neuroinflammation (16). Recently, considerable progress has been made in understanding the pathogenesis, diagnosis, and treatment of AD. However, there are no effective therapies to prevent or treat the condition until now (17). Prevention via modifiable factors is a promising avenue for slowing down the progression of this disease (18,19).

Growing evidence from cross sectional and longitudinal studies have demonstrated an association between AD and infectious conditions, such as periodontal disease (20). Periodontal disease and its characteristic oral microbial dysbiosis are thought to contribute to neuroinflammation and amyloid protein production via translocation of periodontal pathogens and their components to the brain. The presence of periodontal pathogens, such as Porphyromonas gingivalis and Treponema species, have been found in post-mortem brain tissues of AD patients (21,22). In addition, animal studies have been conducted to determine the effects of periodontitis on pathogen translocation and possible effects on the brain. Periodontal pathogens and their molecules have also been reported in the brain (23,24). Overall, these findings strengthen the hypothesis that periodontal pathogens play an important role in the development of AD. This hypothesis is further supported by research linking the presence of periodontal microorganisms with increased Amyloid P deposition, tau hyperphosphorylation, and glial cell inflammation and activation (25- 27). A recent study showed that treatment with a gingipain inhibitor reduced P. gingivalis infection in the brain of mice and prevented further neurodegeneration and accumulation of pathologic plaques, suggesting that controlling periodontal infections and microorganisms may be an effective way to slow down the development of AD (28).

Nisin. a class I Lantibiotic bacteriocin produced by Lactococcus laciis. has shown efficacy in treating a variety of infectious diseases, including gastrointestinal infections, respiratory tract infections, skin/soft tissue infections, and oral infectious diseases, including periodontal disease (29). In addition, we’ve demonstrated that nisin treatment can decrease the levels of periodontal pathogens in oral biofilms and return their microbial diversity back to control ‘healthy’ levels (30). Nisin can also prevent periodontal disease-related bone loss and inflammation while promoting reparative proliferation and a healthy microbiome (31). Therefore, the objective of this study was to examine the potential role of nisin in modulating brain microbiome dysbiosis, neuroinflammation, and amyloid-P and tau production after polymicrobial periodontal disease.

METHODS

Polymicrobial infection and treatment of mice

A total of 24 eight-week old BALB/cByJ female mice (The Jackson Laboratories, Bar Harbor, ME) were housed in microisolator plastic cages and randomly distributed into 4 groups (6 mice per group). The experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (1ACUC approval number: AN 171564-0 IB). All the mice were given trimethoprim (0.17 mg per ml) and sulfamethoxazole (0.87 mg per ml) daily for 7 days in the drinking water and their oral cavity was rinsed with 0.12% chlorhexidine gluconate (Peridex) mouth rinse to inhibit the native oral microbiota as described previously (32). The polymicrobial inoculum (5 * 10⁹ combined bacteria per ml; l * 10⁹ cells in 0.2 ml per mouse; 2.5x l0⁸ P. gingivalis, 2.5*10⁸ T. denticola. 2.5 x lO⁸ T. forsythia and 2.5 x lO⁸ F. nucleatum) was prepared in a 4% (w/v) carboxymethyl cellulose (CMC) solution and administered topically in the morning for 4 consecutive days every week for a total of 8 weeks as described previously (32). Nisin (300 pg/ml. 0.2 ml per mouse) was administered every day in the evening every⁷ week for a total of 8 weeks. A sterile 4% (w/v) carboxymethyl cellulose solution was administered as the control treatment. At 8 weeks following the polymicrobial infection, the mice were euthanized, and the brain tissues were collected for subsequent assays.

