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Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease

Article in Oral Diseases · August 2007 DOI: 10.1111/j.1601-0825.2006.01291.x · Source: PubMed

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Mariagrazia Perilli Antonella Polimeni Università degli Studi dell'Aquila Sapienza University of Rome

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The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. Oral Diseases (2007) 13, 376–385. doi:10.1111/j.1601-0825.2006.01291.x 2007 The Authors. Journal compilation 2007 Blackwell Munksgaard All rights reserved http://www.blackwellmunksgaard.com ORIGINAL ARTICLE Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease

DN Della Riccia1, F Bizzini1, MG Perilli2, A Polimeni3, V Trinchieri4, G Amicosante2 and MG Cifone1

1Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy; 2Department of Biomedical Sciences and Technologies, University of L’Aquila, L’Aquila, Italy; 3Department of Odontostomatologic Sciences, University ÔLa Sapienza’, Rome, Italy; 4Department of Infectious and Tropical Diseases, University ÔLa Sapienza’, Rome, Italy

OBJECTIVES: To analyze the anti-inflammatory effects Introduction of Lactobacillus brevis extracts on periodontitis patients and to investigate the involved mechanisms in vitro on Lactic acid bacteria (LAB) belong to a variety of genera activated macrophages. used for milk fermentation. The primary metabolic end METHODS: Eight healthy subjects and 21 patients with product from carbohydrate metabolism is lactic acid, chronic periodontitis were enrolled to analyze the effect which in turn preserves milk by providing the acidity of L. brevis-containing lozenges on periodontitis-associ- necessary for a tart flavor and for changes in the ated symptoms and signs. Before and after the treat- structure of casein to achieve syneresis and desired ment, the patients received a complete periodontal functional characteristics. The extensive knowledge on examination. Saliva samples, collected before and after LAB has opened new possibilities for their application treatment, were analyzed for metalloproteinase and ni- (for reviews see Konings et al, 2000; C¸aglar et al, 2005). tric oxide synthase (NOS) activity, immunoglobulin-A Reports of many investigators have confirmed that LAB and their products have beneficial effects on the health (IgA), prostaglandin E2 (PGE2) and c-interferon (IFN-c) levels. Arginine deiminase (AD) and NOS activities were of animals and humans, i.e., protection against enteric determined through a radiometric assay. Metallopro- infections, use as an oral adjuvant, immunopotentiation teinases were assayed by zymogram and Western blot- in malnutrition, and prevention of chemically induced tumors (Bodana and Rao, 1990; Roberfroid, 1998; ting, whereas IgA, PGE2 and IFN-c were assayed by enzyme-linked imunosorbent assay tests. Dugas et al, 1999; Gorbach, 2000). Almost all genera of RESULTS: The treatment led to the total disappearance LAB are able to produce bacteriocins that are primarily or amelioration of all analyzed clinical parameters in all lethal to other strains and species of bacteria form short- patients. This was paralleled to a significant decrease of lived pores in biologic membranes by interactions with different compounds including nisin, lipid II and nitrite/nitrate, PGE2, matrix metalloproteinase, and IFN- c levels in saliva samples. phospholipids, thereby killing the target bacteria (Kon- CONCLUSION: Our results suggest that the anti- ings et al, 2000). In the attempt to better characterize the inflammatory effects of L. brevis could be attributed to metabolic features of some LAB strains, our group has the presence of AD which prevented nitric oxide gen- recently shown the presence of high levels of arginine eration. Our findings give further insights into the deiminase (AD) in Lactobacillus brevis (Di Marzio et al, knowledge of the molecular basis of periodontitis and 2001). As bacterial AD may compete with nitric oxide have a potential clinical significance, giving the experi- synthase (NOS) by using the same substrate, arginine, mental ground for a new innovative, simple and effica- we decided to investigate the ability of L. brevis extracts cious therapeutical approach of periodontal disease. to inhibit NOS activity as well other inflammatory Oral Diseases (2007) 13, 376–385 parameters [c-interferon (IFN-c), prostaglandin E2 (PGE2) and metalloproteinases], known to be associated Keywords: L. brevis; nitric oxide; metalloproteinases; prostaglan- with NOS induction (Cifone et al, 1999; Goodwin et al, din E ; c-interferon 1999; Di Marzio et al, 2001; Ishii et al, 2003). In 2 particular, the chronic periodontal disease was chosen as the known inflammatory model to test the potential Corresponding Author: Dr MG Cifone, Department of Experimental in vivo anti-inflammatory effects of L. brevis. Medicine, University of L’Aquila, Via Vetoio, 10, Coppito 2, 67100 Periodontal disease is a major cause of tooth loss in L’Aquila, Italy. Tel: 0862 433503, Fax: 0862 433523, E-mail: pres- humans and is one of the most prevalent diseases [email protected] associated with bone loss. Following bacterial coloniza- Received 19 November 2006; revised 9 January 2006; accepted 30 January 2006 tion, the gingiva becomes inflamed leading, in some cases, Anti-inflammatory effects of L. brevis DN Della Riccia et al 377 to the destruction of the alveolar bone. Periodontitis has (Centricon 10) from Amicon (Amicon, Millipore Co., two distinct but interconnected etiologic components: Bedford, MA, USA). IFN-c (Endogen, Inc., Walburn, periodontopathic bacteria