10.59987/ads/2011.3-4.10-18
Lactoferrin (Lf), an iron-binding glycoprotein able to chelate two ferric ions per molecule, is a component of human secretions synthesized by exocrine glands and neutrophils in infection/inflammation sites. Lactoferrin in saliva represents an important defence factor against bacterial injuries including those related to Streptococcus mutans and periodontopathic bacteria through its ability to decrease bacterial growth, biofilm development, iron overload, reactive oxygen formation and inflammatory processes. A growing body of research suggests that inflammatory periodontal disease involves a failure of resolution pathways to restore tissue homeostasis. There is an important distinction between anti-inflammation and resolution; anti-inflammation is pharmacologic intervention in inflammatory pathways, whereas resolution involves biologic pathways restoring inflammatory homeostasis. An appropriate regulation of pro-inflammatory cytokine synthesis might be useful in reducing periodontal tissue destruction. Recently, the multifunctional IL-6 is emerging as an important factor able to modulate bone, iron and inflammatory homeostasis. Here, we report an overview of Lf functions as well as for the first time Lf anti-inflammatory ability against periodontitis in in vitro model and observational clinical study. In in vitro model, represented by gingival fibroblasts infected with Prevotella intermedia, Lf exerted a potent anti-inflammatory activity. In the observational clinical trial performed through bovine Lf (bLf) topically administered to volunteers suffering from periodontitis, bLf decreased cytokines, including IL-6 in crevicular fluid, edema, bleeding, pocket depth, gingival and plaque index, thus improving clinical attachment levels. Even if other clinical trials are required, these results provide strong evidence for a instead of an therapeutic potential of this multifunctional natural protein.
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Annali di stomatologia
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Lactoferrin and oral diseases: current status and perspective in periodontitis
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Lactoferrin and oral diseases: current status and perspective in periodontitis
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                                                                                                                            Literature review



    Lactoferrin and oral diseases: current status
    and perspective in periodontitis




                                                                                                                                 li
                                                                                                                              na
    Francesca Berlutti1, BScD                                                  riodontitis in in vitro model and observational clinical
    Andrea Pilloni2, MD                                                        study. In in vitro model, represented by gingival fi-
    Miriam Pietropaoli3, BScD, LD                                              broblasts infected with Prevotella intermedia, Lf exer-




                                                                                                                           io
    Antonella Polimeni2, MD, DDS                                               ted a potent anti-inflammatory activity. In the obser-
    Piera Valenti1, BScD                                                       vational clinical trial performed through bovine Lf (bLf)
                                                                               topically administered to volunteers suffering from pe-




                                                                                                 az
                                                                               riodontitis, bLf decreased cytokines, including IL-6 in
    “Sapienza” University of Rome, Rome,Italy                                  crevicular fluid, edema, bleeding, pocket depth, gingival
    1 Department of Health Sciences
                                                                               and plaque index, thus improving clinical attachment
    and Infectious Diseases                                                    levels.
    2 Department of Oral and Maxillofacial Sciences
                                                                               Even if other clinical trials are required, these results




                                                                                               rn
    3 Microbo srl, Biotechnology Company, Rome, Italy
                                                                               provide strong evidence for a instead of an therapeutic
                                                                               potential of this multifunctional natural protein.

    Corresponding author:                                                      Key words: lactoferrin, oral cavity, periodontitis, in-




                                                                                    te
    Prof. Piera Valenti                                                        flammation.
    Department of Public Health and Infectious Diseases
    “Sapienza” University of Rome
    P.le A.Moro, 5
    00185 Rome, Italy
                                                                        In     Introduction
    Phone: +390649914543
                                                                               Lactoferrin (Lf), an 80-kDa iron-binding glycoprotein be-
    Fax: +390649914626
    E-mail: piera.valenti@uniroma1.it                                          longing to the transferrin family, is a component of human
                                                                               secretions including saliva, and it is synthesized by exo-
                                                         ni
                                                                               crine glands and neutrophils in infection and inflammation
    Summary                                                                    sites (1). Lf, containing 691 amino acid residues, is folded
                                                                               into two homologous lobes (N-lobe residues 1–333, and
                                              io


    Lactoferrin (Lf), an iron-binding glycoprotein able to                     C-lobe residues 345–691) connected by a peptide (resi-
    chelate two ferric ions per molecule, is a component                       dues 334–344), which forms a 3-turn α-helix (Fig. 1). This
    of human secretions synthesized by exocrine glands                         glycoprotein, highly conserved among human, bovine, mou-
                                    iz



    and neutrophils in infection/inflammation sites. Lac-                      se, and porcine species, is able to reversibly chelate two
    toferrin in saliva represents an important defence fac-                    ferric ions per molecule with high affinity (Kd ~ 10−20 M)
    tor against bacterial injuries including those related                     retaining ferric iron to pH values as low as 3.0, whereas
                                                                               transferrin retains ferric iron to pH of about 5.5 (1, 2, 3).
                   Ed




