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European journal of oral sciences

Antimicrobial activity and regulation of CXCL9 and CXCL10 in oral keratinocytes.

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Article Details
Authors
Alison Marshall, Antonio Celentano, Nicola Cirillo, Michele D Mignogna, Michael McCullough, Stephen Porter
Journal
European journal of oral sciences
PM Id
27671889
DOI
10.1111/eos.12293
Table of Contents
Abstract
Antimicrobial Activity And Regulation Of
CXCL9 And CXCL10 In Oral
Material And Methods
Cell-Culture Techniques
RT-PCR
Results
Discussion
26. DAN H, LIU W, WANG J, WANG Z, WU R, CHEN Q, ZENG X,
Abstract
Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-c (IFN-c) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPSstimulated cells. Finally, stimulation with IFN-c enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms. Alison Marshall, Antonio Celentano, Nicola Cirillo, Michele D. Mignogna, Michael McCullough, Stephen Porter University College London, UCL Eastman Dental Institute, London, UK; University Federico II of Naples, Department of Neuroscience, Reproductive and Odontostomatological Sciences, Naples, Italy; The University of Melbourne, Melbourne Dental School and Oral Health CRC, Melbourne, Vic., Australia
Antimicrobial activity and regulation of
CXCL9 and CXCL10 in oral
keratinocytes Marshall A, Celentano A, Cirillo N, Mignogna MD, McCullough M, Porter S. Antimicrobial activity and regulation of keratinocytes. Eur J Oral Sci 2016; 00: 00–00. © 2016 Eur J Oral Sci Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-c (IFN-c) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPSstimulated cells. Finally, stimulation with IFN-c enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms. Alison Marshall1, Antonio Celentano2,3, Nicola Cirillo3, Michele D. Mignogna2, Michael McCullough3, Stephen Porter1 1University College London, UCL Eastman Dental Institute, London, UK; 2University Federico II of Naples, Department of Neuroscience, Reproductive and Odontostomatological Sciences, Naples, Italy; 3The University of Melbourne, Melbourne Dental School and Oral Health CRC, Melbourne, Vic., Australia Antonio Celentano, Department of Neuroscience, Reproductive and Odontostomatological Sciences, University Federico II of Naples Via Pansini 5, Naples 80131, Italy. E-mail: antonio.celentano@unina.it Key words: CCL28; chemokines; CXCL10; CXCL9; Streptococcus sanguinis Accepted for publication July 2016 Bacteria colonize all surfaces of humans, but colonization is particularly dense in the lower gastrointestinal tract and in the oral cavity where streptococci represent a large proportion of the resident microflora. It has previously been shown that bacteria can adhere to and invade host oral epithelial cells (1) and also that there are bacterial receptors present in saliva that can be absorbed onto oral mucosal surfaces (2). Among them, the Toll-like receptors (TLRs), expressed on host cells, are involved in the recognition of conserved bacterial patterns such as lipopolysaccharide (LPS), the cell-wall component of Gram-negative bacteria (3). Following bacterially mediated tissue damage, keratinocytes produce a wide range of molecules, differentially regulated to across epidermal layers, including antimicrobial peptides (AMPs) and proinflammatory cytokines (4). The chemokines monokine, chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10, which are induced by interferon-c (IFN-c) and IFN-c-induced protein-10, respectively, are two chemokines belonging to the CXC family, and both bind the same receptor, namely chemokine (C-X-C motif) receptor (CXCR)3 (5). Chemokines play an important role in directing the migration of specific immune-cell populations and, for some, direct antibacterial and/or antifungal activities have been demonstrated (6–8). As oral epithelial cells are known to produce chemokines (9–11), these may play a direct role in microbial defense. It is known that CXC ELR-negative chemokines can be induced by LPS in some cell types (12) and that these chemokines are expressed during bacterial infections (13). It is also known that LPS is capable of inducing a range of cytokines and chemokines from epithelial cells predominately by signaling through TLR-4 (14). The induction of these chemokines may be triggered in response to an alteration in the microbial flora, which could, in turn, cause an ensuing immune-cell infiltration. We recently found that CXCL9 and CXCL10 are dysregulated in oral inflammatory disease (A. MARSHALL, A. CELENTANO, N. CIRILLO, M. MCCULLOUGH, S. PORTER, personal communication), but nothing is known about their antibacterial activity. Furthermore, while the induction of several different types of cytokine has Eur J Oral Sci 2016; 1–7 DOI: 10.1111/eos.12293 Printed in Singapore. All rights reserved 2016 Eur J Oral Sci European Journal of Oral Sciences been demonstrated in oral epithelial cells after stimulation with LPS (11, 14–17), the roles of CXCL9 and CXCL10 in the infection and immunity of the oral cavity have never been investigated. Therefore, the aim of this paper was to investigate the potential of CXC ELR-negative chemokines to mediate microbicidal activity on the Gram-positive species, Streptococcus sanguinis, one of the most prevalent residents of the oral microflora, and on the expression of chemokines in oral keratinocytes after exposure to infectious and inflammatory stimuli. The chemokine (C-C motif) receptor (CCR)10 ligands – chemokine (C-C motif) ligand (CCL)27 (CTACK) and CCL28 (MEC) – are two C-C chemokines that bind the CCR10 receptor found to exert potent antimicrobial activity against Candida albicans, Gramnegative bacteria, and Gram-positive bacteria (7). We used these two chemokines as positive controls.