adjacent to the periodontal MA, USA) and PGE2 Elisa kits (Assay Designs Inc., tissues, and host-mediated connective tissue-destructive Ann Arbor, MI, USA) were purchased from TEMA responses to the causative bacteria and their metabolic ricerca srl (Bologna, Italy). IgAs Elisa kits were products (Golub et al, 1998). Treatment strategies obtained from IPR (Catania, Italy). against periodontal diseases have evolved with the aim of eliminating specific pathogens or suppressing destruc- Preparation of bacterial extracts tive host response. Although the pathogenesis of the Lactobacillus brevis, obtained from VSL Pharmaceuti- various forms of this disease is not completely under- cals (Gaithersburg, MD, USA) in a pure lyophilized stood, several factors are believed to play an important form (108 colony-forming units per gram), was routinely role, including inflammatory cytokines (McGee et al, grown in MRS broth medium under aerobic conditions. 1998; Graves, 1999), PGE2 (Offenbacher, 1996; Tsai et al, For the determination of AD activity and for in vitro 1998; Kornman, 1999), nitric oxide (Matejka et al, 1999; experiments on macrophages, L. brevis cultures were Takeichi et al, 2000) and salivary immunoglobulin-A washed and resuspended in 10 ml phosphate-buffered (IgA) antibodies (Schenk et al, 1993). Moreover, many of solution (PBS), sonicated (30 min, alternating 10 s the characteristics of periodontal diseases such as inflam- sonication and 10 s pause) with a Vibracell sonicator mation and attachment loss are associated with proteo- (Sonic and Materials Inc., Danbury, CT, USA), centri- lytic events. Indeed, oral damage can result from the fuged at 7500 g for 20 min at 4C and the supernatants release of an array of proteolytic enzymes by colonizing stored at )20C until used (bacterial extract). For bacteria as well as host-derived proteases (Henskens et al, the in vitro experiments, the bacterial extract was 1996; Baron et al, 1999; Korostoff et al, 2000; Travis and added to cell cultures at 0.5 mg/105cells/1 ml)1 (final Potemba, 2000). Selection of an appropriate delivery concentration). system is an important factor and, periodontitis being a For gene expression studies, genomic DNA was Ôlocalized’ disease condition, local drug treatment should isolated from an overnight culture of L. brevis using be preferred. a WIZARD genomic DNA purification kit (Promega, The aims of this study were firstly to analyze the Milan, Italy), according to the manufacturer’s effects of AD-expressing L. brevis-containing lozenges, instructions. both on the clinical signs of chronic periodontitis patients and on the NOS activity levels, matrix metal- Arginine deiminase activity loproteinase (MMP) levels, eicosanoid generation, IgA Arginine deiminase activity was determined in L. brevis 3 levels, IFN-c levels of the saliva. The mechanisms extracts by measuring the conversion of [ H]-L-arginine to 3 underlying the anti-inflammatory effect of L. brevis [ H]-L-citrulline. Samples were homogenized by sonica- extract were investigated in vitro on rat peritoneal tion in homogenization ice-cold buffer (25 mM Tris-HCl, macrophages activated by lipopolysaccharide (LPS). pH 7.4, 1 mM EDTA, 1 mM EGTA) containing protease inhibitors (0.2 mM PMSF, 10 lgml)1 aprotinin, )1 )1 Materials and methods 10 lgml pepstatin A and 10 l gml leupeptin). The homogenates were centrifuged at 4C for 20 min at 11 000 g. The supernatants were passed through an Materials AG50WX-8 Dowex resin (Bio-Rad Laboratories, Mel- ville, NY, USA) to remove endogenous arginine. The Nitrate reductase, L-lactic dehydrogenase (LDH), pyru- protein content was determined by the bicinchoninic acid vic acid, b-nicotinamide adenine dinucleotide phosphate procedure (BCA; Pierce, Rockford, IL, USA) using BSA (NADPH), flavin adenine dinucleotide (FAD), flavin as standard. Enzymatic reactions were conducted at 37C mononucleotide (FMN), (6R)-5,6,7,8-tetrahydrobiop- for 30 min in 50 mM Tris-HCl, pH 7.4, containing 1 mM terin (H4B), L-valine, phenylmethanesulfonyl fluoride CaCl2, 1 mM MgCl2, 1 mM L-citrulline (to inhibit the 3 (PMSF), pepstatin A, leupeptin, aprotinin, ethylene catabolism of [ H]-L-citrulline), 60 mM L-valine, 60 mM glycol-bis (2-aminoethlether)-N,N,N,‘N’-tetraacetic L-ornithine, 60 mM L-lysine (to inhibit nonspecific argi- )1 3 acid (EGTA), ethylenediamine tetraacetic acid (EDTA), nase activity) and 10 lCi ml of [ H]-L-arginine (Amer- 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sham, Bukinghamshire, UK). The reaction was stopped (HEPES), Tris base, gelatin, Triton X-100, Coomassie by the addition of 0.4 ml of stop buffer (50 mM HEPES, brilliant blue and other chemicals were obtained from pH 5.5, 5 mM EDTA). The samples were spotted onto Sigma Aldrich srl (Milan, Italy). L-ornithine hydrochlo- TLC plates and developed in chloroform/methanol/ ride and TPA were purchased from Alexis (San Diego, ammonium hydroxide/water (1:4:2:1, by vol) as solvent CA, USA). L-lisin monohydrochloride was obtained system. After drying, the radiolabeled spots were visual- from Merck (Merck Bioscience GmbH, Schwalback, ized by autoradiography, after spraying EN3HANCER Germany). EN3HANCE spray was obtained from (Perkin-Elmer Life and Analytical Sciences). [3H]-argin- Perkin-Elmer (Perkin-Elmer Life and Analytical ine and [3H]-citrulline, identified by co-chromatography Sciences, Boston, MA, USA). Methanol, acetic acid, with unlabeled standards which were revealed with chloroform and ammonium hydroxide were purchased ninhydrin spray, was scraped, and quantified by liquid from J.T. Baker (Milan, Italy), Microconcentrators scintillation. Where indicated, bacterial extracts were