    to Streptococcus mutans and periodontopathic bac-
    teria through its ability to decrease bacterial growth,                    The iron-binding affinity is high enough that, in the presence
    biofilm development, iron overload, reactive oxygen                        of Lf or transferrin, the concentration of free iron in body
    formation and inflammatory processes.                                      fluids cannot exceed 10–18 M, thus preventing the preci-
    A growing body of research suggests that inflamma-                         pitation of this metal as insoluble hydroxides, inhibiting mi-
    tory periodontal disease involves a failure of resolu-                     crobial growth and hindering the formation of reactive oxy-
    tion pathways to restore tissue homeostasis. There is                      gen species and related inflammatory processes (1).
 IC




    an important distinction between anti-inflammation and                     Lf concentration in human exocrine secretions is reported
    resolution; anti-inflammation is pharmacologic inter-                      in Table 1. The reported concentrations increase in infec-
    vention in inflammatory pathways, whereas resolution                       tion and/or inflammation sites due to the recruitment of neu-
    involves biologic pathways restoring inflammatory ho-                      trophils: 106 neutrophils secrete 15 µg of Lf.
C




    meostasis. An appropriate regulation of pro-inflam-
    matory cytokine synthesis might be useful in reducing                      Antibacterial activity of lactoferrin related
    periodontal tissue destruction. Recently, the multi-                       and unrelated to its iron withholding ability
    functional IL-6 is emerging as an important factor able                    The first function attributed to Lf was antibacterial activi-
©




    to modulate bone, iron and inflammatory homeosta-                          ty depending on its ability to sequester iron necessary for
    sis.                                                                       bacterial growth and survival (5). This action of Lf was con-
    Here, we report an overview of Lf functions as well as                     sidered bacteriostatic, as reversible by the addition of fer-
    for the first time Lf anti-inflammatory ability against pe-                ric iron (6).


    Grants: This work was granted by Sapienza University of Rome, Italy, to AP and PV to perform in vitro anti-inflammatory activity of lactoferrin.
    The observational clinical study was supported by Microbo srl Biotechnology Company, Rome, Italy


    10                                                                                                Annali di Stomatologia 2011; II (3-4): 10-18
    Lactoferrin and oral diseases: current status and perspective in periodontitis


                                                                          Table 1 - Lactoferrin concentration in human secretions.

                                                                          Biological fluids                    Concentration (mg/ml)

                                                                          Colostrum                                      8
                                                                          Milk                                         1.5-4




                                                                                                                        li
                                                                          Tears                                          2
                                                                          Saliva                                       0.008




                                                                                                                     na
                                                                          Joint fluid                                  0.001
                                                                          Vaginal secretion                            0,008
                                                                          Seminal fluid                                0.112
                                                                          Cerebrospinal fluid                       Undetectable




                                                                                                                  io
                                                                          Plasma                                      0.0004




                                                                                           az
    Figure 1 - Structure of lactoferrin. From: Baker & Baker, 2004 (4).   kers of mucosal immunity as immunoglobulins, including
                                                                          IgA, IgM and IgG, α-amylase, lysozyme and Lf are de-
                                                                          tectable in saliva (27). Evidence is constantly increasing
    However, bacterial pathogens are able to overcome iron li-            to support the use of saliva as a non-invasive tool for mo-
    mitation by means of two principal systems. The first is re-




                                                                                         rn
                                                                          nitoring biomarkers in health and pathological human sta-
    presented by the synthesis of small chelators, sideropho-             tus (28).
    res, which bind ferric ion with high affinity and transport it        However, it is important to follow some guidelines prior to
    into bacteria through a specific receptor (7, 8). In addition         saliva collection in order to minimize error variance and
    to the synthesis of siderophores, some highly host-adap-




                                                                              te
                                                                          chances of methodological errors (29, 30). Food or drink
    ted bacterial species as Porphyromonas gingivalis acqui-              intake must be avoided at least 2 h prior to sampling, due
    re iron directly through surface receptors able to specifically       to variations in saliva secretion which in turn will negati-
    bind Lf or other iron binding molecules as hemin and heme             vely affect the results. Food or drink high in sugar content,
    (9, 10, 11), and transport it across the outer membrane. The
                                                                      In  caffeine or acidity can stimulate saliva flow, and acidity will
    iron is then bound by a periplasmic iron-binding protein,             lower mouth pH levels, both compromising antibody–an-
    FbpA, and transported into the bacteria via an inner mem-             tigen binding and enzyme activity thus leading to invalid
    brane complex comprised of FbpB and FbpC (12).                        immunoassay results. In addition, alcohol consumption 24
    An iron-independent bactericidal effect of Lf was also de-            h prior to sampling should be avoided as it may cause in-
                                                        ni
    scribed (13). A direct interaction between Lf and lipopoly-           creased saliva secretion (29, 30, 31).
    saccharide (LPS) of Gram-negative or lipoteichoic acid of             Among salivary proteins, Lf is the most important factor
    Gram-positive bacteria is required for the lethal effect (14,         of natural immunity. Its concentration corresponds to
                                             io