Material and methods
Cell-culture techniques
Cell culture of normal human oral keratinocytes: All normal oral mucosa was obtained from healthy patients attending the Oral Surgery Clinic, Eastman Dental Institute (London, UK) for routine third-molar extraction. Three different normal human oral keratinocyte (NHOK) strains (NHOK1, NHOK2, and NHOK3) were isolated from the excised normal tissue. The samples were cut into pieces of approximately 1 mm3 and cultured at 37°C/5% CO2 in keratinocyte basal medium-2 containing the recommended growth supplements (Biowhittaker, Wokingham, UK). The epithelial cells were then detached using 0.25% trypsin/1 mM EDTA. The viability of the keratinocytes was confirmed by Trypan Blue exclusion. All cell lines/ strains were derived before 2001 and therefore were not subject to ethics committee approval in the UK (18). The study was approved by the internal research committee at the Eastman Dental Institute, University College London (London, UK). H357 cell culture: The oral squamous cell carcinoma cell line, H357, was established by PRIME et al. (19). This cell line was grown in the same medium as described for NHOKs. Bacterial cell culture and antimicrobial assessment All bacterial stocks were maintained frozen at 70°C in trypticase soy broth (TSB) (Becton Dickinson, Oxford Science Park, Oxford, UK) supplemented with 0.6% yeast extract (Oxoid, Basingstoke, UK) and 10% glycerol (BDH Chemical, Dorset, UK). Cultures were checked weekly, both visually and by Gram staining, for contamination with other bacteria. Stocks of Escherichia coli NCTC JM22 and S. sanguinis NCTC 10904 (provided by Dr Rod McNab at the Eastman Dental Institute, University College London) were plated on agar plates containing 3% TSB. They were grown for 48 h at 37°C/5% CO2 and maintained by twice-weekly subculture onto TSB agar plates. Each E. coli colony was collected and resuspended in 50 ml of 3% TSB. For the oral streptococcal species, three streaks of each species on a culture plate were resuspended in 10 ml of 3% TSB. The cultures were shaken, on an orbital shaker, at 250 rpm for 15–18 h at 37°C. Then, 50 ml of the E. coli culture or 2 ml of the oral streptococcal species culture was transferred into 50 ml or 10 ml of 3% TSB, respectively. This was shaken, on an orbital shaker, at 250 rpm for 3.5 h at 37°C. At this point, the culture was adjusted to an optical density (OD) of 1 at 620 nm. To prepare the underlay, 50 ml of 100 mM sodium phosphate buffer, 5 g of agarose [low electroendosmosis (EEO); Sigma, Poole, UK], and 5 ml of 3% TSB were added to 1 l of distilled water. The pH was then adjusted to 7.4 and the agarose was dissolved by heating the solution in the microwave. The solution was dispensed into 50-ml aliquots and autoclaved. The underlay aliquots were then stored at room temperature until required for use in the radial diffusion assays, at which point they were heated in a microwave until fluid and then stored in a 60°C water bath. Eight milliliters of E. coli or 16 ml of the streptococcal species was added to 5 ml of molten underlay and dispensed into a Petri dish, using a leveling tray. When the underlay was set, 3-mm holes were punched in the gel, using 10-ml pipettes (Sarstedt, Leicester, UK). Then, 5 ll of test solution diluted in 0.01% acetic acid was added to the wells. For overlays, 10 g of agarose, low EEO (Sigma), was added to 6% TSB, dispensed into 50-ml aliquots, and autoclaved. The overlay aliquots were then stored at room temperature before use in radial diffusion assays. This plate was incubated for 3 h at 37°C, then 5 ml of overlay was added to the plate and incubated at 37°C overnight. Radial diffusion assays were then performed, adding 5 ll of either recombinant human CXCL9/CXCL10/ CCL27/CCL28 (all Peprotech EC, London, UK) or 0.01% acetic acid to the wells before incubating the plates. The positive control for the assay was 100 