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 378 preincubated for 60 min with 20 mM formamidine any patient, and antibiotics were not taken during the hydrochloride (Fluka Seelze, Germany), an inhibitor of previous 4 months. No patient was subjected to oral AD (Weikmann et al, 1978a,b). hygienics. Before and after the treatment with lozenges, the patients received a periodontal examination including Arginine deiminase gene PCR amplification plaque index (Silness and Lo¨ e, 1964), gingival index, The oligonucleotides were designed on the bases of bleeding on probing, calculus and temperature sensitivity nucleotide sequences of AD genes conserved at EMBL scores. All periodontal disease measurements were per- and GeneBank databases (accession number AJ001330). formed in four quadrants at six sited per tooth for all For polymerase chain reaction (PCR) experiments the teeth, with the exception of plaque index for which four following primers were used: primer arcA1 frame sites were examined. The results were expressed as a mean 5¢-ATTTGCACAAACATTACGAGAC and primer value accompanied by standard error of the mean. The arcA2 reverse 5¢-GTGAAGACTGTATCTAAATG- saliva samples, collected from controls and patients by CAT. Polymerase chain reaction (PCR) to amplify the spitting into an ice-cooled vessel before and after the AD-encoding gene, was performed in a 100 ll volume. treatment, were analyzed for MMP and NOS activity (the The amplification reaction mixture contained 2.5 U of latter determined as nitrite/nitrate levels in the fluid saliva AmpliTaq Gold DNA polymerase (Perkin-Elmer Life fraction and as citrulline levels in the cellular saliva and Analytical Sciences), the reaction buffer provided by fraction), and IgA, PGE2, IFN-c. Five patients, four men the Taq manufacturer (containing 1.5 mM MgCl2), and one women, 29–48 years old (mean age 40 ± 9.6) 200 lM each deoxyribonucleotide triphosphate manifesting periodontitis were also enrolled in the trial (dNTPs), 0.5 lM each primer, and 1 lg of genomic and treated for 4 days with conventional oral antibiotic ) DNA as template. Amplification reactions were carried therapy (spiromycin, 1 · 300 mg day 1) with the aim of out as follows: 12 min at 95 C for activation of comparing the effects of this conventional treatment with AmpliTaq Gold DNA polymerase; denaturation at 94 those exerted by the bacterial extracts of L. brevis. All C for 1 min, annealing at 55 C for 1 min, and extension participants were carefully informed of the aim of the at 72C for 1 min, repeated for 30 cycles. A final DNA investigation and were free to withdraw from the study at extension step at 72C for 7 min was allowed at the end any time. Written consent was obtained from all patients of the termal cycling. Direct sequencing of PCR ampl- prior to the collection of samples. The study was approved icons was performed on both strands by dideoxy-chain by the Institute of Odontoiatric Clinics of the La Sapienza termination method using a dRhodamina Terminator University, Rome, and was conducted in according with Cycle Sequencing Ready reaction Kit and the ABI the principles of the Declaration of Helsinki. PRISM 377 DNA Sequencer (Perkin-Elmer Life and Analytical Sciences). Sequencing was performed on three Animals PCR products derived from three independent reactions. Wistar rats (5–6 weeks old) were purchased from Charles River Breeding Laboratories (Calco, Como, L. brevis-containing lozenges Italy). Each lozenge (VSL Pharmaceuticals) was composed of the following: lyophilized bacteria (200 mg), fructose Rat peritoneal macrophages (400 mg), mannitole (1020 mg), aspartame (40 mg), talc Rat macrophages were obtained by peritoneal washes (60 mg), aerosil 200 (10 mg), Peg 6000 (50 mg), mag- with PBS, centrifuged and resuspended in Roswell Park nesium stearate (20 mg), tartaric acid (100 mg), and Memorial Institute (RPMI) 1640 medium containing orange flavor (100 mg). 2 mM glutamine, 10% heat-inactivated fetal calf serum (FCS) and 100 IU ml)1 penicillin and 100 lgml)1 Healthy volunteers, patients, treatment, and sample streptomycin. The cells were counted and their viability, collection as assessed by Trypan blue dye exclusion, was routinely The study was conducted as a double-blind paired- greater than 98%. Two milliliters of cell suspension ) comparison study, with treatment assignments random- (4 · 106cell ml 1) was then plated on a 12-well tissue ized and balanced to a group of eight healthy volunteers culture plate (Falcon; Becton Dickinson, Buccinasco, (people of our laboratories; male and female, age 24– Milan). The following day, cell cultures were washed 47 years, mean age 33.6 ± 7.96), who were clinically free thrice with medium without FCS and then cultured in of periodontal disease. Twenty-one patients with chronic the same medium for 18 h with or without LPS periodontitis, 16 men and five women, 30–51 years old (100 ng ml)1) in the presence or absence of bacterial (mean age 41.9 ± 7.23) were enrolled to analyze the effect extracts (0.5 mg ml)1). At the end of the treatments, cell of L. brevis-containing lozenges (4 lozenges/day for pellets were used for NOS activity determination and 4 days) on periodontitis-associated symptoms and signs, supernatants, concentrated 10-fold by Centricon C10 from the clinical point of view. Patients in this study (Amicon, Millipore Co.), were used in zymogram displayed a range of periodontal disease from moderate to experiments to detect metalloproteinase activity. severe periodontitis. Pocket depth, measured to the nearest millimeter marking, was >4 mm. Mean loss of Nitrite/nitrate levels attachment, evaluated by measuring the distance from the In aqueous solution, NO reacts rapidly with O2 and cemento-enamel junction to the bottom of the probing accumulates in the culture medium as nitrite and nitrate pocket, was 4.7 mm. No systemic disease was observed in ions. Twenty microliters of saliva samples or superna-