Periodontal bacteria and polymicrobial inoculum

The following periodontal pathogens, namely P. gingivalis FDC 381, T. denticola ATCC 35405, T. forsythia ATCC 43037, and F. nucleatum ATCC 10953, were obtained from ATCC (Manassus, VA) and cultured anaerobically (85% N2, 10% H2, 5% CO2) at 37 oC according to methods described in our previous study (32). P. gingivalis and F. nucleatum were grown for 3 days in Tryptic Soy Broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 5 mg/ml yeast extract, 0.5mg/ml L-cysteine hydrochloride, 5 pg/ml hemin, Ipg/ml menadione and 5% fetal bovine serum (FBS) (Gibco Thermo Fisher Scientific, Waltham, MA). T. denticola was cultured in Oral Treponeme Enrichment Broth medium (Anaerobe systems, Morgan Hill, CA) for 5 days. T. forsythia was grown for 7 days in Try ptic Soy Broth containing 5 mg/ml yeast extract, 0.5mg/ml L-cysteine hydrochloride, 5 pg/ml hemin, 1 pg/ml menadione, 10 pg/ml JV-acetylmuramic acid (Sigma- Aldrich, St. Louis, MO), and 5% FBS. The bacterial concentration was determined quantitatively using a spectrophotometer and each organism was resuspended in phosphate-buffered saline (PBS) at 1 x 10¹⁰ bacteria per ml for experiments.

For the oral polymicrobial infection, P. gingivalis was mixed with an equal volume of T. denticola for 5 min. Subsequently, T. forsythia was added to the culture tubes containing P. gingivalis and T. denticola, and the bacteria were mixed gently for 1 min and allowed to interact for an additional 5 min. Finally, F. nucleatum was added and mixed well with P. gingivalis, T. denticola, and T. forsythia. After 5 min, the four bacterial consortia were mixed thoroughly with an equal volume of sterile 4% (w/v) CMC in PBS, and this mixture was used as the polymicrobial oral inoculum.

Nisin preparation

An ultra-pure (>95%) food grade form of nisin Z (NisinZ® P) was purchased from Handary (S.A., Brussels, Belgium), a primary' manufacturer of nisin in the food industry'. The nisin stock solution was prepared at a concentration of 600 pg/ml in sterile Milli- Q filtered water, that was further filtered using a 0.22 pm syringe filter, and stored at 4°C for a maximum of 5 days for use in experiments (30,31). For oral treatment of mice, the nisin solution was then mixed with an equal volume of sterile 4% CMC and adjusted to the final concentration of 300 pg/ml.

DNA isolation from brain tissues

DNA was extracted from the mouse brain tissues to evaluate microbiological changes following periodontal bacterial challenge and/or nisin treatment using realtime polymerase chain reaction (PCR) and 16s rRNA sequencing. DNA was isolated and purified using the QIAamp® DNA Mini kit (Qiagen. Hilden, Germany) as in our previous study (31,32). The isolated DNA was stored at -20°C until further processing for real-time PCR and 16s rRNA sequencing analysis.

RNA isolation from brain tissues

For RNA stabilization, the mouse brain tissues were treated immediately after sample collection with an RNAlater solution (Invitrogen) at 4 °C overnight. Samples were powdered with a mortar and pestle under continuous liquid nitrogen, and total RNA was then isolated from each sample using an RNeasy Lipid Tissue Mini Kit (QIAGEN). The purity and quantity of the RNA were evaluated using the NanoVue Plus spectrophotometer (Biochrom Ltd.). Subsequently, total RNA was synthesized into cDNA using a High-Capacity⁷ cDNA Reverse Transcription Kit (Applied Biosystems) and according to manufacturer's protocols. Microbiome analysis of brain tissues via 16s rRNA sequencing

The purity and quantity of DNA samples isolated from brain tissues were deemed suitable and met quality control measures for 16s rRNA sequence performed by Novogene, Inc. (en.novogene.com). For the sequencing library preparation, the V4 variable region (515F-806R) of the samples was amplified using specific barcoded primers. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs) and the PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries were generated with the NEBNext® UltraTM DNA Library Prep Kit for Illumina and were then sequenced using the Illumina NovaS eq 6000 System.