lM tetracycline. Images of the plates were taken using AlphaImager software (AlphaInnotech, Cannock, UK) and the zones around the cultures were measured from three different points from the end of the well. IFN-c cell-treatment assay In a modification of the method utilized by ALTENBURG et al. (20), the H357 cells were seeded at 8 9 104 cells per well in a Falcon 6-well plate (Becton Dickinson) in 3 ml of KBM-2 medium containing no hydrocortisone. The cells were incubated for at least 3–5 d until the cell culture was 60–80% confluent. Medium containing 1,000 U ml 1 of IFN-c was added to three wells and control cell-culture medium only was added to the remaining three wells. The cells were incubated for 48 h. The supernatant was extracted, centrifuged, and stored at 70°C. The adherent cells were washed with PBS (Gibco Life Technologies, Paisley, UK) before addition of 0.5 ml of TRI Reagent (Sigma). The suspension was then removed and stored at 70°C. The RNA was isolated as described below. CXCL9 and CXCL10 mRNA transcripts in oral epithelial cells in response to the presence of LPS and IFN-c: mRNA isolation and semiquantitative
RT-PCR
mRNA transcripts for 18S, CXCL9, or CXCL10 were isolated from H357 cells, exposed or not exposed (control) to LPS for 2, 4, 6, or 8 h. The RNA was extracted using TRI Reagent (Sigma) and 2 ml of Pellet Paint Co-precipitant (Novagen, Nottingham, UK) to visualize the RNA pellet. Single-strand cDNA synthesis was performed. Two milliliters of RNA was added to 4 ml of deoxynucleotides (dNTPs) (2.5 mM) (Sigma), 2 ml of random hexamers (50 mm) (Ambion, Austin, TX, USA), and 9.5 ml of distilled H2O (dH2O). Then, 1 ml of RNAaseIN (Ambion), 2 ml of 109 MuLVRT buffer, and 0.5 ml of M-MuLVRT (200 U ml 1) (Boehringer-Mannheim, Lewes, UK) was added and the reaction mix was incubated at 42°C for 1 h. for 18S, CXCL9, and CXCL10 The magnesium concentration was optimized for each primer as follows: 1 ml of cDNA was added to 4 ml of dNTPs (2.5 mM), 5 ml of 109 buffer, 0.225 ml of AmpliTaq (5.0 U ml 1) (PerkinElmer, Waltham, MA, USA), 4 ml of each specific primer (5 mM), and 1.5, 3.0, or 4.5 mM MgCl in each reaction, and dH2O was added to give a final volume of 50 ml. The products were separated on a 2% agarose (GibcoBRL Life Technologies, Paisley, UK) gel and visualized by staining with ethidium bromide (Sigma). Specific bands were visualized by ultraviolet transillumination in a MultiImage Light Cabinet (AlphaInnotech, Cannock, UK), and digital images were acquired and stored using AlphaImager software (AlphaInnotech). The CXCL9 (forward: 50-ccaacaccccacagaagtgc-30; reverse: 50-gccagcacctgctctgagac-30), CXCL10 (forward: 50-gccaatttt gtccacgtgttg-30; reverse: 50-aaagaatttgggccccttgg-30), and 18S ribosomal RNA (forward: 50-tttcggaactgaggccatga-30; reverse: 50-gcatgccagagtctcgttcg-30) primers were generated for use in this study (Genosys-Sigma, Poole, UK). The thermocycler (Techne Genius, Cambridge, UK) parameters utilized were 94°C for 45 s, 57°C for 45 s, and 72°C for 45 s. For each primer the linear range was determined by repeating the above reaction with optimized magnesium concentration for each primer and stopping the reaction at 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 cycles. The mid-point of each linear range was determined by using intensity analysis of the bands with AlphaImager software (AlphaInnotech), and this cycle length was utilized for each primer in subsequent reactions. 18S primer and 18S Competitor primers (Ambion) were combined at ratios of 1:9, 2:8, and 3:7, respectively. For each of the primers CXCL9 and CXCL10, 4 ml of the 18S primer/competitor mix was added to the RT-PCR reaction. The band intensity of the 18S and specific primers was quantified for each primer in each sample using Phoretix 1D software (Phoretix, Newcastle, UK). Unless specified otherwise, all experiments were performed at least in triplicate.