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 379 tants from macrophage cultures was used for nitrite activity was compared with that of partially purified measurement by a colorimetric assay based on the Griess gelatinases (MMP-2 and MMP-9) obtained from the reaction. Briefly, 5 ll of HEPES (50 mM, pH 7.4), 5 ll supernatants of HT1080 human fibrosarcoma cell line of FAD (5 lM), 10 ll NADPH (0.1 mM), 58 llofH2O (ICLC HTL98016, Interlab Cell Line Collection, Genova, and 2 ll of nitrate reductase (0.2 IU ml)1) were added to Italy) stimulated with TPA (10)7M) for 48 h. All 20 ll of samples, which were then incubated for 30 min experiments were conducted with or without EDTA, at 37 C. At the end of the incubation time 10 llof serine or cystein proteinase inhibitors. Bands relative to pyruvic acid (100 mM) and 1 ll of LDH (1500 IU ml)1) enzyme activity were quantified by scanning densito- were added to the samples. After 10 min of incubation at metry using Molecular Analyst PCTM software for the 37 C, samples were mixed with an equal volume of Bio-Rad model 670 scanning densitometer. Griess reagent in a microtiter plate and incubated at room temperature in the dark for 10 min. The OD was Western blot measured at 550 nm using a micro-enzyme-linked imu- Saliva collected from healthy volunteers and patients nosorbent assay (ELISA) reader (Easy Reader EAR 400; were centrifuged at 600 g for 15 min and the superna- Kontron Analytic, London, UK). KNO3 dissolved in tants were assayed for protein using the BCA method H2O was used as standard and H2O as blank. (Pierce). Twenty-eight microliters of each sample (40 lg of protein) was mixed with 7 ll of nonreducing PAGE Nitric oxide synthase activity buffer 5X [0.5 mol/l Tris-HCl (pH 6.8), 28% glycerol, Nitric oxide synthase activity was determined in both 4.4% SDS, 0.55% bromophenol blue] and separated on macrophage extracts and cell fraction of salivary sam- an 8.5% polyacrylamide gel. After electrophoresis, SDS 3 ples by measuring the conversion of [ H]-L-arginine to was removed by washing the gel in 50 mM Tris HCl pH 3 [ H]-L-citrulline as above described for AD assay. The 7.4, 2% Triton X-100 for 15 min at 37C and then with assay, performed with the addition to the reaction buffer the same buffer lacking Triton X-100 for 15 min at 37C. of tetrahydrobiopterin (BH4), 2 lM FAD, 2 lM flavin Afterwards, the gel was equilibrated in electrotransfer adenine mononucleotide, CaCl2 (0.6 mM) and calmod- buffer (25 mM Tris-HCl, 192 mM glycine, 10% meth- ulin (CAM) (0.1 lM), measures both the calcium- anol) for 20 min and transferred to polyvinyl difluoride dependent (constitutive) and calcium-independent (PVDF) membrane (Millipore) for 1 h at 4C at 100V (inducible) isoenzyme activities. Any activity inhibited using a Mini Trans-Blot Cell apparatus (Bio-Rad by AG or detected in the absence of Ca/CAM and in the Laboratories, Hercules, CA, USA). Nonspecific binding presence of the CAM antagonist W13 (100 lM) (to sites were blocked with 10% nonfat dry milk in TBS-T inhibit endogenous CAM), represented iNOS activity. (TBS and 0.05% Tween 20) for 16 h at 4C. The Where indicated, 20 mM formamidine or 10 lM ami- membrane was then incubated for 1 h at 25C with a noguanidine (AG) or 500 lM L-NMMA were added to mouse monoclonal antihuman MMP-9 (Calbiochem- the assay system to measure AD and/or NOS activity. Novabiochem, Darmstadt, Germany) diluted 1:400 in 5% nonfat dry milk in TBS-T. After incubation the Determination of saliva IFN-c, PGE2, and IgA levels membrane was incubated for 1 h at 25C with goat The IFN-c (Endogen Inc., Woburn, MA, USA), PGE2 antimouse-horseradish peroxidase-conjugated IgG (San- (Assay Designs) and IgA (IPR S.p.A., Catania, Italy) ta Cruz Biotechnology, Santa Cruz, CA, USA) diluted levels were determined in the fluid saliva fraction using 1:3000 in 2.5% non-fat dry milk in TBS-T. Immunore- ELISA kits, according to the manufacturer’s instruc- activity was assessed by chemiluminescence reaction tions. Where indicated, PGE2 levels were also assessed using ECL Western blotting detection system (Pierce). in macrophage culture medium. Statistical analysis Substrate gel electrophoresis (zymogram) The differences in the analyzed parameters between the SDS-PAGE zymograms containing 0.1% gelatin were control and patient groups were tested by using the performed as described previously (Thorgeirsson and Student’s t-test. The statistical significance of the differ- MacKay, 1992) using approximately 2 lg of sample ences in clinical and laboratory parameters between T0 protein from saliva samples or cultured macrophage and T1 was analyzed using the paired sample t-test. supernatants to reveal the gelanolytic activity. Gelano- The STATPAC program was used to perform statistical lytic activity secreted by cultured macrophages in basal analysis and statistical difference set at P < 0.05. or stimulated conditions was analyzed using equal 10-fold concentrated sample volume (10–20 ll). Follow- Results ing electrophoresis, gels were rinsed in 50 mM Tris-HCl (pH 7.4) containing 2% Triton X-100 followed by Arginine deiminase activity in L. brevis extracts 50 mM Tris-HCl (pH 7.4) and incubated overnight for Arginine deiminase activity was analyzed in bacterial gelatinases, in a buffer containing 50 mM Tris-HCl (pH extracts as described in the Materials and methods 7.4), 0.2 M NaCl, 5 mM CaCl2, and 1% Triton X-100 at section. The enzymatic assay, based on the conversion 37C. Enzyme activity was detected following staining of L-arginine to L-citrulline in appropriate conditions with 0.1% Coomassie brilliant blue in a mixture of allowing to exclude NOS and arginase activity, showed acetic acid:methanol:water (1:3:6), destained in the same that AD activity was present at high levels in L. brevis mixture without dye and dried. Sample collagenase (Figure 1a). The generation of citrulline by L. brevis

