Interplay of zinc deficiency, epithelial microdamage, and Escherichia coli infection in a mouse model of oral lichen planus

Interplay of zinc deficiency, epithelial microdamage, and Escherichia coli infection in a mouse model of oral lichen planus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Interplay of zinc deficiency, epithelial microdamage, and Escherichia coli infection in a mouse model of oral lichen planus Youngnim Choi, Phuc Vo, Joo-Young Park, Hyeong-jin Kim, Hye-Jung Yoon, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3913717/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oral lichen planus (OLP) is a chronic T-cell-mediated inflammatory mucosal disease of unknown etiology. The lack of suitable animal models has hampered understanding of its etiopathogenesis. This study aimed to elucidate the contribution of bacterial infection and zinc deficiency (ZD) to the pathogenic processes underlying OLP by developing a murine model. After subjecting to standard or zinc-deficient diets, C57BL/6 mice underwent labial mucosal microdamage via scratching, followed by oral administration of OLP-isolated Escehrichia coli 7.2. Scratching alone triggered bacterial translocation to the epithelium and lamina propria, upregulated Mmp9 , increased immune responses in the cervical lymph nodes, and augmented CD4 + T-cell recruitment to labial mucosae. E. coli infection intensified these responses, in strong synergism with ZD, which shifted the Th response from Th1 to Th17 dominance. Repeated scratching plus E. coli infection amplified T-cell recruitment, even without ZD, leading to the development of severe inflammatory foci in the labial mucosa, characterized by colloid bodies and disrupted basement membranes. Interestingly, Th1 blockade during E. coli infection hindered bacterial clearance in the epithelium and caused detachment of the epithelium from the underlying lamina propria with dense inflammatory infiltrates. This suggests that the Th1/IFNγ pathway may not be a suitable therapeutic target for OLP. In conclusion, OLP-like histopathology in the oral mucosa was induced through E. coli infection, synergized by repeated epithelial microdamage, ZD, or Th1 blockade. This animal model provides a valuable platform for exploring specific hypotheses related to OLP pathogenesis and potential therapeutic targets. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Oral lichen planus (OLP) is a prevalent oral mucosal disease that affects approximately 0.5–4% of the general population 1 . Clinical presentations of OLP encompass six distinct types—reticular, papular, plaque, atrophic, erosive, and bullous—often affecting regions such as the buccal mucosae, tongue, and gingivae. OLP is characterized as a chronic inflammatory condition primarily mediated by T cells with histopathological features, including a band-like subepithelial and intra-epithelial infiltration of T cells, liquefaction of the basal cell layer of epithelia with colloid bodies, and saw-tooth form rete pegs 2 . Despite extensive research, the etiology of OLP remains elusive, with several potential triggers, including genetic and psychological factors, systemic medications, trauma, and microbial infections 2 . Notably, the mechanisms underlying T-cell activation and recruitment are still poorly understood. A previous study revealed an increased presence of bacteria in the basal cell layer of epithelium and lamina propria at OLP lesions, along with a positive correlation between bacterial quantities and T-cell infiltration 3 . Moreover, the presence of immunologic synapse-like structures between infected epithelial cells and T cells suggests that T cells may be responding to bacterial antigens presented by infected cells 3 . Comparative analysis of bacterial communities within OLP lesions unveiled an enrichment of Escherichia coli , a common gut commensal bacterium, though pathogenic varieties exist 4 . Although E. coli strains isolated from OLP tissues lack the major virulence factors of pathogenic E. coli , they efficiently invade into and survive within human oral keratinocytes (HOKs) 4 , 5 . These findings suggest the possibility that infection with the OLP-isolated E. coli could trigger T-cell activation and infiltration in OLP. Transcriptomic data analysis of OLP has identified an enrichment of gene signatures associated with barrier defects, wound healing, and response to infection. Conversely, genes linked to response to zinc ions were found to be decreased in OLP cases 6 . Zinc deficiency (ZD) can induce immune dysfunction and increase susceptibility to infection by disrupting epithelial barriers, suppressing phagocyte function, reducing the activities of natural killer cells and cytotoxic T lymphocytes (CTLs), and dampening Th1 cell responses 7 . Notably, several studies have reported significant reductions in serum and/or saliva zinc levels in OLP patients 8 , 9 . Intriguingly, zinc stands out as a trace element that has demonstrated therapeutic effects in OLP lesions through both topical and systemic supplementation 10 – 12 . Nevertheless, the precise role of ZD in the pathogenesis of OLP remains unclear. The absence of suitable animal models for OLP poses a significant obstacle to research aimed at comprehending its etiopathogenesis and exploring novel therapeutic avenues. This study aims to elucidate the contribution of bacterial infection and ZD to the pathogenic processes underlying OLP by developing a murine model. In this model, we harness the intricate interplay between ZD, mechanical epithelial microdamage, and the oral infection of an OLP-isolated E. coli strain. RESULTS Effect of ZD on E. coli invasion and survival in HOKs To investigate the impact of ZD on bacterial invasion and survival, primary HOKs were infected with an OLP-isolated strain E. coli 7.2, a human gut commensal-derived laboratory strain E. coli K-12 MG1655, and the oral commensal species Streptococcus salivarius in the absence or presence of N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), a zinc chelator. The concentration of TPEN (1 µM) chosen for the study did not affect HOK viability (Fig. 1 a). TPEN did not affect the invasion rates, which were determined by flow cytometry (Fig. 1 b). However, both E. coli strains significantly increased their intracellular survival under ZD, while S. salivarius did not survive (Fig. 1 c). In exploring the potential involvement of reactive oxygen species (ROS) production in the increased intracellular survival of E. coli with TPEN, the levels of intracellular ROS were quantified using 2′,7-dichlorofluorescein diacetate (DCF-DA), a cell-permeant indicator sensitive to oxidation by ROS. While HOKs significantly increased the ROS in the presence of TPEN alone, as previously reported 13 , 14 , E. coli , unlike most bacteria, notably suppressed ROS production in HOKs (Fig. 1 d). Recent research has established zinc poisoning by phagolysosomal accumulation of free zinc as a mechanism for killing intracellular bacteria in macrophages 15 , 16 . To investigate if a similar mechanism occurs in HOKs, intracellular free zinc was visualized using a zinc-specific fluorescent probe, FluoZin-3. E. coli infection significantly elevated the intracellular zinc signals that colocalized with endosomal E. coli , while infection under ZD neutralized this effect (Fig. 1 e). The increase in intracellular free zinc accompanied the regulation of zinc transporter genes in HOKs: upregulation of SLC30A1 encoding ZnT1 that can deliver zinc into vesicle lumen and downregulation of SLC39A1/ SLC39A2 encoding ZIP1/ZIP2 that can deliver zinc from the vesicle lumen to cytoplasm. HOKs also upregulated genes involved in zinc detoxification, such as metal-regulatory transcription factor 1 ( MTF1 ), metallothionein 1 ( MT1 ), and MT2 . However, these changes in gene expression were not observed under ZD conditions (Fig. 1 f). These results collectively suggest that HOKs employ zinc toxicity, rather than relying on ROS, to combat intracellular E. coli , and zinc deprivation mitigates this effect, allowing more bacteria to survive within HOKs. Impact of ZD, epithelial microdamage, and E. coli infection on immune response in the cervical lymph nodes of mice To explore the impact of ZD on the immune response to oral E. coli infection, the C57BL/6 mice were subjected to either a control diet (CD) or a zinc-deficient diet (ZDD) for one month. In our preliminary experiments, oral administration of E. coli did not cause bacterial invasion into oral tissues, probably due to the keratinized nature of mouse oral mucosa. To facilitate E. coli invasion into the epithelial cells, we introduced a mucosal microdamage model by scratching the labial mucosa using a dental stone bur. Mice from each diet were divided into three groups: non-scratching (NS, n = 6/group), scratching alone (S, n = 7/group), or scratching followed by infection with 10 9 viable cells of E. coli 7.2 (S + I, n = 7/group). The mice were sacrificed either three days or 21 days after a single episode of scratching and infection, and the latter additionally received repeated E. coli 7.2 inoculations without scratching (Fig. 2 a). The successful induction of ZD by ZDD was confirmed by serum zinc level upon sacrifice (Fig. 2 b). When the impact of ZD on immune cell composition in the cervical lymph nodes, mesenteric lymph nodes, and spleen was analyzed by flow cytometry (Fig. S1 ), ZDD resulted in the specific enlargement of the cervical lymph nodes primarily due to T-cell expansion in the NS group (Figs. 2 b and S2). Both scratching and infection amplified this ZDD-induced expansion of immune cells, including lymphocytes, dendritic cells, monocytes, and granulocytes. The degree of expansion was reduced at Day 21 compared to Day 3 despite the repeated oral inoculation of E. coli 7.2, confirming that E. coli 7.2 does not infect mouse oral mucosae without microdamage (Fig. 2 b). Moreover, ZDD reduced the Th1/Th17 ratio, particularly in the cervical lymph nodes of the NS group due to a reduction in Th1 cells (Figs. 2 c and S2). While Th1 cells decreased only under ZD, scratching increased regulatory T (Treg) cells, and E. coli infection increased Th17 cells. Consequently, the Th1/Th17 ratio synergistically decreased with ZD and E. coli infection, while the Th17/Treg ratio increased solely due to E. coli infection, particularly at Day 3 (Fig. 2 d). These results indicate that E. coli infection under ZD conditions altered T-cell responses. Synergistic effects of E. coli infection and ZDD on bacterial invasion and inflammation in labial mucosae The impact of E. coli infection and ZD on bacterial invasion of labial mucosae was investigated using in situ hybridization targeting bacterial 16S rRNA. Scratching alone increased eubacterial invasion into both the epithelium and lamina propria, particularly under ZD conditions at both Day 3 and Day 21. E. coli infection further amplified bacterial presence in tissues only at Day 3 (Figs. 3 a and 3 b). Additionally, examination of E. coli invasion using an E. coli -specific probe revealed a similar pattern with less signal intensity and statistical significance (Fig. S3). For bacteria to reach the lamina propria, the basement membranes need to be compromised. As matrix metalloproteinase-9 (MMP-9) can break down key components of the basement membrane, such as type IV collagen and laminin 17 , its expression was examined. In the mouse labial tissues, scratching alone upregulated expression of the Mmp9 gene, further enhanced by E. coli infection, particularly under ZD conditions (Fig. 3 c). To relate the expression of the Mmp9 gene to that of protein, HOK in vitro infection system was utilized. E. coli infection and ZD synergistically upregulated MMP9 mRNA and protein. However, active MMP-9 production occurred solely upon E. coli infection (Fig. 3 d). These results indicate that E. coli infection and ZD can induce barrier destruction. Histological analysis using hematoxylin and eosin (H&E) staining demonstrated that mice on ZDD had more infiltrating cells three days after E. coli infection compared to mice on CD, mostly getting resolved by Day 21. However, E. coli -infected mice under ZD conditions exhibited a higher number of remaining cells near the basal cell layer with several colloid bodies, indicating the initial stage of OLP lesion development (Fig. 3 e). Impact of ZDD and E. coli infection on CD4 + T-cell infiltration and Th1 vs. Th17 responses To gain a deeper understanding of the molecular mechanisms underlying the increased bacterial invasion and inflammatory cell infiltration, we conducted transcriptomic analysis on lip tissues from mice infected with E. coli under either CD or ZDD (Fig. 4 a). Gene set enrichment analysis (GSEA) revealed that genes expressed in the ZDD group were significantly enriched in processes related to keratinocyte differentiation, keratinization, oxidative stress-induced cell death, DNA replication, inflammatory response, chemotaxis of immune cells, and antimicrobial humoral response via antimicrobial peptides. In contrast, processes associated with antigen processing and presentation, as well as the activation of NK and NKT cells, were enriched in the CD group (Fig. 4 b). Notably, the CD group also showed enrichment in the positive regulation of Th1 cell response and IFNγ production, along with the negative regulation of IL-17 production, mirroring the findings in the cervical lymph nodes. These data suggested that mice under CD primarily responded to E. coli infection with cellular immunity, while mice under ZDD responded with armamentaria against extracellular bacteria, leading to increased inflammation and immune cell infiltration. To validate the transcriptomic analysis results at the protein level, we