    15, 16). Furthermore, it has been demonstrated that the bin-          1.23 mg/l in gingival crevicular fluids (GCF) and to 8.96 and
    ding of Lf to the lipid A of LPS (17, 18), induces a release          7.11 mg/l in unstimulated and stimulated saliva, respec-
    of LPS. This bactericidal activity of Lf appears to be loca-          tively. The Lf concentration in oral cavity is related to dif-
                                   iz



    ted in the N-terminal region as its derivative cationic pep-          ferent fluid samples to be assayed. In fact, unstimulated
    tide (residues 17-41), called lactoferricin (Lfcin), is several       saliva mainly derives from 70% submandibular, 20% pa-
    fold more active than the intact protein (19, 20, 21). More           rotid, and 2% sublingual glands, while stimulated saliva
    recently, the 1-11 residues in N-terminal region of Lf has
                  Ed




                                                                          mainly derives from 30% submandibular and 60% paro-
    shown to possess an antibacterial activity against Strep-             tid glands (28). Moreover, the concentration of this gly-
    tococcus mutans through its binding with bacterial DNA (22).          coprotein in GCF is influenced by the amount of the ex-
    Moreover, the Lf antibacterial activity related to its capa-          creted fluid which, in turn, depends from physiological or
    bility to release LPS can be annulled by high calcium con-            pathological status of the subject. The Lf concentration,
    centration in the culture media (23). As Lf is also able to           measured /site, clearly shows that its levels increase from
    bind Ca(II) through the carboxylate groups of the sialic acid         36 ng/site in healthy humans to 63 and 90 ng/site in gin-
 IC




    residues, present on two glycan chains, it cannot be ruled            gival and periodontal diseases, while its concentration cal-
    out that the release of LPS from Gram-negative bacteria               culated /ml is similar in health and pathological conditions
    can be also due to this additional binding property of Lf (23).       (32).
                                                                          Even if the detection of Lf as well as of other proteins in
C




    Lactoferrin in saliva                                                 saliva, easily accessible source of potential local and sy-
    Saliva is composed of 98% water and its pH is around 6.64             stemic biomarkers of health and pathological status, the
    (24, 25). Saliva composition consists of hormones, pep-               different amount of salivary flux in calculating the real con-
    tides, electrolytes, mucus, antibacterial compounds and               centration of the proteins must be considered.
©




    various enzymes (26). Steroid hormones detectable in sa-              This is a very critical point which can explain the conflic-
    liva comprise cortisol, androgens including testosterone              ting data reported by different Authors on Lf oral concen-
    and dehydroepiandrosterone, oestrogens and progeste-                  tration. Of note, several defence proteins (e.g. lysozyme,
    rone as well as aldosterone. Furthermore, saliva is rich              Lf and histatin-1) were found significantly more abundant
    in organic constituents as proteins, albumin, urea, uric acid,        in old (55–65 years old) than in young female (20–30 ye-
    lactate, and creatinine (26). Inorganic compounds are also            ars old) subjects (33). These results, demonstrating that
    present in saliva; Na+, K+ and Ca2+ are the main cations              Lf synthesis in the saliva of females is age dependent, are
    and Cl− and HCO3− the main anions (26). Furthermore, mar-             in sharp contrast with the well ascertained data showing

    Annali di Stomatologia 2011; II (3-4): 10-18                                                                                      11
    F. Berlutti et al.


    that Lf synthesis is under the control of steroid hormones        both a physiological process and a serious problem that
    (34) and consequently, it decreases in menopausal and             can lead to oral illness. Efforts to control microbial adhe-
    postmenopausal periods.                                           sion by anti-adhesive new materials or compounds have
    Summarizing, the changes of the Lf concentration depend           had modest success once applied to the patient. Conse-
    not only from significant changes of saliva samples (un-          quently, it could be very helpful to discover other com-
    stimulated, stimulated) but they are also age-dependent.          pounds able to hinder microbial adhesion. The ability of Lf,




                                                                                                                     li
    It was reported that Lf levels in GCF of patients with gin-       in both apo- and iron-saturated form, to inhibit the adhe-
    givitis, adult periodontitis (chronic periodontitis), and lo-     sion of S. mutans to hydroxyapatite (HA), mimicking tooth




                                                                                                                  na
    calized juvenile periodontitis (aggressive periodontitis) are     surface (47), may represent an interesting function. The
    similar, but higher than in normal subjects (32, 35, 36). Ano-    demonstration that Lf inhibits the adhesion of S. mutans
    ther study indicates that Lf level in GCF correlates with the     to a salivary film and HA through residues 473–538 of its
    clinical severity of periodontal diseases and the number          C-lobe (48), further helped to understand this activity, which




                                                                                                               io
    of polymorphonuclear leucocytes (37). A decrease in Lf le-        is unrelated to its iron binding properties. Both apo- and
    vels was observed in GCF and saliva after surgical pe-            iron-saturated bLf also inhibit adhesion of free and ag-
    riodontal treatment in chronic periodontitis (38). Similar Lf     gregated S. mutans cells to a dental polymer when Lfs were
    decrease in GCF and peripheral blood after oral hygiene           pre-coated to dental polymer or bound to both dental po-