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 380 (a) (b) )

–1 45 40 35

prot min 30 –1 25 20 15 665 bp 10 5 0 Citrulline (pmol mg

L. brevis

+ L-NAME Markers

+ Formamidine

+ Aminoguanidine (c) 5’ aaagaggttt ctagcgatgt cttcgatggc gtccgccgtc gtccgaggga aaccccaatt gctaagacgt gtcggacaa gacgagctcg tcgccccctt cgatgtggga ttcgtggttc cggtcgcgcc atacctcaac ttgccccgca aaccgtgggt ggtggttgat gatgtattcc gtgatcaggg Figure 1 Arginine deiminase expression in attcacgttg acgggccggg aaagtcatct tgttaatgga gatcccgtca Lactobacillus brevis extracts. (a) Arginine ccaatggagg cctgcggatc cgcgtgaag taagcgtttg gcattgggtc deiminase activity in bacterial extracts in the cattaagaac ggccagtccg tattttcgga aacggattgc agatccggtt presence or absence of formamidine, a specific gggcgatgtc aatgtcagtc ccccgcaccc cttcgatgat cttgtcaacc inhibitor of arginine deiminase, L-NMMA, a atttcctgcg gggtcatttg ggtagaggta gtcatataag gcgtcgtggg nitric oxide synthase (NOS) inhibitor, and tggtacccgc aacgtaacca gattcggcga tcatccgg 3’ aminoguanidine, a relatively specific inhibitor of the inducible form of NOS. The results represent the mean values of duplicate deter- (d) L. sakei L D M V E K I M A G V R T N E I D I K S K minations and are representative of one of L. brevis M E T V D K I I E G V R G T D I D I A Q P three experiments. SD values were ever lower than 10% of mean value. *P < 0.001 when L. sakei A L I D V S A ------compared with L. brevis extracts alone. (b) L. brevis D L Q S V S E N T D W P F L M E T D P M E Analysis of arginine deiminase gene, after L. sakei - - N L Y F T R D P A A S M G D G L T I N PCR amplification performed on genomic L. brevis T P N A Y F T R D P Q A S I G D G I S I N DNA, through 1.2% agarose gel electro- phoresis. (c) Analysis of the direct sequencing L. sakei K M - - T F E A R Q R E S M F M E V I M Q of the PCR-obtained fragment amplimer L. brevis K M E T T F P A R Q R E S L I T E Y I I N (arcA1-2). (d) The sequence of a 124 amino L. sakei H H P R F A N Q G A Q V W R D R D H I D R acid residue encoded by the PCR-obtained L. brevis H H P R F A G O – V E V W R D R N H E S H fragment, corresponding to arginine deimi- nase protein. The stringent homology (52%) L. sakei M E G G D E L I L S D K V L A I G I S with the arginine deiminase of L. sakei is L. brevis I E G G D E L V L S D H V L A I G V S shown

extract could be attributed to AD activity, as it was 124 amino acid residues which showed several conserved observed in the absence of NOS cofactors (NADPH, structural elements typical of AD genes. Comparing FAD, FMN, and BH4) and it was not affected either by amino acids residues of the AD of L. brevis and AD of L-valine, an arginase inhibitor, or by L-NMMA, a NOS L. sakei (Figure 1d), a stringent homology (52%) was inhibitor, and aminoguanidine, a relatively specific found. Although the AD gene is scarcely conserved, inhibitor of the inducible form of NOS. Moreover, the including the internal regions, our results clearly abrogation of citrulline generation observed in the demonstrated the presence of this gene in L. brevis. presence of formamidine, a specific inhibitor of AD, definitively established that the conversion of arginine to Clinical evaluation citrulline was due to the presence of high levels of this The scores for gingival inflammation, plaque, tartar, enzyme in L. brevis extracts. temperature sensitivity, and bleeding on probing before The presence of AD in L. brevis was further con- and after the treatment with L. brevis lozenges are firmed through the structural analysis of the gene reported in Table 1. All clinical parameters that were encoding the enzyme. A couple of primers designed on analyzed reached statistically significant differences the basis of consensus sequences known to be an between patients before treatment (T0) and the same internal region of the AD encoding gene was used to after treatment with L. brevis containing lozenges (T1). attempt PCR amplification. The PCR, performed on On the mean, gingival inflammation was rated as genomic DNA of L. brevis, yielded an amplimer of moderate/diffuse by 20 patients at enrollment, and mild 665 bp (Figure 1b) which was subjected to direct by one subject. After treatment, almost all patients sequencing. The sequencing of the PCR fragment, showed no gingival inflammation; two individuals named arcA1-2, resulted in a completely identified complained of a mild inflammation, while just one 478 bp sequence (Figure 1c) encoding a polypeptide of patient had a still moderate gingival inflammation. The