performed immunofluorescence using anti-CD4, IFNγ, and IL-17 antibodies to assess the infiltration of Th1 vs. Th17 cells in the labial mucosae. Scratching alone induced the recruitment of CD4 + T cells, with similar levels between the CD and ZDD groups. E. coli infection further heightened CD4 + T-cell infiltration, which was exacerbated under ZD (Figs. 4 c and 4 d). These infiltrated cells were detected both in the epithelium and the lamina propria. In the CD group, the recruited CD4 + T cells were Th1-dominant, while in the ZDD group, they were Th17-dominant (Figs. 4 c, 4 e, and 4 f). This comprehensive analysis suggests that differential immune responses to E. coli infection were elicited in the CD and ZDD groups, impacting the balance between Th1 and Th17 cell populations in the context of the oral mucosa. Impact of repeated scratching and infection on T-cell infiltration The repeated oral administration of E. coli 7.2 without scratching failed to infect mouse oral mucosae. To replicate the chronic inflammatory nature of OLP, we investigated whether repeated scratching and infection would induce a more robust T-cell response and heightened inflammation. Following one month of controlled diets, mice (n = 5/group) underwent two (2S + I) or three (3S + I) rounds of scratching and infection at 10-day intervals, and these groups were compared with the NS group, which was sacrificed concurrently with the 3S + I group (Fig. 5 a). Analysis of immune cell populations from the cervical lymph nodes in the 2S + I group yielded results similar to those of mice infected only once (Fig. S4). Histological examination revealed a more prevalent lymphocytic infiltration throughout the section of the labial mucosae compared to the single infection. Several mice, both from the CD and ZDD groups, developed severe inflammatory foci in the epithelium and adjacent lamina propria, closely mimicking OLP-like histopathology (Figs. 5 b and S5). Moreover, the characteristic "saw-tooth" shaped rete pegs tended to be more prominent upon repeated scratching and infection (Fig. S5). To confirm T-cell infiltration, CD3 immunohistochemical staining was conducted. Regardless of dietary conditions, mice typically exhibited only a few T cells in the epithelium and lamina propria throughout the section, which significantly increased with scratching and infection (Figs. 5 b and 5 c). Repeated scratching and infection presented a notable trend of increasing T-cell infiltration only in mice fed with CD, partially counteracting the effects of ZD upon three-time infection (Fig. 5 c). Additionally, silver staining revealed destruction of the basement membrane in areas exhibiting inflammatory foci (Fig. 5 b). Impact of Th1 blockade on bacterial clearance and inflammation in labial mucosae To elucidate the role of Th1 cells in controlling E. coli infection and OLP-like histopathology, Th1 differentiation was blocked by injecting anti-IFNγ and anti-IL-12 antibodies during E. coli infection (Fig. 6 a). Flow cytometric analysis confirmed a significant reduction in Th1 cells in the cervical lymph nodes, although the decrease in the Th1/Th17 ratio did not reach statistical significance (Fig. 6 b). Th1 blockade impeded bacterial clearance in the labial mucosae, particularly in the epithelium (Figs. 6 c and 6 d). Moreover, CD3 immunohistochemistry revealed a significant increase in T-cell infiltration into the lamina propria in the Th1 blockade group (Figs. 6 e and 6 f). Notably, the number of T cells in the lamina propria showed a strong positive correlation with the bacterial quantities in the epithelium rather than with those in the lamina propria (Fig. 6 g). Interestingly, four out of the five mice in the Th1 blockade group developed lesions characterized by epithelial detachment from the underlying lamina propria. These lesions were accompanied by dense inflammatory infiltrates, including T cells, and destruction of the basement membrane, closely resembling one of the key features of OLP pathology (Figs. 6 e and S6). DISCUSSION OLP is a chronic inflammatory mucosal disorder characterized by T cell-mediated immune responses, but the lack of suitable animal models has limited the ability to understand its etiology and pathogenesis. This study marks a groundbreaking step forward by introducing the first successful animal model that replicates several vital aspects of OLP, including T-cell infiltration into the epithelium and subepithelial lamina propria, the presence of colloid bodies, induction of Mmp9 , basement membrane destruction and detachment of the epithelium from the underlying lamina propria, bacterial invasion of tissue, and skewed Th17 responses. The interplay of ZD, epithelial microdamage, and E. coli infection was required to develop OLP-like histopathology in mice. ZD led to reductions in Th1 cells and the Th1/Th17 ratio in the cervical lymph nodes, which aligns with previous findings 18 , 19 . Although ZD has been linked to enhanced susceptibility to bacterial infection 7 , ZD alone did not cause any bacterial invasion or inflammation in the labial mucosa despite the enlarged cervical lymph nodes, highlighting the roles of epithelial microdamage and infection with specific microorganisms such as E. coli in the current model. It is crucial to recognize that OLP typically presents later in life when individuals, particularly postmenopausal women, begin to experience mucosal thinning, higher susceptibility to masticatory trauma, decreased muscle tone, and xerostomia due to chronic adult diseases and medications, which all contribute to increased friction between the buccal mucosa and gingiva 20 , 21 . No enhancements in bacterial invasion of labial mucosa on Day 21 compared to Day 3 despite the repeated E. coli oral inoculations confirmed our preliminary results that E. coli does not infect oral tissues in mice with intact epithelium. As opposed to in humans, the buccal and labial mucosae of mice are keratinized, potentially offering a more robust barrier function than those in humans 22 , 23 . In this context, the introduction of microdamage to the epithelium through scratching played a fundamental role. Scratching alone triggered bacterial translocation to the epithelium and lamina propria, upregulation of Mmp9 , increased immune responses in the cervical lymph nodes, and increased CD4 + T-cell recruitment to labial mucosa. As shown in HOKs infected with E. coli , the bacteria translocated to the epithelium might have induced the production of active MMP9 protein from the keratinocytes. MMP9 can further negatively impact the integrity of the epithelium by reducing the expression of junctional proteins and destructing the basement membrane 24 – 26 , leading to bacterial invasion into the lamina propria and T-cell recruitment. Notably, E. coli infection exacerbated Mmp9 expression, bacterial invasion of labial mucosae, immune response in the cervical lymph nodes, and CD4 + T-cell recruitment, surpassing the effect of scratching alone. The effect of E. coli infection on T-cell infiltration and inflammation of labial mucosa increased with repeated scratching and infection. The heightened inflammatory response at infection sites might be attributed to E. coli 's ability to augment the Th17 cell population and the Th17/Treg ratio, as documented previously 27 . Furthermore, E. coli infection displayed a stronger synergism with ZD, leading to enhanced Mmp9 expression, bacterial invasion of tissues, and T-cell infiltration, along with a reduced Th1/Th17 ratio in the cervical lymph nodes, compared to the effects of scratching alone. Scratching would have caused translocation of commensals that have low invasive ability, as previously reported 28 . In contrast, E. coli causes intracellular infection in keratinocytes. As observed in HOKs, zinc poisoning rather than ROS played a pivotal role in the control of E. coli infection in oral keratinocytes, and intracellular survival of E. coli in HOKs increased under ZD conditions. Additionally, Th cells that infiltrated the oral mucosa in response to E. coli infection were shifted from Th1-dominance to Th17-dominance under ZD conditions. Blockade of Th1 differentiation during E. coli infection in the mice fed with CD significantly impeded bacterial clearance in the epithelium, suggesting the importance of Th1 cells in the control of intracellular infection. Importantly, GSEA revealed that the mice fed with ZDD responded to E. coli infection with a humoral immune response characterized by the production of antimicrobial peptides, while not effective against intracellular bacteria, whereas the mice fed with CD induced mostly cell-mediated immunity, emphasizing the importance of skewed immune responses within the context of OLP. Furthermore, the oral mucosa of the mice infected with E. coli under ZD conditions upregulated genes associated with keratinocyte differentiation and keratinization, mirroring findings in human OLP lesions 6 . All these results underscore the pivotal roles of the interplay between E. coli and ZD in the current model and provide insights into the therapeutic effects of zinc supplementation reported in OLP 10 – 12 . Both Th1 and Th17 cells are increased in OLP lesions compared to healthy controls 29 , 30 . It coincides with the finding in this study that E. coli -infected mice from both the CD and ZDD groups exhibited higher levels of both Th1 and Th17 cells than the NS group. In the past, a dominance of Th1 over Th2 was documented in OLP lesions and peripheral blood T cells 31 , 32 . More recently, the predominance of Th17 cells has been consistently reported, particularly in erosive OLP 33 – 35 . However, the specific roles played by each subset of Th cells in the pathogenesis of OLP remain speculative 36 . Th1 cells are believed to play crucial roles in the pathogenesis of OLP through the production of inflammatory cytokine TNFα and assisting in the activation of CD8 + T cells. Interestingly, blocking Th1 differentiation during E. coli infection in the current model induced the detachment of the epithelium from the underlying lamina propria with dense inflammatory infiltrate, along with the complete destruction of the basement membrane. This appears to represent an earlier stage of erosive OLP lesions. It is not clear why a similar phenotype did not develop in the mice infected with E. coli under ZD conditions, despite similar reductions in Th1 cells; this could be presumably attributed to anti-IFNγ antibodies administered during E. coli infection which may have neutralized the effector function of Th1 cells, as well as differentiation. This suggests that the Th1/IFNγ pathway may not be a suitable therapeutic target for OLP. It is essential to acknowledge the limitations of this study, as the multifaceted nature of OLP cannot be entirely reproduced in a single animal model. Although we attempted to measure the thickness of the keratin layer based on the enrichment of genes involved in keratinization, the thicknesses of the keratin layer and the epithelium significantly varied even within a single mouse, depending on the location. Further, our model did not address the infiltration of CD8 + T cells, a cell type abundantly present in OLP lesions and believed to mediate keratinocyte apoptosis 1 , 37 . Additionally, the degree of T-cell infiltration observed in the current model is less severe than that observed in OLP patients. Notably, only a few T cells are typically distributed in the labial mucosa of mice maintained under specific pathogen-free conditions, whereas human lesions develop over months to years. Nevertheless, this animal model serves as a valuable platform for investigating specific hypotheses related to the pathogenesis and potential therapeutic targets of OLP. It demonstrates that OLP-like histopathology in the oral mucosa can be induced through E. coli infection when combined with repeated epithelial microdamage, ZD, or Th1 blockade. The exploration of the mechanisms underlying these factors offers an opportunity to enhance our comprehension of OLP's pathogenesis and opens avenues for the investigation of potential therapeutic interventions. MATERIALS AND METHODS Study design Our study aimed to elucidate the contribution of bacterial infection and ZD in the pathogenesis of OLP using a murine model. The effect of ZD on the intracellular survival of E. coli was examined in HOKs in vitro in the presence of TPEN, a zinc chelator. ZD was induced in the mice by feeding them a ZDD for one month. Further, E. coli infection of the labial mucosae was induced by oral administration of E. coli following epithelial microdamage. We evaluated the effects of ZD, epithelial microdamage, and E. coli infection on the Th-cell response in the cervical lymph nodes, bacterial invasion of labial mucosae, Mmp9 expression, Th1 vs. Th17 infiltration, and inflammation in the labial mucosae. Additionally, we analyzed the effects of repeated epithelial microdamage plus E. coli infection and Th1 blockade during E. coli infection on bacterial clearance, T-cell infiltration, mucosal inflammation, and basement membrane disruption. In all the experiments, the animals were randomly assigned to different groups without blinding. However, all histological analyses were blindly performed without identifying the group to which each image belonged. The first animal experiment, presented in Figs. 2 and 3 , was conducted in three independent experiments, and the results were combined. The sample sizes for the following experiments were determined as n = 5/group based on the mean and standard deviation of Th1 cells in the NS groups fed with either CD or ZDD, a parameter that showed the least difference among various comparisons. No samples or animals were excluded from our analyses, although the