                                                                                       az
    procedures in experimental gingivitis using healthy vo-           lymer and bacterial cells (41). Apo-Lf but not iron-satura-
    lunteers was also detected (39). Lf is not synthesized in         ted Lf also inhibits the attachment on HA of Prevotella ni-
    the healthy gingival tissues and elevated Lf levels in the        grescens by binding to both HA and bacteria (49). Apo-
    GCF of inflamed tissues originate from invading inflam-           Lf reduces the initial attachment of the commensal Strep-




                                                                                     rn
    matory cells (40). Thus, Lf is released from neutrophils in       tococcus gordonii by iron sequestration, but not that of pe-
    GCF in response to the inflammatory condition of perio-           riodontopathogens Fusobacterium nucleatum and P. gin-
    dontitis as a potential host defence factor against perio-        givalis. Interestingly, the initial attachment of mixed po-
    dontopathic bacteria and may be a good marker of pe-              pulations of S. gordonii/F. nucleatum and S. gordonii/P. gin-




                                                                          te
    riodontal diseases.                                               givalis is significantly reduced in the presence respect to
                                                                      that observed in the absence of Lf (50). In other studies,
    Influence of iron and lactoferrin on the lifestyle                Lf has been shown to inhibit the adhesion of A. actino-
    of oral microbiota                                                mycetemcomitans and P. intermedia to reconstituted ba-
    In human saliva, the iron content ranges from 0.1 to 1.0
                                                                  In  sement membrane, through ionic binding, and P. intermedia
    µM depending on meals but it can increase for gingival blee-      to bacterial adhesins by a specific binding of Lf (51).
    ding due to infection and inflammatory processes. During          The different nature of abiotic surfaces, the varying mi-
    the infection and inflammatory processes, the recruitment         crobial adhesion mechanisms and the different in vitro ex-
    of neutrophils increases saliva Lf concentration from 20 to
                                                     ni
                                                                      perimental conditions could explain the different results ob-
    60 µg/ml (41).                                                    tained for inhibition of bacterial adhesion by apo- or iron-
    Therefore, saliva represents an interesting model to in-          saturated Lf, which in some cases requires only ionic bin-
    vestigate the influence of iron and Lf concentrations on bac-     ding to biomaterials, and in others specific binding to bac-
                                           io


    terial infections. As matter of fact, the different ratio bet-    terial structures, or both.
    ween iron and Lf plays an important role in the lifestyle of
    several bacteria (42, 43) by inducing aggregation and bio-        Cell surfaces
                                 iz



    film development (41). In particular, in a saliva pool well       The ability of microbes to adhere, colonize and form bio-
    defined for iron and Lf content, apo-Lf (iron unsaturated         film on host cells is a crucial step for the development and
    form) was found to enhance S. mutans aggregates and bio-          persistence of infections.
                  Ed




    film formation, whereas iron-saturated Lf decreased ag-           A large number of Gram-positive and Gram-negative bac-
    gregation and biofilm development (41). Similar behaviour         teria possess specific adhesins that mediate the adhesion
    has been recently described in the periodontopathogen Ag-         process on epithelial host cells. Lf and Lfcin are all able
    gregatibacter (Actinobacillus) actinomycetemcomitans: iron        to bind to bacterial Gram-negative and Gram-positive sur-
    limitation up-regulates its biofilm genes contributing to bio-    faces, as well as to host cells, by binding to glycosami-
    film formation (44).                                              noglycans (GAGs) (52) and in particular to heparan sul-
    The reported data suggest that to assess the effect of Lf         phate (HS) (53) and the inhibition of bacterial adhesion se-
 IC




    in oral cavity it is necessary to evaluate preliminarily the      ems generally to be mediated by Lf binding to both bac-
    iron content of saliva. In fact, in periodontitis patients, the   terial and host cell surfaces (1).
    high iron concentration and the presence of hemin, which          Evidences of anti-adhesive function of Lf against oral pe-
    can form complexes with Lf, together with Lf degradation          riodontopathogens was shown by Alugupally (1997) (51)
C




    by bacterial and human enzymes (45, 46), could be re-             who demonstrated that the Lf-dependent inhibition of the
    sponsible, in vivo, for the lack or reduced activity of Lf even   adhesion of A. actinomycetemcomitans, P. intermedia and
    if its concentration is increased following infection and in-     P. nigrescens to fibroblasts can involve binding of lactoferrin
    flammation.                                                       to both the bacteria and host cells.
©




                                                                      Recently, the Lf influence on S. mutans adhesion has been
    Inhibition of bacterial adhesion on abiotic                       tested in vitro on epithelial cells. Lf at physiological con-
    and cell surfaces by lactoferrin                                  centration (20 µg/ml) is able to significantly decrease the
                                                                      adhesion efficiency of S. mutans to epithelial cells (Fig. 2).
    Abiotic surfaces
    Microbial adhesion and subsequent colonization, resulting         Inhibition of microbial invasion
    in biofilm formation on abiotic surfaces such as dental sur-      of host cells by lactoferrin
    faces and medical devices as dental prosthesis, represents        Some mucosal pathogenic bacteria are capable not only

    12                                                                                     Annali di Stomatologia 2011; II (3-4): 10-18
    Lactoferrin and oral diseases: current status and perspective in periodontitis




                                                                                                                         li
                                                                                                                      na
                                                                                                                   io
                                                                                             az
                                                                                           rn
    Figure 2 - Bovine lactoferrin in inhibiting adhesion efficiency of Streptococcus mutans on epithelial cells.