Results
Production of CXCL9 and CXCL10 mRNA transcripts in an oral epithelial cell line in response to IFN-c The production of CXCL9 and CXCL10 in oral mucosal keratinocytes was first assessed over time in preliminary experiments using the keratinocyte cell lineH357 (Fig. S1). The expression of CXCL10 mRNA was detected in H357 cellsasearlyas3 haftertreatmentwithIFN-candappeared to peak at 24 h. In contrast, the control cells showed virtuallyundetectablelevelsofmRNAoverthesametimeperiod. The patterns shownbyCXCL9 andCXCL10mRNAtranscriptswere similar, beingbiphasicpatterns that showedan initial rapid induction ofmRNA in the stimulated cells followed by a second peak at 24/48 h. Thus, the expression of CXCL9/CXCL10 in H357 cells can be significantly enhanced by IFN-c in a time-dependent manner, with a peakafter48 h(Fig. 1).Similarresultswereobtainedinprimary NHOKs (Fig. 2). These data were confirmed at the proteinlevelbyELISA(datanotshown).Theseresults indicate that the expression of CXCL9 and CXCL10 in oral epithelial cells is significantly enhanced by IFN-c in a timedependentmanner. Production of CXCL9 and CXCL10 mRNA transcripts in an oral epithelial cell line in response to LPS CXCL10 mRNA was expressed in control cells (no exposure to LPS), but was enhanced by stimulation with LPS, 2, 4, and 6 h after exposure. This expression subsequently decreased after 8 h of stimulation (Fig. 3). In contrast, CXCL9 mRNA was not expressed in either control or LPS-stimulated cells at any of the timepoints tested.
Antimicrobial effect of CXCL9, CXCL10, CCL27, or
CCL28 on S. sanguinis and E. coli CCL27 and CCL28 were used as positive controls. The clear zones of bacterial growth depletion as a result of the antimicrobial activity of the chemokines are indicated in Fig. 4 and Fig. 5. All chemokines investigated demonstrated a level of antimicrobial activity at the tested concentration (Table 1). It was found that CXCL9 was the most effective chemokine against both S. sanguinis and E. coli; CCL27 and CXCL10 had a less effective antimicrobial action against S. sanguinis than against E. coli; and CCL28 had more effective antimicrobial action against S. sanguinis than against E. coli. These results clearly demonstrate that the epithelial-derived chemokines CXCL9 and CXCL10 exert antimicrobial activity.
