Myeloid dendritic cells stimulated by thymic stromal lymphopoietin promote Th2 immune responses and the pathogenesis of oral lichen planus

<div><p>Oral lichen planus (OLP) is a chronic inflammatory disease characterized by subepithelial T-cell infiltration. Recent studies reported that specific T helper (Th) subsets, especially Th2 cells, are involved in the pathogenesis of OLP. Thymic stromal lymphopoietin (TSLP) is mainly secreted by epithelial cells and potently activates myeloid dendritic cells (mDCs) to induce Th2-mediated inflammation. Here, we investigated the expression of TSLP and related molecules in OLP. Buccal mucosa specimens from patients with OLP, hyperkeratosis, and ulcer were analyzed by immunohistochemistry for expression of TSLP, its receptor (TSLPR), and inflammatory cells. TSLP was detected in/around the epithelium of patients with OLP and hyperkeratosis, whereas TSLPR, CD11c (mDC), and GATA3 (Th2) were strongly expressed in the subepithelial layer only in OLP patients. Double immunofluorescence staining showed that TSLPR expression mainly co-localized with CD11c. Moreover, the number of CD11c- and GATA-3 positive cells was correlated in OLP patients. In lesions selectively extracted by laser microdissection, the mRNA expression of Th2 (IL-4, MDC, TARC, GATA3)- and Th17 (IL-17, RORγt)-related molecules in OLP patients was significantly higher than in other groups. These results suggest that CD11c⁺ mDCs expressing TSLPR contribute to aberrant Th2 immune responses and the pathogenesis of OLP via TSLP stimulation.</p></div

Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

<div><p>Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by <i>Candida albicans</i>. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of <i>C. albicans</i>, though the ratio of <i>C. dubliniensis</i> was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of <i>C. dubliniensis</i> were positively correlated with treatment duration. These results suggest that <i>C. dubliniensis</i> and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.</p></div

mRNA expression of Th-related cytokines, chemokines, and transcription factors in the selective BM specimens by using Laser Microdissection (LMD).

<p><b>A</b>, Hematoxylin and eosin (HE)-stained BM specimens (a). The infiltration of inflammatory cells was indicated by a green line (b). The preselected area was extracted by using a guided laser beam after LMD. (c). Scale bars, 500 μm. <b>B</b>, mRNA expression levels of Th-related cytokines, chemokines, and transcription factors in BM samples from patients with OLP (n = 15), hyperkeratosis (n = 5) and ulcer (n = 5). mRNA expression levels of Th-related cytokines, chemokines, and transcription factors were estimated quantitatively as described in the Methods section. Statistical significance of differences between groups was determined by Kruskal-Wallis tests (**<i>P</i> < 0.01, *<i>P</i> < 0.05).</p

Correlation between the number of CD11c⁺ and GATA3⁺ cells in BM specimens.

<p>The number of these positive cells in BM samples from patients with oral lichen planus (OLP) (n = 15), hyperkeratosis (n = 5) and ulcer (n = 5) was calculated from immunohistochemical staining as described in the Methods section. Statistical significance of differences between groups was determined by Spearman’s rank correlation (<i>p</i> < 0.05).</p

Association of Th2-related molecules with clinical characteristics in 43 patients with OLP.

<p>Association of Th2-related molecules with clinical characteristics in 43 patients with OLP.</p

Expression and localization of TSLPR-expressing cells in the BM specimens.

<p><b>A</b>, Representative images of paraffin sections stained with CD11c and CD68 antibodies (brown). Counterstaining with Mayer’s hematoxylin was subsequently performed (blue). Scale bars, 100 μm. <b>B</b>, Double immunofluorescence staining performed with anti-CD11c (green) or anti-CD68 (green), anti-TSLPR (red), and DAPI for staining nuclei (blue). White arrows indicate co-localizing staining of cells (yellow). Scale bars, 100 μm. <b>C</b>, The number and ratio of merged cells to TSLPR⁺ cells from 15 patients with OLP calculated by double immunofluorescence staining. Statistical significance of differences between groups was determined by Mann—Whitney <i>U</i> tests (*<i>P</i> < 0.05).</p

Localization of Thymic Stromal Lymphopoietin (TSLP) in Buccal Mucosa (BM) specimens from patients with Oral Lichen Planus (OLP).

<p><b>A</b>, Representative images of paraffin sections stained with hematoxylin and eosin (a-c) and TSLP antibodies (brown) (d-f). Comparative examination was performed between lesion (b, e) and normal section (c, f). Counterstaining with Mayer’s hematoxylin was subsequently performed (blue). Scale bars, 100 μm. <b>B</b>, Distribution of TSLP and TSLPR in BM specimens from representative patients with OLP, hyperkeratosis and ulcer. Counterstaining was performed with Mayer's hematoxylin (blue). Scale bars, 100 μm. <b>C</b>, The number of TSLP⁺ and TSLPR⁺ cells in BM specimens from patients with OLP (n = 15), hyperkeratosis (n = 5) and ulcer (n = 5). The number of these positive cells was calculated from immunohistochemical staining as described in the Methods section. Statistical significance of differences between groups was determined by Kruskal-Wallis tests (**<i>P</i> < 0.01, *<i>P</i> < 0.05).</p