transcriptomic analysis was performed only on randomly chosen three mice per group from two groups. Bacteria culture E. coli K-12 MG1655 (American Type Culture Collection, Manassas, VA, USA) and E. coli 7.2, a strain isolated from an OLP tissue 4 , were cultured in Luria-Bertani broth (BD Biosciences, Franklin Lakes, NJ, USA) at 37°C under aerobic conditions without shaking. Streptococcus salivarius KCTC 5512 (Korean Collection for Type Culture, Seoul, Korea) and Fusobacterium nucleatum ATCC 51191 (ATCC) were cultured in a brain heart infusion broth supplemented with 5 µg/ml hemin (Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/ml vitamin K 3 (Sigma-Aldrich) under anaerobic conditions (10% CO 2 , 10% H 2 , and 80% N 2 ) at 37°C. Cell culture Primary HOK cells (ScienCell, Carlsbad, CA, USA) were cultured on a poly-L-lysine-coated dish in an oral keratinocyte medium (OKM; ScienCell) supplemented with manufacturer’s growth factors and penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 . The effect of various concentrations of TPEN (Sigma-Aldrich) on HOK viability was determined using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Bacterial invasion of HOKs and intracellular survival HOKs were seeded into 24-well (1 x 10 5 /well) or 48-well (6 x 10 4 /well) plates and incubated overnight. The cells were infected with bacteria labeled with 1 µM pHrodoRed (Invitrogen, Carlsbad, CA, USA) at a multiplicity of infection (MOI) of 1000 in antibiotics-free OKM for 2 hours in the presence or absence of 1 µM TPEN (Sigma-Aldrich). For invasion analysis, the infected cells were detached using trypsin-EDTA, stained with trypan blue, and subjected to flow cytometric analysis using a FACSCalibur (BD Biosciences). For the measurement of intracellular survival of bacteria, the infected cells were subjected to antibiotics protection assay using gentamicin by culturing for additional 24 hours in the absence or presence of TPEN, and the number of colony-forming units grown on agar plates was counted. Measurement of ROS production HOKs (2 x 10 4 ) were seeded into 96-well flat bottom black plates and incubated with 10 µM of DCF-DA (Sigma-Aldrich) at 37 o C for 30 minutes. After washing, the cells were infected with E. coli 7.2, E. coli K-12 MG1655, S. salivarius , and F. nucleatum at MOI 1000 for 24 hours, in the absence or presence of 1 µM TPEN. The DCF fluorescence intensity was measured at the indicated time points at excitation and emission wavelengths of 485 and 535 nm, respectively, using a fluorometric plate reader (Synergy H1-Multi Mode Microplate Reader, BioTek, Winooski, VT, USA). Intracellular Zn2 + measurements HOKs were seeded into 24-well black plates for microscopy and infected with pHrodoRed-labeled E. coli 7.2 at MOI 1000 for 2 hours. After fixation, the cells were stained with 1 µM FluoZin-3 AM (Invitrogen) and observed under an LSM 980 confocal laser scanning microscope (Carl Zeiss Inc. Oberkochen, Germany), recorded, and analyzed using the Zen software (Carl Zeiss, Inc.). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA was isolated from HOKs or mouse lip tissues using an easy-BLUETM total RNA extraction kit (iNtRON Biotechnology, Gyeonggi-do, Korea) in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using 2 µg of total RNA, oligo dT, and M-MLV reverse transcriptase (Promega, Madison, WI, USA) in 30 µl of the reaction mixture at 42 o C for 1 hour. Real-time PCR was performed in 20 µl of the mixture, including primer pairs (400 nM each), cDNA (2 µl), and 10 µl of Power SYBR® Green Mastermix (Applied Biosystems, Waltham, MA, USA) under the following condition: initial denaturation at 95 o C for 4 minutes, followed by 40 cycles of denaturation (95 o C, 15 seconds), annealing (60 o C, 15 seconds), and elongation (72 o C, 33 seconds) using QuantStudio 3 Real-Time PCR Systems (Applied Biosystems, Waltham, Massachusetts, USA). The gene expression levels were normalized with the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) gene and calculated using the 2 −ΔΔCT method. The primers used in this study are listed in Table S1 . Measurement of MMP-9 protein The quantity of total and active MMP-9 protein from HOK supernatant was measured using Fluorokine E human active MMP-9 assay kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s protocol. The fluorescence intensity was measured at excitation and emission wavelengths of 320 and 405 nm, respectively, using a fluorometric plate reader (Synergy H1-Multi Mode Microplate Reader, BioTek). Animal experiment The animal studies were conducted after approval by the Seoul National University Animal Care and Use Committee (SNU-191111-1-5 and SNU-210916-4-1) in accordance with the Animal Protection Act in Korea. Mice were maintained under specific pathogen-free conditions at 12-hour light, 12-hour dark cycle in the Laboratory Animal Facility at the School of Dentistry, Seoul National University. Eight-week-old female C57BL/6 mice were put on either CD or ZDD for 4–8 weeks. Mouse chows with normal and decreased zinc levels (30.4 ppm and 1.2 ppm, respectively) were purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The labial mucosae of mice were microdamaged by rotating a dental stone bur (Dura-Green Stones, Shank HP, Shape FL1, Shofu Inc., Kyoto, Japan) 20 times, following which the mice were orally given 10 9 cells of viable E. coli 7.2 in 50 µl PBS containing 1.5% carboxymethylcellulose (Tokyo Chemical Industry, Tokyo, Japan) according to the indicated experimental schemes. The number of rotations was determined in preliminary experiments to ensure that it does not cause traumatic ulcers on the mucosa while allowing E. coli invasion. After sacrifice, the serum, cervical and mesenteric lymph nodes, spleen, and lip tissue were collected. The lip tissue was cut along the midline; one half was stored at -80°C, and the other half was subjected to tissue processing. Th1 blockade model Th1 blockade in vivo was carried out two days before E. coli infection by injecting anti-IFNγ antibodies (200 µg/mouse; clone XMG1.2, BioXCell, Lebanon, NH, USA) and anti-IL-12p75 antibodies (300 µg/mouse; clone R2-9A5, BioXCell) intraperitoneally; this was repeated every three days for the entire duration of the experiment. Isotype control antibodies (clone HRPN and clone LTF-2, BioXCell) were used in the control group. Measurement of Zn 2+ in serum The Zn 2+ concentration in mice sera was measured with a commercial zinc assay kit (BioAssay Systems, Hayward, CA, USA) in accordance with the manufacturer’s instructions. Flow cytometry Single-cell suspensions isolated from the cervical lymph nodes, mesenteric lymph nodes, and spleen were prepared in DPBS containing 2.5% fetal bovine serum (FBS) and incubated with anti-CD16/CD32 antibodies to block the Fc receptor for 20 minutes at 4°C. For surface staining, the cells were stained with fluorescence-labeled antibodies for 45 minutes at 4°C. For intracellular staining of cytokines, the cells were stimulated with 50 ng/ml PMA, 1 µM ionomycin (Sigma-Aldrich), and GolgiPlug™ (BD Biosciences) in RPMI medium for 4 hours. The cell suspension was then fixed and permeabilized using a Foxp3/ Transcription Factor staining kit (Invitrogen) before being stained with fluorescence-labeled antibodies for 45 minutes at room temperature. The antibodies used in this experiment are listed in Table S2. The stained samples were run by LSRFortessa X-20 (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences). Live cells were gated by the exclusion of dead cells based on forward scatter and viability dye staining. Tissue processing and histological analyses Paraffin-embedded 4.5-µm thick sections were stained with H&E to examine overall inflammation of the labial mucosae. The basement membrane was visualized using the Jones’ methenamine silver stain 38 . To assess bacterial presence in tissue, in situ hybridization using digoxigenin-labeled universal and E. coli -specific probes targeting bacterial 16S rRNA was performed as previously described 4 . Four areas per sample were photographed at 400x magnification. Bacterial signals were quantified using the ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). The distribution of T cells was determined by immunohistochemical staining of CD3. The sections were tile scanned at 200x magnification using an Automated Upright Microscope System (Leica Biosystems, Wetzlar, Germany). The number of CD3-positive cells in the epithelium and lamina propria in the entire section was counted and expressed as the number of cells per unit length by measuring the length of tissue, using the ImageJ software. The distribution of Th1 and Th17 cells in labial mucosae was examined by immunofluorescence staining for CD4, IFNγ, and IL-17 on 10-µm thick frozen sections. A secondary antibody was used to amplify the CD4 signal. The samples were examined under an LSM 980 confocal laser scanning microscope (Carl Zeiss, Inc.), and five areas per sample were photographed at 400x magnification. The signal intensity and colocalization coefficients of images were analyzed using the ZEN software (Carl Zeiss, Inc.). The antibodies used for immunohistochemistry and immunofluorescence are listed in Table S2. Two sections per sample were blindly analyzed for all histological analyses. Transcriptomic analysis Total RNA was extracted from the lip tissue of mice, and its quality was assessed using an Agilent TapeStation 4000 system (Agilent Technologies, Santa Clara, CA, USA). High-throughput sequencing and sequence analysis were conducted by eBiogen Inc. (Seoul, Korea). In brief, library construction was performed using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria), and high-throughput sequencing was performed using a NextSeq 500 (Illumina, Inc., San Diego, California, USA). The heat map of total genes was generated using R (version 3.5.1) ( http://www.r-project.org/ ). GO biological process pathway enrichment analyses were performed with the GSEA ( http://software.broadinstitute.org/gsea/index.jsp ) as previously described 39 . The raw transcriptomic data are accessible through the BioProject database of the National Center for Biotechnology Information under accession number PRJNA1039963. Statistical analysis Data were analyzed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA) and tested for normal distribution using the D'agostino & Pearson test. A two-tailed unpaired Student’s t-test was used to determine the statistical significance between the two independent groups. A two-way analysis of variance (ANOVA) was used to determine the main effects of scratching (vs. non-scratching) and ZD or infection (vs. non-infection) and ZD. Bonferroni’s multiple comparison test was applied to examine statistical significance among multiple groups by diet (ZD vs. CD). The skewed data were handled through log transformation and subjected to ANOVA analysis. A p -value < 0.05 was considered statistically significant. Declarations Acknowledgments This research was supported by grants from the National Research Foundation of Korea (2018R1A5A2024418 and 2020R1A2C2007038). Conflict of interests Authors declare no competing interests. Author contributions: Conceptualization: YC Methodology: J-YP, H-JY Investigation: VTDP, HK, S-HC Visualization: SFB, MJM, JLS, EH Funding acquisition: YC Supervision: YC Writing – original draft: VTDP, YC Writing – review & editing: VTDP, J-YP, HK, H-JY, S-HC, YC References Deng, X., Wang, Y., Jiang, L., Li, J. & Chen, Q. Updates on immunological mechanistic insights and targeting of the oral lichen planus microenvironment. Front. Immunol. 13 , 1023213 (2022). Roopashree, M. R. et al . Pathogenesis of oral lichen planus--a review. J. Oral Pathol. Med. 39 , 729-734 (2010). Choi, Y. S. et al . The presence of bacteria within tissue provides insights into the pathogenesis of oral lichen planus. Sci. Rep. 6 , 29186 (2016). Baek, K. et al . Characterization of intratissue bacterial communities and isolation of Escherichia coli from oral lichen planus lesions. Sci. Rep. 10 , 3495 (2020). Min, H., Baek, K., Lee, A., Seok, Y. J. & Choi, Y. Genomic characterization of four Escherichia coli strains isolated from oral lichen planus biopsies. J. Oral Microbiol. 13 , 1905958 (2021). Vo, P. T. et al . Gene signatures associated with barrier dysfunction and infection in oral lichen planus identified by analysis of transcriptomic data. PLoS. One 16 , e0257356 (2021). Gammoh N. Z. & Rink, L. in Nutrition and Immunity, (eds Mahmoudi, M. & Rezaei, N.) pp. 127-158. (Springer International Publishing, 2019). Gholizadeh, N. & Sheykhbahaei. N. Micronutrients Profile in Oral Lichen Planus: a Review Literature. Biol. Trace Elem. Res. 199 , 912-924 (2021). Bao, Z. X., Yang, X. W., Shi, J. & Liu, L. X. Serum zinc levels in 368 patients with oral mucosal diseases: A preliminary study. Med. Oral Patol. Oral Cir. Bucal 21 , e335-340 (2016). Mehdipour, M. et al . Comparison of the Effect of Mouthwashes with and without Zinc and Fluocinolone on the Healing Process of Erosive Oral Lichen Planus. J. Dent. Res. Dent. Clin. Dent. Prospects 4 , 25-28 (2010). Chaitanya, N. C. et al. Zinc Therapy in Treatment of Symptomatic Oral Lichen Planus. Indian Dermatol. Online J. 10 , 174-177 (2019). Suvarna, C. et al. A Comparative Evaluation on the Effect of Oral Zinc 50 mg with or without 0.1% Triamcinolone Orabase on Oral Lichen Planus. Int. J. Appl. Basic Med. Res. 10 , 54-58 (2020). Aimo, L., Cherr, G. N. & Oteiza, P. I. Low extracellular zinc increases neuronal oxidant production through nadph oxidase and nitric oxide synthase activation. Free Radic. Biol. Med. 48 , 1577-1587 (2010). Ho, E. & Ames, B. N. Low intracellular zinc induces oxidative DNA damage, disrupts p53, NFkappa B, and AP1 DNA binding, and affects DNA repair in a rat glioma cell line. Proc. Natl. Acad. Sci. U S A 99 , 16770-16775 (2002). Botella, H. et al. Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10 , 248-259 (2011). Stocks, C. J. et al. Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli . J. Leukoc. Biol. 109 , 287-297 (2021). Luchian, I., Goriuc, A., Sandu, D. & Covasa, M. The Role of Matrix Metalloproteinases (MMP-8, MMP-9, MMP-13) in Periodontal and Peri-Implant Pathological Processes. Int. J. Mol. Sci. 23 , (2022). Prasad, A S. Effects of Zinc Deficiency on Th1 and Th2 Cytokine Shifts. J. Infect. Dis. 182 , S62-S68 (2000). Kulik, L., Maywald, M., Kloubert, V., Wessels, I. & Rink, L. Zinc deficiency drives Th17 polarization and promotes loss of Treg cell function. J. Nutr. Biochem. 63 , 11-18 (2019). Lamster, I. B., Asadourian, L., Del Carmen, T. & Friedman, P. K. The aging mouth: differentiating normal aging from disease. Periodontol. 2000 72 , 96-107 (2016). Lauritano, D. et al . Oral lichen planus clinical characteristics in Italian patients: a retrospective analysis. Head Face Med. 12 , 18 (2016). Treuting, P. M. & Morton, T. H. in Comparative Anatomy and Histology, (eds Treuting, P. M. & Dintzis, S. M.) pp. 95-110. (Academic Press, 2012). Jones, K. B. & Klein, O. D. Oral epithelial stem cells in tissue maintenance and disease: the first steps in a long journey. Int. J. Oral Sci. 5 , 121-129 (2013). Vermeer, P. D. et al. MMP9 modulates tight junction integrity and cell viability in human airway epithelia. Am. J. Physiol. Lung Cell Mol. Physiol. 296 , L751-762 (2009). Al-Sadi, R. et al. Matrix Metalloproteinase-9 (MMP-9) induced disruption of intestinal epithelial tight junction barrier is mediated by NF-κB activation. PLoS One 16 , e0249544 (2021). Furuyama, A., Hosokawa, T. & Mochitate, K. Interleukin-1beta and tumor necrosis factor-alpha have opposite effects on fibroblasts and epithelial cells during basement membrane formation. Matrix Biol. 27 , 429-440 (2008). Wang, J. et al. Escherichia coli enhances Th17/Treg imbalance via TLR4/NF-κB signaling pathway in oral lichen planus. Int. Immunopharmacol. 119 , 110175 (2023). Liu, M. & Choi, Y. A murine periodontitis model using coaggregation between human pathogens and a predominant mouse oral commensal bacterium. J. Periodontal Implant Sci. 52 , 141-154 (2022). Weber, B. et al. Distinct interferon-gamma and interleukin-9 expression in cutaneous and oral lichen planus. J. Eur. Acad. Dermatol. Venereol. 31 , 880-886 (2017). Xie, S., Ding, L., Xiong, Z. & Zhu, S. Implications of Th1 and Th17 cells in pathogenesis of oral lichen planus. J. Huazhong Univ. Sci. Technolog. Med. Sci. 32 , 451-457 (2012). Wang, Y., Zhou, J., Fu, S., Wang, C. & Zhou, B. A Study of Association Between Oral Lichen Planus and Immune Balance of Th1/Th2 Cells. Inflammation 38 , 1874-1879 (2015). Lu, R. et al. Expression of T-bet and GATA-3 in peripheral blood mononuclear cells of patients with oral lichen planus. Arch. Oral Biol. 56 , 499-505 (2011). Husein-ElAhmed, H. & Steinhoff, M. Potential role of INTERLEUKIN-17 in the pathogenesis of oral lichen planus: a systematic review with META-analysis. J. Eur. Acad. Dermatol. Venereol. 36 , 1735-1744 (2022). Piccinni, M. P. et al. Potential pathogenetic role of Th17, Th0, and Th2 cells in erosive and reticular oral lichen planus. Oral Dis. 20 , 212-218 (2014). Wang, H. et al. Overexpression and varied clinical significance of Th9 versus Th17 cells in distinct subtypes of oral lichen planus. Arch. Oral Biol. 80 , 110-116 (2017). Wang, H. et al. Role of distinct CD4(+) T helper subset in pathogenesis of oral lichen planus. J. Oral Pathol. Med. 45 , 385-393 (2016). El-Howati, A., Thornhill, M. H., Colley, H. E. & Murdoch, C. Immune mechanisms in oral lichen planus. Oral Dis. 29 , 1400-1415 (2023). Popov, H., Stoyanov, G. S. & Ghenev, P. Modified Silver Impregnation Method for Basal Membranes in Renal Biopsies. Cureus 14 , e30171 (2022). Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34 , 267-273 (2003). 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3913717","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":270922735,"identity":"67214788-9b9d-4082-8156-756d3afd7fbc","order_by":0,"name":"Youngnim Choi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYPACCQZ+BJNYLZINJGphYDA4QKwWg+M9Zg+/7rCINj5/9pgEQ40dg+TsAwS0nDljbix7RiJ32428NAmGY8kM0nwJBLTcyDGTlmwDaeExk2BgO8Agx0PIYTAtm/vPALX8I1KL5Eeglg0MOWYSjG0HGKQJaZE8c6xMmhGoZcaNHGOLxL5kHskeAlr4jjdvk/zZVpfb33/G8MaHb3ZyEmcIaFE4wMDADHdKAgMDIWcxMMg3MDAw/iCobBSMglEwCkY0AABpSDxGJd+hPAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6496-5560","institution":"Seoul National University School of Dentistry","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Youngnim","middleName":"","lastName":"Choi","suffix":""},{"id":270922736,"identity":"9e3b98b7-e6c4-4d53-ba87-d6fe9a635eb5","order_by":1,"name":"Phuc Vo","email":"","orcid":"","institution":"Seoul National University School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Phuc","middleName":"","lastName":"Vo","suffix":""},{"id":270922737,"identity":"b666d0d6-401e-4272-ac20-82c597d389eb","order_by":2,"name":"Joo-Young Park","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Joo-Young","middleName":"","lastName":"Park","suffix":""},{"id":270922738,"identity":"251b4c54-6daf-4dfc-8e06-0814c050347c","order_by":3,"name":"Hyeong-jin Kim","email":"","orcid":"","institution":"Seoul National University School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyeong-jin","middleName":"","lastName":"Kim","suffix":""},{"id":270922739,"identity":"57afb3ea-53e6-41fe-8bad-241520794106","order_by":4,"name":"Hye-Jung Yoon","email":"","orcid":"https://orcid.org/0000-0002-3534-3295","institution":"Seoul National University, School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hye-Jung","middleName":"","lastName":"Yoon","suffix":""},{"id":270922740,"identity":"1a4d4d62-2f05-4f9f-bab7-863a532839c7","order_by":5,"name":"Sung-Ho Chang","email":"","orcid":"","institution":"Seoul National University School of Dentistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung-Ho","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-01-31 11:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3913717/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3913717/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50717516,"identity":"1ae7007b-ac2b-4bd1-9e10-fa3be1cdf43e","added_by":"auto","created_at":"2024-02-06 09:10:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2947846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZinc deficiency increases intracellular survival of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in HOKs due to impaired zinc poisoning\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Primary HOKs were plated and their viability was measured using a CCK-8 assay kit after treatment with various concentrations of TPEN for 24 hours. (\u003cstrong\u003eb\u003c/strong\u003e) HOKs were infected with pHrodoRed-labeled \u003cem\u003eE. coli \u003c/em\u003e7.2, \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655, and \u003cem\u003eS. salivarius\u003c/em\u003e at an MOI of 1000 for 2 hours in the absence or presence of 1 μM TPEN, and then the cells were analyzed by flow cytometry. The left panel presents a typical flow cytometric result, and the right panel presents the mean ± standard error of mean (SEM) of the results from three independent experiments performed in triplicates. (\u003cstrong\u003ec\u003c/strong\u003e) HOKs were infected with bacteria for 2 hours and further cultured for 22 hours in the presence of gentamicin. The cells were then lysed, and the lysates were plated on the agar plates. The colony-forming units (CFUs) indicate the number of bacteria that survived intracellularly. The data present mean ± SEM of the three independent experiments performed in triplicates. **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 versus VC by Student’s t-test. (\u003cstrong\u003ed\u003c/strong\u003e) Left panel: HOKs preincubated with DCF-DA were infected with \u003cem\u003eE. coli\u003c/em\u003e 7.2 for 2 hours in the absence or presence of 1 mM TPEN, and then the DCF fluorescence intensity was measured. Right panel: HOKs preincubated with DCF-DA were infected with various bacteria, and then the DCF fluorescence intensity was measured at the indicated time. The data present mean ± SEM from two independent experiments performed in triplicates. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 versus control by Student’s t-test. VC, vehicle control; NI, non-infected; Ec, \u003cem\u003eE. coli\u003c/em\u003e; Ss, \u003cem\u003eS. salivarius\u003c/em\u003e; Fn, \u003cem\u003eF. nucleatum\u003c/em\u003e. (\u003cstrong\u003ee\u003c/strong\u003e) Left panel: HOKs were infected with pHrodoRed-labeled \u003cem\u003eE. coli\u003c/em\u003e 7.2 at an MOI of 1000 for 2 hours in the absence or presence of 1 μM TPEN. The cells were fixed and stained with zinc-specific fluorescent probe FluoZin-3 AM (FZ3, green) and Hochest 33342 (blue) and then examined by confocal microscopy. Scale bar: 20 μm. Right panel: The intensity of FZ3 was quantified, and the data present mean ± SEM of two independent experiments performed in triplicates. (\u003cstrong\u003ef\u003c/strong\u003e) HOKs were infected with \u003cem\u003eE. coli\u003c/em\u003e 7.2 for 2 hours and subjected to total RNA extraction immediately or after further culture in the presence of gentamicin for 16 hours. The relative mRNA levels of the indicated genes were analyzed by qRT-PCR. The data present mean ± SEM of two independent experiments performed in triplicates. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 versus NI by Student’s t-test.\u003c/p\u003e","description":"","filename":"IMG00001.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/8854e6ff73935622ee84db07.png"},{"id":50718210,"identity":"b750455f-0325-4fa6-b132-6318df8ee6bd","added_by":"auto","created_at":"2024-02-06 09:18:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2942169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZinc deficiency reduces Th1/Th17 ratio, while \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection increases Th17/Treg ratio in the cervical lymph nodes \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental scheme. Mice were subjected to a standard control diet (CD) or zinc-deficient diet (ZDD) for one month. The labial mucosae of the mice were microdamaged using a dental stone bur, and then the mice received oral administration of \u003cem\u003eE. coli\u003c/em\u003e 7.2 every other day for 3 weeks. (\u003cstrong\u003eb\u003c/strong\u003e) Zinc concentration in mice serum after one month of controlled diets. (\u003cstrong\u003ec\u003c/strong\u003e) Leukocyte population in the cervical lymph nodes was analyzed by flow cytometry (n = 6 for NS; for S and S+I, n = 7 for the number of total cells, CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, and CD8\u003csup\u003e+\u003c/sup\u003e T cells; n = 5 for the number of B cells, dendritic cells, monocytes, granulocytes). (\u003cstrong\u003ed\u003c/strong\u003e) Frequency of Th1, Th2, Th17, Treg and the ratio of Th1/Th17 and Th17/Treg (n = 6 for NS; for S and S+I, n = 7). The data present mean ± SD from three independent experiments. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 versus CD by Student’s t-test. s, p \u0026lt; 0.05; ss, p \u0026lt; 0.01; sss, p \u0026lt; 0.001 versus NS; i, p \u0026lt; 0.05; ii, p \u0026lt; 0.01; iii, p \u0026lt; 0.001 versus S; z, p \u0026lt; 0.05; zz, p \u0026lt; 0.01; zzz, p \u0026lt; 0.001 versus CD by two-way ANOVA with Bonferroni’s multiple comparison test.\u003c/p\u003e","description":"","filename":"IMG00002.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/8220155fc84bdf47ecd922d6.png"},{"id":50718209,"identity":"0465ae2b-41b1-49ca-91d1-315720a17aa7","added_by":"auto","created_at":"2024-02-06 09:18:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10651978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and zinc deficiency synergistically enhance bacterial invasion, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMmp9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression, and inflammatory infiltration into labial mucosa \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Paraffin-embedded sections of the labial tissues obtained from the experimentpresented in Figure 2 were subjected to \u003cem\u003ein situ\u003c/em\u003ehybridization using a digoxigenin-labeled eubacterial probe targeting bacterial 16S rRNA. The violet color indicates positive signals. Scale bar: 50 μm. (\u003cstrong\u003eb\u003c/strong\u003e) The mean intensity of bacterial signals per ROI in the epithelium and lamina propria was analyzed using the ImageJ software. The data present mean ± SEM of four images per mouse and multiple mice, obtained from three independent experiments (n = 6 for NS; n = 7 for S and S+I). ss, p \u0026lt; 0.01; sss, p \u0026lt; 0.001 versus NS; i, p \u0026lt; 0.05; ii, p \u0026lt; 0.01; iii, p \u0026lt; 0.001 versus S; z, p \u0026lt; 0.05; zz, p \u0026lt; 0.01; zzz, p \u0026lt; 0.001 versus CD by two-way ANOVA with Bonferroni’s multiple comparison test. (\u003cstrong\u003ec\u003c/strong\u003e) The total RNA of lip tissues obtained from the mice sacrificed three days after scratching and infection (n = 5/group) was extracted, and the relative mRNA level of the \u003cem\u003eMmp9\u003c/em\u003e was determined by qRT-PCR. The data present mean ± SD. sss, p \u0026lt; 0.001 versus NS; iii, p \u0026lt; 0.001 versus S; z, p \u0026lt; 0.05 versus CD by two-way ANOVA with Bonferroni’s multiple comparison test. (\u003cstrong\u003ed\u003c/strong\u003e) HOKs were infected with \u003cem\u003eE. coli\u003c/em\u003e 7.2 for 2 hours and further cultured in the presence of gentamicin for 22 hours. Total cellular RNA was extracted, and the relative mRNA level of the \u003cem\u003eMMP9\u003c/em\u003e was determined by qRT- PCR. Cell supernatant was collected, and the total and active MMP-9 were measured using a commercial kit. The data present mean ± SEM of two independent experiments performed in triplicates. iii, p \u0026lt; 0.001 versus NI; z, p \u0026lt; 0.05 versus VC by two-way ANOVA with Bonferroni’s multiple comparison test. ND: not detected. (\u003cstrong\u003ee\u003c/strong\u003e) Sections of labial tissues were stained with hematoxylin and eosin (H\u0026amp;E). Under high magnification, yellow and red arrows depict infiltrated lymphocytes and colloid bodies, respectively. