Boterberg, Tom

Bracke, Marc

De Ryck, Tine

De Schrijver, Joachim

Kerckhof, Frederiek-Maarten

Van De Wiele, Tom

Vanhoecke, Barbara

English

bracke, marc

Ghent University Academic Bibliography

Effects of Irradiation on Epithelial Wound Healing and Microbial Diversity in anin-vitro Oral Mucosa ModelTine De Ryck1, Tom Boterberg2, Frederiek-Maarten Kerckhof3, Joachim De Schrijver4, Marc Bracke1, Tom Van de Wiele3* and Barbara Vanhoecke31Laboratory of Experimental Cancer Research (LECR), Ghent University, 9000 Ghent, Belgium2Department of Radiation Oncology, Ghent University Hospital, 9000 Ghent, Belgium3Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, 9000 Ghent, Belgium4Laboratory of Bioinformatics and Computational Genomics (Biobix), Ghent University, 9000 Ghent, Belgium*Corresponding author: Tom Van de Wiele, Laboratory of Microbial Ecology and Technology (LabMET), Coupure Links 653 Building A, 9000 Ghent, Belgium, Tel: +329 264 59 12; Fax: +32 9 264 62 48; E-mail: tom.vandewiele@ugent.beReceived date: Feb 09, 2015, Accepted date: Mar 16, 2015, Publication date: Mar 19, 2015Copyright: © 2015 Ryck TD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.AbstractThe impact of irradiation on host-microbe crosstalk is still underexplored. By use of an in vitro oral mucosa model,we show an impact of irradiation on epithelial wound healing depending on the microbial composition andfunctionality. 454-pyrosequencing analyses pointed to a slight increase in abundance of Rothia, Granulicatella andGemella in our model after irradiation. Further research is needed to unravel the effects of irradiation on the oralmicrobiota and the host-microbe interactions more in detail.Keywords: Host-microbe; Irradiation; 454-pyrosequencing; In vitrooral mucosa model; Wound healingAbbreviations:PBS: Phosphate Buffered Saline; BHI: Brain Heart Infusion; CFU:Colony Forming Units; PCR-DGGE: Polymerase Chain reaction-Denaturing Gradient Gel ElectrophoresisIntroductionOral mucositis is an important side effect of cancer therapies. In91% of the patients with head and neck cancer, radiotherapy results inthe development of mucositis [1]. The pathobiological process ofmucositis can be subdivided in 5 phases: the initiation phase, theprimary damage response followed by signal amplification, theulceration and the healing phase [2,3]. From these, the healing phase ishighly variable between patients and is probably the least understood[2,4]. Although differences in treatment schedules, irradiation dosesand individual radiation sensitivity may be responsible for thedifferences in wound healing, the composition of the oral microbiomeis also thought to be one of the determining factors. An oral coremicrobiome has been described for healthy individuals withStreptococcus being a predominant genus; yet, genera like Neisseria,Prevotella, Veillonella or Haemophilus can also become highlyabundant. At species level, this interindividual variation is even larger[5]. A recent review describes important shifts in the core oralmicrobiome due to radiotherapy, with an increased abundance ofGram-negative species and Lactobacillus spp. [6]. Irradiation has thusa clear impact on the host and on the oral microbiome, however it isunknown to what extent irradiation affects the host-microbe crosstalk,particularly during the wound healing phase of mucositis. Here, weused an in vitro oral mucosa model [7] to study the impact of oralmicrobes on the dynamics of the wound healing process followingirradiation more in detail.Materials and MethodsEpithelial cell lineTR146 cells, an oral squamous carcinoma cell line derived from alocal lymph node metastasis was obtained from Clare HallLaboratories (Cancer Research UK). The Dulbecco’s modified Eagle’scell culture Medium (DMEM) (Gibco, Belgium) was supplementedwith 