Quantification of periodontal pathogens by real-time PCR

Absolute quantification by standard real-time PCR was used to evaluate the abundance of the periodontal pathogens in the brain tissue samples. Four periodontal pathogens used for the polymicrobial infection were measured by PCR using TaqMan primers and probes (Invitrogen) corresponding to the 16S rRNA gene as in our previous study (31, 32). Ten-fold serial dilutions of the DNA of known concentration were used to construct standard curves for quantification of the periodontal pathogens. The amplification was conducted using a QuantStudio 3 Real Time PCR system (Thermo Fisher Scientific) with a final reaction volume of 20 pL including TaqMan Fast Advanced Master Mix (Applied Biosystems). DNA (15 ng/ pL), primers, and probes. The optimized thermal cycling conditions were as follows: 95 °C for 10 min followed by 50 cycles of denaturing at 95 °C for 15 s, annealing and extension at 60 °C for 1 min. Data were analyzed using QuantStudioTM Design & Analysis Software vl.4.3 (Thermo).

Real-time PCR evaluation of gene expression from brain tissues To evaluate the immune cytokine profiles from brain tissues, relative gene expression was measured by real-time PCR using the following TaqMan primers and probes (TaqMan Gene Expression Assays; Applied Biosystems): interleukin 10 (III ft Mm00434228_ml), IL-6 (116; Mm00446190_ml), tumor necrosis factor-a (Tnf; Mm00443258_ml ). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Mm999999 l 5_g l ) was used as a housekeeping gene to normalize the amount of mRNA present in each reaction. PCR was performed in 20 pl reaction mixtures containing the TaqMan Fast Advanced Master Mix, cDNA template (20 ng/pl well), primers, and probes. The optimized thermal cycling conditions were as follows: 20 min at 95°C, followed by 40 cycles per 1 min at 95°C, and 20 min at 60°C. To compare the expression levels among different samples, the relative expression level of the genes was calculated by the comparative cycle threshold (AACT) method using QuantStudioTM Design & Analysis Software.

Determination of the 42 amino acid form of amyloid-P (A042), total Tan, and phosphorylated Tau (pS199) in brain tissues by enzyme-linked immuno-sorbent assay (ELISA)

Brain tissues were harvested and frozen at -80 °C until ready for use. A 42, total tau and phosphorylated tau (pS199) levels were assessed utilizing a commercially available ELISA kit (Invitrogen, Ap42: #KMB3441, total tau: #KMB7011, pS199: #KMB7041). Briefly, brain tissue was homogenized in eight volumes of 5M guanidine-HCL/50mM Tris-HCL at pH 8.0, and then mixed and incubated for 3 h at room temperature. After the incubation, the homogenates were diluted 10-fold with cold PBS with 10 protease inhibitor cocktail (Sigma) and subsequently centrifuged at 16,000 xg for 20 min at 4 °C. The supernatant was transferred to new tubes and further diluted with standard diluent buffer. Afterwards, levels of A 42, tau and phosphorylated tau (pS 199) in the samples were quantitatively assessed by a sandwich ELISA as per the manufacturer’s directions. Data were expressed in pg/rnL of homogenate. Statistical analysis

SPSS 21.0 statistical software (IBM, Chicago, IL, USA) was used for the statistical analyses. The comparison of bacterial numbers, immune profile-related gene expression, and Ap42, tau and tau (pS 199) levels were analyzed using ANOVA and Tukey’s test for multiple comparison among 4 groups. ANOVA and Tukey’s test for multiple comparison were also used for analysis of a-diversity indices, including Chaol, Shannon and Simpson index. In addition, t-test was performed to evaluate the Analysis of Similarity (ANOSIM) between two groups. A p value less than 0.05 was considered to be significant.

Oral polymicrobial infection/periodontal disease shifts the brain microbiome composition and nisin reverses the change