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 381 Table 1 Effect of the treatment with Lactobacillus brevis-containing lozenges on gingival inflammation, plaque and calculus, temperature sensitivity, and bleeding in periodontitis patients Healthy controls HT1080

T0 T1 P-value 250 Gingival inflammation 2.24 ± 0.12 0.14 ± 0.10 <0.0001 150 Plaque 2.10 ± 0.14 1.10 ± 0.12 <0.0001 92 100 Calculus 1.62 ± 0.21 1.29 ± 0.16 <0.0001 75 Temperature sensitivity 2.29 ± 0.14 0.43 ± 0.11 <0.0001 72 Bleeding 1.38 ± 0.19 0.05 ± 0.05 <0.0001 50 37 tolerability was considered as very good by all patients. Overall, the clinical evaluation of both the dentist and the patient overlapped, and the results confirmed recovery in 18 patients and improvement in three individuals. The antibiotic treatment for 4 days did 250 150 not significantly modify signs and symptoms (data not 92 100 shown), as expected. 75 72 Effects of L. brevis-containing lozenges on saliva sample 50 37 NOS activity, PGE2, IFN-c, and IgA levels The levels of nitrites and nitrates and those of PGE2, IFN-c, and IgA, determined in the fluid saliva fraction of healthy controls and periodontitis patients, as described in Materials and methods in an unblinded fashion, are reported in Table 2. Periodontitis patients’ saliva had a significantly higher level of nitrites/nitrates Patients

when compared with normal subjects (P < 0.01). In the HT1080 same Table are also reported the results obtained when Figure 2 Salivary matrix metalloproteinase (MMP) activity in healthy periodontitis patients’ saliva was analyzed for nitrite/ subjects and periodontitis patients. Gelatinase activity in the soluble nitrate levels before (T0) and after (T1) treatment with salivary fractions of representative healthy subjects and periodontitis L. brevis-containing lozenges. In all patients, the treat- patients is shown. The electrophoretic mobility of MMP-9 and MMP- ment was associated with a significant reduction in 2, obtained from supernatants of HT1080 cell, is indicated on the left- hand side. Molecular weights of the standard are indicated on the right nitrite and nitrate levels. Similar results were obtained when the conversion of radiolabeled arginine to citrul- line was analyzed in the cell saliva fraction (data not shown). In addition the PGE2 and IFN-c levels were amounts of MMPs than the control samples, thus significantly higher in patients with periodontitis than in confirming previous reports. Figure 2 shows gelatinase controls (P < 0.01). Interestingly, treatment with activity present in five different control salivary samples L. brevis-containing lozenges was associated with a (panel a) as well as in five different patients’ salivary strong decrease in the levels of these inflammation- samples (panel b), representative of all examined cases. associated molecules (P < 0.01). In contrast, IgA levels, Enzymatic activity was compared with that of gelatin- which were significantly reduced in patients compared ases obtained from the supernatants of the HT1080 cell with controls (P < 0.05), were not significantly mod- line stimulated with tissue plasminogen activator (TPA) ified by the treatment. (10)7 M) for 48 h (the 72 kDa type IV collagenase, gelatinase A, MMP-2, and the 92 kDa type IV collag- Effects of L. brevis-containing lozenges on saliva sample enase, gelatinase B, MMP-9). The zymogram of healthy MMP activity and levels control salivas (Figure 2a) shows bands with relative The measurements of MMP activity in the soluble molecular weights of 92 kDa, of about 120 kDa and fraction of saliva samples disclosed that the salivary higher than 200 kDa. Aside from a band of about samples of periodontitis patients contained higher 37 kDa, little or no enzymatic activity was visible at

Table 2 Effect of the treatment with Lactobacillus brevis-containing lozenges on laboratory parameters in periodontitis patients

)1 )1 )1 Samples Nitrite/nitrate (lM) PGE2 (ng ml ) IFN-c (pg ml ) IgA (lgml )

HC 4.33 ± 1.31 P < 0.0001 0.32 ± 0.08 10.06 ± 0.84 885.62 ± 118.28 T0 38.86 ± 6.07* P < 0.0001 1.64 ± 0.1* 28.23 ± 2.11* 309.76 ± 143.06*P < 0.05 T1 9.39 ± 1.47** 0.46 ± 0.07** 8.82 ± 4.74**1.04 466.51 ± 237.1

HC, healthy controls; T0, before treatment; T1, after treatment. *P ¼ T0 vs HC; **P ¼ T1 vs T0.