10% heat-inactivated fetal bovine serum (Greiner bio-one,Belgium), 22.8 µg/mL penicillin-streptomycin (5000 U/mL; Gibco,Belgium) and 2.5 µg/mL amphotericin B (Bristol-MyersSquibb,Belgium). Cells were cultured at 37°C and 10% CO2 and regularlycontrolled to be mycoplasma free (MycoAlert Mycoplasma Detectionkit; Lonza, USA).IrradiationA single 6 MV photon beam was generated using a linearaccelerator (SLi-18, Elekta, UK). The 24-well plates were placed on topof a 1.5 cm Perspex (polymethylmethacrylate) plate to compensate forthe build-up effect and irradiated from below. The model wasirradiated with a total dose of 10 Gy at an instantaneous dose rate of430 cGy/min.Oral swab collectionBefore sampling, the oral cavity of a healthy volunteer wasextensively flushed with drinking water. A sterile cotton swab was usedto rub the buccal mucosa 10 times before submersion in 2 mL ofsynthetic saliva buffer. Briefly, an organic buffer (500 mg/L ureum), aninorganic buffer (2.987 g/L KCl, 2.96 g/L NaH2PO4, 0.667 g/L KSCN,1.9 g/L Na2SO4, 0.993 g/L NaCl and 10.8 mM NaOH) and an additivebuffer (483 mg/L amylase, 167 mg/L mucin and 50 mg/L uric acid)were autoclaved and mixed (4:3:3) immediately before use. Afterincubating the oral swabs for 15 min at 37°C, the microbialsuspensions were snap frozen in liquid nitrogen and stored at -80°CNuclear Medicine & RadiationTherapy Ryck et al., J Nucl Med Radiat Ther 2015, 6:2http://dx.doi.org/10.4172/2155-9619.1000218Short Communication Open AccessJ Nucl Med Radiat TherISSN:2155-9619 JNMRT, an open access journal Volume 6 • Issue 2 • 1000218until the experiments were performed. For all experiments, a differentoral swab was taken from the same healthy volunteer.Oral mucosa modelTo co-culture microbiota and TR146 epithelial cells in non-infectious conditions, we used our recently published oral mucosamodel [7]. Briefly, 75 µL of an agar/mucin solution was spotted on theporous membrane (pore size 0.4 µm) of a 24-well plate Transwell®system (Corning inc., USA) and allowed to solidify for at least 30 min.After thawing at room temperature, 20 µL of the microbial samples(~3.2 log/filter) were brought on top of the agar/mucin layer. In thebasal compartment, a confluent layer of TR146 cells was generated byseeding 300 000 TR146 cells into a well after labelling with DiI celllabelling solution (Life technologies Europe, Belgium). Cells wereallowed to attach and grow for 3 days prior to wounding.Wound healing assayTR146 monolayers were scratched using a sterile plastic pipette tip.Floating cells were removed by replacing the medium with freshserum-free, antibiotics-free DMEM. Filters containing the microbiotaand the agar/mucin layer were placed on top of the wounded cellsprior to irradiation. By means of an automated fluorescencemicroscope (Zeiss Axiovert 200M), pictures of 4 selected points alongthe wounds in each well were taken immediately after irradiation (0 h)and 24 h after incubation at 37°C and 5% CO2. Migration of the cellsinto the wounded area in the presence and absence of oral microbiotawas assessed by comparing the micrographs after 0 and 24 h (Figures1A and 2A). The relative surface (%) of the wound was determined bydividing the wound surface after 24 h by the wound surface at time 0.For this we made use of a custom-made macro in ImageJ [7]. For eachwell, the mean value of the wound surface was calculated (n=1) andused for further analysis. In total, 5 independent experiments wereperformed, each with 2 to 4 wells per condition.Microbial enumerationTo enumerate the microbiota at the start of the experiment, 10-folddilutions of the oral swab suspension were prepared in sterile PBS andplated on BHI agar plates (Sigma, Belgium) using the microdilutionplating technique.After 24 h of co-culture, microbiota in the apical compartment weresuspended