In order to assess effects of the periodontal polymicrobial infection/periodontal disease and nisin treatment on the brain microbiome, the microbial composition and abundance were analyzed by 16s rRNA sequencing and Metastat analysis at the phylum and genus level. At the phylum level, the relative abundance of Firmicutes and Proteobacteria were higher in the infected mice than in the healthy control mice, whereas the proportion of Actinobacteria, Bacteroidetes and Cyanobacteria were lower; although Proteobacteria did not reach statistical significance (Fig. 14A). In contrast, nisin treatment alone or in the context of infection shifted the microbial composition by increasing the relative abundance of Proteobacteria but decreasing the proportion of Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria (Fig. 14A). At the genus level, the proportion of Stenotrophomonas and Pseudomonas significantly increased in the infection group compared to the control group (P<0.05). whereas the relative abundance of Acinetobacter , Sphingobium, Massilia, Branchybacterium and Segetibacter decreased (Fig. 14B). Furthermore, the administration of nisin recovered the disease-associated changes by significantly increasing the abundance of Acinetobacter (P<0.05), and reducing the proportion of Stenotrophomonas , Pseudomonas and Methylobacterium (P<0.05) (Fig. 14B). Nisin similarly abrogated the oral microbial dysbiosis triggered by the periodontal disease, as previously reported (31, 33)

Oral polymicrobial infection/periodontal disease shifts the brain microbiome diversity and community structure, and nisin reverses the change

In order to assess the changes in brain bacterial diversity' following oral polymicrobial infection and nisin treatment, the Chaol estimator, Shannon index and Simpson index were analyzed based on the numbers of OTUs in the brain tissues. As showTi in Figure 2A, there was no significant difference in the community richness among the four groups (P>0.05). As for the Shannon and Simpson indices, the bacterial diversity score of the infection + nisin group yvas significantly lower than that of the control, infection and nisin group (P<0.001) (Fig. 15B, 2C). To evaluate the overall changes in the microbiome, we further performed Principal Coordinates Analysis (PCoA) based on the Weighted Unifrac distance and analysis of similarities (ANOSIM) (Fig. 15D, 2E). Interestingly, we found that the brain microbiome of the control and nisin group were in the middle state between the infection and infection + nisin group (Fig. 15D). The microbiome composition of the control, nisin, and infection + nisin group were significantly different from that of the infection group (P=0.007 with R=0.296, P=0.008 with R=0.352 and P=0.002 with R=0.967, respectively) (Fig. 15E). In addition, the microbiome composition of the control group was similar to that of the nisin group (P=0.322 with R=0.035) (Fig. 15E). Furthermore, nisin treatment in infected mice induced a shift in the brain microbiome, which yvas significantly different from that of the control and nisin group (P=0.006 with R=0.509 and P=0.009 with R=0.7, respectively) (Fig. 15E).

Nisin attenuates the periodontal pathogen burden in the brain following oral polymicrobial infection/periodontal disease In order to further assess the presence of periodontal bacteria in the brain tissues, the number of periodontal pathogens was measured in the brain samples using RT-PCR in a manner of absolute quantification. The detection frequency of the four periodontal pathogens is shown in Figure 3A. P. gingivalis could be detected in all four groups with different frequencies. T. forsythia was detected in the infection, nisin, and nisin + infection group, but not in the control group. F nucleatum was detected in the infection, nisin + infection group, but not in the control and nisin group. The presence of T. denticola was not detected in any of the groups. The copy number for P. gingivalis in the control group was significantly lower than that in the infection group (P<0.05) (Fig. 16B). Although the P. gingivalis copy number was lower in the infection + nisin group than in the infection group, this did not reach statistical significance (Fig. 16B). However, nisin treatment significantly reduced the T. forsythia copy number in the infection+nisin group compared to the infection group (P<0.05) (Fig. 16C). Interestingly, the F. nucleatum levels were higher in the infection + nisin group than that in the infection group, but the difference was not statistically significant (Fig. 16D). When taken in aggregate, the copy numbers for T. forsythia were highest among all 4 pathogens in the infection group (Fig. 16E).

Oral polymicrobial infection/periodontal disease induces neuroinflammation in the brain and nisin reverses the change

To evaluate the ability of an oral polymicrobial infection to induce inflammation in the brain and the ability of nisin to relieve this neuroinflammation, we performed gene expression analysis to evaluate the immune cytokine profiles in the brain tissues. The mRNA expression of pro-inflammatory cytokines, including IL- ip, IL-6 and TNF-a were significantly higher in the infection group than that in the control and nisin group (P<0.05) (Fig. 17). After nisin treatment, the expression of IL- 1 and IL-6 mRNA in the infected group decreased significantly to a level similar to that in the control group and nisin group (P<0.05). Nisin treatment also significantly reduced the expression of TNF-a mRNA in the infected group compared to the infection group (Fig. 17).