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"IMG00003.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/5625f219f3133287c498496d.png"},{"id":50717519,"identity":"0854b110-0638-4235-b58e-d875cdf95513","added_by":"auto","created_at":"2024-02-06 09:10:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4587903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection under zinc deficiency increases CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T-cell recruitment to labial mucosa and shifts the T-cell response from the Th1-dominant to Th17-dominant response \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Heat map of transcriptomic data from lip tissues obtained 3 days after the scratching and \u003cem\u003eE. coli\u003c/em\u003e infection (n = 3). The color change from red to green represents the change from upregulation to downregulation. (\u003cstrong\u003eb\u003c/strong\u003e) Gene Set Enrichment Analysis (GSEA) results of CD vs. ZDD with nominal p \u0026lt; 0.05. Normalized Enrichment Score (NES) value indicates enrichment of Gene Ontology (GO) biological term in CD (blue) and in ZDD (green). AMP: antimicrobial peptides. (\u003cstrong\u003ec\u003c/strong\u003e) Immunofluorescence staining of CD4 (red), IFNg (green), IL-17 (magenta), and Hochest 33342 (blue) in labial tissues of mice, obtained three days after scratching alone or scratching plus \u003cem\u003eE. coli\u003c/em\u003e infection \u0026nbsp;(n = 5/group). Scale bar, 50 μm. Quantification of CD4 signal intensity (\u003cstrong\u003ed\u003c/strong\u003e), IFNg/CD4 colocalization (\u003cstrong\u003ee\u003c/strong\u003e), and IL-17/CD4 colocalization (\u003cstrong\u003ef\u003c/strong\u003e) was performed using the Zen software. Data present mean ± SEM. sss, p \u0026lt; 0.001 versus NS; iii, p \u0026lt; 0.001 versus S; z, p \u0026lt; 0.05; zz, p \u0026lt; 0.01; zzz, p \u0026lt; 0.001 versus CD by two-way ANOVA with Bonferroni’s multiple comparison test.\u003c/p\u003e","description":"","filename":"IMG00004.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/191010aedf774d6a65a5214b.png"},{"id":50717523,"identity":"2d330c39-ce74-40a1-9ab9-0a4750d970c4","added_by":"auto","created_at":"2024-02-06 09:10:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11710463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeated scratching and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection aggravates T-cell infiltration and basement membrane destruction \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental scheme (n = 5/group). (\u003cstrong\u003eb\u003c/strong\u003e) Histological analyses of labial mucosa by H\u0026amp;E staining, immunohistochemistry of CD3, and the Jones’ methenamine silver staining. Since the overall histology of mice did not show significant differences between the 2S+I and 3S+I groups, one representative mouse from each of the CD and ZDD groups is presented. Scale bars, 100 μm (\u003cstrong\u003ec\u003c/strong\u003e) Number of CD3\u003csup\u003e+\u003c/sup\u003e T cells detected in the epithelium and lamina propria of the entire labial mucosa. The data present mean ± SD. ii, p \u0026lt; 0.01; iii, p \u0026lt; 0.001 versus NS; z, p \u0026lt; 0.05 versus CD by two-way ANOVA with Bonferroni’s multiple comparison test.\u003c/p\u003e","description":"","filename":"IMG00005.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/0322452197ac2f7aef2b856c.png"},{"id":50717522,"identity":"e4d7e4a4-07e1-485d-8df2-48e222c8383e","added_by":"auto","created_at":"2024-02-06 09:10:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7956143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking of Th1 cell differentiation during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection impairs bacterial clearance in the epithelium \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental scheme. For Th1 blockade, the mice were intraperitoneally injected with anti-IFNg and anti-IL-12p75 antibodies two days before the first infection, and this was repeated on Day 1, Day 4, and Day 7. The control mice were injected with isotype control antibodies. (\u003cstrong\u003eb\u003c/strong\u003e) Frequencies of the Th1 and Th17 cells and the Th1/Th17 ratio in the cervical lymph nodes, mesenteric lymph nodes, and spleen (n = 5/group). The data present mean ± SD. **, p \u0026lt; 0.01 versus the control group by Student’s t-test. Abs, antibodies. (\u003cstrong\u003ec\u003c/strong\u003e) Sections of lip tissues were subjected to \u003cem\u003ein situ\u003c/em\u003e hybridization using an eubacterial probe targeting bacterial 16S rRNA. The violet color indicates positive signals. (\u003cstrong\u003ed\u003c/strong\u003e) The mean intensity of bacterial signals per ROI in the epithelia and lamina propria was analyzed using the ImageJ software. (\u003cstrong\u003ee\u003c/strong\u003e) Histological analyses of labial mucosae by H\u0026amp;E staining, immunohistochemistry of CD3, and the Jones’ methenamine silver staining. Red and blue arrows indicate colloid body and the site of epithelial detachment, respectively. Scale bar, 50 μm. (\u003cstrong\u003ef\u003c/strong\u003e) Number of CD3\u003csup\u003e+\u003c/sup\u003e T cells detected in the epithelium and lamina propria of the entire labial mucosa. The data present mean ± SD. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 versus the control group by Student’s t-test. Abs, antibodies. (\u003cstrong\u003eg\u003c/strong\u003e) Pearson correlations between the number of CD3\u003csup\u003e+\u003c/sup\u003e T cells in the lamina propria and the quantities of bacteria in the epithelium or lamina propria.\u003c/p\u003e","description":"","filename":"IMG00006.png","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/cecff45bda0ae62d33707934.png"},{"id":55500148,"identity":"f36bafd1-9906-4a06-9579-58cc22fbaaf8","added_by":"auto","created_at":"2024-04-29 09:43:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3345954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/bd2444cb-504c-4f84-bd11-a21468090b2b.pdf"},{"id":50717517,"identity":"76e66d6a-b08f-40b5-b45a-ab0337599e68","added_by":"auto","created_at":"2024-02-06 09:10:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1611079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3913717/v1/9d40a79dfb799e9c12d9bcad.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Interplay of zinc deficiency, epithelial microdamage, and Escherichia coli infection in a mouse model of oral lichen planus","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOral lichen planus (OLP) is a prevalent oral mucosal disease that affects approximately 0.5\u0026ndash;4% of the general population\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Clinical presentations of OLP encompass six distinct types\u0026mdash;reticular, papular, plaque, atrophic, erosive, and bullous\u0026mdash;often affecting regions such as the buccal mucosae, tongue, and gingivae. OLP is characterized as a chronic inflammatory condition primarily mediated by T cells with histopathological features, including a band-like subepithelial and intra-epithelial infiltration of T cells, liquefaction of the basal cell layer of epithelia with colloid bodies, and saw-tooth form rete pegs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite extensive research, the etiology of OLP remains elusive, with several potential triggers, including genetic and psychological factors, systemic medications, trauma, and microbial infections\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Notably, the mechanisms underlying T-cell activation and recruitment are still poorly understood. A previous study revealed an increased presence of bacteria in the basal cell layer of epithelium and lamina propria at OLP lesions, along with a positive correlation between bacterial quantities and T-cell infiltration\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, the presence of immunologic synapse-like structures between infected epithelial cells and T cells suggests that T cells may be responding to bacterial antigens presented by infected cells\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Comparative analysis of bacterial communities within OLP lesions unveiled an enrichment of \u003cem\u003eEscherichia coli\u003c/em\u003e, a common gut commensal bacterium, though pathogenic varieties exist\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Although \u003cem\u003eE. coli\u003c/em\u003e strains isolated from OLP tissues lack the major virulence factors of pathogenic \u003cem\u003eE. coli\u003c/em\u003e, they efficiently invade into and survive within human oral keratinocytes (HOKs)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These findings suggest the possibility that infection with the OLP-isolated \u003cem\u003eE. coli\u003c/em\u003e could trigger T-cell activation and infiltration in OLP.\u003c/p\u003e \u003cp\u003eTranscriptomic data analysis of OLP has identified an enrichment of gene signatures associated with barrier defects, wound healing, and response to infection. Conversely, genes linked to response to zinc ions were found to be decreased in OLP cases\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Zinc deficiency (ZD) can induce immune dysfunction and increase susceptibility to infection by disrupting epithelial barriers, suppressing phagocyte function, reducing the activities of natural killer cells and cytotoxic T lymphocytes (CTLs), and dampening Th1 cell responses\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, several studies have reported significant reductions in serum and/or saliva zinc levels in OLP patients\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Intriguingly, zinc stands out as a trace element that has demonstrated therapeutic effects in OLP lesions through both topical and systemic supplementation\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the precise role of ZD in the pathogenesis of OLP remains unclear.\u003c/p\u003e \u003cp\u003eThe absence of suitable animal models for OLP poses a significant obstacle to research aimed at comprehending its etiopathogenesis and exploring novel therapeutic avenues. This study aims to elucidate the contribution of bacterial infection and ZD to the pathogenic processes underlying OLP by developing a murine model. In this model, we harness the intricate interplay between ZD, mechanical epithelial microdamage, and the oral infection of an OLP-isolated \u003cem\u003eE. coli\u003c/em\u003e strain.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEffect of ZD on\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003einvasion and survival in HOKs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of ZD on bacterial invasion and survival, primary HOKs were infected with an OLP-isolated strain \u003cem\u003eE. coli\u003c/em\u003e 7.2, a human gut commensal-derived laboratory strain \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655, and the oral commensal species \u003cem\u003eStreptococcus salivarius\u003c/em\u003e in the absence or presence of N,N,N\u0026prime;,N\u0026prime;-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), a zinc chelator. The concentration of TPEN (1 \u0026micro;M) chosen for the study did not affect HOK viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). TPEN did not affect the invasion rates, which were determined by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, both \u003cem\u003eE. coli\u003c/em\u003e strains significantly increased their intracellular survival under ZD, while \u003cem\u003eS. salivarius\u003c/em\u003e did not survive (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn exploring the potential involvement of reactive oxygen species (ROS) production in the increased intracellular survival of \u003cem\u003eE. coli\u003c/em\u003e with TPEN, the levels of intracellular ROS were quantified using 2\u0026prime;,7-dichlorofluorescein diacetate (DCF-DA), a cell-permeant indicator sensitive to oxidation by ROS. While HOKs significantly increased the ROS in the presence of TPEN alone, as previously reported\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eE. coli\u003c/em\u003e, unlike most bacteria, notably suppressed ROS production in HOKs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eRecent research has established zinc poisoning by phagolysosomal accumulation of free zinc as a mechanism for killing intracellular bacteria in macrophages\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. To investigate if a similar mechanism occurs in HOKs, intracellular free zinc was visualized using a zinc-specific fluorescent probe, FluoZin-3. \u003cem\u003eE. coli\u003c/em\u003e infection significantly elevated the intracellular zinc signals that colocalized with endosomal \u003cem\u003eE. coli\u003c/em\u003e, while infection under ZD neutralized this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The increase in intracellular free zinc accompanied the regulation of zinc transporter genes in HOKs: upregulation of \u003cem\u003eSLC30A1\u003c/em\u003e encoding ZnT1 that can deliver zinc into