Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K.

Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-ß) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

The diagnostic utility of submandibular gland sonography and labial salivary gland biopsy in IgG4-related dacryoadenitis and sialadenitis: Its potential application to the diagnostic criteria.

Objectives: In this study, we investigated the diagnostic utility of submandibular gland (SMG) sonography and labial salivary gland (LSG) biopsy as a less invasive procedure for diagnosing IgG4-related dacryoadenitis and sialadenitis (IgG4-DS)Methods: Sixty-eight patients with suspected IgG4-DS by presenting swelling of elevated serum IgG (>1747 mg/dl) and/or swelling glands underwent SMG sonography, LSG biopsy and measurement for serum IgG4. SMG sonographic diagnosis was determined by the following characteristic changes; 'hypoechoic areas of a nodal pattern with high vascularity' and/or 'hypoechoic areas of a reticular pattern in the superficial part'.Results: Thirty-one patients were diagnosed with IgG4-DS, 5 with IgG4-RD unaccompanied by lacrimal and salivary gland lesions, 28 with Sjögren's syndrome, and 4 with malignant lymphoma. The sensitivity, specificity, and accuracy of SMG sonography and LSG biopsy were 100%, 83.8%, 91.2% and 64.5%, 73.8%, 75.0%, respectively. Moreover, those of SMG sonography and LSG biopsy combined with serum IgG4 concentration (>135 mg/dl) were 100%, 94.6%, 97.1% and 64.5%, 91.9%, 79.4%, respectively.Conclusion: LSG biopsy needs to be extremely careful to diagnose IgG4-DS because of its low sensitivity. SMG sonography is sufficient for the diagnosis of IgG4-DS, especially when combined with serologic analysis. Thus, SMG sonography could adapt to the diagnostic criteria of IgG4-DS as a non-invasive method.

Activated M2 Macrophages Contribute to the Pathogenesis of IgG4-Related Disease via Toll-like Receptor 7/Interleukin-33 Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a unique inflammatory disorder in which Th2 cytokines promote IgG4 production. In addition, recent studies have implicated the Toll-like receptor (TLR) pathway. This study was undertaken to examine the expression of TLRs in salivary glands (SGs) from patients with IgG4-RD. METHODS: SGs from 15 patients with IgG4-RD, 15 patients with Sjögren's syndrome (SS), 10 patients with chronic sialadenitis, and 10 healthy controls were examined histologically. TLR family gene expression (TLR-1 through TLR-10) was analyzed by DNA microarray in the submandibular glands (SMGs). Up-regulation of TLRs was confirmed in SGs from patients with IgG4-RD. Finally, the phenotype of human TLR-7 (huTLR-7)-transgenic C57BL/6 mice was assessed before and after stimulation with TLR agonist. RESULTS: In patients with IgG4-RD, TLR-4, TLR-7, TLR-8, and TLR-9 were overexpressed. Polymerase chain reaction validated the up-regulation of TLR-7 in IgG4-RD compared with the other groups. Immunohistochemical analysis confirmed strong infiltration of TLR-7-positive cells in the SGs of patients with IgG4-RD. Double immunohistochemical staining showed that TLR-7 expression colocalized with CD163+ M2 macrophages. After in vitro stimulation with a TLR-7 agonist, CD163+ M2 macrophages produced higher levels of interleukin-33 (IL-33), which is a Th2-activating cytokine. In huTLR-7-transgenic mice, the focus and fibrosis scores in SMGs, pancreas, and lungs were significantly higher than those in wild-type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL-33 in huTLR-7-transgenic mice was distinctly increased upon stimulation with a TLR-7 agonist (P < 0.05). CONCLUSION: TLR-7-expressing M2 macrophages may promote the activation of Th2 immune responses via IL-33 secretion in IgG4-RD.

The expansion in lymphoid organs of IL-4+ BATF+ T follicular helper cells is linked to IgG4 class switching in vivo.

