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Role of oral flora in chemotherapy-induced oral mucositis in vivo
Archives of Oral Biology 101 (2019) 51–56
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Role of oral flora in chemotherapy-induced oral mucositis in vivo ⁎
N. Gupta, S.Y. Quah, J.F. Yeo, J. Ferreira, K.S. Tan , C.H.L. Hong
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T
Faculty of Dentistry, National University of Singapore, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Oral mucositis Antineoplastic therapy Oral flora Germ-free Inflammation
Objective: To determine if commensal oral microflora impacts the severity of chemotherapy-induced oral mucositis (OM). Design: Specific-pathogen-free (SPF) and germ-free Swiss Webster mice in the experimental groups were dosed with 5-fluorouracil (5-FU) to induce OM. Mice in the control group received phosphate buffered saline. Comparative analyses of the epithelial thickness and cell proliferation/turnover rates, as well as the expression levels of metalloproteinases and pro-inflammatory mediators in the oral mucosa between the control and experimental groups were determined by histopathological and immunohistochemical analyses. Results: 5-FU-treated SPF and germ-free mice showed characteristic features of OM with reduced oral epithelial thickness, presence of inflammatory cells in the connective tissues, and increased levels of expression of metalloproteinases and pro-inflammatory cytokines compared to the respective control groups. When 5-FU-treated SPF and germ-free mice were compared, 5-FU-treated germ-free mice exhibited less severe epithelial destruction with higher expression of the cell proliferation marker Ki67, coupled with lower expression levels of metalloproteinases and pro-inflammatory cytokine in the oral mucosa. Conclusion: This study provides the first histopathological evidence that oral flora has a detrimental effect on chemotherapy-induced OM in vivo.
1. Introduction Cancer therapy-induced oral mucositis (OM) is a common complication in patients undergoing chemotherapy or radiation therapy. OM affects 40–100% of patients undergoing standard and/or myeloablative chemotherapy, and 100% of those undergoing head and neck radiotherapy (Elting, Cooksley, Chambers, & Garden, 2007). The painful symptoms of OM often contribute to poor quality of life, the need for parenteral nutrition, intravenous analgesics and extended hospitalizations. The cost of management of OM symptoms and complications is estimated to be USD$25,000 per patient (Elting et al., 2003, 2007; Sonis et al., 2004). The current understanding of OM pathophysiology comprises of five stages i.e. initiation, primary damage response, damage amplification, ulceration, and healing (Sonis, 2004). Although the oral microbiota is rich and diverse encompassing bacterial species that are implicated in inflammatory oral diseases (Kilian et al., 2016), the current model of OM pathophysiology does not consider the potential role of oral flora in the pathogenesis of OM. Elsewhere in intestinal mucositis and other gut inflammatory disorders, it has been theorized that gut bacteria contribute to the disease process by influencing intestinal inflammation,
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permeability and epithelial repair (Cani et al., 2008; Hamad, Fragkos, & Forbes, 2013; Vasconcelos et al., 2016; Yeung et al., 2015). As oral and intestinal mucosa are part of the same alimentary tract, the concepts of host-microbiome interactions in intestinal mucositis may well be extrapolated to the pathogenesis of OM. Several clinical studies have reported changes in oral microbiota from a largely Gram positive to a Gram negative consortium during cancer therapy (Napenas, Brennan, Bahrani-Mougeot, Fox, & Lockhart, 2007; de Mendonca et al., 2012; Laheij et al., 2012; Stringer & Logan, 2015; Vanhoecke, De Ryck, Stringer, Van de Wiele, & Keefe, 2015). In an animal study of radiation-induced OM, ulcerated epithelium was found to contain a higher abundance of oral microbiota compared to uninjured epithelium where the peak of bacterial colonization also coincided with greater OM severity (Sonis, 2009). However, these observed changes in oral microflora may be causal or consequential, and differentiating the two is difficult. Thus, in this study, the impact of commensal oral microbiota on the severity of chemotherapy-induced OM was determined. For the first time, analysis of OM severity was evaluated in specific-pathogen-free (SPF) and germ-free in vivo models by comparing the differences in histopathologic and innate immune responses between the two groups. Outcomes obtained herein provide
Corresponding authors at: Faculty of Dentistry, National University of Singapore, 9 Lower Kent Ridge Road, 119085, Singapore. E-mail addresses: [email protected] (K.S. Tan), [email protected] (C.H.L. Hong).
https://doi.org/10.1016/j.archoralbio.2019.03.008 Received 4 January 2019; Received in revised form 6 March 2019; Accepted 10 March 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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the evidence that oral flora has a significant detrimental impact on the severity of OM.
abundance of the 16S rRNA gene. Sequences of the primers used were, MMP-3 (forward 5′-ACATGGAGACTTTGTCCCTTTTG; reverse 5′-TTGG CTGAGTGGTAGAGTCCC); MMP-9 (forward 5′-CTGGACAGCCAGACAC TAAAG; reverse 5′-CTCGCGGCAAGTCTTCAGAG); IL-1β (forward 5′-CAACCAACAAGTGATATTCTCCATG; reverse 5′- GATCCACACTCTC CAGCTGCA); TNF-α (forward 5′-CATCTTCTCAAAATTCGAGTGACAA; reverse 5′-TGGGAGTAGACAAGGTACAACCC; MPO (forward 5′- AGTT GTGCTGAGCTGTATGGA; reverse 5′-CGGCTGCTTGAAGTAAAACAGG; 16S rRNA (forward 5′-CTTAGAGGGACAAAGGGCG; reverse 5′ -ACGC TGAGCCAGTCAGTGTA).
2. Material and methods 2.1. Animals Hamsters and mice have been used as animal models to investigate the pathogenesis of OM (Sonis, Tracey, Shklar, Jenson, & Florine, 1990; Bertolini, Sobue, Thompson, & Dongari-Bagtzoglou, 2017). In this study, we employed murine model of OM as germ-free hamsters are unavailable. Ten-week old SPF and germ-free Swiss Webster mice were obtained from Taconic Biosciences (Rensselaer, NY, USA). Germ-free mice were maintained in sterile environment in positive pressure isolators. Germ-free status was validated by obtaining oral swabs from the oral cavity of the animals and cultured on tryptic soy agar supplemented with 5% sheep blood. All mice were housed under standard conditions with environmental temperature of 22 °C, 50% humidity, 12hour light/dark cycle with access to food and water ad libitum. The animal study was carried out according to the Institutional Animal Care and Use Committee of the National University of Singapore (protocol number R15-1106).
2.5. Immunohistochemistry (IHC)
SPF and germ-free mice (n = 12/group) were injected with 70 mg/ kg body weight of 5-FU intraperitoneally (i.p.) once a day from days 1 through 5, and days 8 and 9. The control SPF and germ-free mice received sterile phosphate buffered saline (PBS) i.p. Mice were euthanized on day 10, and tongues were harvested and fixed in 10% buffered formalin (Sigma-Aldrich).