vesicle lumen and downregulation of \u003cem\u003eSLC39A1/ SLC39A2\u003c/em\u003e encoding ZIP1/ZIP2 that can deliver zinc from the vesicle lumen to cytoplasm. HOKs also upregulated genes involved in zinc detoxification, such as metal-regulatory transcription factor 1 (\u003cem\u003eMTF1\u003c/em\u003e), metallothionein 1 (\u003cem\u003eMT1\u003c/em\u003e), and \u003cem\u003eMT2\u003c/em\u003e. However, these changes in gene expression were not observed under ZD conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThese results collectively suggest that HOKs employ zinc toxicity, rather than relying on ROS, to combat intracellular \u003cem\u003eE. coli\u003c/em\u003e, and zinc deprivation mitigates this effect, allowing more bacteria to survive within HOKs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of ZD, epithelial microdamage, and\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003einfection on immune response in the cervical lymph nodes of mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the impact of ZD on the immune response to oral \u003cem\u003eE. coli\u003c/em\u003e infection, the C57BL/6 mice were subjected to either a control diet (CD) or a zinc-deficient diet (ZDD) for one month. In our preliminary experiments, oral administration of \u003cem\u003eE. coli\u003c/em\u003e did not cause bacterial invasion into oral tissues, probably due to the keratinized nature of mouse oral mucosa. To facilitate \u003cem\u003eE. coli\u003c/em\u003e invasion into the epithelial cells, we introduced a mucosal microdamage model by scratching the labial mucosa using a dental stone bur. Mice from each diet were divided into three groups: non-scratching (NS, n\u0026thinsp;=\u0026thinsp;6/group), scratching alone (S, n\u0026thinsp;=\u0026thinsp;7/group), or scratching followed by infection with 10\u003csup\u003e9\u003c/sup\u003e viable cells of \u003cem\u003eE. coli\u003c/em\u003e 7.2 (S\u0026thinsp;+\u0026thinsp;I, n\u0026thinsp;=\u0026thinsp;7/group). The mice were sacrificed either three days or 21 days after a single episode of scratching and infection, and the latter additionally received repeated \u003cem\u003eE. coli\u003c/em\u003e 7.2 inoculations without scratching (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The successful induction of ZD by ZDD was confirmed by serum zinc level upon sacrifice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the impact of ZD on immune cell composition in the cervical lymph nodes, mesenteric lymph nodes, and spleen was analyzed by flow cytometry (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), ZDD resulted in the specific enlargement of the cervical lymph nodes primarily due to T-cell expansion in the NS group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and S2). Both scratching and infection amplified this ZDD-induced expansion of immune cells, including lymphocytes, dendritic cells, monocytes, and granulocytes. The degree of expansion was reduced at Day 21 compared to Day 3 despite the repeated oral inoculation of \u003cem\u003eE. coli\u003c/em\u003e 7.2, confirming that \u003cem\u003eE. coli\u003c/em\u003e 7.2 does not infect mouse oral mucosae without microdamage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eMoreover, ZDD reduced the Th1/Th17 ratio, particularly in the cervical lymph nodes of the NS group due to a reduction in Th1 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and S2). While Th1 cells decreased only under ZD, scratching increased regulatory T (Treg) cells, and \u003cem\u003eE. coli\u003c/em\u003e infection increased Th17 cells. Consequently, the Th1/Th17 ratio synergistically decreased with ZD and \u003cem\u003eE. coli\u003c/em\u003e infection, while the Th17/Treg ratio increased solely due to \u003cem\u003eE. coli\u003c/em\u003e infection, particularly at Day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results indicate that \u003cem\u003eE. coli\u003c/em\u003e infection under ZD conditions altered T-cell responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynergistic effects of\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003einfection and ZDD on bacterial invasion and inflammation in labial mucosae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe impact of \u003cem\u003eE. coli\u003c/em\u003e infection and ZD on bacterial invasion of labial mucosae was investigated using \u003cem\u003ein situ\u003c/em\u003e hybridization targeting bacterial 16S rRNA. Scratching alone increased eubacterial invasion into both the epithelium and lamina propria, particularly under ZD conditions at both Day 3 and Day 21. \u003cem\u003eE. coli\u003c/em\u003e infection further amplified bacterial presence in tissues only at Day 3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Additionally, examination of \u003cem\u003eE. coli\u003c/em\u003e invasion using an \u003cem\u003eE. coli\u003c/em\u003e-specific probe revealed a similar pattern with less signal intensity and statistical significance (Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor bacteria to reach the lamina propria, the basement membranes need to be compromised. As matrix metalloproteinase-9 (MMP-9) can break down key components of the basement membrane, such as type IV collagen and laminin\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, its expression was examined. In the mouse labial tissues, scratching alone upregulated expression of the \u003cem\u003eMmp9\u003c/em\u003e gene, further enhanced by \u003cem\u003eE. coli\u003c/em\u003e infection, particularly under ZD conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). To relate the expression of the \u003cem\u003eMmp9\u003c/em\u003e gene to that of protein, HOK \u003cem\u003ein vitro\u003c/em\u003e infection system was utilized. \u003cem\u003eE. coli\u003c/em\u003e infection and ZD synergistically upregulated \u003cem\u003eMMP9\u003c/em\u003e mRNA and protein. However, active MMP-9 production occurred solely upon \u003cem\u003eE. coli\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These results indicate that \u003cem\u003eE. coli\u003c/em\u003e infection and ZD can induce barrier destruction.\u003c/p\u003e \u003cp\u003eHistological analysis using hematoxylin and eosin (H\u0026amp;E) staining demonstrated that mice on ZDD had more infiltrating cells three days after \u003cem\u003eE. coli\u003c/em\u003e infection compared to mice on CD, mostly getting resolved by Day 21. However, \u003cem\u003eE. coli\u003c/em\u003e-infected mice under ZD conditions exhibited a higher number of remaining cells near the basal cell layer with several colloid bodies, indicating the initial stage of OLP lesion development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of ZDD and\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003einfection on CD4\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eT-cell infiltration and Th1 vs. Th17 responses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo gain a deeper understanding of the molecular mechanisms underlying the increased bacterial invasion and inflammatory cell infiltration, we conducted transcriptomic analysis on lip tissues from mice infected with \u003cem\u003eE. coli\u003c/em\u003e under either CD or ZDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Gene set enrichment analysis (GSEA) revealed that genes expressed in the ZDD group were significantly enriched in processes related to keratinocyte differentiation, keratinization, oxidative stress-induced cell death, DNA replication, inflammatory response, chemotaxis of immune cells, and antimicrobial humoral response via antimicrobial peptides. In contrast, processes associated with antigen processing and presentation, as well as the activation of NK and NKT cells, were enriched in the CD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Notably, the CD group also showed enrichment in the positive regulation of Th1 cell response and IFNγ production, along with the negative regulation of IL-17 production, mirroring the findings in the cervical lymph nodes. These data suggested that mice under CD primarily responded to \u003cem\u003eE. coli\u003c/em\u003e infection with cellular immunity, while mice under ZDD responded with armamentaria against extracellular bacteria, leading to increased inflammation and immune cell infiltration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the transcriptomic analysis results at the protein level, we performed immunofluorescence using anti-CD4, IFNγ, and IL-17 antibodies to assess the infiltration of Th1 vs. Th17 cells in the labial mucosae. Scratching alone induced the recruitment of CD4\u003csup\u003e+\u003c/sup\u003e T cells, with similar levels between the CD and ZDD groups. \u003cem\u003eE. coli\u003c/em\u003e infection further heightened CD4\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, which was exacerbated under ZD (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These infiltrated cells were detected both in the epithelium and the lamina propria. In the CD group, the recruited CD4\u003csup\u003e+\u003c/sup\u003e T cells were Th1-dominant, while in the ZDD group, they were Th17-dominant (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThis comprehensive analysis suggests that differential immune responses to \u003cem\u003eE. coli\u003c/em\u003e infection were elicited in the CD and ZDD groups, impacting the balance between Th1 and Th17 cell populations in the context of the oral mucosa.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImpact of repeated scratching and infection on T-cell infiltration\u003c/h2\u003e \u003cp\u003eThe repeated oral administration of \u003cem\u003eE. coli\u003c/em\u003e 7.2 without scratching failed to infect mouse oral mucosae. To replicate the chronic inflammatory nature of OLP, we investigated whether repeated scratching and infection would induce a more robust T-cell response and heightened inflammation. Following one month of controlled diets, mice (n\u0026thinsp;=\u0026thinsp;5/group) underwent two (2S\u0026thinsp;+\u0026thinsp;I) or three (3S\u0026thinsp;+\u0026thinsp;I) rounds of scratching and infection at 10-day intervals, and these groups were compared with the NS group, which was sacrificed concurrently with the 3S\u0026thinsp;+\u0026thinsp;I group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of immune cell populations from the cervical lymph nodes in the 2S\u0026thinsp;+\u0026thinsp;I group yielded results similar to those of mice infected only once (Fig. S4). Histological examination revealed a more prevalent lymphocytic infiltration throughout the section of the labial mucosae compared to the single infection. Several mice, both from the CD and ZDD groups, developed severe inflammatory foci in the epithelium and adjacent lamina propria, closely mimicking OLP-like histopathology (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and S5). Moreover, the characteristic \"saw-tooth\" shaped rete pegs tended to be more prominent upon repeated scratching and infection (Fig. S5).\u003c/p\u003e \u003cp\u003eTo confirm T-cell infiltration, CD3 immunohistochemical staining was conducted. Regardless of dietary conditions, mice typically exhibited only a few T cells in the epithelium and lamina propria throughout the section, which significantly increased with scratching and infection (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Repeated scratching and infection presented a notable trend of increasing T-cell infiltration only in mice fed with CD, partially counteracting the effects of ZD upon three-time infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, silver staining revealed destruction of the basement membrane in areas exhibiting inflammatory foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImpact of Th1 blockade on bacterial clearance and inflammation in labial mucosae\u003c/h2\u003e \u003cp\u003eTo elucidate the role of Th1 cells in controlling \u003cem\u003eE. coli\u003c/em\u003e infection and OLP-like histopathology, Th1 differentiation was blocked by injecting anti-IFNγ and anti-IL-12 antibodies during \u003cem\u003eE. coli\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Flow cytometric analysis confirmed a significant reduction in Th1 cells in the cervical lymph nodes, although the decrease in the Th1/Th17 ratio did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Th1 blockade impeded bacterial clearance in the labial mucosae, particularly in the epithelium (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Moreover, CD3 immunohistochemistry revealed a significant increase in T-cell infiltration into the lamina propria in the Th1 blockade group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Notably, the number of T cells in the lamina propria showed a strong positive correlation with the bacterial quantities in the epithelium rather than with those in the lamina propria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Interestingly, four out of the five mice in the Th1 blockade group developed lesions characterized by epithelial detachment from the underlying lamina propria. These lesions were accompanied by dense inflammatory infiltrates, including T cells, and destruction of the basement membrane, closely resembling one of the key features of OLP pathology (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and S6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOLP is a chronic inflammatory mucosal disorder characterized by T cell-mediated immune responses, but the lack of suitable animal models has limited the ability to understand its etiology and pathogenesis. This study marks a groundbreaking step forward by introducing the first successful animal model that replicates several vital aspects of OLP, including T-cell infiltration into the epithelium and subepithelial lamina propria, the presence of colloid bodies, induction of \u003cem\u003eMmp9\u003c/em\u003e, basement membrane destruction and detachment of the epithelium from the underlying lamina propria, bacterial invasion of tissue, and skewed Th17 responses. The interplay of ZD, epithelial microdamage, and \u003cem\u003eE. coli\u003c/em\u003e infection was required to develop OLP-like histopathology in mice.