                                                                                te
    Legend: Epifluorescence optical microscopy images of epithelial cells infected with Streptococcus mutans, stained with Ba-
    cLight®LIVE/DEAD viability probe, in the absence (Panel A) or in the presence (Panel B) of bovine lactoferrin (20 µg/ml).



    of adhering, but also of entering into non-professional pha-
                                                                      In    chanisms that underlie pathogenicity and the interactions
    gocytes, such as epithelial cells. Inside host cells, bacte-            between pathogenic and non-pathogenic microbes coe-
    ria are in a protective niche in which they can replicate and           xisting on humans. These motivations are especially per-
    persist, thus avoiding host defences. Virulence determi-                tinent in the case of the human oral flora, which compri-
    nants, such as surface proteins able to bind host cells, play           ses at least 400 to 700 different bacterial species (55). Oral
                                                         ni

    a key role in the entry process inside the host cells. Lf has           microorganisms constitute a complex and dynamical
    been shown to inhibit the entry of facultative intracellular            community, responsible for two important oral infectious
    bacteria, both Gram-negative and Gram-positive (1).                     diseases affecting virtually all humans: gingival and pe-
                                            io


    Recently, the capability of S. mutans to enter inside gin-              riodontal diseases. Differently from gingivitis, confined to
    gival fibroblast cells has been reported (54). Preliminary              gingival mucosa, periodontitis involves periodontium de-
    experiments show the anti-invasive activity of Lf against               generation, alveolar bone resorption and gingival epithe-
                                   iz



    S. mutans infecting gingival fibroblasts.                               lium migration along tooth surface, and the resulting pe-
                                                                            riodontal pockets.
    Anti-inflammatory activity of lactoferrin in oral diseases              It is well know that periodontal disease is associated to an
                  Ed




    Understanding the role of microbial communities in human                inflammatory process that occurs in the tissues surroun-
    health is emerging as a fundamental and fascinating mi-                 ding the teeth in response to the accumulation of sub-
    crobiological challenge. In the battle against infectious di-           gingival bacterial plaque, mainly constituted by anaerobic
    seases, finally there is the awareness that the discovery               Gram-negative facultative intracellular pathogens and
    of novel connections between infection, inflammation                    their LPS, one of the factors responsible for the inflam-
    and human diseases is a pivotal tool in the research of no-             matory reaction (56, 57, 58). Differently from commensal
    vel antibacterial drugs.                                                bacteria, intracellular bacteria induce the over-expression
 IC




    The increasingly frequent threats of bacterial resistance               of several pro-inflammatory cytokines (59, 60, 61). In par-
    to antibiotics reinforce the necessity to understand the me-            ticular, increased levels of pro-inflammatory cytokines have
C




    Table 2 - Synthesis of pro-inflammatory cytokines by gingival fibroblasts uninfected or infected with Prevotella intermedia in the
    absence or in the presence of bovine lactoferrin.

                                                   Uninfected cells                                           Infected cells
©




                                         None                         bLf                            None                         bLf

    IL-1β (ng/ml)                     1.050±150                  1.000 ± 97                       4.800±421                     900±180
    IL-6 (ng/ml)                        90±21                      90±23                           930±105                       83±38
    IL-8 (pg/ml)                      2.100±241                  2.150±210                        9.450±750                    5.800±550
    TNF-α (pg/ml)                       22±14                      22±12                           120±65                         18±8


    Annali di Stomatologia 2011; II (3-4): 10-18                                                                                           13
    F. Berlutti et al.


                                                                                        tentially toxic-free iron (68) as well as to sequester LPS
    Table 3 - Clinical parameters and pro-inflammatory cytokine                         thus neutralizing its biological activity (69, 70). Moreover,
    levels in gingival crevicular fluids before and after bovine                        bLf is also able to inhibit biofilm development (42, 43) thus
    lactoferrin topical administration.                                                 decreasing inflammation due to the exopolisaccharides for-
                                                                                        ming biofilm (71).
                              Baseline                 After 4 weeks                    Recently, in several clinical trials on women suffering from




                                                                                                                                       li
                                                 of topical administration              anaemia, oral administration bLf has been found to be ef-
                                                   of bovine lactoferrin                fective in decreasing serum levels of IL-6, a key factor of




