Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Migration‐stimulating factor as a novel biomarker in salivary gland tumours

J Oral Pathol Med (2011) 40 : 747–754 Background: The identification of novel stratification biomarkers would benefit the clinical management of patients with salivary gland tumours. Migration‐stimulating factor (MSF) is a potent stimulator of cell invasion, matrix remodelling and angiogenesis. The aim of this study was to determine whether MSF was expressed in salivary gland tumours and its potential value as a diagnostic biomarker. Methods: Paraffin‐embedded archival specimens of small salivary gland tumours were stained with an MSF‐specific antibody. The specimens included 27 malignant and seven benign tumours; histologically normal salivary gland adjacent to the tumour was present in 16 specimens. MSF expression was assessed by consensus of 2–4 independent observers according to various indices, including ‘overall MSF grade’, ‘percentage of area stained’ and ‘intensity of the staining’. The motogenic effect of MSF on a salivary gland tumour cell line, HSG, was examined in the transmembrane assay. Results: Overall MSF expression increased significantly in a step‐wise fashion from normal salivary gland to benign and malignant tumours (P = 0.04–0.0001); with moderate/strong positive specimens representing 6%, 33% and 74% of the normal, benign and malignant specimens, respectively. MSF was heterogeneously expressed in both carcinoma and stromal cell compartments, its expression being higher in malignant than benign tumours regarding various MSF indices. In tissue culture studies, exogenous MSF stimulated the migration of HSG cells. Conclusions: These immunohistochemical and functional studies suggest that MSF expression is a potentially useful biomarker of salivary gland tumour progression.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Toll-like receptor activity in Recurrent Aphthous Ulceration

Background:  Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, virus, fungal or host tissues. Activation of TLRs causes the production of cytokines that mediate inflammatory responses and drive T helper (Th) 1 and 2 cell development. As an exaggerated Th1 immune response is supposed to be involved in pathogenesis of Recurrent Aphthous Ulceration (RAU), we suggest that RAU patients may have an imbalance in TLR pathways.
Methods:  To study the function of TLR activation ex vivo , peripheral blood mononuclear cells (PBMCs) from RAU patients ( n  = 17) and controls ( n  = 17) were exposed to TLR2 [lipoteichoic acid (LTA), heat-killed Listeria monocytogenes (HKLM) and PamC3CSK4], TLR3 [Poly(I:C)], TLR4 [lipopolysaccharide (LPS)], TLR5 (flagellin) and TLR7 (imiquimod) ligands, and the time course of supernatant tumor necrosis factor-α (TNF-α) levels was quantified by enzyme-linked immunosorbent assay. In addition, serological and salivary TNF-α and soluble CD14 levels were quantified. The TNF-α produced by PBMCs in contact with each TLR ligand and autologous serum or saliva at the same time was also investigated. The data were analyzed by statistical multivariate tests.
Results:  The control group had a higher response to LTA, whereas RAU had a higher response to HKLM. LTA and LPS interfered with the salivary stimulation of the RAU PBMC and HKLM with the stimulation of the control. Autologous serum was capable of inhibiting TLR2 responsiveness to LTA and enhancing LPS stimulation. Salivary and serological levels of sCD14 and TNF-α were not significantly different.
Conclusion:  Recurrent Aphthous Ulceration patients have an anomalous activity of the TLR2 pathway that probably influences the stimulation of an abnormal Th1 immune response.     

Salivary and plasma levels of Toll-like receptor 2 and Toll-like receptor 4 in chronic periodontitis

Background: This cross‐sectional study was planned to investigate whether patients with chronic periodontitis exhibit different salivary or plasma concentrations of Toll‐like receptor (TLR) 2 and TLR4 compared to subjects who are clinically healthy. Methods: Whole saliva and plasma samples were obtained and full‐mouth clinical periodontal measurements were recorded from 22 otherwise healthy patients with chronic periodontitis and 21 systemically and periodontally healthy control subjects. Salivary and plasma TLR2 and TLR4 levels were determined by enzyme‐linked immunoassays. Data were tested statistically using Mann‐Whitney U test. Results: The healthy group exhibited significantly lower values in all clinical measurements (P <0.001). The salivary TLR2 levels were similar in the two study groups (P >0.05). The patients with chronic periodontitis exhibited significantly higher salivary TLR4 (P <0.01) and plasma TLR2 and TLR4 levels (P <0.05). Conclusion: The present findings support a hypothesis that inflammation increases expression of TLRs which leads to an increased detection of TLRs in saliva and plasma, which could be useful as a diagnostic test for periodontal diseases.     