Slides were deparaffinized, rehydrated followed by antigen retrieval using citrate buffer solution (pH 6.0, 10 mM). Subsequently, slides were incubated with 3% hydrogen peroxide (Thermo Scientific, Waltham, MA, USA) for 10 min, followed by blocking with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. Slides were incubated with primary antibodies against Ki67 (Thermo Scientific), IL1β (Abcam, Cambridge, United Kingdom), TNF-α (Abcam), MMP-3 and -9 (Abcam) and MPO (Abcam) overnight at 4 °C. Subsequently, tissues were incubated with biotinylated goat secondary antibody (Thermo Scientific) and streptavidin horseradish peroxidase consecutively (Thermo Scientific) at room temperature. Thereafter, the chromogenic substrate diaminobenzidine tetra hydrochloride (DAB) (Thermo Scientific) was applied. Finally, tissues were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Quantitative analysis of the respective immunolabelled tissues were carried out in a blinded fashion by determining the number of positively stained cells out of the total number of cells per field at 400X magnification using Image J software version 1.46 r (NIH, Bethesda, MD, USA).
2.3. Histopathologic examination
2.6. Statistical analysis
Formalin fixed tongue tissues were embedded in paraffin blocks. Sections of 5 μm thickness were prepared and stained with hematoxylin and eosin (H&E). Tissues were observed under Leica DMI8 microscope (Leica Microsystems, Wetzlar, Germany) for morphological alterations and presence of inflammatory infiltrates. Epithelial thickness was measured using the Image J software version 1.46 r (NIH, Bethesda, MD, USA). Modified histological grading of OM was carried out, as described previously (Sunavala‐Dossabhoy, Abreo, Timiri Shanmugam, & Caldito, 2015). The criteria used to grade the microscopic changes were as follows : 0, normal mucosa; 1, focal or diffuse alteration of basal cell layer with nuclear atypia and ≤ 2 dyskeratotic squamous cells; 2, epithelial lining (2–4 cell layer) and/or ≥ 3 dyskeratotic squamous cells in the epithelium; 3a, loss of epithelium without a break in keratinization or presence of atrophied epithelium with thinning of keratinized layer; 3b, Subepithelial vesicle or bullous formation; 4, complete loss of epithelial and keratinized cell layers; ulcerations. The histological examination was performed in a blinded fashion.
Statistical analysis was performed using GraphPad Prism version 6 (San Diego, CA, USA). Results are presented as mean + SD. For data involving two experimental groups, Student’s t-test was carried out. For multiple group analysis, ANOVA was used. Differences with pvalue < 0.05 were considered statistically significant.
2.2. 5-fluorouracil administration
3. Results 3.1. Epithelial thickness and turnover in SPF and germ-free mice Oral epithelium of PBS-treated SPF and germ-free mice showed classical histological features manifested by intact keratinized stratified squamous epithelium, conical filiform papillae, and absence of inflammatory infiltrates in the connective tissues (Fig. 1). In contrast, the 5-FU-treated SPF and germ-free mice exhibited damage of the oral epithelium (Fig. 1B & F). The oral epithelium of 5-FU-treated SPF mice showed epithelial atrophy with irregular epithelial stratification, nuclear aberrations, and areas of obliterated filiform papillae with desquamative keratinization (Fig. 1B). However, the histological changes in the oral epithelium of the germ-free mice were less evident (Fig. 1F). Inflammatory infiltrates consisting of neutrophils, macrophages and Tlymphocytes were present within the connective tissues of the oral mucosa of 5-FU-treated SPF and germ-free mice (Fig. 1D & H). However, the 5-FU-treated germ-free mice exhibited milder inflammation compared to the SPF mice. Quantitative analysis of the epithelium thickness revealed that the oral epithelium of 5-FU-treated SPF mice was significantly thinner compared to 5-FU-treated germ-free mice (Fig. 2A). Further, histological grading of the severity of OM demonstrated that 5-FU-treated SPF mice suffered from Grade 3 OM, while the germ-free counterpart had Grade 1 OM (Fig. 2B). In addition, the number of Ki67-expressing cells in the basal epithelium of 5-FU-treated SPF and germ-free mice was
2.4. RNA extraction and qPCR analysis Total RNA was extracted from tongue tissues using GeneAll RNA extraction kit (Seoul, Korea). Extracted RNA was treated with RQ1 DNAseI (Promega, Medison, MI, USA) to remove residual DNA, prior to reverse transcription to cDNA using iScript Reverse transcription SuperMix (BioRad, Hercules, CA, USA). All procedures were carried out according to the manufacturers’ protocols. qPCR analysis was carried out using the cDNAs in a CFX Connect Real-Time Detection System (BioRad). A qPCR reaction mixture consisted of 1 μL of cDNA, 10 μL of iTaq Universal SYBR Green Supermix (BioRad), 10 pmol of the respective forward and reverse primers, in a final volume of 20 μL. Thermal cycling was carried out for 40 cycles, after which the expression of the respective target mRNA was normalized to the relative 52
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Fig. 1. Histopathological comparisons of the oral mucosa tissue of PBS- and 5-FU-treated SPF and germ-free mice. H & E staining of the oral epithelium of (A) PBStreated and (B) 5-FU-treated SPF mice. H & E staining of the connective tissues of (C) PBS-treated and (D) 5-FU-treated SPF mice. H & E staining of the oral epithelium of (E) PBS-treated and (F) 5-FU-treated germ-free mice. H & E staining of the connective tissues of (G) PBS-treated and (H) 5-FU-treated germ-free mice. Arrow heads indicate representative inflammatory cells. Epi: epithelium. Magnification 400 × . Scale bar: 100 μm.
activation of inflammatory mediators and other signaling pathways (Bailey et al., 1994; Louis et al., 2000; Saarialho-Kere et al., 1996). We found that the expression of both MMP-3 and -9 were significantly higher in 5-FU-treated SPF mice compared to 5-FU-treated germ-free mice (Fig. 3A & B). IHC analysis was carried out to determine the location and cell types expressing MMP-3 and -9 in the oral mucosa. We found that immune infiltrates in the connective tissues were the predominant cell types expressing MMP-3 and -9 (Fig. 3C & F). On quantitative assessment, the number of positively stained MMP-3 and -9 cells were significantly higher in 5-FU-treated SPF mice compared to the germ-free animals (Fig. 3G & H).
significantly lower compared to PBS-treated SPF and germ-free mice (Fig. 2C–G). Macroscopically, no visible ulcerations were observed in the oral cavity of both the 5-FU-treated SPF and germ-free animals. This may be likely due to the highly keratinized nature of oral epithelium in mice, thus rendering it less susceptible to mucosa breakage (Gibson, Bowen, & Keefe, 2010; Bowen, Gibson, & Keefe, 2011). Furthermore, in our study, no mechanical trauma was introduced to the oral mucosa to elicit OM.