in 200 µL of sterile PBS before plating 10-fold dilutions asdescribed above.454 pyrosequencingDNA extraction and 454 pyrosequencing analysis of the apicalcompartment was performed as described earlier [7]. On average, 12907 number of reads were generated by pyrosequencing for eachsample. After processing the results, operational taxonomic units wereclassified using the RDP database.StatisticsStatistical analyses were performed with SPSS statistics 22. Afternormality check with the Shapiro-Wilk assay, an ANOVA, Student’sT, Kruskal Wallis or Mann Whitney-U test was used. Bonferronicorrection was performed to correct for multiple comparisons.All other techniques used for this study are described in detail in DeRyck et al. [7].Results and DiscussionIn patients suffering from oral mucositis, the healing phasespontaneously starts 2-3 weeks after completion of radiotherapy.Signals originating from the extracellular matrix promote woundhealing by stimulating migration, proliferation and differentiation ofthe epithelium [2]. We previously showed our in vitro oral mucosamodel to be useful for the study of effects of oral microbiota on thewound healing capacity of different oral-derived cell lines [7]. In non-irradiated conditions, epithelial recovery was significantly reducedwith 12-16% in the presence of microbiota (Figures 1B and 2B, 0GY;p<0.001). After irradiation however, our experimental data could becategorized in 2 different sets.In 3 of the 5 independent experiments (set 1) the inhibitory effect ofthe microbiota on wound healing was more pronounced afterirradiation as compared to the non-irradiated condition (Figure 1B,10Gy; p<0.001). In contrast, no effect of the microbiota afterirradiation could be noticed in the other 2 independent experiments(set 2; Figure 2B, 10Gy; p=1). Interestingly, plate counts revealed alower number of microbiota in set 2 after 24 h of co-culture comparedto set 1 (p=0.053). We hypothesized that this might be the result of adifference in microbial load and/or composition in the oral cavity ofthe healthy volunteer at the time of sampling. While the difference inmicrobial concentrations between the 2 experimental datasets was notinfluenced by irradiation (Table 1; pset1:0Gy-10Gy=0.979;pset2:0Gy-10Gy=0.800), our data suggest that microbial growth is animportant determining factor for the wound healing process afterirradiation.Microbial Counts (logCFU/Filter)0 Gy 10 GySet 1 7.29 ± 0.51 7.31 ± 0.74Set 2 5.88 ± 1.55 5.77 ± 1.08Table 1: Microbial enumeration of the apical compartment after 24 hof co-culture with TR146 cells in irradiated (10 Gy) or non-irradiated(0 Gy) conditions (mean ± SD).Irradiation has previously been shown to prolong the lag phase forfoodborne pathogens delaying the stationary phase [8]. As aconsequence, microbial metabolites might be present atconcentrations that are too low to have an effect on the epithelial cells.A microbial metabolite that was present in our in vitro model was D-lactic acid [7]. Here we show that oral microbiota increase the levels ofD-lactic acid after 24 h in both datasets (Figures 1C and 2C; set1:p0Gy<0.001 and p10Gy<0.001; set2: p0Gy=0.167 and p10Gy=0.445), butthat irradiation did not have an effect on this increase (pset1= 0.308;pset2=1). Therefore, D-lactic acid present in the co-culture mediumcould not explain the difference in effect on wound healing of bothsets. Furthermore, the epithelial wound healing was not affected inpresence of exogenous D-lactic acid up to 5000 µg/mL (data notshown).Citation: Ryck TD, Boterberg T, Kerckhof FM, Schrijver JD, Bracke M, et al. (2015) Effects of Irradiation on Epithelial Wound Healing andMicrobial Diversity in an in-vitro Oral Mucosa Model. J Nucl Med Radiat Ther 6: 218. doi:10.4172/2155-9619.1000218Page 2 of 4J Nucl Med Radiat TherISSN:2155-9619 