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration‐dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca²⁺]_i in HSG cells, whereas specific agonists of the B2 receptor had a Ca²⁺ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin‐mediated increase in cytosolic Ca²⁺, whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca²⁺. Bradykinin did not affect a carbachol‐induced rise of [Ca²⁺]_i and did not modulate translocation of aquaporin‐5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor‐α (TNF‐α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca²⁺ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein–coupled receptors.     

Correlation of Toll‐Like Receptor 4, Interleukin‐18, Transaminases,and Uric Acid in Patients With Chronic Periodontitis and Healthy Adults

Background: Because of the potential association between periodontal disease and inflammation, the purpose of the present study is to examine the level of Toll‐like receptor 4 (TLR‐4), interleukin‐18 (IL‐18), and uric acid as markers of the inflammatory host response in the plasma and saliva of healthy individuals and patients with periodontitis. In addition, routine biochemical parameters such as fasting glucose, insulin, total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, triglycerides, alanine transaminase (ALT), and aspartate transaminase (AST) were measured. The authors also wanted to check whether patients with chronic periodontitis (CP) exhibit different modulations in salivary and/or plasma concentrations of these parameters compared with clinically healthy individuals. Methods: Saliva and plasma samples were collected from 40 patients with CP and 20 healthy individuals. TLR‐4 and IL‐18 measurements were done using commercially available enzyme‐linked immunosorbent assay kits. Total, HDL, and LDL cholesterol; triglycerides; fasting glucose; AST; and ALT levels were analyzed on a biochemistry analysis system using specific kits. Non‐parametric tests were used for certain parameters in the statistical analyses because the data did not follow Gaussian distribution. Results: Significant differences were observed in plasma and salivary TLR‐4 and IL‐18 levels, along with clinical measurements such as plaque index and probing depth, in patients with CP (P <0.001). The plasma level of TLR‐4 was found to be increased from 0.99 to 3.28 ng/mL in patients with CP. Salivary TLR‐4 levels also showed a slightly higher increase in the diseased state (12.44 to 29.97 ng/mL). A significant increase of ≈46% was recorded in the plasma IL‐18 level. However, salivary IL‐18 levels rose up to >5‐fold in the patients with CP compared with healthy individuals. The level of plasma uric acid was found to be highly significantly increased compared with control individuals. HDL cholesterol and triglyceride also showed significant differences (P <0.02 and P <0.03, respectively). Plasma glucose, total cholesterol, LDL cholesterol, and insulin levels did not show any significant difference. There was only a slight increase in plasma AST and ALT levels between diseased and healthy states (22.55 versus 25.50 IU/L and 12.35 versus 15.95 IU/L, respectively). However, salivary AST and ALT levels showed a ≈6‐fold rise in the patients with CP compared with the healthy individuals. Cross‐correlation analysis in the periodontitis disease group showed a significant association of plasma AST, salivary AST, and salivary ALT with uric acid level. Conclusions: Based on this study, the authors believe that TLR‐4, IL‐18, and uric acid could have a role in the inflammatory pathology of periodontitis. These parameters are suggested to be useful in the prognosis and diagnosis of CP. However, the mechanistic association of these parameters with inflammatory pathology of patients with periodontitis needs to be further elucidated in a higher number of samples.     

Autoantibodies in Sjögren's syndrome patients acutely inhibit muscarinic receptor function

Oral Diseases (2012) 18 , 132–139 Objectives: Autoantibodies from the sera of Sjögren’s syndrome patients (SS IgG) have been suggested to inhibit muscarinic receptor function. However, the acute nature of such an inhibitory effect remains controversial. In this study, we investigated the acute effects of SS IgG on muscarinic receptor function in human submandibular gland (HSG) cells. Methods: The effects of autoantibodies on muscarinic receptor function were studied using microspectrofluorimetry, whole‐cell patch clamp, immunofluorescence confocal microscopy, and a co‐immunoprecipitation assay. Results: Carbachol (CCh) was found to consistently increase intracellular calcium concentration ([Ca²⁺]_i) and activate K⁺ current in HSG cells. However, pretreatment of the cells with SS IgG for 5 or 30 min significantly attenuated these responses, with a substantially more prominent effect after 30 min of treatment. Like CCh, adenosine 5′‐triphosphate (ATP) also increased [Ca²⁺]_i and activated K⁺ currents in HSG cells, although pretreatment with SS IgG did not affect the cellular response to ATP. CCh was found to reorganize α‐fodrin in HSG cells in a Ca²⁺‐dependent manner. However, pretreatment with SS IgG prevented the cytoskeletal reorganization of α‐fodrin induced by CCh. Conclusions: SS IgG acutely and reversibly inhibited muscarinic receptor function, thereby inhibiting the Ca²⁺ mobilization necessary for the activation of K⁺ currents and α‐fodrin reorganization in HSG cells.     