3.2. Matrix metalloproteinases expression in SPF and germ-free mice Matrix metalloproteinases (MMPs) are involved in mucosal damage,
Fig. 2. Severity of oral mucositis in 5-FU-treated SPF and germ-free mice. (A) oral epithelium thickness in PBS- and 5-FU-treated SPF and germ-free mice, (B) Histological grading of oral mucositis in 5-FU-treated SPF and germ-free mice. Ki67 immunostaining of the oral epithelium of (C) PBS- and (D) 5-FU-treated SPF mice, and (E) PBS- and (F) 5-FU-treated germ-free mice. Arrow heads indicate positively stained cells within the basal epithelial layer. (G) Quantitative analysis of Ki67stained positive cells in the oral mucosa of PBS- and 5-FU-treated SPF and germ-free mice. ***p < 0.001. 53
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Fig. 3. Expression of MMPs in the oral mucosa of 5-FU-treated SPF and germ-free mice. (A) MMP-3 and (B) MMP-9 in 5-FU-treated SPF and germ-free mice. Immunostaining of MMPs in the connective tissue of oral mucosa tissues (C) MMP-3 immunostaining in 5-FU-treated SPF mice, (D) MMP-3 immunostaining in 5-FUtreated germ-free mice (E) MMP-9 immunostaining in 5-FU-treated SPF mice, (F) MMP-9 immunostaining in 5-FU-treated germ-free mice. Arrow heads indicate positively stained cells. Quantitative analysis of (G) MMP-3 in 5-FU-treated SPF and germ-free mice, and (H) MMP-9 in 5-FU-treated SPF and germ-free mice. Magnification 400 × . Scale bar: 100 μm. ***p < 0.001.
Bacterial species such as Porphyromonas gingivalis, Porphyromonas micra, Treponema denticola, and Fusobacterium nucleatum were found to be associated with OM-related ulcerations in hematopoietic stem cell transplant patients (Laheij et al., 2012). In another clinical study, taxon Capnocytophaga was found to be abundant in OM and proposed to be an important bacterial species in OM progression (Napenas et al., 2007). However, it remains unclear if these reported perturbations in oral microbiota are clinically significant since the impact of bacteria in the severity of OM is uncertain. To confirm the role of oral flora in OM, in this study, the severity of chemotherapy-induced OM was compared in conventional (SPF) and germ-free in vivo models. The oral cavity of germ-free mice are free of all microorganisms. However, SPF mice harbor bacteria of the genera Enterococcus and Streptococcus, Lactobacillus, Staphylococcus, Propionibacterium. Among these the predominant species reported include Lactobacillus murinus, Staphylococcus aureus, Streptococcus faecalis, Staphylococcus sciuri and Escherichia coli (Trudel, St. Amand, Bareil, Cardinal, & Lavoie, 1986). In addition under immunosuppressed conditions such as cancer therapy, Candida species is capable of inducing candidiasis in mice (Costa, Pereira, Junqueira, & Jorge, 2013). Unlike other disease processes where the use of germ-free models has become a powerful tool to explore the interplay between host and microorganisms (Yi & Li, 2012), there has not been a study to confirm the biological significance of the oral flora and the changes observed during OM.
3.3. Pro-inflammatory cytokines expression in SPF and germ-free mice The expression of pro-inflammatory cytokines i.e. IL-1β, TNF-α and MPO in the oral mucosa of SPF mice were significantly higher compared to germ-free mice (Fig. 4A–C). IHC analysis revealed that the cells expressing IL-1β, TNF-α and MPO were localized within the connective tissues of the oral mucosa in both 5-FU-treated SPF and germ-free mice (Fig. 4D–I). IL-1β and TNF-α in 5-FU-treated SPF mice were predominantly expressed by macrophages and T-lymphocytes while MPO was primarily associated with neutrophil granulocytes. On quantitative analysis, levels of IL-1β, TNF-α, and MPO were significantly higher in oral mucosa of 5-FU-treated SPF mice compared to germ-free mice (Fig. 4J–L). 4. Discussion Antineoplastic chemotherapy leads to multiple OM biological events resulting in the damage of oral mucosa integrity due to impairment of the turnover of basal keratinocytes. Interestingly, accompanying changes in oral flora have been reported during OM (Vasconcelos et al., 2016). In OM, clinical observational studies have shown that Gramnegative bacteria increased in the oral cavity during chemotherapy with corresponding decrease in levels of Gram-positive microorganisms (Laheij et al., 2012; Napenas et al., 2007; Stokman et al., 2003). 54
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Fig. 4. Expression of pro-inflammatory mediators in the oral mucosa of 5-FU-treated SPF and germ-free mice. (A) IL-1β, (B) TNF-α and (C) MPO in 5-FU-treated SPF and germ-free mice. Immunostaining of cells expressing (D–E) IL-1β, (F–G) TNF-α and (H–I) MPO in the connective tissues of the oral mucosa in SPF and germ-free mice. Quantitative analysis (J) IL-1β, (K) TNF-α and (L) MPO stained positive cells in 5-FU-treared SPF and germ-free mice. Magnification 400 × . Scale 100 μm. ***p < 0.001.