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 382 molecular weights lower than 92 kDa. The profile of multiple forms of gelatinases found in patients’ saliva is shown in Figure 2b. In addition to the same bands of 250 controls, a strong gelatinolytic activity at molecular 150 weights lower than 92 kDa could be seen in all patients’ salivas. In some cases (i.e., lanes 2 and 3) the high 100 molecular weight activities of gelatinase were almost 75 absent, being prevailing below 92 kDa. These latter species mainly migrated with a connecting smear, being evident as separate bands only at the 37–50 kDa region. 50 The gelatinolytic activity detected by zimogram appeared to be due to the presence of MMPs, as protease activity was completely abrogated by the C T0 T1 T0 T1 T0 T1 addition of EDTA, but not by the addition of serine Patient n. 456 and cysteine proteinase inhibitors to the incubation buffer (data not shown). The treatment with lozenges led Figure 4 Effects of Lactobacillus brevis-containing lozenges on saliva sample matrix metalloproteinase levels. Western blot analysis of to the near disappearance of these MMP bands in all gelatinase expression in one representative healthy control and three patients’ samples. Figure 3 shows the gelatinolytic activ- periodontitis patients’ saliva samples before and after treatment with ity of five representative periodontitis patients before lozenges containing L. brevis. C, healthy control; T0, before treatment; (T0) and after (T1) the treatment with experimental T1, after treatment. Molecular mass markers are indicated on the left- lozenges. The profile of multiple forms of gelatinases hand side was similar in all patients. The treatment led to the near disappearance of the low molecular weight bands and 92 kDa were detected. Western blotting with specific restored the control profile of gelatinolytic activity. antibodies against human MMP-2, MMP-3, MMP-1, Western blot experiments demonstrated that the bands and MMP-8 showed no immunoreactivity, suggesting revealed by zymograms were due mainly to the enzymatic that these MMPs were not detectable in the saliva of either activity of MMP-9. Figure 4 shows a Western blot of one controls or patients (data not shown). representative control and three representative patients before and after treatment with experimental lozenges. In Effect of L. brevis extracts on macrophage NOS activity the saliva of patients antibody against MMP-9 revealed Rat peritoneal macrophages incubated in vitro in the some high molecular weight forms (>200 kDa) together presence of LPS for 18 h were induced to express iNOS with bands at the 180 kDa region and at 120 kDa. Other activity (Figure 5a). NOS activity was inhibited by the molecular weights ranging from 92 to 64 kDa and NOS inhibitors AG and L-NMMA. In the presence of approximately at 54 and 44 kDa were seen in the saliva bacterial extracts, NOS activity disappeared and argin- of patients with chronic periodontitis. After treatment the ine was converted in citrulline through bacterial AD, enzymatic pattern resembled that of healthy controls. as demonstrated either by the unsensitivity of this Indeed, a relevant reduction in high molecular weight conversion to NOS inhibitor treatment (Figure 5a) or bands was observed and little or no MMP-9 species below by the disappearance of enzymatic activity in the presence of the AD inhibitor, formamidine (data not shown). Similar results were obtained with L. fermentum 250 extracts, but not with Streptococcus thermophilus,a 150 LAB in which the AD activity was not detectable (not 100 92 shown). 75 These results suggest that bacterial AD was able to 72 50 totally utilize the arginine present in the medium thus 37 preventing its uptake by macrophages and consequently NOS activity.

Effect of L. brevis extracts on macrophage PGE2 release When rat peritoneal macrophages were cultured for up to 18 h with LPS, a gradual accumulation of PGE in T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 2 the supernatants occurred (fivefold increase over that in HT1080 45 7 36 control cells) (Figure 5b). This effect was almost totally Patient n. abrogated in the presence of L. brevis extracts as Figure 3 Effects of Lactobacillus brevis-containing lozenges on saliva occurred when the cells were previously treated with sample matrix metalloproteinase (MMP) activity. Zymography com- L-NMMA or aminoguanidine. paring gelatinolitic activities in saliva of five periodontitis patients before treatment (T0) with gelatinolitic activities in saliva of the same Effect of L. brevis extracts on MMP production by patients after treatment with L. brevis-containing lozenges (T1). The electrophoretic mobility of MMP-9 and MMP-2, obtained from macrophages in the presence of LPS supernatants of HT1080 cell line, is indicated on the left-hand side. To investigate the L. brevis effect on MMP production, Molecular weights of the standard are indicated on the right-hand side activated rat peritoneal macrophages were treated with

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 383 (a) 3 Control

) L. brevis –1 2.5 LPS LPS + L. brevis 2 prot min –1 1.5

1

0.5 Citrulline (pmol mg 0 None L-NAME Aminoguanidine Formamidine

(b)60 Control (c) L. brevis LPS LPS+ L.brevis 50 L. brevis Lactobacillus brevis LPS+ L-NMMA Figure 5 Effect of LPS+ AG cells) extracts on LPS-activated macrophages. 6 40 LPS Effect of L. brevis extracts on iNOS activity HT 1080 Control LPS + L. brevis (a), PGE2 levels (b) and MMP activity (c)in 30 rat peritoneal macrophages incubated in vitro MMP9 in the presence or absence of LPS for 18 h. 20