Combined effects of hydrogen sulfide and lipopolysaccharide on osteoclast differentiation in rats

Background: Lipopolysaccharide (LPS) stimulates osteoclast differentiation through toll‐like receptors (TLRs) 2 and 4, and hydrogen sulfide (H₂S) induces osteoclast differentiation. If H₂S activates TLRs, H₂S may enhance the effects of LPS on osteoclast differentiation. The purpose of the present study is to examine the combined effects of sodium hydrogen sulfide (NaHS, an H₂S donor drug) and LPS on osteoclast differentiation and TLR expression in rat periodontal tissue. Methods: Twenty‐eight male Wistar rats (8 weeks old) were divided into four groups (n = 7 per group): a control (no treatment) group and three experimental groups (NaHS group, LPS group, and a combination [NaHS + LPS] group). At 1 day after topical application of NaHS and/or Porphyromonas gingivalis LPS into the gingival sulcus of first molars, the number of tartrate‐resistant acid phosphate (TRAP)‐positive osteoclasts in the periodontal tissue was counted. Expression of TLR2 and TLR4 mRNAs and proteins in the gingival was also assessed. Results: The number of TRAP‐positive osteoclasts was significantly higher in the combination group than in any other group (P <0.01). The combination group had 11.0‐fold higher TLR4 mRNA levels than the control group. TLR4 protein levels were also higher in the combination group than in the NaHS or LPS group. However, the TLR2 mRNA and protein levels were not significantly different in the combination group and the LPS group. Conclusion: In rat periodontal tissue, NaHS and LPS had an additive effect on osteoclast differentiation through activation of the TLR4 pathway but not the TLR2 pathway.     

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

MutSα expression predicts a lower disease-free survival in malignant salivary gland tumors: an immunohistochemical study

Background Appropriate DNA replication is vital to maintain cell integrity at the genomic level. Malfunction on DNA repair mechanisms can have implications related to tumor behavior. Our aim was to evaluate the expression of key complexes of the DNA mismatch-repair system MutSα (hMSH2-hMSH6) and MutSβ (hMSH2-hMSH3) in a panel comprising the most common benign and malignant salivary gland tumors (SGT), and to determine their association with disease-free survival.Material and Methods Ten cases of normal salivary gland (NSG) and 92 of SGT (54 benign and 38 malignant) were retrieved. Immunohistochemistry was performed for hMSH2, hMSH3, hMSH6. Scanned slides were digitally analyzed based on the percentage of positive cells with nuclear staining. Cases were further classified in MutSαhigh and MutSβhigh based on hMSH2-hMSH6 and hMSH3-hMSH6 expression, respectively.Results hMSH3 expression was lower in malignant SGT compared to NSG and benign cases. Adenoid cystic carcinoma (ACC) cases with perineural invasion presented a lower percentage of hMSH3 positive cells. hMSH6 was downregulated in both benign and malignant SGT compared to NSG. Malignant SGT cases with MutSαhigh expression had lower disease-free survival compared to MutSαlow cases. A 10.26-fold increased risk of presenting local recurrence was observed.Conclusions Our findings suggest that a lack of hMSH3 protein function is associated with a more aggressive phenotype (malignancy and perineural invasion) and that MutSα overexpression predicts a poor clinical outcome in malignant SGT. Key words:Salivary Gland Neoplasms, salivary gland cancer, DNA-repair, biomarkers, prognosis.     

Immunohistochemical analysis of P53 and bcl-2 in benign and malignant salivary glands tumors