observed elevated levels of expression of MMP-3 and -9 in the basal epithelium, lamina propria and submucosa of 5-FU dosed SPF mice. MMP-3 could contribute to mucosal damage by disruption of cell-to-cell and cell-to-ECM through degradation of collagens, fibronectin, laminin, aggrecan, insulin-like growth factor binding protein-3 and serpins. The active enzyme also activates MMP-1, -8, -9, and -13 thus amplifying the process of tissue damage (Song, Park, & Sung, 2013). Our results are consistent with a study carried out by Al‐Azri et al. (2015)) where the authors found that MMP-3 and -9 is up-regulated by irinotecan in all layers of epithelium, lamina propria and submucosa of tongue tissue in mice. As tissue injury progresses during the pathogenesis of OM, the tight junction proteins such as E-cadherin, occludin and claudin-1 are downregulated which could further weaken the barrier function of oral mucosa. This allows for increased permeability and conduit of bacterial products (Pedroso et al., 2015; Xu et al., 2014) leading to advanced tissue damage. In this study, we found that in 5-FU-treated SPF mice, the damage to the oral epithelium was more severe with elevated levels of mediators of innate immune responses such as IL-1β, TNF-α, and MPO compared to 5-FU-treated germ-free mice. Furthermore, the proliferative potential of the basal epithelium was compromised in SPF mice compared to the germ-free mice. These results are consistent with the findings from an intestinal mucositis study by Pedroso et al. (2015) where the authors reported that CPT-11-treated SPF models showed greater damage to the epithelial structure and thickness, increased inflammatory infiltrates and loss of cell differentiation in SPF mice compared to the germ-free mice resulting in an increased intestinal permeability. The oral epithelium, fibroblasts, endothelial cells and infiltrating immune cells express a repertoire of pattern recognition receptors (PRRs) including such as toll-like receptor (TLR)-2, -4, -9, and nucleotide-binding oligomerization domain-containing protein-1 (NOD-1) and -2 (Piccinini & Midwood, 2010). Therefore, damage of oral mucosa barrier function could intensify inflammation by allowing translocation of bacterial products and toxins to cells in the submucosa region. In addition, damage-associated molecular patterns (DAMPs)
Initiation of OM begins either as a direct consequence of the toxicity of chemotherapy agents or harmful effects of cellular reactive oxygen species (ROS) which leads to oxidative stress. High levels of ROS induce DNA strand breaks, lipid peroxidation, protein mis-folding, aggregation and degradation thus affecting normal cell proliferation/turnover (Conklin, 2004). This is evident in our results where 5-FU-treated SPF mice showed hallmark histopathological features of OM where proliferation of oral mucosa cells was significantly reduced compared to the control mice. Oxidative stress induces the activation of transcription factor NF-κB leading to increased expression of pro-inflammatory cytokines (Sonis, 2004). Indeed, we found that the pro-inflammatory cytokines, IL-1β and TNF-α are expressed by cells in the oral epithelium, endothelium and mucosal connective tissues in 5-FU-treated SPF mice. Similar results were reported by Bertolini et al. (2017) where the tongue tissues of mice dosed with 5-FU showed epithelial atrophy with reduced number of Ki67 stained positive cells and increased expression of pro-inflammatory cytokines. ROS itself also elicits endothelial dysfunction causing vasodilation enhancing migration of leukocytes from the blood vessels into the extracellular matrix (ECM). Neutrophils which are one of the principal cell types in innate immune response of OM, express MPO when activated (Selders, Fetz, Radic, & Bowlin, 2017). We observed elevated levels of MPO in the connective tissues of the oral mucosa in 5-FU-treated SPF mice. MPO, which is released from neutrophil’s azurophilic granules, catalyzes the production of hypochlorous acid causing oxidative damage to host tissues (Manicone & McGuire, 2008). One of the consequences of tissue damage is the activation of MMPs. MMPs, also known as matrixins, are group of enzymes that are present in the extracellular environment of cells and are responsible for the degradation of most extracellular matrix (ECM) proteins during organogenesis, growth and normal tissue turnover. Under normal physiological conditions, the expression of MMPs is low in oral mucosa tissues (Al‐Azri et al., 2015). MMP-3 (stromelysin) is expressed by fibroblasts, endothelial cells, macrophages, vascular smooth muscle cells and keratinocytes in response to stimuli (Maksymowych et al., 2007). We 55
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which are biomolecules released extracellularly as a result of cellular stress and tissue injury would further intensify the inflammatory response. One such DAMP is the high mobility group box-1 protein (HMGB-1) which is recognized by toll-like receptors (TLRs) especially TLR-2 and TLR-4, and receptor for advanced glycation end products (RAGE) (Diener, Al‐Dasooqi, Lousberg, & Hayball, 2013). HMGB-1 released by necrotic cells induces both innate and adaptive immunity response by activating antigen-presenting cells to counter tissue damage caused by bacterial infiltration. It can also act like a chemokine or cytokine by ligation with specific receptors such as TLR-2, TLR-4 and TLR-9 (Diener et al., 2013). Thus, PAMPs, DAMPs and their overlapping receptors could further drive the immune response in OM. In summary, the present study demonstrates the detrimental impact of oral microbiota in worsening chemotherapy-induced OM. To our knowledge, this is the first study comparing the severity of OM in germfree and SPF in vivo models. Future research needs to be formulated to observe if the degree of oral microflora colonization and/or enrichment of specific bacterial species play a more significant role in OM pathogenesis. Answers to these questions will facilitate efforts to drive novel therapeutic interventions for OM.
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Funding This research was supported by National University Health System (grant number R221-000-088-733). Conflict of interest statement The authors declare no conflict of interest. Author contributions Conceptualization: K.S.T. & C.H.L.H.; Study design: N.G., Y.J.F., J.F., K.S.T. & C.H.L.H. Data acquisition: N.G., S.Y.Q., K.S.T.; Data analysis and interpretation: N.G., Y.J.F., J.F., K.S.T. & C.H.L.H; Statistical analysis: N.G.; K.S.T; Manuscript preparation: N.G., K.S.T. & C.H.L.H.; Manuscript editing: N.G., S.Y.Q., J.F.Y., J.F., K.S.T. & C.H.L.H. All authors have read and approved the final article. References Al‐Azri, A. R., Gibson, R. J., Bowen, J. M., Stringer, A. M., Keefe, D. M., & Logan, R. M. (2015). Involvement of matrix metalloproteinases (MMP‐3 and MMP‐9) in the pathogenesis of irinotecan‐induced oral mucositis. Journal of Oral Pathology and Medicine, 44, 459–467. Bailey, C., Hembry, R., Alexander, A., Irving, M., Grant, M., & Shuttleworth, C. (1994). Distribution of the matrix metalloproteinases stromelysin, gelatinases A and B, and collagenase in Crohn’s disease and normal intestine. Journal of Clinical Pathology, 47, 113–116. Bertolini, M., Sobue, T., Thompson, A., & Dongari-Bagtzoglou, A. (2017). Chemotherapy induces oral mucositis in mice without additional noxious stimuli. Translational Oncology, 10, 612–620. Bowen, J. M., Gibson, R. J., & Keefe, D. (2011). Animal models of mucositis: Implications for therapy. The Journal of Supportive Oncology, 9, 161–168. Cani, P. D., Rodrigo, B., Knauf, C., Waget, A., Neyrinck, A. M., Delzenne, N. M., ... Burcelin, R. (2008). Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes, 57, 1470–1481. Conklin, K. A. (2004). Chemotherapy-associated oxidative stress: Impact on chemotherapeutic effectiveness. Integrative Cancer Therapies, 3(4), 294–300. Costa, A. C., Pereira, C. A., Junqueira, J. C., & Jorge, A. O. (2013). Recent mouse and rat methods for the study of experimental oral candidiasis. Virulence, 4(5), 391–399. de Mendonca, R. M., de Araujo, M., Levy, C. E., Morari, J., Silva, R. A., Yunes, J. A., ... Brandalise, S. R. (2012). Prospective evaluation of HSV, Candida spp., and oral bacteria on the severity of oral mucositis in pediatric acute lymphoblastic leukemia. Supportive Care in Cancer : Official Journal of the Multinational Association of Supportive Care in Cancer, 20, 1101–1107. Diener, K. R., Al‐Dasooqi, N., Lousberg, E. L., & Hayball, J. D. (2013). The multifunctional alarmin HMGB1 with roles in the pathophysiology of sepsis and cancer. Immunology and Cell Biology, 91, 443–450. Elting, L. S., Cooksley, C., Chambers, M., Cantor, S. B., Manzullo, E., & Rubenstein, E. B. (2003). The burdens of cancer therapy. Clinical and economic outcomes of
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Role of oral flora in chemotherapy-induced oral mucositis in vivo ⁎
N. Gupta, S.Y. Quah, J.F. Yeo, J. Ferreira, K.S. Tan , C.H.L. Hong
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T
Faculty of Dentistry, National University of Singapore, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Oral mucositis Antineoplastic therapy Oral flora Germ-free Inflammation
Objective: To determine if commensal oral microflora impacts the severity of chemotherapy-induced oral mucositis (OM). Design: Specific-pathogen-free (SPF) and germ-free Swiss Webster mice in the experimental groups were dosed with 5-fluorouracil (5-FU) to induce OM. Mice in the control group received phosphate buffered saline. Comparative analyses of the epithelial thickness and cell proliferation/turnover rates, as well as the expression levels of metalloproteinases and pro-inflammatory mediators in the oral mucosa between the control and experimental groups were determined by histopathological and immunohistochemical analyses. Results: 5-FU-treated SPF and germ-free mice showed characteristic features of OM with reduced oral epithelial thickness, presence of inflammatory cells in the connective tissues, and increased levels of expression of metalloproteinases and pro-inflammatory cytokines compared to the respective control groups. When 5-FU-treated SPF and germ-free mice were compared, 5-FU-treated germ-free mice exhibited less severe epithelial destruction with higher expression of the cell proliferation marker Ki67, coupled with lower expression levels of metalloproteinases and pro-inflammatory cytokine in the oral mucosa. Conclusion: This study provides the first histopathological evidence that oral flora has a detrimental effect on chemotherapy-induced OM in vivo.