Distinct T follicular helper (TFH) subsets that influence specific class-switching events are assumed to exist, but the accumulation of isotype-specific TFH subsets in secondary lymphoid organs (SLOs) and tertiary lymphoid organs has not been hitherto demonstrated. IL-4-expressing TFH cells are surprisingly sparse in human SLOs. In contrast, in IgG4-related disease (IgG4-RD), a disorder characterized by polarized Ig class switching, most TFH cells in tertiary and SLOs make IL-4. Human IL-4+ TFH cells do not express GATA-3 but express nuclear BATF, and the transcriptomes of IL-4-secreting TFH cells differ from both PD1hi TFH cells that do not secrete IL-4 and IL-4-secreting non-TFH cells. Unlike IgG4-RD, IL-4+ TFH cells are rarely found in tertiary lymphoid organs in Sjögren's syndrome, a disorder in which IgG4 is not elevated. The proportion of CD4+IL-4+BATF+ T cells and CD4+IL-4+CXCR5+ T cells in IgG4-RD tissues correlates tightly with tissue IgG4 plasma cell numbers and plasma IgG4 levels in patients but not with the total plasma levels of other isotypes. These data describe a disease-related TFH subpopulation in human tertiary lymphoid organs and SLOs that is linked to IgG4 class switching.

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

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.

Interleukin-33 produced by M2 macrophages and other immune cells contributes to Th2 immune reaction of IgG4-related disease.

IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and marked infiltration of IgG4-positive cells in multiple organs. Interleukin-33 (IL-33) is a recently described cytokine that is secreted by damaged epithelial cells, macrophages, and dendritic cells, and potently activates helper T type 2 (Th2) immune responses, which have been suggested to play a major role in IgG4 production of IgG4-RD. Here, we assessed the expression of IL-33 and related molecules in the salivary glands (SGs) of patients with IgG4-RD versus that in patients with Sjögren's syndrome (SS) and controls. Expression of IL-33 and its receptor (ST2) was strongly detected around ectopic germinal centers (GCs) in the SGs from patients with IgG4-RD, whereas IL-33 was expressed only in epithelial cells in patients with SS and controls. Moreover, IL-33 and CD68+/CD163+ macrophages were mainly distributed around ectopic GCs in patients with IgG4-RD. Double immunofluorescence staining showed that IL-33 expression co-localized with CD68+/CD163+ macrophages. Finally, mRNA expression levels of IL-33 showed a positive correlation to those of Th2 cytokines (IL-4 and IL-13) in patients with IgG4-RD. Our data suggest that IL-33 produced by M2 macrophages might contribute to the pathogenesis of IgG4-RD via aberrant activation of Th2 immune responses.

Lesional CD4+ IFN-γ+ cytotoxic T lymphocytes in IgG4-related dacryoadenitis and sialoadenitis.

OBJECTIVES: IgG4-related disease (IgG4-RD) is a chronic, systemic, inflammatory condition of unknown aetiology. We have recently described clonally expanded circulating CD4+ cytotoxic T lymphocytes (CTLs) in IgG4-RD that infiltrate affected tissues where they secrete interleukin (IL)-1ß and transforming growth factor -ß1 (TGF-ß1). In this study, we sought to examine the role of CD4+ CTLs in the pathogenesis of IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) and to determine whether these cells secrete interferon-gamma (IFN-γ) at lesional sites. METHODS: Salivary glands of 25 patients with IgG4-DS, 22 patients with Sjögren's syndrome (SS), 12 patients with chronic sialoadenitis (CS) and 12 healthy controls were analysed in this study. Gene expression analysis was performed on submandibular glands (SMGs) from five patients with IgG4-DS, three with CS and three healthy controls. Infiltrating CD4+ CTLs were examined by quantitative multicolour imaging in tissue samples from 20 patients with IgG4-DS, 22 patients with SS, 9 patients with CS and 9 healthy controls. RESULTS: In IgG4-DS tissues, nine genes associated with CD4+ CTLs were overexpressed. The expression of granzyme A (GZMA) mRNA was significantly higher in samples from patients with IgG4-RD compared with corresponding tissues from SS and healthy controls. Quantitative imaging showed that infiltrating CD4+ GZMA+ CTLs were more abundant in patients with IgG4-DS than in the other groups. The ratio of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS correlated with serum IgG4 concentrations and the number of affected organs. A large fraction of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS secreted IFN-γ. CONCLUSIONS: The pathogenesis of IgG4-DS is associated with tissue infiltration by CD4+GZMA+ CTLs that secrete IFN-γ.

Molecular analysis of fungal populations in patients with oral candidiasis using next-generation sequencing.

Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections.