Where indicated, the effect of L-NMMA, PGE2 (ng/10 10 aminoguanidine (AG), and formamidine is MMP2 shown 0 bacterial extracts in the presence or absence of LPS and enzymatic activity (Di Marzio et al, 2001), we checked cell culture supernatants were analyzed by zymography the presence of the AD gene. The analysis of a fragment for MMP-9 and MMP-2 levels. Compared with macr- obtained by PCR unambiguously revealed the presence ophages in culture medium alone, LPS increased MMP- of this gene, thus confirming our previous findings. 9 activity, whereas MMP-2 was not detectable. The Nitric oxide is known to be an important inflamma- presence of L. brevis extract in the culture medium led to tory mediator, and is involved in the pathophysiology of a relevant decrease in MMP-9 activity, either when several inflammatory disorders. An increase in NO added alone or in the presence of stimuli. Similarly, a production has been also demonstrated in periodontitis total inhibition of MMP activity was observed in the as its role in the inflammatory response of periodontal presence of iNOS inhibitors. A representative zymogra- tissues has been suggested (Matejka et al, 1998, 1999; phy is illustrated in Figure 5c. Enzymatic activity was Lappin et al, 2000; Hirose et al, 2001; Kendall et al, compared with that of partially purified gelatinases 2001; Lohinai et al, 2001; Daghigh et al, 2002). These (MMP-2 and MMP-9) obtained from the supernatants findings are consistent with the hypothesis suggesting of HT1080 cell line stimulated with TPA (10)7M) for that treatment with agents able to block the production 48 h. The bands revealed by zymograms could be of NO or its effects might be therapeutically valuable for attributed to MMPs as they disappear with EDTA but periapical lesions (Lohinai et al, 1998; Paquette and not by the addition of other proteinase inhibitors to the Williams, 2000; Daghigh et al, 2002). Considering the incubation buffer (data not shown). role of NO in inflammatory events in general and in periodontitis in particular, we analyzed the potential L. brevis Discussion beneficial effects of -based treatment in patients with periodontal disease. Of note, the proposed experi- Periodontitis is characterized by extensive destruction of mental treatment led to the total disappearance or the gingival tissues and associated supporting structures amelioration of all analyzed clinical parameters (gingi- of the teeth. In the present work, we first analyzed the val inflammation, plaque, calculus, temperature sensi- potential anti-inflammatory action in vivo of L. brevis- tivity, and bleeding on probing) in almost all 21 patients containing lozenges in 21 adult patients with periodontal (recovery in 18 patients and improvement in three disease. Previously, our group reported the presence of individuals). The beneficial effects of the treatment on arginine-deiminase in L. brevis, a strain belonging to clinical signs and symptoms were paralleled by a strong LAB (Di Marzio et al, 2001). The presence of high levels and statistically significant decrease in oral NOS activ- of this enzyme, which metabolize arginine to citrulline ity. Besides its direct role as an inflammatory modulator, and ammonia, aids L. brevis extracts to inhibit NO NO represents a multifunctional messenger molecule generation, by competing with NOS for the same able to modulate the production of inflammatory substrate, arginine. To confirm the presence of AD in cytokines (Cifone et al, 1999), PGE2 (Goodwin et al, L. brevis, previously shown through the assay of 1999), and MMPs (Maeda et al, 1998; Tronc et al, 2000;

Oral Diseases Anti-inflammatory effects of L. brevis DN Della Riccia et al 384 Hirai et al, 2001). On the other hand, inflammatory exclude the possibility that L. brevis extract-based cytokines such as IFN-c, tumor necrosis factor-a and treatment, besides the anti-inflammatory effects repor- interleukin-1-a have been shown to be significantly ted here, is also able to antagonize the growth of specific higher in periodontitis patients than in healthy controls periodontopathic pathogens. (Takeichi et al, 2000; Hirose et al, 2001; Tervahartiala Taken together, our findings give further insights into et al, 2001; Ukai et al, 2001). Moreover, the generation the knowledge of the molecular basis of periodontitis of PGE2, a potent eicosanoid lipid mediator derived and have a potential clinical significance, representing from phospholipase-released arachidonic acid that is the experimental ground for a new innovative, simple involved in numerous homeostatic biologic functions and efficacious therapeutical approach of periodontal and inflammation, has been shown to be strongly disease. incremented in periodontal lesions (Cavanaugh et al, 1998; Leibur et al, 1999; Paquette and Williams, 2000; Acknowledgements Tsilingaridis et al, 2003). Furthermore, MMP levels have been previously shown to play a critical role in the The authors thank Gasperina De Nuntiis (Department of inflammatory process leading to alveolar bone and Experimental Medicine, University of L’Aquila, L’Aquila, connective tissue loss in periodontal disease (Makela Italy) for technical assistance and Miss Florence Pryen for et al, 1994; Ding et al, 1995; Ejeil et al, 2003; Sorsa et al, careful language revision of the manuscript. This work was 2004). Indeed, one of the most salient features of financially supported by grant ÔPROGETTO DI INTERESSE periodontitis and gingivitis is the increase in the levels of GENERALE DI ATENEO’ from the University of L’Aquila. bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. Bacterial pathogens involved in References periodontal diseases partly trigger and induce host cells Baron AC, Gansky SA, Ryder MI et al (1999). Cysteine to elevate their secretion of MMP. Proteolytic enzymes protease inhibitory activity and levels of salivary cystatins in released by the host cells and/or by pathogen bacteria whole saliva of periodontally diseased patients. J Periodon- are associated with tissue destruction and MMP have tal Res 34: 437–444. the primary role in this process as they can degrade most Bodana AR, Rao DR (1990). Antimutagenic activity of milk of the extracellular matrix components (Ding et al, fermented by Streptococcus thermophilus and Lactobacillus 1995). bulgaricus. J Dairy Sci 73: 3379–3384. Altogether, these observations prompted us to ana- C¸aglar E, Kargul B, Tanboga I (2005). Bacteriotherapy and lyze the potential effects of L. brevis extracts containing probiotics’ role on oral health. Oral Dis 11: 131–137. Cavanaugh PFJR, Meredith MP, Buchanan W et al (1998). lozenges on IFN-c,PGE2, and MMP salivary levels in periodontitis patients. Our findings clearly showed that Coordinate production of PGE2 and IL-1 beta in the gingival crevicular fluid of adults with periodontitis: its all these inflammatory-associated factors were drastic- relationship to alveolar bone loss and disruption by twice ally reduced in periodontal disease patients after the daily treatment with ketorolac tromethamine oral rinse. L. brevis-based treatment. However, no significant J Periodontal Res 33: 75–82. effects were observed on salivary IgA levels after the Cifone MG, D’alo` S, Parroni R et al (1999). Interleukin-2- proposed treatment. activated rat natural killer cells express inducible nitric oxide In the attempt to define the mechanisms underlying synthase that contributes to cytotoxic function and inter- the anti-inflammatory action of L-brevis-based treat- feron-gamma production. Blood 93: 3876–3884. ment, a set of experiments were performed by using rat Daghigh F, Borghaei RC, Thornton RD et al (2002). 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