1. Introduction Cancer therapy-induced oral mucositis (OM) is a common complication in patients undergoing chemotherapy or radiation therapy. OM affects 40–100% of patients undergoing standard and/or myeloablative chemotherapy, and 100% of those undergoing head and neck radiotherapy (Elting, Cooksley, Chambers, & Garden, 2007). The painful symptoms of OM often contribute to poor quality of life, the need for parenteral nutrition, intravenous analgesics and extended hospitalizations. The cost of management of OM symptoms and complications is estimated to be USD$25,000 per patient (Elting et al., 2003, 2007; Sonis et al., 2004). The current understanding of OM pathophysiology comprises of five stages i.e. initiation, primary damage response, damage amplification, ulceration, and healing (Sonis, 2004). Although the oral microbiota is rich and diverse encompassing bacterial species that are implicated in inflammatory oral diseases (Kilian et al., 2016), the current model of OM pathophysiology does not consider the potential role of oral flora in the pathogenesis of OM. Elsewhere in intestinal mucositis and other gut inflammatory disorders, it has been theorized that gut bacteria contribute to the disease process by influencing intestinal inflammation,
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permeability and epithelial repair (Cani et al., 2008; Hamad, Fragkos, & Forbes, 2013; Vasconcelos et al., 2016; Yeung et al., 2015). As oral and intestinal mucosa are part of the same alimentary tract, the concepts of host-microbiome interactions in intestinal mucositis may well be extrapolated to the pathogenesis of OM. Several clinical studies have reported changes in oral microbiota from a largely Gram positive to a Gram negative consortium during cancer therapy (Napenas, Brennan, Bahrani-Mougeot, Fox, & Lockhart, 2007; de Mendonca et al., 2012; Laheij et al., 2012; Stringer & Logan, 2015; Vanhoecke, De Ryck, Stringer, Van de Wiele, & Keefe, 2015). In an animal study of radiation-induced OM, ulcerated epithelium was found to contain a higher abundance of oral microbiota compared to uninjured epithelium where the peak of bacterial colonization also coincided with greater OM severity (Sonis, 2009). However, these observed changes in oral microflora may be causal or consequential, and differentiating the two is difficult. Thus, in this study, the impact of commensal oral microbiota on the severity of chemotherapy-induced OM was determined. For the first time, analysis of OM severity was evaluated in specific-pathogen-free (SPF) and germ-free in vivo models by comparing the differences in histopathologic and innate immune responses between the two groups. Outcomes obtained herein provide
Corresponding authors at: Faculty of Dentistry, National University of Singapore, 9 Lower Kent Ridge Road, 119085, Singapore. E-mail addresses: [email protected] (K.S. Tan), [email protected] (C.H.L. Hong).
https://doi.org/10.1016/j.archoralbio.2019.03.008 Received 4 January 2019; Received in revised form 6 March 2019; Accepted 10 March 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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the evidence that oral flora has a significant detrimental impact on the severity of OM.
abundance of the 16S rRNA gene. Sequences of the primers used were, MMP-3 (forward 5′-ACATGGAGACTTTGTCCCTTTTG; reverse 5′-TTGG CTGAGTGGTAGAGTCCC); MMP-9 (forward 5′-CTGGACAGCCAGACAC TAAAG; reverse 5′-CTCGCGGCAAGTCTTCAGAG); IL-1β (forward 5′-CAACCAACAAGTGATATTCTCCATG; reverse 5′- GATCCACACTCTC CAGCTGCA); TNF-α (forward 5′-CATCTTCTCAAAATTCGAGTGACAA; reverse 5′-TGGGAGTAGACAAGGTACAACCC; MPO (forward 5′- AGTT GTGCTGAGCTGTATGGA; reverse 5′-CGGCTGCTTGAAGTAAAACAGG; 16S rRNA (forward 5′-CTTAGAGGGACAAAGGGCG; reverse 5′ -ACGC TGAGCCAGTCAGTGTA).
2. Material and methods 2.1. Animals Hamsters and mice have been used as animal models to investigate the pathogenesis of OM (Sonis, Tracey, Shklar, Jenson, & Florine, 1990; Bertolini, Sobue, Thompson, & Dongari-Bagtzoglou, 2017). In this study, we employed murine model of OM as germ-free hamsters are unavailable. Ten-week old SPF and germ-free Swiss Webster mice were obtained from Taconic Biosciences (Rensselaer, NY, USA). Germ-free mice were maintained in sterile environment in positive pressure isolators. Germ-free status was validated by obtaining oral swabs from the oral cavity of the animals and cultured on tryptic soy agar supplemented with 5% sheep blood. All mice were housed under standard conditions with environmental temperature of 22 °C, 50% humidity, 12hour light/dark cycle with access to food and water ad libitum. The animal study was carried out according to the Institutional Animal Care and Use Committee of the National University of Singapore (protocol number R15-1106).
2.5. Immunohistochemistry (IHC)
SPF and germ-free mice (n = 12/group) were injected with 70 mg/ kg body weight of 5-FU intraperitoneally (i.p.) once a day from days 1 through 5, and days 8 and 9. The control SPF and germ-free mice received sterile phosphate buffered saline (PBS) i.p. Mice were euthanized on day 10, and tongues were harvested and fixed in 10% buffered formalin (Sigma-Aldrich).