Nisin abrogates the deposition of Ap42, Tan, and phosphorylated Tan in the brain triggered by an oral polymicrobial infection/ periodontal disease

To evaluate the ability of an oral polymicrobial infection to trigger brain pathological changes and nisin’ s ability to reverse these changes, enzyme-linked immunosorbent assays (ELISA) were performed to evaluate the levels of A042, Tau, and phosphorylated Tau in brain tissue homogenates. As shown in Fig. 18, the concentrations of Ap42, Tau, and Tau (pS199) were significantly higher in the infection group compared to the control group (PO.OOl). Nisin treatment markedly reduced the A042. total Tau, and phosphorylated Tau deposition in the brain in the infection group (P<0.05).

In the present study, we investigated the ability of an oral polymicrobial infection/periodontal disease to induce changes in the brain microbiome and key molecular biomarkers of neurodegenerative disease and the ability of nisin to mitigate these changes. We hypothesized that nisin could prevent the periodontal pathogen mediated neurodegeneration as a direct consequence of its ability to reverse the changes in the brain microbiome, immune profiles, and pathologic protein deposition. To test this hypothesis, we used BALB/cByJ mice, a common mouse strain, to establish periodontal disease by oral infection with four key periodontal pathogens. Different mouse models of periodontal infection have been used to investigate the role of periodontal disease in the pathogenesis of AD (24,28,34-37). Most of the models used P. gingival i or its lipopolysaccharide as the only pathogenic bacteria or virulence factor. However, infection with a single periodontal pathogen does not recapitulate the polymicrobial nature of periodontal disease. Studies have indicated that the host immune responses to a polymicrobial infection are different from responses to a monoinfection (38,39). Our previous studies demonstrated that an oral polymicrobial infection in BALB/cByJ mice led to significant alveolar bone loss, a heightened antibody response to the periodontal pathogens, and an elevated cytokine immune response, indicating that this model is a representative and useful model of periodontal disease (31-33). Therefore, this polymicrobial mouse model of periodontal disease is an improvement over mono-infection models, and thus useful for evaluating the relationship between periodontal disease and systemic diseases and the effect of treatment modalities.

Over the last decade, a growing body of scientific evidence has demonstrated that alterations in the oral microbiota play an important role in the initiation and progression of AD. Oral microbiota could be transported to the brain through the blood stream in patients with periodontitis (40). Among the oral microorganisms, P. gingivalis, T. forsythia, F. nucleatum and T. denticola have been implicated in the development of AD (21,22,41). P. gingivalis is the most documented periodontal pathogen in AD related studies. The presence of P. gingivalis and its virulence factors, including lipopolysaccharide and gingipains, were confirmed in this context in both human and animal studies (24,28.42). In the present study, P. gingivalis was detected in all six mice in the infection group, which further confirmed the translocation of P. gingivalis from oral cavity to the brain. However, P. gingivalis was also detected in four of the six control mice at the conclusion of the study. The reason for this result may be that the mice were not housed in a gnotobiotic or specific pathogen free (SPF) grade facility, and thus there may have been potential environmental contamination; although the animals had an initial antimicrobial wash out period. However, the quantitative PCR results showed that the number of P. gingivalis copies in the infection group were significantly higher than that in the control group, indicating that oral infection did result in an increased load of P. gingivalis in the brain tissues. In contrast, T. denticola was not detected in the brain samples in our study, which is inconsistent with former studies (43,44). Miklossy first proposed that some spirochetes derived from the oral cavity were associated with AD (41). Riviere et al further demonstrated that several types of oral Treponema species, including T. denticola have been found in AD brain samples (21).