                                                                                                                                    na
                                                                                        iron homeostasis, thus restoring the physiological distri-
    PPD                        2.6±0.2                       0.5±0.8                    bution of iron between tissues and blood (72, 73, 74). As
    GI                        0.8 ±0.1                       0.5±0.1                    matter of fact, the iron deficiency in blood (iron deficien-
    PlI                       0.8 ±0.1                       0.4±0.2                    cy anaemia) is related to iron overload in tissues as well
    BOP (%)                       32                            0




                                                                                                                                 io
                                                                                        as to high levels of serum IL-6 (73).
    CAL (mm)                  1.50±0.7                      0.50 ±0.2                   In addition to the local tooth and gum effects, periodon-
    IL-1β (ng/ml)              138±87                         95 ±74                    tal disease has been shown to influence various systemic
    IL-6 (ng/ml)              1.35±0.8                      0.64±0.32                   disorders and diseases as cardiovascular disease (75), and




                                                                                                         az
    IL-8 (pg/ml)               94±65                          35±12                     diabetes mellitus (76). Moreover, significant associations
    TNF-α (pg/ml)               31±21                          3±2                      between periodontal disease and preterm low birth weight
                                                                                        have been demonstrated (77). Recently, the prevention of
    Legend: The clinical parameters and pro-inflammatory cytokine concentrations
    are reported as mean values ± standard deviation deriving from a total of twenty-
                                                                                        preterm delivery, mainly associated to high levels of IL-6,




                                                                                                       rn
    six teeth (2 teeth/subject). PPD: probing pocket depth; GI: gingival index; PlI:    has been obtained through bLf ability to decrease serum
    plaque index; BOP: bleeding on probing; CAL: clinical attachment level.             and cervicovaginal IL-6 (78).
                                                                                        As there is not ideal therapeutic approach to cure perio-
                                                                                        dontitis, the anti-inflammatory efficacy of oral administra-




                                                                                            te
                                                                                        tion of bLf encourages new therapeutically approaches to
    been detected in the inflamed gingival tissue and GCF at                            cure periodontal diseases.
    infected and inflamed sites (62, 63, 64).
     Although inflammation is an essential component of the                             In vitro and in vivo influence of bovine lactoferrin on
    host defence response to bacterial challenge, excessive
                                                                                    In  pro-inflammatory cytokine synthesis
    cytokine production from host cells results in consequent                           As reported in the previous section, IL-6 is involved not only
    periodontal tissue destruction. Therefore, chronic and pro-                         in bone resorption but also in iron homeostasis, where its
    gressive infection by anaerobic Gram-negative intracellular                         over-expression induces iron overload in tissues and se-
    pathogens inducing the inflammatory process is respon-
                                                                  ni
                                                                                        cretions and iron deficiency in blood (79). Consequently,
    sible for alveolar bone destruction. In fact, the bone is a                         we believe that the difficulty in resolving inflammatory pe-
    continuous remodelling between osteoblast (bone for-                                riodontal disease also involves the failure of restoring iron
    mation) and osteoclast (bone resorption), and its ho-                               homeostasis strictly related to inflammation through the
                                                      io


    meostasis is well controlled. Among soluble regulation fac-                         over-expression of IL-6 (79). Therefore our opinion is that
    tors influencing bone homeostasis, cytokines play an im-                            the resolution of inflammation in the context of periodon-
    portant role. It is well demonstrated that the pro-inflam-                          tal disease should involve the decrease of iron overload
                                          iz



    matory cytokines having bone-resorption activity, act                               in oral tissues and secretions.
    osteoblasts and stimulate osteoclastogenesis (i.e. bone re-                         In physiological conditions the availability of free iron in tis-
    sorption ) through up-regulation of receptor activator of nu-                       sues and secretions do not exceed 10-18 M, while in pa-
                     Ed




    clear factor (NFkB) ligand (RANKL) and down-regulation                              thological conditions the high concentration of free iron in-
    of osteoprotegerin in osteoblasts (65, 66, 67).                                     duces microbial multiplication, biofilm development, reactive
    Taken together these data reinforce the evidence that ap-                           oxygen species, cell damage and inflammation (1).
    propriate regulation of cytokine production by immuno-                              A strategy addressed to decrease iron overload could con-
    modulators might be useful in reducing periodontal tissue                           tribute to inhibit microbial growth and biofilm development
    destruction such as alveolar bone resorption. There is an                           as well as formation of reactive oxygen species, cell da-
    important distinction between anti-inflammation and re-                             mage and related inflammation.
 IC




    solution; anti-inflammation is pharmacologic intervention                           Here, we first report the anti-inflammatory activity of ad-
    in inflammatory pathways, whereas resolution involves bio-                          ded bLf to infected gingival fibroblasts.
    logic pathways restoring inflammatory homeostasis. In-                              To mimic the in vivo environment, the synthesis of inter-
    terestingly, the resolution of inflammation is an active, well-                     leukin 1 (IL-1 ), IL-6, interleukin 8 (IL-8) and tumor necrosis
C




    orchestrated return of tissue homeostasis. A growing body                           factor-α (TNF-α) by human gingival fibroblasts infected or
    of research suggests that inflammatory periodontal disease                          not with P. intermedia, an anaerobic Gram-negative in-
    involves a failure of resolution pathways to restore ho-                            tracellular pathogen associated to periodontitis, has been
    meostasis.                                                                          detected. The infection of monolayers was performed in the
©