Slides were deparaffinized, rehydrated followed by antigen retrieval using citrate buffer solution (pH 6.0, 10 mM). Subsequently, slides were incubated with 3% hydrogen peroxide (Thermo Scientific, Waltham, MA, USA) for 10 min, followed by blocking with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. Slides were incubated with primary antibodies against Ki67 (Thermo Scientific), IL1β (Abcam, Cambridge, United Kingdom), TNF-α (Abcam), MMP-3 and -9 (Abcam) and MPO (Abcam) overnight at 4 °C. Subsequently, tissues were incubated with biotinylated goat secondary antibody (Thermo Scientific) and streptavidin horseradish peroxidase consecutively (Thermo Scientific) at room temperature. Thereafter, the chromogenic substrate diaminobenzidine tetra hydrochloride (DAB) (Thermo Scientific) was applied. Finally, tissues were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Quantitative analysis of the respective immunolabelled tissues were carried out in a blinded fashion by determining the number of positively stained cells out of the total number of cells per field at 400X magnification using Image J software version 1.46 r (NIH, Bethesda, MD, USA).
2.3. Histopathologic examination
2.6. Statistical analysis
Formalin fixed tongue tissues were embedded in paraffin blocks. Sections of 5 μm thickness were prepared and stained with hematoxylin and eosin (H&E). Tissues were observed under Leica DMI8 microscope (Leica Microsystems, Wetzlar, Germany) for morphological alterations and presence of inflammatory infiltrates. Epithelial thickness was measured using the Image J software version 1.46 r (NIH, Bethesda, MD, USA). Modified histological grading of OM was carried out, as described previously (Sunavala‐Dossabhoy, Abreo, Timiri Shanmugam, & Caldito, 2015). The criteria used to grade the microscopic changes were as follows : 0, normal mucosa; 1, focal or diffuse alteration of basal cell layer with nuclear atypia and ≤ 2 dyskeratotic squamous cells; 2, epithelial lining (2–4 cell layer) and/or ≥ 3 dyskeratotic squamous cells in the epithelium; 3a, loss of epithelium without a break in keratinization or presence of atrophied epithelium with thinning of keratinized layer; 3b, Subepithelial vesicle or bullous formation; 4, complete loss of epithelial and keratinized cell layers; ulcerations. The histological examination was performed in a blinded fashion.
Statistical analysis was performed using GraphPad Prism version 6 (San Diego, CA, USA). Results are presented as mean + SD. For data involving two experimental groups, Student’s t-test was carried out. For multiple group analysis, ANOVA was used. Differences with pvalue < 0.05 were considered statistically significant.
2.2. 5-fluorouracil administration
3. Results 3.1. Epithelial thickness and turnover in SPF and germ-free mice Oral epithelium of PBS-treated SPF and germ-free mice showed classical histological features manifested by intact keratinized stratified squamous epithelium, conical filiform papillae, and absence of inflammatory infiltrates in the connective tissues (Fig. 1). In contrast, the 5-FU-treated SPF and germ-free mice exhibited damage of the oral epithelium (Fig. 1B & F). The oral epithelium of 5-FU-treated SPF mice showed epithelial atrophy with irregular epithelial stratification, nuclear aberrations, and areas of obliterated filiform papillae with desquamative keratinization (Fig. 1B). However, the histological changes in the oral epithelium of the germ-free mice were less evident (Fig. 1F). Inflammatory infiltrates consisting of neutrophils, macrophages and Tlymphocytes were present within the connective tissues of the oral mucosa of 5-FU-treated SPF and germ-free mice (Fig. 1D & H). However, the 5-FU-treated germ-free mice exhibited milder inflammation compared to the SPF mice. Quantitative analysis of the epithelium thickness revealed that the oral epithelium of 5-FU-treated SPF mice was significantly thinner compared to 5-FU-treated germ-free mice (Fig. 2A). Further, histological grading of the severity of OM demonstrated that 5-FU-treated SPF mice suffered from Grade 3 OM, while the germ-free counterpart had Grade 1 OM (Fig. 2B). In addition, the number of Ki67-expressing cells in the basal epithelium of 5-FU-treated SPF and germ-free mice was
2.4. RNA extraction and qPCR analysis Total RNA was extracted from tongue tissues using GeneAll RNA extraction kit (Seoul, Korea). Extracted RNA was treated with RQ1 DNAseI (Promega, Medison, MI, USA) to remove residual DNA, prior to reverse transcription to cDNA using iScript Reverse transcription SuperMix (BioRad, Hercules, CA, USA). All procedures were carried out according to the manufacturers’ protocols. qPCR analysis was carried out using the cDNAs in a CFX Connect Real-Time Detection System (BioRad). A qPCR reaction mixture consisted of 1 μL of cDNA, 10 μL of iTaq Universal SYBR Green Supermix (BioRad), 10 pmol of the respective forward and reverse primers, in a final volume of 20 μL. Thermal cycling was carried out for 40 cycles, after which the expression of the respective target mRNA was normalized to the relative 52
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Fig. 1. Histopathological comparisons of the oral mucosa tissue of PBS- and 5-FU-treated SPF and germ-free mice. H & E staining of the oral epithelium of (A) PBStreated and (B) 5-FU-treated SPF mice. H & E staining of the connective tissues of (C) PBS-treated and (D) 5-FU-treated SPF mice. H & E staining of the oral epithelium of (E) PBS-treated and (F) 5-FU-treated germ-free mice. H & E staining of the connective tissues of (G) PBS-treated and (H) 5-FU-treated germ-free mice. Arrow heads indicate representative inflammatory cells. Epi: epithelium. Magnification 400 × . Scale bar: 100 μm.
activation of inflammatory mediators and other signaling pathways (Bailey et al., 1994; Louis et al., 2000; Saarialho-Kere et al., 1996). We found that the expression of both MMP-3 and -9 were significantly higher in 5-FU-treated SPF mice compared to 5-FU-treated germ-free mice (Fig. 3A & B). IHC analysis was carried out to determine the location and cell types expressing MMP-3 and -9 in the oral mucosa. We found that immune infiltrates in the connective tissues were the predominant cell types expressing MMP-3 and -9 (Fig. 3C & F). On quantitative assessment, the number of positively stained MMP-3 and -9 cells were significantly higher in 5-FU-treated SPF mice compared to the germ-free animals (Fig. 3G & H).
significantly lower compared to PBS-treated SPF and germ-free mice (Fig. 2C–G). Macroscopically, no visible ulcerations were observed in the oral cavity of both the 5-FU-treated SPF and germ-free animals. This may be likely due to the highly keratinized nature of oral epithelium in mice, thus rendering it less susceptible to mucosa breakage (Gibson, Bowen, & Keefe, 2010; Bowen, Gibson, & Keefe, 2011). Furthermore, in our study, no mechanical trauma was introduced to the oral mucosa to elicit OM.