JNMRT, an open access journal Volume 6 • Issue 2 • 1000218Figure 1: Effects of irradiation on microbe-epithelial interactions inthe first set of observations. A: Representative examples of themicrographs of the wound healing capacity of TR146 cells at 0 hand 24 h in the absence or presence of oral microbiota, both inirradiated (10 Gy) or non-irradiated (0 Gy) conditions B: Woundhealing capacity of TR146 cells in the absence or presence of oralmicrobiota (% of open wound after 24 h, mean + SD; *p<0.05), bothin irradiated (10 Gy) or non-irradiated (0 Gy) conditions. C:Concentrations of D-lactate in the co-culture medium in theabsence or presence of oral microbiota, both in irradiated (10 Gy)or non-irradiated (0 Gy) conditions (mean + SD; *p<0.05).Figure 2: Effects of irradiation on microbe-epithelial interactions inthe second set of observations. A: Representative examples of themicrographs of the wound healing capacity of TR146 cells at 0 hand 24 h in the absence or presence of oral microbiota, both inirradiated (10 Gy) or non-irradiated (0 Gy) conditions B: Woundhealing capacity of TR146 cells in the absence or presence of oralmicrobiota (% of open wound after 24 h, mean + SD; *p<0.05), bothin irradiated (10 Gy) or non-irradiated (0 Gy) conditions. C:Concentrations of D-lactate in the co-culture medium in theabsence or presence of oral microbiota, both in irradiated (10 Gy)or non-irradiated (0 Gy) conditions (mean + SD; *p<0.05).Next, we evaluated to what extent irradiation induces shifts in themicrobial composition of the oral biofilm after 24 h in our in vitro oralmucosa model. To this end, PCR-DGGE was used as a quick screeningtechnique. Although shifts in microbial composition were noted uponirradiation, this was only observed in 50% of the experiments (Figure3A). In all samples, an identical GC-poor group of microbiota could bedetected (arrow-marked on Figure 3A), most probably representingthe oral core microbiome.454 pyrosequencing analysis of the microbiota co-incubated with anepithelial monolayer for 24 or 48 h, showed that irradiation induces asmall increase (<1%) in Rothia, Granulicatella and Gemella species(Figure 3B; pRothia-24h=0.196, pGranulicatella-24h=0.062,pGemella-24h=0.233, pRothia-48h=0.220, pGranulicatella-48h=0.572,pGemella-48h=0.207). Unlike Granulicatella and Gemella that areomnipresent in the oral cavity, Rothia is mostly linked with toothsurfaces [9]. Interestingly, Rothia species were reported to becomemore abundant in head and neck cancer patients during radiotherapy,whereas Granulicatella species were shown to decrease afterirradiation [10,11]. Therefore Granulicatella species are not likely to bethe causing agents of post-radiation diseases [11]. In contrast, differentcases of Rothia bacteraemia have been reported of which 36% could belinked with oral mucositis [12].Figure 3: Effects of irradiation on the microbial composition. A:DGGE profiling of the oral microbiota present in the apicalcompartment after 24 h of co-culture with TR146 epithelial cells inirradiated (10 Gy) or non-irradiated (0 Gy) conditions. Indicated(arrow) is a group of GC-poor bacteria present in all samples(n=24) B: Pyrosequencing analysis of the oral microbiota culturedfor 24 or 48 h in co-culture with TR146 epithelial cells in irradiatedor non-irradiated conditions. Results of 454 pyrosequencing of 16SrDNA assigned to genus are shown.In summary, our study shows that irradiation can affect the host-microbe crosstalk during wound healing, depending on thecomposition and functionality of the microbiota. A slight, althoughnot significant, increase in abundance of Rothia after irradiation couldbe noticed in our in vitro system, which might be correlated withmucositis-related local and/or systemic infections observed in vivo.Citation: Ryck TD, Boterberg T, Kerckhof