The diagnostic utility of labial salivary gland biopsy in IgG4-related disease.

OBJECTIVE: For the definitive diagnosis of IgG4-related disease (IgG4-RD), biopsies of local lesions are recommended so as to exclude other diseases, including lymphoma and cancer. However, performing biopsies of underlying organs is technically difficult. In this study, we examined the diagnostic utility of labial salivary gland (LSG) biopsy as a less invasive procedure. METHODS: Sixty-six patients with suspected IgG4-RD by clinical findings or high serum IgG4 underwent LSG biopsy. We examined the relationship between the number of IgG4-positive plasma cells in LSG and clinical findings. RESULTS: The final diagnosis was 45 patients with IgG4-RD, 12 with Sjögren's syndrome, four with suspected Sjögren's syndrome, three with malignant lymphoma, one with systemic lupus erythematosus, and one with Warthin's tumor. The sensitivity, specificity, and accuracy of LSG biopsy were 55.6%, 100.0%, and 70.0%, respectively. Forty-five IgG4-RD patients were divided into two groups: 1) 25 with lesions of salivary glands (IgG4-RD S+) and 2) 20 without these lesions (IgG4-RD S-). Seventeen of 25 (68.0%) IgG4-RD S + and 8 of 20 (40.0%) IgG4-RD S - patients were positive for LSG biopsy. In the IgG4-RD S - patients, the mean number of affected organs and serum IgG4 in the positive cases for LSG biopsy were significantly higher than in the negative cases. CONCLUSION: A solo LSG biopsy is insufficient for the diagnosis of IgG4-RD because of its low sensitivity. However, LSG biopsy combined with clinical findings, including serum IgG4 and number of affected organs, may contribute towards a diagnosis of IgG4-RD patients with affected underlying organs.

DNA Microarray Analysis of Submandibular Glands in IgG4-Related Disease Indicates a Role for MARCO and Other Innate Immune-Related Proteins.

IgG4-related disease (IgG4-RD) is a novel systemic disease entity characterized by elevated serum IgG4 and tissue infiltration of IgG4-positive plasma cells accompanied by severe fibrosis. Although recent studies demonstrated that innate immune cells including monocytes and macrophages might promote local fibrosis and IgG4 production, the pathological mechanism remains unclear. In this study, we sought to identify the disease-associated genes, especially innate immune molecules. Gene expression was analyzed by DNA microarray in submandibular glands (SMGs) from patients with IgG4-RD (n = 5), chronic sialoadenitis (CS) (n = 3), and controls (n = 3). Differentially expressed genes (DEGs) were validated by real-time polymerase chain reaction (PCR) and immunohistochemical staining in IgG4-RD (n = 18), CS (n = 4), Sjögren syndrome (n = 11), and controls (n = 10). Gene expression patterns in the 3 groups were quite different from each other by the pvclust method and principal components analysis. In IgG4-RD, 1028 upregulated genes and 692 downregulated genes were identified as DEGs (P < 0.05). Gene Ontology (GO) term analysis indicated that the upregulated DEGs in IgG4-RD encoded proteins involved in T/B cell activation and chemotaxis. PCR validated significantly higher expression of macrophage receptor with collagenous structure (MARCO), a pattern-recognition receptor, in IgG4-RD compared with the other groups (P < 0.01). Immunohistochemical analysis confirmed that the expression pattern of MARCO was similar to that of the M2 macrophage marker CD163. MARCO was identified as a disease-associated molecule in IgG4-RD by DNA microarray. Moreover, M2 macrophages might contribute to the initiation of IgG4-RD via MARCO.

Cytokine profiles contribute to understanding the pathogenic difference between Good syndrome and oral lichen planus: two case reports and literature review.

