Immunohistochemical detection of Mycoplasma salivarium in oral lichen planus tissue

Background Oral lichen planus (OLP) is a T‐cell‐mediated inflammatory disease; however, its exact etiology is unknown. Hyperkeratosis is often observed in OLP lesions. Previous studies have revealed the localization of Mycoplasma salivarium in the epithelial cells of oral leukoplakia with hyperkeratosis. Herein, we investigated the presence of M. salivarium in OLP tissue by immunohistochemistry to determine the causative factor of OLP. Methods Forty‐one formalin‐fixed, paraffin‐embedded samples obtained from 31 patients with OLP were examined. Ten samples of normal‐appearing oral mucosa were used as controls. Immunohistochemistry (IHC) was performed using anti‐M. salivarium monoclonal antibodies. Results and Conclusions Mycoplasma salivarium was detected in the epithelium and lymphocyte infiltrate area in 24 of 41 OLP samples (58.5%). The bacteria were intracellularly localized in epithelial cells, while it was unclear whether they were also localized in lymphocyte cells or in the extracellular spaces among the lymphocytes in the subepithelial lymphocyte infiltrate area. Little or no staining was observed in the epithelium in the normal‐appearing mucosa samples. Sawtooth rete ridge formation was observed in 21 OLP samples (51.2%), and a significant positive correlation between sawtooth rete ridge formation and IHC positivity was demonstrated. However, the role of M. salivarium in the epithelium and lamina propria of OLP tissue remains unknown.

degeneration of the basal cells is induced by apoptosis, which is primarily triggered by CD8+ T cells. 8 However, its precise mechanism is unclear, and the antigen that triggers the inflammatory response of the T cells remains unknown.
Several causative factors of OLP have been proposed, including infectious viruses and bacteria, dental materials, drugs, and autoimmunity. 6 With regard to dental materials and drugs, the presence of oral mucosal lesions has been reported, which are improved or eliminated by removing the dental restorative materials or drug withdrawal. 9 It is difficult to distinguish these lesions from OLP because they are clinically and histopathologically similar to OLP. However, these lesions are regarded as different than OLP and are referred to as "oral lichenoid lesions (OLLs)". 9 The association of various infectious agents, such as viruses and bacteria, with OLP has been studied. Some viruses, including human papilloma virus (HPV) and hepatitis C virus (HCV), have been detected at higher rates in patients with OLP than in control subjects. However, the relationship between infection with these viruses and OLP is controversial. 10 Plaque control can improve the symptoms of OLP, suggesting an association of some oral bacteria with OLP. 11 Recently, several pathogenic periodontal bacteria were detected at higher levels in patients with OLP than in non-OLP patients. 12 However, the correlation between these bacteria and OLP is also unclear.
Recently, Choi et al. 13 reported the presence of bacteria throughout the epithelium and lamina propria of OLP tissues, and they proposed that intracellular bacteria trigger T-cell infiltration and provide target antigens. However, the species of bacteria detected in the OLP tissues was not clarified.
Mycoplasmas are the smallest free-living bacteria capable of selfreplication. Among the 16 mycoplasmas isolated from humans, M. salivarium is the most common species isolated from the oral cavity. 14 It preferentially resides in dental plaques and gingival sulci, similar to pathogenic periodontal bacteria. 15 Mizuki et al. 16  Hyperkeratosis is regarded as a histopathological feature of OLP.
Wickham striae appear in reticular types of OLP, corresponding to the focal regions of hyperkeratosis or parakeratosis. 17  Ten FFPE samples containing normal-appearing oral mucosa, which were obtained by excising cysts, benign tumors, or other diseases, were used as controls. The mean age of the 10 patients (four males and six females) was 59 years (range: 31-79 years).
Specimens were cut into 4-lm-thick serial sections and mounted onto MAS-coated slides (Matsunami, Osaka, Japan). The slides were deparaffinized, rehydrated, and used for hematoxylin and eosin staining and IHC.

| Histopathological diagnosis of OLP
Histopathological diagnosis was performed using slides stained with hematoxylin and eosin, according to the histopathological criteria of OLP by Eisenberg. 4 Samples with findings of liquefaction degeneration of the basal cells, a subepithelial dense band-like lymphocytic infiltrate, and a normal epithelial maturation pattern, which are essential features for the histological diagnostic criteria of OLP, were diagnosed as OLP.
After the histological diagnosis, 41 samples obtained from 31 patients were selected for further analysis.  Tables 1 and 2.

| DISCUSSION
OLP is a T-cell-mediated inflammatory disease. However, the exact antigen that triggers the inflammatory response of the T cells is unknown, 18 although some infectious microorganisms have been proposed as an etiological factor of OLP.
Associations between OLP and viruses, including Herpes simplex 1, Epstein-Barr virus, cytomegalovirus, Herpes virus 6, HPV, and HCV, have been studied. 10 In particular, HPV has been detected in OLP tissues, but the detection rates have varied greatly among investigations. 10 In contrast, many investigators have shown a significantly higher risk of HCV infection in patients with OLP than in control subjects, although geographic regional differences among the studies were found. 19 Some studies also suggested that patients with HCV are more likely to develop OLP.
However, the mechanism involved in OLP development in patients with HCV is not clear, and the localization of HCV in OLP lesions is controversial. 19 In Although sawtooth rete ridge formation is recognized to be less prevalent in OLP than in the lichen planus of the skin, it was reportedly observed in 30% of OLP cases. 3  This study represents preliminary research to examine the presence of M. salivarium in OLP tissues by IHC. Credible data demonstrating the pathogenesis of OLP could not be obtained. In addition, this study has several limitations, including the small number of OLP samples. However, we believe that the findings obtained from this study will be useful to elucidate the etiological factors and pathogenesis of OLP in the future. Further studies are necessary to clarify the role of M. salivarium in OLP.

ACKNOWLEDG EMENTS
We thank Professor Yasunori Takeda  for help with the microscopy experiments.

CONFLI CT OF INTEREST
The authors declare no conflict of interests associated with this manuscript.