FM, Schrijver JD, Bracke M, et al. (2015) Effects of Irradiation on Epithelial Wound Healing andMicrobial Diversity in an in-vitro Oral Mucosa Model. J Nucl Med Radiat Ther 6: 218. doi:10.4172/2155-9619.1000218Page 3 of 4J Nucl Med Radiat TherISSN:2155-9619 JNMRT, an open access journal Volume 6 • Issue 2 • 1000218Further research is necessary to determine which species are likely tohave a negative impact on the recovery of the epithelium afterirradiation and to develop new strategies to combat infectionsassociated with radiotherapy-induced mucositis.AcknowledgementsWe want to thank Karlien Van Wesemael for routinely checking theepithelial cells for mycoplasma contamination. Tine De Ryck wasfinancially supported by an FWO project grant (G.0712.10 N). TomVan de Wiele received funding an FWO research grant (1426012N)and the GOA project (BOF12/GOA/008). Barbara Vanhoecke’sresearch leading to these results has received funding from the SeventhFramework Programme (FP7/2011) under grant agreement n° 299169(Mucositis Platform). Frederiek-Maarten Kerckhof received fundingof BOF (BOF09/GOA/005).References1. Elting LS, Cooksley CD, Chambers MS, Garden AS (2007) Risk,outcomes, and costs of radiation-induced oral mucositis among patientswith head-and-neck malignancies. Int J Radiat Oncol Biol Phys 68:1110-1120.2. Sonis ST (2007) Pathobiology of oral mucositis: novel insights andopportunities. J Support Oncol 5: 3-11.3. Sonis ST (2004) The pathobiology of mucositis. Nat Rev Cancer 4:277-284.4. Wygoda a, Skladowski K, Rutkowski T, Hutnik M, Golen M, et al. (2012)Acute mucosal radiation reactions in patients with head and neck cancer.Patterns of mucosal healing on the basis of daily examinations.Strahlenther Onkol 188: 686–691.5. Bik EM, Long CD, Armitage GC, Loomer P, Emerson J, et al. (2010)Bacterial diversity in the oral cavity of 10 healthy individuals. ISME J 4:962-974.6. Vanhoecke B, De Ryck T, Stringer A, Van de Wiele T, Keefe D (2015)Microbiota and their role in the pathogenesis of oral mucositis. Oral Dis21: 17-30.7. De Ryck T, Grootaert C, Jaspaert L, Kerckhof FM, Van Gele M, et al.(2014) Development of an oral mucosa model to study host-microbiomeinteractions during wound healing. Appl Microbiol Biotechnol 98:6831-6846.8. Aguirre JS, Rodríguez MR, García de Fernando GD (2011) Effects ofelectron beam irradiation on the variability in survivor number andduration of lag phase of four food-borne organisms. Int J Food Microbiol149: 236-246.9. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining thenormal bacterial flora of the oral cavity. J Clin Microbiol 43: 5721-5732.10. Hu YJ, Shao ZY, Wang Q, Jiang YT, Ma R, et al. (2013) Exploring thedynamic core microbiome of plaque microbiota during head-and-neckradiotherapy using pyrosequencing. PLoS One 8: e56343.11. Hu YJ, Wang Q, Jiang YT, Ma R, Xia WW, et al. (2013) Characterizationof oral bacterial diversity of irradiated patients by high-throughputsequencing. Int J Oral Sci 5: 21-25.12. Ramanan P, Barreto JN, Osmon DR, Tosh PK (2014) Rothia bacteremia:a 10-year experience at Mayo Clinic, Rochester, Minnesota. J ClinMicrobiol 52: 3184-3189. Citation: Ryck TD, Boterberg T, Kerckhof FM, Schrijver JD, Bracke M, et al. (2015) Effects of Irradiation on Epithelial Wound Healing andMicrobial Diversity in an in-vitro Oral Mucosa Model. J Nucl Med Radiat Ther 6: 218. doi:10.4172/2155-9619.1000218Page 4 of 4J Nucl Med Radiat TherISSN:2155-9619 JNMRT, an open access journal Volume 6 • Issue 2 • 1000218

Effects of irradiation on epithelial wound healing and microbial diversity in an in-vitro oral mucosa model

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Effects of irradiation on epithelial wound healing and microbial diversity in an in-vitro oral mucosa model

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