In our previous study, we found that it was relatively hard for T. denticola to colonize the oral cavity compared with the other three bacteria (32). At the end of the experimental period, only two of the six mice in the infection group had detectable T. denticola, and the proportion of T. denticola among total bacteria was very low. Therefore, we surmise that the limited number of T. denticola that colonized the oral cavi ty were not sufficient to disseminate and infect the brain. Although the IgG levels to T. forsythia and F. nucleatum are elevated in AD patients (45), these two periodontal pathogens have not been detected in brain tissues. Our study, for the first time, demonstrates the possibility⁷ that T. forsythia and F. nucleatum can be transported from the oral cavity⁷ to the brain. The T. forsythia bacterial load in the brain of infected mice was higher than that of P. gingivalis, suggesting that T. forsythia may possess specific virulence factors that promote its translocation to the brain. Therefore, T. forsythia may be another important periodontal pathogen that links periodontal infection with AD pathology⁷. F. nucleatum was also detected in the brain of the infected mice but at extremely low levels compared to that of P. gingivalis and T. forsythia. Therefore, the role of F. nucleatum in AD pathology may be similar to that of its role in dental plaque formation (46), that is, it may act as a "microbial bridge" to communicate with other bacteria.

In addition to individual bacteria, dysbiosis can also contribute to AD pathogenesis. Gut and oral dysbiosis have been implicated in AD development and progression (34,47-49). However, there is no direct evidence supporting a correlation between alterations in the microbial profile of the oral cavity⁷ and AD brain. By using 16s rRNA sequencing, we found that the microbiome composition of the brain of the periodontally-infected mice was significantly different from that of the control mice. The relative abundance of Firmicutes and Proteobacteria was significantly higher in the brain after polymicrobial-periodontal infection, and the proportions of Actinobacteria, Bacteroidetes and Cyanobacteria were much lower. These altered dominant phyla may not possess periodontal pathogenicity, and may not originate from the oral cavity, but these changes could be the result of oral microbial alterations. Therefore, periodontal infection may not only lead to the transfer of periodontal pathogens from oral cavity to brain, but more importantly, it may also contribute to changes in the microbiome composition of the brain.

The potential for a periodontal infection to promote an increase in brain proinflammatory cytokines, including IL-1 , IL-6 and TNF-a, which ultimately promote the production of Ap and hyperphosphorylation of tau, resulting in neurodegeneration and AD has been postulated (50). Therefore, restoring the composition of the oral microbiome could be a helpful approach and possible therapeutic intervention strategy' for AD. Recently, our group demonstrated that nisin can prevent and disrupt oral biofilms, decrease the amount of oral pathogens within oral biofilms, and return the diversity and composition of diseases-associated oral biofilms back to control levels, demonstrating that nisin can modulate pathogenic oral biofilms towards health in vitro (30). In this study, the effect of nisin treatment on the translocation of periodontal pathogens to the brain was also evaluated. We found that although treatment with the nisin bacteriocin did not significantly change the bacterial load of P. gingivalis and F. nucleatum, it did reduce the number of T. forsythia in the brain of infected mice, suggesting that the antibacterial effect of nisin was varied among different bacteria in this community setting with different modulating mechanisms. Our study further discovered that nisin treatment resulted in significant alterations in the brain microbiome. Nisin treatment dramatically increased the relative abundance of Proteobacteria but decreased the proportions of Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria. A study on the bacterial diversity of subgingival plaque showed that the predominant species in the subgingival plaque included Proteobacteria. Actinobacteria, Bacteroidetes. Firmicutes, Fusobacteria and Spirochetes, and the latter five phyla were closely related to periodontal disease pathogenesis ( 1). The results of our study indicate that the dominant bacterial species of the brain bacterial community' in periodontally infected mice is similar to that of the oral microbiota. In addition, nisin reduced the relative abundance of several species in the brain, including Firmicutes, Aciinobacieria. Bacteroidetes and Fusobaclerla, which may be the consequence of nisin’ s ability to alter the oral microbial community (Figure 19; 31, 33).