Discussion
Chemokines are known to have antimicrobial effects but little is known of the action of chemokines derived from the oral mucosal epithelium. The present study is the first to examine the expression of CXC ELR-positive chemokines by oral epithelial cells when stimulated with bacteria-derived products such as LPS. The present study has established that a cell line of oral origin is capable of expressing CXCL9 and CXCL10 mRNAs when stimulated with LPS. The production of these chemokines was also enhanced by inflammatory stimuli such as IFN-c. Expression of CXCL10 mRNA was increased in H357 cells after stimulation with LPS, whereas expression of CXCL9 mRNA was not induced over the same time-period. Previously, we have shown that CXCL10 can act as a potent chemoattractant of lymphocytes; hence, this local production of CXCL10 by oral epithelial cells in response to LPS could have important effects on oral inflammation, perhaps crucially in the initial stages of inflammation. Previous studies have also shown that LPS treatment alone can induce or enhance expression of CXCL10 mRNA in several different cell types (12, 21–23). However, in contrast to the present findings, LPS stimulation did not induce production of CXCL10 from cultured skin keratinocytes (24), suggesting perhaps that oral keratinocytes are more responsive than cutaneous keratinocytes to LPS stimulation. This difference in expression may reflect the high bacterial load in the oral cavity. The rapid expression of CXCL10 mRNA observed in the present study is in accordance with that of murine macrophages (17, 25) although LPS stimulation may be more transient than IFN-c relative to stimulation of CXCL10 (10). This short-term effect may be essential to avoid over-stimulation of CXCL10 in response to resident bacteria in proximity to the epithelium. Interleukin-10 is known to be able to down-regulate production of LPS-induced CXCL10 in macrophages (22). This cytokine is present within oral lichen planus lesions and is increased in serum and saliva from patients with oral lichen planus (26), and thus may act to down-regulate expression of CXCL10 in oral inflammation. There are few reports of LPS-induced production of CXCL9. The present study revealed that CXCL9 mRNA was not expressed by H357 cells when stimulated with LPS. In contrast to the present study, CXCL9 mRNA was found to be expressed in LPS-stimulated murine dendritic cells (12); however, in another study, this chemokine was not induced in the same murine cell line by LPS, despite induction of CXCL9 by IFN-c (25). It is possible that CXCL9 displays a delayed response, in comparison with CXCL10, as induction of CXCL9 mRNA in lung tissue of mice treated intravenously with LPS shows a later induction than CXCL10 mRNA, and is never expressed at the same levels as CXCL10 (25). Many studies report that LPS and IFN-c act synergistically to induce the production of high levels of CXCL10 mRNA, for example, in breast carcinoma cells (21, 22). It is then possible that CXCL10 levels could be enhanced in oral inflammation where there is both LPS and IFN-c, perhaps through the enhancement of specific TLRs (27). Gram-positive bacteria, such as S. sanguinis, contain components other than LPS that are known to stimulate chemokine release from various cell types and it would be interesting to determine whether these are also capable of stimulating production of CXC ELRnegative chemokines in oral epithelium. Many Grampositive bacterial components act on a different Tolllike receptor, TLR-2, which is functionally expressed on keratinocytes (28). However, TLR-2 agonists do not induce production of CXCL10 in macrophages (29) or dendritic cells (30) in vitro. This suggests that TLR-2 agonistic bacterial products would also not induce CXCL10 in epithelial cells. Therefore, only products bound by TLR-4 would be influential in up-regulating CXCL10 production in epithelial cells. In addition, as LPS-mediated CXCL10 production is TLR-4 dependent, this strongly suggests that oral keratinocytes bear functional TLR-4, and therefore stimulation of epidermal cells with LPS is not caused by TLR-2 agonist contaminants in LPS preparations, as previously suggested (28). Chemokine modulation in oral cells by bacterial products is thus complex and many different factors, including T-cell contact, may play a factor in chemokine induction during an immune response. The present studies suggest that in certain circumstances, bacterial products could stimulate oral epithelial cells to produce an inflammatory response through TLR-4 agonists (perhaps after continuous stimulation with TLR-2) and this may induce epithelially derived CXCL10-mediated inflammation. This inflammation would presumably be characterized by activated memory T-cell infiltration localized under the basal epithelium, reminiscent of the pathology of oral lichen