    At present, there is no ideal therapeutic approach to cure                          absence or presence of bLf at 20 µg/ml concentration cor-
    periodontitis and achieve an optimal periodontal tissue re-                         responding to that detected in saliva of healthy subjects
    generation.                                                                         and found ineffective against P. intermedia growth. The ab-
    Recently, the milk derivative bovine Lf (bLf) is emerging                           sence of antibacterial activity of bLf in our in vitro model
    as an important regulator of iron and inflammatory ho-                              is pivotal to exclude the different cytokine synthesis rela-
    meostasis exerting a potent effect in decreasing inflam-                            ted to the different number of viable bacteria. For this pur-
    matory host responses. The bLf ability in decreasing in-                            pose semi-confluent monolayers were infected with P. in-
    flammatory processes is related to its ability to bound po-                         termedia at a multiplicity of infection 100 bacteria per cell

    14                                                                                                       Annali di Stomatologia 2011; II (3-4): 10-18
    Lactoferrin and oral diseases: current status and perspective in periodontitis


    in the absence or in the presence of 20 µg/ml of bLf. Af-             according to the World Medical Association Declaration of
    ter infection, the supernatants from uninfected and infec-            Helsinki.
    ted cells with or without bLf were collected and IL-1 , IL-           At each visit, GCFs to be analyzed were collected from two
    6, IL-8 and TNF-α concentrations were determined using                teeth affected by periodontitis/subject. All subjects were cli-
    standard enzyme-linked immunosorbent assays (ELISA)                   nically evaluated in the following periodontal measurements:
    (Table 2).                                                            number of teeth present, plaque index (PlI) (80), gingival




                                                                                                                         li
    The results clearly demonstrated that bLf added to unin-              index (G I) (81), probing depth (PD), clinical attachment
    fected monolayers did not modify cytokine synthesis, whi-             level (CAL), and bleeding on probing (BOP) (82). Two te-




                                                                                                                      na
    le it significantly decreased the synthesis of IL-1 , IL-6, IL-       eth affected by periodontitis were examined per subject.
    8 and TNF-α by gingival fibroblasts infected with P. inter-           PlI, GI, PD, CAL and BOP (%) were measured at four si-
    media.                                                                tes around each tooth.
    These in vitro results have encouraged us to carry out an             All enrolled subjects were directed to topically place 100




                                                                                                                   io
    observational preclinical study on volunteers suffering from          mg of lyophilized bLf two times a day for 4 weeks after meal
    mild chronic periodontitis. The calibration was performed             and correct oral hygiene as well as after extensive washings
    before the study with 5 volunteer subjects among the re-              until the arrest of the putative gum bleeding. In particular,
    searchers involved in the study of bLf functions. Then other          100 mg of lyophilized bLf, contained in a pocket, were de-




                                                                                           az
    volunteers suffering from mild chronic periodontitis among            posited in an little container in order to be adsorbed on soft
    patients were enrolled at a private practice. Pregnant wo-            bristles of the appropriate toothbrush and applied through
    men as well as the subjects with other concomitant di-                a soft brush on the gums, particularly on the bleeding si-
    seases were excluded from this study as well as smokers,              tes.




                                                                                         rn
    subjects with ascertained allergic reactions to cow’s milk,           Periodontal clinical parameters consisting in PPD, GI, PlI,
    and subjects receiving antibiotic treatment within the pre-           BOP (%) and cytokine concentrations in GCFs were as-
    vious 3 months.                                                       sayed in all subjects before (baseline) and after 1, 2 and
    As a result, 13 subjects (7 female and 6 men, age range               4 weeks of therapy. The parameters at the baseline and




                                                                              te
    42–63 years) suffering from mild chronic periodontitis were           after 4 weeks of bLf topical treatment are reported as cu-
    enrolled in this observational preclinical trial after the un-        mulated mean values because the total number of subjects
    derstanding and written consent. The trial was conducted              in each group (seven women and six men) is very low (Ta-
                                                                   In
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                                   iz
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©




    Figure 3 - Putative lactoferrin mechanism in inhibiting bone resorption. High levels of IL-6 induce bone resorption through the osteo-
    clast activation induced by the osteoblast-mediated production of RANKL (Panel A). The topical administration of bLf by decreasing IL-
    6 levels and probably RANKL may induce both the inhibition of osteoclast activation and the activation and proliferation of osteoblast
    (Panel B). Legend: IL-6: interleukin-6; bLf: bovine lactoferrin; RANK: receptor activator of nuclear factor NFkB; RANKL: RANK ligand.