\u003c/p\u003e \u003cp\u003eZD led to reductions in Th1 cells and the Th1/Th17 ratio in the cervical lymph nodes, which aligns with previous findings\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although ZD has been linked to enhanced susceptibility to bacterial infection\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, ZD alone did not cause any bacterial invasion or inflammation in the labial mucosa despite the enlarged cervical lymph nodes, highlighting the roles of epithelial microdamage and infection with specific microorganisms such as \u003cem\u003eE. coli\u003c/em\u003e in the current model.\u003c/p\u003e \u003cp\u003eIt is crucial to recognize that OLP typically presents later in life when individuals, particularly postmenopausal women, begin to experience mucosal thinning, higher susceptibility to masticatory trauma, decreased muscle tone, and xerostomia due to chronic adult diseases and medications, which all contribute to increased friction between the buccal mucosa and gingiva\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. No enhancements in bacterial invasion of labial mucosa on Day 21 compared to Day 3 despite the repeated \u003cem\u003eE. coli\u003c/em\u003e oral inoculations confirmed our preliminary results that \u003cem\u003eE. coli\u003c/em\u003e does not infect oral tissues in mice with intact epithelium. As opposed to in humans, the buccal and labial mucosae of mice are keratinized, potentially offering a more robust barrier function than those in humans\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this context, the introduction of microdamage to the epithelium through scratching played a fundamental role. Scratching alone triggered bacterial translocation to the epithelium and lamina propria, upregulation of \u003cem\u003eMmp9\u003c/em\u003e, increased immune responses in the cervical lymph nodes, and increased CD4\u003csup\u003e+\u003c/sup\u003e T-cell recruitment to labial mucosa. As shown in HOKs infected with \u003cem\u003eE. coli\u003c/em\u003e, the bacteria translocated to the epithelium might have induced the production of active MMP9 protein from the keratinocytes. MMP9 can further negatively impact the integrity of the epithelium by reducing the expression of junctional proteins and destructing the basement membrane\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, leading to bacterial invasion into the lamina propria and T-cell recruitment.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eE. coli\u003c/em\u003e infection exacerbated \u003cem\u003eMmp9\u003c/em\u003e expression, bacterial invasion of labial mucosae, immune response in the cervical lymph nodes, and CD4\u003csup\u003e+\u003c/sup\u003e T-cell recruitment, surpassing the effect of scratching alone. The effect of \u003cem\u003eE. coli\u003c/em\u003e infection on T-cell infiltration and inflammation of labial mucosa increased with repeated scratching and infection. The heightened inflammatory response at infection sites might be attributed to \u003cem\u003eE. coli\u003c/em\u003e's ability to augment the Th17 cell population and the Th17/Treg ratio, as documented previously\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, \u003cem\u003eE. coli\u003c/em\u003e infection displayed a stronger synergism with ZD, leading to enhanced \u003cem\u003eMmp9\u003c/em\u003e expression, bacterial invasion of tissues, and T-cell infiltration, along with a reduced Th1/Th17 ratio in the cervical lymph nodes, compared to the effects of scratching alone. Scratching would have caused translocation of commensals that have low invasive ability, as previously reported\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eE. coli\u003c/em\u003e causes intracellular infection in keratinocytes. As observed in HOKs, zinc poisoning rather than ROS played a pivotal role in the control of \u003cem\u003eE. coli\u003c/em\u003e infection in oral keratinocytes, and intracellular survival of \u003cem\u003eE. coli\u003c/em\u003e in HOKs increased under ZD conditions. Additionally, Th cells that infiltrated the oral mucosa in response to \u003cem\u003eE. coli\u003c/em\u003e infection were shifted from Th1-dominance to Th17-dominance under ZD conditions. Blockade of Th1 differentiation during \u003cem\u003eE. coli\u003c/em\u003e infection in the mice fed with CD significantly impeded bacterial clearance in the epithelium, suggesting the importance of Th1 cells in the control of intracellular infection. Importantly, GSEA revealed that the mice fed with ZDD responded to \u003cem\u003eE. coli\u003c/em\u003e infection with a humoral immune response characterized by the production of antimicrobial peptides, while not effective against intracellular bacteria, whereas the mice fed with CD induced mostly cell-mediated immunity, emphasizing the importance of skewed immune responses within the context of OLP. Furthermore, the oral mucosa of the mice infected with \u003cem\u003eE. coli\u003c/em\u003e under ZD conditions upregulated genes associated with keratinocyte differentiation and keratinization, mirroring findings in human OLP lesions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. All these results underscore the pivotal roles of the interplay between \u003cem\u003eE. coli\u003c/em\u003e and ZD in the current model and provide insights into the therapeutic effects of zinc supplementation reported in OLP\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBoth Th1 and Th17 cells are increased in OLP lesions compared to healthy controls\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. It coincides with the finding in this study that \u003cem\u003eE. coli\u003c/em\u003e-infected mice from both the CD and ZDD groups exhibited higher levels of both Th1 and Th17 cells than the NS group. In the past, a dominance of Th1 over Th2 was documented in OLP lesions and peripheral blood T cells\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. More recently, the predominance of Th17 cells has been consistently reported, particularly in erosive OLP\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, the specific roles played by each subset of Th cells in the pathogenesis of OLP remain speculative\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Th1 cells are believed to play crucial roles in the pathogenesis of OLP through the production of inflammatory cytokine TNFα and assisting in the activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells. Interestingly, blocking Th1 differentiation during \u003cem\u003eE. coli\u003c/em\u003e infection in the current model induced the detachment of the epithelium from the underlying lamina propria with dense inflammatory infiltrate, along with the complete destruction of the basement membrane. This appears to represent an earlier stage of erosive OLP lesions. It is not clear why a similar phenotype did not develop in the mice infected with \u003cem\u003eE. coli\u003c/em\u003e under ZD conditions, despite similar reductions in Th1 cells; this could be presumably attributed to anti-IFNγ antibodies administered during \u003cem\u003eE. coli\u003c/em\u003e infection which may have neutralized the effector function of Th1 cells, as well as differentiation. This suggests that the Th1/IFNγ pathway may not be a suitable therapeutic target for OLP.\u003c/p\u003e \u003cp\u003eIt is essential to acknowledge the limitations of this study, as the multifaceted nature of OLP cannot be entirely reproduced in a single animal model. Although we attempted to measure the thickness of the keratin layer based on the enrichment of genes involved in keratinization, the thicknesses of the keratin layer and the epithelium significantly varied even within a single mouse, depending on the location. Further, our model did not address the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells, a cell type abundantly present in OLP lesions and believed to mediate keratinocyte apoptosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Additionally, the degree of T-cell infiltration observed in the current model is less severe than that observed in OLP patients. Notably, only a few T cells are typically distributed in the labial mucosa of mice maintained under specific pathogen-free conditions, whereas human lesions develop over months to years.\u003c/p\u003e \u003cp\u003eNevertheless, this animal model serves as a valuable platform for investigating specific hypotheses related to the pathogenesis and potential therapeutic targets of OLP. It demonstrates that OLP-like histopathology in the oral mucosa can be induced through \u003cem\u003eE. coli\u003c/em\u003e infection when combined with repeated epithelial microdamage, ZD, or Th1 blockade. The exploration of the mechanisms underlying these factors offers an opportunity to enhance our comprehension of OLP's pathogenesis and opens avenues for the investigation of potential therapeutic interventions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur study aimed to elucidate the contribution of bacterial infection and ZD in the pathogenesis of OLP using a murine model. The effect of ZD on the intracellular survival of \u003cem\u003eE. coli\u003c/em\u003e was examined in HOKs \u003cem\u003ein vitro\u003c/em\u003e in the presence of TPEN, a zinc chelator. ZD was induced in the mice by feeding them a ZDD for one month. Further, \u003cem\u003eE. coli\u003c/em\u003e infection of the labial mucosae was induced by oral administration of \u003cem\u003eE. coli\u003c/em\u003e following epithelial microdamage. We evaluated the effects of ZD, epithelial microdamage, and \u003cem\u003eE. coli\u003c/em\u003e infection on the Th-cell response in the cervical lymph nodes, bacterial invasion of labial mucosae, \u003cem\u003eMmp9\u003c/em\u003e expression, Th1 vs. Th17 infiltration, and inflammation in the labial mucosae. Additionally, we analyzed the effects of repeated epithelial microdamage plus \u003cem\u003eE. coli\u003c/em\u003e infection and Th1 blockade during \u003cem\u003eE. coli\u003c/em\u003e infection on bacterial clearance, T-cell infiltration, mucosal inflammation, and basement membrane disruption. In all the experiments, the animals were randomly assigned to different groups without blinding. However, all histological analyses were blindly performed without identifying the group to which each image belonged. The first animal experiment, presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, was conducted in three independent experiments, and the results were combined. The sample sizes for the following experiments were determined as n\u0026thinsp;=\u0026thinsp;5/group based on the mean and standard deviation of Th1 cells in the NS groups fed with either CD or ZDD, a parameter that showed the least difference among various comparisons. No samples or animals were excluded from our analyses, although the transcriptomic analysis was performed only on randomly chosen three mice per group from two groups.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacteria culture\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655 (American Type Culture Collection, Manassas, VA, USA) and \u003cem\u003eE. coli\u003c/em\u003e 7.2, a strain isolated from an OLP tissue\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, were cultured in Luria-Bertani broth (BD Biosciences, Franklin Lakes, NJ, USA) at 37\u0026deg;C under aerobic conditions without shaking. \u003cem\u003eStreptococcus salivarius\u003c/em\u003e KCTC 5512 (Korean Collection for Type Culture, Seoul, Korea) and \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e ATCC 51191 (ATCC) were cultured in a brain heart infusion broth supplemented with 5 \u0026micro;g/ml hemin (Sigma-Aldrich, St. Louis, MO, USA) and 1 \u0026micro;g/ml vitamin K\u003csub\u003e3\u003c/sub\u003e (Sigma-Aldrich) under anaerobic conditions (10% CO\u003csub\u003e2\u003c/sub\u003e, 10% H\u003csub\u003e2\u003c/sub\u003e, and 80% N\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003ePrimary HOK cells (ScienCell, Carlsbad, CA, USA) were cultured on a poly-L-lysine-coated dish in an oral keratinocyte medium (OKM; ScienCell) supplemented with manufacturer\u0026rsquo;s growth factors and penicillin-streptomycin at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. The effect of various concentrations of TPEN (Sigma-Aldrich) on HOK viability was determined using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBacterial invasion of HOKs and intracellular survival\u003c/h2\u003e \u003cp\u003eHOKs were seeded into 24-well (1 x 10\u003csup\u003e5\u003c/sup\u003e/well) or 48-well (6 x 10\u003csup\u003e4\u003c/sup\u003e/well) plates and incubated overnight. The cells were infected with bacteria labeled with 1 \u0026micro;M pHrodoRed (Invitrogen, Carlsbad, CA, USA) at a multiplicity of infection (MOI) of 1000 in antibiotics-free OKM for 2 hours in the presence or absence of 1 \u0026micro;M TPEN (Sigma-Aldrich). For invasion analysis, the infected cells were detached using trypsin-EDTA, stained with trypan blue, and subjected to flow cytometric analysis using a FACSCalibur (BD Biosciences). For the measurement of intracellular survival of bacteria, the infected cells were subjected to antibiotics protection assay using gentamicin by culturing for additional 24 hours in the absence or presence of TPEN, and the number of colony-forming units grown on agar plates was counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of