J Oral Pathol Med (2010) 39 : 48–55 Background: Salivary glands tumors are relatively uncommon neoplasm with widely variable histopathologic and biologic characteristics. Alteration in some proto‐oncogenes and tumor suppressor genes may lead to the development and progression of these tumors. Aims: The purpose of this study was to analyze the immunohistochemical expression of P₅₃ and bcl‐2 in some salivary gland tumors in relation to tumor size, histologic grade, and extent of invasion. Setting and design: The sample consisted of 22 formalin‐fixed paraffin embedded blocks of benign and malignant salivary glands tumors. Material and method: P₅₃ and bcl‐2 immunoreactivity was semi‐quantitatively evaluated in at least 1000 cells examined under the microscope at 40× magnification and recorded as percentage of P₅₃ or bcl‐2 positive tumor cells over the total number of cells examined in the same area. Percentage scores were subsequently categorized using the 5% cut‐off point for positive staining. Results and conclusion: Pleomorphic adenoma (PA) showed negative expression of P₅₃ and bcl‐2 in 70% of cases, whereas all malignant salivary glands tumors were positive for P₅₃ and bcl‐2. P₅₃ positive/bcl‐2 positive immunostaining reaction was week in small size PA and was totally absent in larger lesions. However, all malignant tumors expressed P₅₃, with the highest record in low‐grade adenocarcinoma (76%) and the lowest score was observed in both low‐grade carcinoma in PA and adenocystic carcinoma. bcl‐2 immunostaining was also assessed, the highest recorded score was in high‐grade adeno cystic carcinoma (90%) and the lowest in both low‐grade carcinoma ex‐PA and low‐grade cystic mucoepidermoid carcinoma (3%). P₅₃/bcl‐2 immunostaining reactivity could be helpful in demonstrating salivary glands tumor behavior in terms of progression and extent of invasion.     

Nerve growth factor and tyrosine kinase A in human salivary adenoid cystic carcinoma: expression patterns and effects on in vitro invasive behavior.

PURPOSE: Perineural invasion is a frequent occurrence in salivary adenoid cystic carcinoma (ACC) and may prevent complete surgical resection. Studies have indicated that nerve growth factor (NGF) and its high-affinity receptor tyrosine kinase A (TrkA) may play a role in perineural invasion in several malignancies in which perineural invasion is observed. The present study was conducted to investigate the expression of NGF and TrkA in salivary ACC and to examine the effects of NGF on adhesion, migration and invasion capacities of a salivary ACC cell line (SACC-83) in vitro. PATIENTS AND METHODS: Expression of NGF and TrkA was explored using immunohistochemistry in paraffin-embedded tissues of 32 cases of salivary ACC. The effects of NGF on in vitro adhesion, migration, and invasion capacities of the SACC-83 cell line were examined using an MTT assay and a modified Boyden chamber assay respectively. RESULTS: In ACC specimens, 31 (96.9%) and 32 (100%) tumors showed immunoreactivity for NGF and TrkA respectively. Significant correlations were found between NGF/TrkA expression levels and perineural invasion (P < .05). In cell adhesion assay, the percent adherences of SACC-83 cells co-cultured with 25 ng/ml NGF at 1.5 hours and 5, 25 ng/ml NGF at 6 hours were significantly higher than that co-cultured with 0 ng/ml NGF (P < .05). However, high concentration of NGF (500 ng/ml) resulted in a significant inhibition of invasion (P < .05). CONCLUSION: Overexpression of NGF and TrkA in human salivary ACC tissues may constitute a reason for perineural invasion in salivary ACC.     

Recurrent aphthous ulcers—a Toll‐like receptor–mediated disease?

J Oral Pathol Med (2012) 41 : 158–164 Background: Recurrent aphthous ulcer (RAU) is characterized by acute and painful inflammatory ulcerations, which heal spontaneously but tend to recur. Many pathogens have been proposed as causative agents, but none has been consistently proven. According to our hypothesis, RAU is an autoinflammatory disorder triggered by pathogen‐associated molecular patterns (PAMPs) shared by different pathogenic and commensal microbes. Methods: PAMP‐reactive Toll‐like receptors (TLRs) were mapped in oral epithelium in healthy controls compared to RAU. Results: In controls, the superficial epithelium formed a TLR^?, a PAMP non‐reactive physical barrier zone, but all TLRs were found deeper in the epithelium, usually restricted to suprabasal and basal cell layers. In RAU, the epithelial TLR polarity was lost: TLRs 1, 2, 5, 7, and 8 were found throughout the epithelium, but also TLRs 4, 6, and 10 extended higher up than normally, whereas TLR‐3 was almost lost in RAU. In RAU lesions, connective tissue stroma was heavily infiltrated by TLR⁺ inflammatory cells. Conclusions: Normal TLR architecture prevents inflammatory responses against normal microbes but still contains a deep TLR⁺, PAMP‐reactive dormant defense zone. In RAU, the TLR⁺, PAMP‐reactive zone extends to surface or subsurface exposed to microbial PAMPs. TLR reactivity is further enhanced by recruitment of inflammatory leukocytes forming a new deep line of defense. The organization of the TLR system in healthy mucosa and its changes in RAU are compatible with active pathogenic involvement of TLRs, which together with the typical clinical picture and course suggest that RAU is a TLR‐mediated disease.