    Annali di Stomatologia 2011; II (3-4): 10-18                                                                                       15
    F. Berlutti et al.


    ble 3). Therefore, the data summarized in Table 3 corre-                         6. Tomita M, Takase M, Bellamy W, Shimamura S. A review: the ac-
    spond to the mean values deriving from a total of 13 sub-                           tive peptide of lactoferrin. Acta Paediatr Jpn 1994; 36: 585–91.
    jects and 26 teeth.                                                              7. Vorland LH, Ulvatne H, Andersen J, Haukland H, Rekdal O, Svend-
                                                                                        sen JS, Gutteberg TJ. Lactoferricin of bovine origin is more active
    From the data reported in Table 3, it can be observed that
                                                                                        than lactoferricins of human, murine and caprine origin. Scand J
    bLf topical administration decreased the concentrations of                          Infect Dis 1998; 30: 513–7.
    pro-inflammatory cytokines in GCF, thus decreasing oe-




                                                                                                                                        li
                                                                                     8. Elass-Rochard E, Legrand D, Salmon V, Roseanu A, Trif M, Tobias
    dema and inflammation. Moreover, after four weeks of to-                            PS, Mazurier J, Spik G. Lactoferrin inhibits the endotoxin interac-
    pical bLf therapy, the absence of bleeding, the reduction




                                                                                                                                     na
                                                                                        tion with CD14 by competition with the lipopolysaccharide- bind-
    of pocket depth, gingival and plaque index as well as an                            ing protein. Infect Immun 1998; 66: 486–91.
    improvement of clinical attachment level were observed.                          9. Gao JL, Nguyen KA, Hunter N. Characterization of a hemophore-
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    jects receiving topical administration of bLf.                                      2010;285:40028-38.
                                                                                    10. Dashper SG, Hendtlass A, Slakeski N, Jackson C, Cross KJ, Brown-




                                                                                                                                  io
    In conclusion, Lf is a component of saliva and an impor-
                                                                                        field L, Hamilton R, Barr I, Reynolds EC. Characterization of a nov-
    tant defence factor against bacterial injuries (1) including                        el outer membrane hemin-binding protein of Porphyromonas gin-
    those related to S. mutans (54) and to other representa-                            givalis. J Bacteriol. 2000;182:6456-62.
    tive periodontopathic bacteria as A. actinomycetemco-




                                                                                                       az
                                                                                    11. Hendtlass A, Slakeski N, Jackson C, Cross KJ, Brownfield L, Hamil-
    mitans, P. gingivalis, and P. intermedia (83). Periodonto-                          ton R, Barr I, Reynolds EC. Characterization of a novel outer mem-
    pathic bacteria reside as a biofilm in supragingival and sub-                       brane hemin-binding protein of Porphyromonas gingivalis. J Bac-
    gingival plaque and Lf is able to reduce their biofilm de-                          teriol 2000;182:6456-62.
    velopment (84).                                                                 12. Nibbering PH, Ravensbergen E, Welling MM, van Berkel LA, van




                                                                                                     rn
    Even if in a small-scale clinical study, oral administration                        Berkel PH, Pauwels EK, Nuijens JH. Human lactoferrin and pep-
                                                                                        tides derived from its N terminus are highly effective against in-
    of bLf has been found to reduce P. gingivalis and P. inter-
                                                                                        fections with antibiotic resistant bacteria. Infect Immun 2001; 69:
    media in the subgingival plaque of chronic periodontitis pa-                        1469-76.
    tients thus suggesting that the inhibitory effects of Lf on                     13. Appelmelk BJ, An YQ, Geerts M, Thijs BG, de Boer HA, MacLaren




                                                                                         te
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    seases (83).                                                                    14. Brandenburg K, Jurgens G, Muller M, Fukuoka S, Koch MHJ. Bio-
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                                                                                In      vation by lactoferrin. Biol Chem 2001; 382: 1215–25.
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                                                                                        of the hydrophobic domain of polymyxin B nonapeptide: effect on
    flammatory cytokine synthesis might be useful in reducing
                                                                                        outer-membrane permeabilization and lipopolysaccharide neu-
    periodontal tissue destruction. Recently, among pro-in-                             tralization. Mol Pharmacol 2002; 62: 1036–42.
                                                               ni
    flammatory cytokines, the multifunctional IL-6 is emerging                      16. Ellison RT, Laforce FM, Giehl TJ, Boose DS, Dunn BE. Lactofer-
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                                                   io


    periodontitis and achieve optimal periodontal tissue re-                        17. Nikaido H, Vaara M. Molecular basis of bacterial outer membrane
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                                        iz



                                                                                        tonini G. Ca2+ binding to bovine lactoferrin enhances protein sta-
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                                                                                        bility and influences the release of bacterial lipopolysaccharide.
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                    Ed




    tent effect of bLf topically administered results in an im-                         timicrobial peptides. Eur J Biochem 2001; 268: 5589–600.
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    sorption (Figure 3).                                                                64: 159–169.
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                                                                                        Kavase K. Potent antibacterial peptides generated by pepsin of
    vide strong evidence for a role of bLf in curing periodon-
 IC




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C




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©




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          Biochem Cell Biol 2011, in press.                                                 Antimicrob Agents Chemother 2009;53:3308-16.




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    18                                                                                                          Annali di Stomatologia 2011; II (3-4): 10-18