ROS production\u003c/h2\u003e \u003cp\u003eHOKs (2 x 10\u003csup\u003e4\u003c/sup\u003e) were seeded into 96-well flat bottom black plates and incubated with 10 \u0026micro;M of DCF-DA (Sigma-Aldrich) at 37\u003csup\u003eo\u003c/sup\u003eC for 30 minutes. After washing, the cells were infected with \u003cem\u003eE. coli\u003c/em\u003e 7.2, \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655, \u003cem\u003eS. salivarius\u003c/em\u003e, and \u003cem\u003eF. nucleatum\u003c/em\u003e at MOI 1000 for 24 hours, in the absence or presence of 1 \u0026micro;M TPEN. The DCF fluorescence intensity was measured at the indicated time points at excitation and emission wavelengths of 485 and 535 nm, respectively, using a fluorometric plate reader (Synergy H1-Multi Mode Microplate Reader, BioTek, Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular Zn2\u003csup\u003e+\u003c/sup\u003e measurements\u003c/h2\u003e \u003cp\u003eHOKs were seeded into 24-well black plates for microscopy and infected with pHrodoRed-labeled \u003cem\u003eE. coli\u003c/em\u003e 7.2 at MOI 1000 for 2 hours. After fixation, the cells were stained with 1 \u0026micro;M FluoZin-3 AM (Invitrogen) and observed under an LSM 980 confocal laser scanning microscope (Carl Zeiss Inc. Oberkochen, Germany), recorded, and analyzed using the Zen software (Carl Zeiss, Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eRNA was isolated from HOKs or mouse lip tissues using an easy-BLUETM total RNA extraction kit (iNtRON Biotechnology, Gyeonggi-do, Korea) in accordance with the manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized using 2 \u0026micro;g of total RNA, oligo dT, and M-MLV reverse transcriptase (Promega, Madison, WI, USA) in 30 \u0026micro;l of the reaction mixture at 42\u003csup\u003eo\u003c/sup\u003eC for 1 hour. Real-time PCR was performed in 20 \u0026micro;l of the mixture, including primer pairs (400 nM each), cDNA (2 \u0026micro;l), and 10 \u0026micro;l of Power SYBR\u0026reg; Green Mastermix (Applied Biosystems, Waltham, MA, USA) under the following condition: initial denaturation at 95\u003csup\u003eo\u003c/sup\u003eC for 4 minutes, followed by 40 cycles of denaturation (95\u003csup\u003eo\u003c/sup\u003eC, 15 seconds), annealing (60\u003csup\u003eo\u003c/sup\u003eC, 15 seconds), and elongation (72\u003csup\u003eo\u003c/sup\u003eC, 33 seconds) using QuantStudio 3 Real-Time PCR Systems (Applied Biosystems, Waltham, Massachusetts, USA). The gene expression levels were normalized with the glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) gene and calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of MMP-9 protein\u003c/h2\u003e \u003cp\u003eThe quantity of total and active MMP-9 protein from HOK supernatant was measured using Fluorokine E human active MMP-9 assay kit (R\u0026amp;D Systems, Minneapolis, MN, USA) in accordance with the manufacturer\u0026rsquo;s protocol. The fluorescence intensity was measured at excitation and emission wavelengths of 320 and 405 nm, respectively, using a fluorometric plate reader (Synergy H1-Multi Mode Microplate Reader, BioTek).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiment\u003c/h2\u003e \u003cp\u003e The animal studies were conducted after approval by the Seoul National University Animal Care and Use Committee (SNU-191111-1-5 and SNU-210916-4-1) in accordance with the Animal Protection Act in Korea. Mice were maintained under specific pathogen-free conditions at 12-hour light, 12-hour dark cycle in the Laboratory Animal Facility at the School of Dentistry, Seoul National University. Eight-week-old female C57BL/6 mice were put on either CD or ZDD for 4\u0026ndash;8 weeks. Mouse chows with normal and decreased zinc levels (30.4 ppm and 1.2 ppm, respectively) were purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The labial mucosae of mice were microdamaged by rotating a dental stone bur (Dura-Green Stones, Shank HP, Shape FL1, Shofu Inc., Kyoto, Japan) 20 times, following which the mice were orally given 10\u003csup\u003e9\u003c/sup\u003e cells of viable \u003cem\u003eE. coli\u003c/em\u003e 7.2 in 50 \u0026micro;l PBS containing 1.5% carboxymethylcellulose (Tokyo Chemical Industry, Tokyo, Japan) according to the indicated experimental schemes. The number of rotations was determined in preliminary experiments to ensure that it does not cause traumatic ulcers on the mucosa while allowing \u003cem\u003eE. coli\u003c/em\u003e invasion. After sacrifice, the serum, cervical and mesenteric lymph nodes, spleen, and lip tissue were collected. The lip tissue was cut along the midline; one half was stored at -80\u0026deg;C, and the other half was subjected to tissue processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTh1 blockade model\u003c/h2\u003e \u003cp\u003eTh1 blockade \u003cem\u003ein vivo\u003c/em\u003e was carried out two days before \u003cem\u003eE. coli\u003c/em\u003e infection by injecting anti-IFNγ antibodies (200 \u0026micro;g/mouse; clone XMG1.2, BioXCell, Lebanon, NH, USA) and anti-IL-12p75 antibodies (300 \u0026micro;g/mouse; clone R2-9A5, BioXCell) intraperitoneally; this was repeated every three days for the entire duration of the experiment. Isotype control antibodies (clone HRPN and clone LTF-2, BioXCell) were used in the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Zn\u003csup\u003e2+\u003c/sup\u003e in serum\u003c/h2\u003e \u003cp\u003eThe Zn\u003csup\u003e2+\u003c/sup\u003e concentration in mice sera was measured with a commercial zinc assay kit (BioAssay Systems, Hayward, CA, USA) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions isolated from the cervical lymph nodes, mesenteric lymph nodes, and spleen were prepared in DPBS containing 2.5% fetal bovine serum (FBS) and incubated with anti-CD16/CD32 antibodies to block the Fc receptor for 20 minutes at 4\u0026deg;C. For surface staining, the cells were stained with fluorescence-labeled antibodies for 45 minutes at 4\u0026deg;C. For intracellular staining of cytokines, the cells were stimulated with 50 ng/ml PMA, 1 \u0026micro;M ionomycin (Sigma-Aldrich), and GolgiPlug\u0026trade; (BD Biosciences) in RPMI medium for 4 hours. The cell suspension was then fixed and permeabilized using a Foxp3/ Transcription Factor staining kit (Invitrogen) before being stained with fluorescence-labeled antibodies for 45 minutes at room temperature. The antibodies used in this experiment are listed in Table S2. The stained samples were run by LSRFortessa X-20 (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences). Live cells were gated by the exclusion of dead cells based on forward scatter and viability dye staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTissue processing and histological analyses\u003c/h2\u003e \u003cp\u003eParaffin-embedded 4.5-\u0026micro;m thick sections were stained with H\u0026amp;E to examine overall inflammation of the labial mucosae. The basement membrane was visualized using the Jones\u0026rsquo; methenamine silver stain\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To assess bacterial presence in tissue, \u003cem\u003ein situ\u003c/em\u003e hybridization using digoxigenin-labeled universal and \u003cem\u003eE. coli\u003c/em\u003e-specific probes targeting bacterial 16S rRNA was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Four areas per sample were photographed at 400x magnification. Bacterial signals were quantified using the ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). The distribution of T cells was determined by immunohistochemical staining of CD3. The sections were tile scanned at 200x magnification using an Automated Upright Microscope System (Leica Biosystems, Wetzlar, Germany). The number of CD3-positive cells in the epithelium and lamina propria in the entire section was counted and expressed as the number of cells per unit length by measuring the length of tissue, using the ImageJ software.\u003c/p\u003e \u003cp\u003eThe distribution of Th1 and Th17 cells in labial mucosae was examined by immunofluorescence staining for CD4, IFNγ, and IL-17 on 10-\u0026micro;m thick frozen sections. A secondary antibody was used to amplify the CD4 signal. The samples were examined under an LSM 980 confocal laser scanning microscope (Carl Zeiss, Inc.), and five areas per sample were photographed at 400x magnification. The signal intensity and colocalization coefficients of images were analyzed using the ZEN software (Carl Zeiss, Inc.). The antibodies used for immunohistochemistry and immunofluorescence are listed in Table S2. Two sections per sample were blindly analyzed for all histological analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the lip tissue of mice, and its quality was assessed using an Agilent TapeStation 4000 system (Agilent Technologies, Santa Clara, CA, USA). High-throughput sequencing and sequence analysis were conducted by eBiogen Inc. (Seoul, Korea). In brief, library construction was performed using the QuantSeq 3\u0026prime; mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria), and high-throughput sequencing was performed using a NextSeq 500 (Illumina, Inc., San Diego, California, USA). The heat map of total genes was generated using R (version 3.5.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). GO biological process pathway enrichment analyses were performed with the GSEA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://software.broadinstitute.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"http://software.broadinstitute.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as previously described\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The raw transcriptomic data are accessible through the BioProject database of the National Center for Biotechnology Information under accession number PRJNA1039963.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA) and tested for normal distribution using the D'agostino \u0026amp; Pearson test. A two-tailed unpaired Student\u0026rsquo;s t-test was used to determine the statistical significance between the two independent groups. A two-way analysis of variance (ANOVA) was used to determine the main effects of scratching (vs. non-scratching) and ZD or infection (vs. non-infection) and ZD. Bonferroni\u0026rsquo;s multiple comparison test was applied to examine statistical significance among multiple groups by diet (ZD vs. CD). The skewed data were handled through log transformation and subjected to ANOVA analysis. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the National Research Foundation of Korea (2018R1A5A2024418 and 2020R1A2C2007038).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: YC\u003c/p\u003e\n\u003cp\u003eMethodology: J-YP, H-JY\u003c/p\u003e\n\u003cp\u003eInvestigation: VTDP, HK, S-HC\u003c/p\u003e\n\u003cp\u003eVisualization: SFB, MJM, JLS, EH\u003c/p\u003e\n\u003cp\u003eFunding acquisition: YC\u003c/p\u003e\n\u003cp\u003eSupervision: YC\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: VTDP, YC\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: VTDP, J-YP, HK, H-JY, S-HC, YC\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDeng, X., Wang, Y., Jiang, L., Li, J. \u0026amp; Chen, Q. 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The lack of suitable animal models has hampered understanding of its etiopathogenesis. This study aimed to elucidate the contribution of bacterial infection and zinc deficiency (ZD) to the pathogenic processes underlying OLP by developing a murine model. After subjecting to standard or zinc-deficient diets, C57BL/6 mice underwent labial mucosal microdamage via scratching, followed by oral administration of OLP-isolated \u003cem\u003eEscehrichia coli\u003c/em\u003e 7.2. Scratching alone triggered bacterial translocation to the epithelium and lamina propria, upregulated \u003cem\u003eMmp9\u003c/em\u003e, increased immune responses in the cervical lymph nodes, and augmented CD4\u003csup\u003e+\u003c/sup\u003e T-cell recruitment to labial mucosae. \u003cem\u003eE. coli\u003c/em\u003e infection intensified these responses, in strong synergism with ZD, which shifted the Th response from Th1 to Th17 dominance. Repeated scratching plus \u003cem\u003eE. coli\u003c/em\u003e infection amplified T-cell recruitment, even without ZD, leading to the development of severe inflammatory foci in the labial mucosa, characterized by colloid bodies and disrupted basement membranes. Interestingly, Th1 blockade during \u003cem\u003eE. coli\u003c/em\u003e infection hindered bacterial clearance in the epithelium and caused detachment of the epithelium from the underlying lamina propria with dense inflammatory infiltrates. This suggests that the Th1/IFNγ pathway may not be a suitable therapeutic target for OLP. In conclusion, OLP-like histopathology in the oral mucosa was induced through \u003cem\u003eE. coli\u003c/em\u003e infection, synergized by repeated epithelial microdamage, ZD, or Th1 blockade. This animal model provides a valuable platform for exploring specific hypotheses related to OLP pathogenesis and potential therapeutic targets.\u003c/p\u003e","manuscriptTitle":"Interplay of zinc deficiency, epithelial microdamage, and Escherichia coli infection in a mouse model of oral lichen planus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 09:10:29","doi":"10.21203/rs.3.rs-3913717/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92d2703b-8957-42be-9af1-8324d52d13eb","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-29T09:35:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-06 09:10:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3913717","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3913717","identity":"rs-3913717","version":["v1"]},"buildId":"veTbxFhMMB0_faC6-Wkog","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}