3.2. Matrix metalloproteinases expression in SPF and germ-free mice Matrix metalloproteinases (MMPs) are involved in mucosal damage,
Fig. 2. Severity of oral mucositis in 5-FU-treated SPF and germ-free mice. (A) oral epithelium thickness in PBS- and 5-FU-treated SPF and germ-free mice, (B) Histological grading of oral mucositis in 5-FU-treated SPF and germ-free mice. Ki67 immunostaining of the oral epithelium of (C) PBS- and (D) 5-FU-treated SPF mice, and (E) PBS- and (F) 5-FU-treated germ-free mice. Arrow heads indicate positively stained cells within the basal epithelial layer. (G) Quantitative analysis of Ki67stained positive cells in the oral mucosa of PBS- and 5-FU-treated SPF and germ-free mice. ***p < 0.001. 53
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Fig. 3. Expression of MMPs in the oral mucosa of 5-FU-treated SPF and germ-free mice. (A) MMP-3 and (B) MMP-9 in 5-FU-treated SPF and germ-free mice. Immunostaining of MMPs in the connective tissue of oral mucosa tissues (C) MMP-3 immunostaining in 5-FU-treated SPF mice, (D) MMP-3 immunostaining in 5-FUtreated germ-free mice (E) MMP-9 immunostaining in 5-FU-treated SPF mice, (F) MMP-9 immunostaining in 5-FU-treated germ-free mice. Arrow heads indicate positively stained cells. Quantitative analysis of (G) MMP-3 in 5-FU-treated SPF and germ-free mice, and (H) MMP-9 in 5-FU-treated SPF and germ-free mice. Magnification 400 × . Scale bar: 100 μm. ***p < 0.001.
Bacterial species such as Porphyromonas gingivalis, Porphyromonas micra, Treponema denticola, and Fusobacterium nucleatum were found to be associated with OM-related ulcerations in hematopoietic stem cell transplant patients (Laheij et al., 2012). In another clinical study, taxon Capnocytophaga was found to be abundant in OM and proposed to be an important bacterial species in OM progression (Napenas et al., 2007). However, it remains unclear if these reported perturbations in oral microbiota are clinically significant since the impact of bacteria in the severity of OM is uncertain. To confirm the role of oral flora in OM, in this study, the severity of chemotherapy-induced OM was compared in conventional (SPF) and germ-free in vivo models. The oral cavity of germ-free mice are free of all microorganisms. However, SPF mice harbor bacteria of the genera Enterococcus and Streptococcus, Lactobacillus, Staphylococcus, Propionibacterium. Among these the predominant species reported include Lactobacillus murinus, Staphylococcus aureus, Streptococcus faecalis, Staphylococcus sciuri and Escherichia coli (Trudel, St. Amand, Bareil, Cardinal, & Lavoie, 1986). In addition under immunosuppressed conditions such as cancer therapy, Candida species is capable of inducing candidiasis in mice (Costa, Pereira, Junqueira, & Jorge, 2013). Unlike other disease processes where the use of germ-free models has become a powerful tool to explore the interplay between host and microorganisms (Yi & Li, 2012), there has not been a study to confirm the biological significance of the oral flora and the changes observed during OM.
3.3. Pro-inflammatory cytokines expression in SPF and germ-free mice The expression of pro-inflammatory cytokines i.e. IL-1β, TNF-α and MPO in the oral mucosa of SPF mice were significantly higher compared to germ-free mice (Fig. 4A–C). IHC analysis revealed that the cells expressing IL-1β, TNF-α and MPO were localized within the connective tissues of the oral mucosa in both 5-FU-treated SPF and germ-free mice (Fig. 4D–I). IL-1β and TNF-α in 5-FU-treated SPF mice were predominantly expressed by macrophages and T-lymphocytes while MPO was primarily associated with neutrophil granulocytes. On quantitative analysis, levels of IL-1β, TNF-α, and MPO were significantly higher in oral mucosa of 5-FU-treated SPF mice compared to germ-free mice (Fig. 4J–L). 4. Discussion Antineoplastic chemotherapy leads to multiple OM biological events resulting in the damage of oral mucosa integrity due to impairment of the turnover of basal keratinocytes. Interestingly, accompanying changes in oral flora have been reported during OM (Vasconcelos et al., 2016). In OM, clinical observational studies have shown that Gramnegative bacteria increased in the oral cavity during chemotherapy with corresponding decrease in levels of Gram-positive microorganisms (Laheij et al., 2012; Napenas et al., 2007; Stokman et al., 2003). 54
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Fig. 4. Expression of pro-inflammatory mediators in the oral mucosa of 5-FU-treated SPF and germ-free mice. (A) IL-1β, (B) TNF-α and (C) MPO in 5-FU-treated SPF and germ-free mice. Immunostaining of cells expressing (D–E) IL-1β, (F–G) TNF-α and (H–I) MPO in the connective tissues of the oral mucosa in SPF and germ-free mice. Quantitative analysis (J) IL-1β, (K) TNF-α and (L) MPO stained positive cells in 5-FU-treared SPF and germ-free mice. Magnification 400 × . Scale 100 μm. ***p < 0.001.