planus. The chemokine CXCL9 was shown to be a potent antimicrobial agent against both S. sanguinis and E. coli, as was CXCL10, but to a lesser degree than CXCL9, confirming the results of the study by COLE et al. (6). Although only one oral bacterial species was tested in the present study, the results hint that the antimicrobial properties of these chemokines may assist in countering bacterial growth in the oral cavity. The choice of using S. sanguinis as a model has been driven by the delicate role played by this bacterium in maintaining the balance of the oral flora. Streptococcus sanguinis is commonly found in healthy tissues as a pioneer colonizer, and it is implicated in modulating the virulence of bacterial biofilms (31). Furthermore, significant inhibitory effects of the intracellular proteins produced by S. sanguinis on the growth and the morphology of many other components of the oral flora, such as Prevotella intermedia, Porphyromonas gingivalis, C. albicans, and Candida tropicalis, and their biofilms, have been demonstrated (32, 33). This allows us to conjecture and expect that a series of chain effects of the antimicrobial activity of CXCL9 and CXCL10 would occur in vivo. As previously shown for the two chemokines, CCL27 and CCL28, for which a wide spectrum of antimicrobial activity is well established, the action of CXCL9 and CXCL10 expected against other microorganisms of the oral cavity should follow a similar pattern (7). The low production of CXCL9 by oral epithelial cells following stimulation with IFN-c or LPS could potentially be a means of avoiding an overactive antimicrobial response as a result of the potent antimicrobial activity of CXCL9. In our study, both CCL27 and CCL28 exerted antimicrobial properties against E. coli and S. sanguinis. The chemokine CCL28 has been shown to have microbicidal activity against a wide range of bacteria (both Gram-negative and Gram-positive) and yeasts (7, 34), and our findings confirmed that this chemokine was also effective against the oral commensal, S. sanguinis. This is only the second report of the antimicrobial effect of CCL28 upon enterobacterial E. coli after the study reported by BERRI et al. in 2014 (34). The production of CXCL10 by oral epithelial cells, in inflammatory conditions, such as oral lichen planus, may be induced by resident bacteria that contain TLR4 agonists. If this inflammation resulted from constant TLR-2 stimulation, this may cause a CXCL10-based inflammation and a resultant influx of memory Thelper 1 CD4+ cells. Furthermore, CXCL10 is antimicrobial at high concentrations. It may be up-regulated by the presence of bacteria, thus playing a role in the defense of oral epithelial cells. The antimicrobial activity of the chemokines tested is likely to be an important mechanism in the homeostasis of oral bacterial colonization. Any IFN-c in the oral epithelial area (which could presumably be produced by the infiltrating T-helper 1 cells) may synergize with TLR-4 agonists to cause an increased inflammatory state. Further studies are still warranted to confirm these novel findings.
26. DAN H, LIU W, WANG J, WANG Z, WU R, CHEN Q, ZENG X,
ZHOU Y. Elevated IL-10 concentrations in serum and saliva from patients with oral lichen planus. Quintessence Int 2011; 42: 157–63. 27. UEHARA A, SUGAWARA S, TAKADA H. Priming of human oral epithelail cells by interferon-c to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J Med Microbiol 2002; 51: 626–635. 28. KAWAI K, SHIMURA H, MINAGAWA M, ITO A, TOMIYAMA K, ITO M. Expression of functional toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci 2002; 30: 185–194. 29. TOSHCHAKOV V, JONES BW, PERERA PY, THOMAS K, CODY MJ, ZHANG S, WILLIAMS BR, MAJOR J, HAMILTON TA, FENTON MJ, VOGEL SN. TLR4, but not TLR2, mediates IFNbeta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol 2002; 3: 392–8. 30. RE F, STROMINGER JL. Toll-like receptor (TLR2) and TLR4 differentially activate human dendritic cells. J Biol Chem 2001; 276: 37692–37699. 31. LEE SH. Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens. J Microbiol 2015; 53: 553–60. 32. MA S, LI H, YAN C, WANG D, LI H, XIA X, DONG X, ZHAO Y, SUN T, HU P, GUAN W. Antagonistic effect of protein extracts from Streptococcus sanguinis on pathogenic bacteria and fungi of the oral cavity. Exp Ther Med 2014; 7: 1486– 1494. 33. PEYRET-LACOMBE A, BRUNEL G, WATTS M, CHARVERON M, DUPLAN H. TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response. Cytokine 2009; 46: 201–10. 34. BERRI M, VIRLOGEUX-PAYANT I, CHEVALEYRE C, MELO S, ZANELLO G, SALMON H, MEURENS F. CCL28 involvement in mucosal tissues protection as a chemokine and as an antibacterial peptide. Dev Comp Immunol 2014; 44: 286–90. Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. 18S, CXCL9 and CXCL10 mRNA expression in the H357 cell line.
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