Importantly, we found that nisin treatment downregulates the expression of proinflammatory cytokines and reduces the deposition of Ap42 and phosphorylated tau proteins in the brain of periodontally infected mice. The “inflammation hypothesis of AD” proposed by Krstic and Knuesel (52) is one of most important hypotheses in the pathogenesis of AD. Neuroinflammation is an inflammatory response to injury or infection in the central nervous system (CNS). It is well known that microglia and astrocytes may be activated during this process, which produce excessive proinflammatory cytokines, especially IL-ip, IL-6, TNF-a, and additional Ap formation (53,54). Therefore, our study indicates that nisin relieves inflammation in the brain, thereby reducing the production of Ap42 and phosphorylated tau protein, which are two characteristic pathological changes of AD brains.

The disclosure provided herein shows that an oral polymicrobial-periodontal infection/periodontal disease can promote translocation of periodontal pathogens, including P. gingivalis, T. forsythia and F. nucleatum from the oral cavity to the brain, and thereby induce a shift in the composition of the brain microbiome, produce neuroinflammation via production of proinflammatory cytokines, and produce Ap and phosphorylated tau proteins in the brains of mice. Importantly, we provide evidence that nisin can effectively abrogate these changes by altering the composition of the brain microbiome after periodontal infection, mitigating inflammatory cytokine release, and reducing the Ap load and hyperphosphorylation of tau; demonstrating a potential role for nisin in the prevention and treatment of AD.

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Claims

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All publications mentioned herein (e.g. those listed numerically herein) are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. The following references include descriptions of methods and materials in this field of technology.

CONCLUSION

This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. CLAIMS:

1. A method of inhibiting development of and/or ameliorating liver steatosis in a subject, the method comprising administering to the subject a pharmaceutical composition comprising nisin in amounts selected to be sufficient to inhibit the development of liver steatosis in the subject.

2. The method of claim 1, wherein the pharmaceutical composition further comprises at least one of: a lipid, a probiotic, a nanoparticle, an anti-inflammatory agent, and a polymeric scaffold.

3. The method of claim 1, wherein the pharmaceutical composition further compnses an anti-inflammatory agent.

4. The method of claim 1, wherein the pharmaceutical composition is administered orally.

5. The method of claim 1, wherein, in a murine model of liver steatosis, amounts of nisin in the composition are selected to be sufficient to decrease amounts of lipid vesicles observable via histology as compared to control mice not administered nisin by at least 10%.

6. The method of claim 1, wherein the subject is selected to be a patient diagnosed with a periodontal disease.

7. The method of claim 1, wherein the subject is selected to be a patient diagnosed with a steatohepatitis.

8. The method of claim 1, wherein the subject is selected to be a patient diagnosed with a nonalcoholic fatty liver disease.

9. The method of claim 1, wherein the subject is selected to be a patient diagnosed with gastrointestinal inflammation or diabtes.

10. A method of inhibiting development of and/or ameliorating brain neuroinflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising nisin in amounts selected to be sufficient to inhibit the development and/or progression of brain neuroinflammation in the subject.

11. The method of claim 10. wherein the pharmaceutical composition further compnses at least one of: a lipid, a probiotic, a nanoparticle, an anti-inflammatory agent, and a polymeric scaffold.

12. The method of claim 10, wherein the pharmaceutical composition further comprises a further anti-inflammatory agent.

13. The method of claim 10, wherein the pharmaceutical composition is administered orally .

14. The method of claim 10, wherein, amounts of nisin administered are selected to be sufficient to decrease expression of TNF-a mRNA in the subject.

15. The method of claim 10. wherein, amounts of nisin administered are selected to be sufficient to reduce phosphorylated Tau deposition in the subject.

16. The method of claim 10, wherein the subject is selected to be a patient diagnosed with a neuroinfl ammatory disease.

17. The method of claim 16, wherein the subject is selected to be a patient diagnosed with Alzheimer's disease. 18. The method of claim 10, wherein the subject is selected to be a patient diagnosed with periodontal disease and Alzheimer’s disease or Liver diseases.

19. Use of nisin in the manufacture of a medicament for treating liver steatosis. 20. Use of nisin in the manufacture of a medicament for treating Alzheimer’s disease.