observed elevated levels of expression of MMP-3 and -9 in the basal epithelium, lamina propria and submucosa of 5-FU dosed SPF mice. MMP-3 could contribute to mucosal damage by disruption of cell-to-cell and cell-to-ECM through degradation of collagens, fibronectin, laminin, aggrecan, insulin-like growth factor binding protein-3 and serpins. The active enzyme also activates MMP-1, -8, -9, and -13 thus amplifying the process of tissue damage (Song, Park, & Sung, 2013). Our results are consistent with a study carried out by Al‐Azri et al. (2015)) where the authors found that MMP-3 and -9 is up-regulated by irinotecan in all layers of epithelium, lamina propria and submucosa of tongue tissue in mice. As tissue injury progresses during the pathogenesis of OM, the tight junction proteins such as E-cadherin, occludin and claudin-1 are downregulated which could further weaken the barrier function of oral mucosa. This allows for increased permeability and conduit of bacterial products (Pedroso et al., 2015; Xu et al., 2014) leading to advanced tissue damage. In this study, we found that in 5-FU-treated SPF mice, the damage to the oral epithelium was more severe with elevated levels of mediators of innate immune responses such as IL-1β, TNF-α, and MPO compared to 5-FU-treated germ-free mice. Furthermore, the proliferative potential of the basal epithelium was compromised in SPF mice compared to the germ-free mice. These results are consistent with the findings from an intestinal mucositis study by Pedroso et al. (2015) where the authors reported that CPT-11-treated SPF models showed greater damage to the epithelial structure and thickness, increased inflammatory infiltrates and loss of cell differentiation in SPF mice compared to the germ-free mice resulting in an increased intestinal permeability. The oral epithelium, fibroblasts, endothelial cells and infiltrating immune cells express a repertoire of pattern recognition receptors (PRRs) including such as toll-like receptor (TLR)-2, -4, -9, and nucleotide-binding oligomerization domain-containing protein-1 (NOD-1) and -2 (Piccinini & Midwood, 2010). Therefore, damage of oral mucosa barrier function could intensify inflammation by allowing translocation of bacterial products and toxins to cells in the submucosa region. In addition, damage-associated molecular patterns (DAMPs)
Initiation of OM begins either as a direct consequence of the toxicity of chemotherapy agents or harmful effects of cellular reactive oxygen species (ROS) which leads to oxidative stress. High levels of ROS induce DNA strand breaks, lipid peroxidation, protein mis-folding, aggregation and degradation thus affecting normal cell proliferation/turnover (Conklin, 2004). This is evident in our results where 5-FU-treated SPF mice showed hallmark histopathological features of OM where proliferation of oral mucosa cells was significantly reduced compared to the control mice. Oxidative stress induces the activation of transcription factor NF-κB leading to increased expression of pro-inflammatory cytokines (Sonis, 2004). Indeed, we found that the pro-inflammatory cytokines, IL-1β and TNF-α are expressed by cells in the oral epithelium, endothelium and mucosal connective tissues in 5-FU-treated SPF mice. Similar results were reported by Bertolini et al. (2017) where the tongue tissues of mice dosed with 5-FU showed epithelial atrophy with reduced number of Ki67 stained positive cells and increased expression of pro-inflammatory cytokines. ROS itself also elicits endothelial dysfunction causing vasodilation enhancing migration of leukocytes from the blood vessels into the extracellular matrix (ECM). Neutrophils which are one of the principal cell types in innate immune response of OM, express MPO when activated (Selders, Fetz, Radic, & Bowlin, 2017). We observed elevated levels of MPO in the connective tissues of the oral mucosa in 5-FU-treated SPF mice. MPO, which is released from neutrophil’s azurophilic granules, catalyzes the production of hypochlorous acid causing oxidative damage to host tissues (Manicone & McGuire, 2008). One of the consequences of tissue damage is the activation of MMPs. MMPs, also known as matrixins, are group of enzymes that are present in the extracellular environment of cells and are responsible for the degradation of most extracellular matrix (ECM) proteins during organogenesis, growth and normal tissue turnover. Under normal physiological conditions, the expression of MMPs is low in oral mucosa tissues (Al‐Azri et al., 2015). MMP-3 (stromelysin) is expressed by fibroblasts, endothelial cells, macrophages, vascular smooth muscle cells and keratinocytes in response to stimuli (Maksymowych et al., 2007). We 55
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which are biomolecules released extracellularly as a result of cellular stress and tissue injury would further intensify the inflammatory response. One such DAMP is the high mobility group box-1 protein (HMGB-1) which is recognized by toll-like receptors (TLRs) especially TLR-2 and TLR-4, and receptor for advanced glycation end products (RAGE) (Diener, Al‐Dasooqi, Lousberg, & Hayball, 2013). HMGB-1 released by necrotic cells induces both innate and adaptive immunity response by activating antigen-presenting cells to counter tissue damage caused by bacterial infiltration. It can also act like a chemokine or cytokine by ligation with specific receptors such as TLR-2, TLR-4 and TLR-9 (Diener et al., 2013). Thus, PAMPs, DAMPs and their overlapping receptors could further drive the immune response in OM. In summary, the present study demonstrates the detrimental impact of oral microbiota in worsening chemotherapy-induced OM. To our knowledge, this is the first study comparing the severity of OM in germfree and SPF in vivo models. Future research needs to be formulated to observe if the degree of oral microflora colonization and/or enrichment of specific bacterial species play a more significant role in OM pathogenesis. Answers to these questions will facilitate efforts to drive novel therapeutic interventions for OM.
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Funding This research was supported by National University Health System (grant number R221-000-088-733). Conflict of interest statement The authors declare no conflict of interest. Author contributions Conceptualization: K.S.T. & C.H.L.H.; Study design: N.G., Y.J.F., J.F., K.S.T. & C.H.L.H. Data acquisition: N.G., S.Y.Q., K.S.T.; Data analysis and interpretation: N.G., Y.J.F., J.F., K.S.T. & C.H.L.H; Statistical analysis: N.G.; K.S.T; Manuscript preparation: N.G., K.S.T. & C.H.L.H.; Manuscript editing: N.G., S.Y.Q., J.F.Y., J.F., K.S.T. & C.H.L.H. All authors have read and approved the final article. References Al‐Azri, A. R., Gibson, R. J., Bowen, J. M., Stringer, A. M., Keefe, D. M., & Logan, R. M. (2015). Involvement of matrix metalloproteinases (MMP‐3 and MMP‐9) in the pathogenesis of irinotecan‐induced oral mucositis. Journal of Oral Pathology and Medicine, 44, 459–467. Bailey, C., Hembry, R., Alexander, A., Irving, M., Grant, M., & Shuttleworth, C. (1994). Distribution of the matrix metalloproteinases stromelysin, gelatinases A and B, and collagenase in Crohn’s disease and normal intestine. Journal of Clinical Pathology, 47, 113–116. Bertolini, M., Sobue, T., Thompson, A., & Dongari-Bagtzoglou, A. (2017). Chemotherapy induces oral mucositis in mice without additional noxious stimuli. Translational Oncology, 10, 612–620. Bowen, J. M., Gibson, R. J., & Keefe, D. (2011). Animal models of mucositis: Implications for therapy. The Journal of Supportive Oncology, 9, 161–168. Cani, P. D., Rodrigo, B., Knauf, C., Waget, A., Neyrinck, A. M., Delzenne, N. M., ... Burcelin, R. (2008). Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes, 57, 1470–1481. Conklin, K. A. (2004). Chemotherapy-associated oxidative stress: Impact on chemotherapeutic effectiveness. Integrative Cancer Therapies, 3(4), 294–300. Costa, A. C., Pereira, C. A., Junqueira, J. C., & Jorge, A. O. (2013). Recent mouse and rat methods for the study of experimental oral candidiasis. Virulence, 4(5), 391–399. de Mendonca, R. M., de Araujo, M., Levy, C. E., Morari, J., Silva, R. A., Yunes, J. A., ... Brandalise, S. R. (2012). Prospective evaluation of HSV, Candida spp., and oral bacteria on the severity of oral mucositis in pediatric acute lymphoblastic leukemia. Supportive Care in Cancer : Official Journal of the Multinational Association of Supportive Care in Cancer, 20, 1101–1107. Diener, K. R., Al‐Dasooqi, N., Lousberg, E. L., & Hayball, J. D. (2013). The multifunctional alarmin HMGB1 with roles in the pathophysiology of sepsis and cancer. Immunology and Cell Biology, 91, 443–450. Elting, L. S., Cooksley, C., Chambers, M., Cantor, S. B., Manzullo, E., & Rubenstein, E. B. (2003). The burdens of cancer therapy. Clinical and economic outcomes of
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