We described and analyzed the pathogenic difference between Good syndrome (GS) and oral lichen planus (OLP) in oral mucosa. Good syndrome (GS) is a rare disease characterized by B and T cell immunodeficiency associated with hypogammaglobulinemia and thymoma. GS patients frequently develop oral lichenoid lesions with lymphocytic infiltration beneath the basal layer. Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa characterized by destruction of basal cells by Langerhans cells, macrophages, and T lymphocytes. Although the histological features of the lesions of both diseases are very similar, the pathogenesis of GS in the oral mucosa remains unknown. In this study, we thus investigated the expression of infiltrating lymphocyte subsets (CD3, CD20, CD4, and CD8) and T helper (Th) cytokines including interferon (IFN)-γ (Th1 type), interleukin (IL)-4 (Th2 type), IL-17 (Th17 type), and IL-10 (regulatory T cell type) by immunohistochemistry in buccal mucosa specimens from 2 GS patients compared with 15 OLP patients. All patients showed a predominance of CD3 T cells over CD20 B cells, and CD4 Th cells over CD8 cytotoxic T cells. This polarization was especially prominent in GS. IFN-γ and IL-10 were strongly detected in the infiltrating lymphocytes of all patients. However, IL-4 and IL-17 were detected in OLP patients only. These results suggest that the pathogenesis of GS is different from that of OLP. GS is a unique inflammatory disorder characterized by dysfunction of Th2 and Th17 immune reactions via abnormal T-B cell interaction.

Rapidly progressive symptom development of pulmonary arterial hypertension: a case report of Trousseau syndrome.

Trousseau syndrome is most commonly defined as a hypercoagulability syndrome associated with mucin-producing adenocarcinoma. We report here a rare case of Trousseau syndrome presenting as pulmonary arterial hypertension. The patient complained of cough and increasing exertional dyspnea. Rapidly progressive symptom development of pulmonary arterial hypertension accompanied by right heart failure was observed, and the patient died on hospital day 2. An autopsy revealed Krukenberg tumors on both ovaries and a signet-ring cell gastric carcinoma. In the lungs there was tumor embolism with signet-ring cells to some extent, but the peripheral pulmonary arteries were occupied primarily by pulmonary embolism with platelets, fibroblasts, and fibrotic organized thrombi.

Prevention of ventricular extrasystole by mexiletine in patients with normal QT intervals is associated with a reduction of transmural dispersion of repolarization.

BACKGROUND: Antiarrhythmic potential of mexiletine in patients with congenital and acquired long-QT syndrome (LQTS) has been attributed to a reduction of transmural dispersion of repolarization (TDR). A similar mechanism could be involved in the antiarrhythmic activity of the drug in patients with normal QT intervals, but the issue remains to be investigated. METHODS AND RESULTS: We analyzed 24-h Holter ECG recordings from 17 patients in sinus rhythm showing premature ventricular complexes (PVCs) with normal QT intervals (age, 62+/-10 years, mean+/-S.D.). Treatment of the patients with oral mexiletine (300 mg/day for 21-40 days) resulted in a significant reduction of PVCs (from 13899+/-18887 to 6949+/-12822 beats/24 h, p<0.01). Rate-dependent behavior of ventricular repolarization was analyzed by plotting QT intervals (QT(peak), QT(end)), and the interval from T-wave peak to T-wave end (TPE) against preceding respective RR intervals of sinus beats. Both the QT(peak) and QT(end) tended to be shortened by mexiletine at RR intervals from 600 ms to 1000 ms, although the changes did not reach statistical significances. TPE, which reflects TDR, was shortened significantly at relatively long RR intervals (by 14+/-9% at RR of 900 ms, p<0.05). There was a linear relationship between the percentage shortening of TPE and the percentage reduction of PVCs (r=0.86, p<0.04). TPE> or =70 ms was significantly associated with PVC suppression >75% with an odds ratio of 0.60 (95% confidence interval 0.36-0.98, per 1 ms increment). CONCLUSION: Inhibitory effect of mexiletine against PVCs in patients with normal QT intervals is mediated at least in part by a reduction of TDR. Mexiletine may be effective in patients exhibiting longer baseline TPE.

Lateral difference and interhemispheric transfer on arm-positioning movement between right and left handers.

We investigated the transfer of an arm-positioning movement between the right and left arms of right and left handers. 30 male (15 strong right handers and 15 strong left handers) subjects were asked to perform a constrained criterion movement, 12 cm in length, with right or left arm and a test movement at estimated 6-, 12-, or 24-cm length with the contralateral arm. In the right handers, the constant error of the left arm test movement was near zero, and that of the right arm indicated overshooting. In the left handers, the constant errors of the left arm test movement were farther from zero than those of right arm test movement. Left handers as well as right handers showed manual asymmetry on positioning movement. A plausible explanation for the manual asymmetry on the arm-positioning task is related to interhemispheric transfer of spatial information on positioning movement.