Targeting TNF-α suppresses the production of MMP-9 in human salivary gland cells

Objective: Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine that plays an essential role in inflammation and apoptosis. Our previous study suggested that TNF-α-induced activation of matrix metalloproteinase-9 (MMP-9) resulted in the destruction of acinar tissue in the salivary glands of patients with Sjögren’s syndrome (SS) via disruption of the acinar cell-basement membrane. Recently, a wide array of biological agents has been designed to inhibit TNF, including etanercept and adalimumab. In this study, we demonstrate the suppressive effect of anti-TNF agents on TNF-α-induced MMP-9 production in NS-AV-AC, an immortalized human salivary gland acinar cell line. Materials and Methods: NS-AV-AC cells were treated with etanercept or adalimumab after TNF-α treatment. MMP-9 production and enzymatic activity were, respectively, visualized by real-time PCR and ELISA assay, and evaluated by gelatin zymography, and apoptosis was evaluated by DNA fragmentation assay. Results: TNF-α induced the production of MMP-9 in NS-SV-AC cells. However, this production was greatly inhibited by treatment with etanercept or adalimumab. In addition, TNF-α-induced DNA fragmentation was prevented by treatment with etanercept or adalimumab. Conclusions: These results may indicate that anti-TNF agents would have therapeutic efficacy for preventing destruction of the acinar structure in the salivary glands of patients with SS

耐糖性酵母の特性解明及び生育制御に関する研究

京都大学0048新制・論文博士博士(農学)乙第8014号論農博第1791号新制||農||638(附属図書館)学位論文||H4||N2513(農学部図書室)UT51-92-U250(主査)教授 熊谷 英彦, 教授 小清水 弘一, 教授 鬼頭 誠学位規則第4条第2項該当Doctor of Agricultural ScienceKyoto UniversityDFA

ビスコクラウリン型アルカロイドであるセファランチンは、1次性シェーグレン症候群患者の唾液分泌量を著明に増加させる

Objective: Our previous findings suggested that the suppression of tumor necrosis factoralpha (TNF-α)-induced matrix metalloproteinase (MMP)-9 production by the biscoclaurine alkaloid cepharanthine could prevent the destruction of the acinar structure in the salivary glands of murine Sjögren's syndrome. Here, we examined the effect of cepharanthine on the salivary secretion in primary Sjögren's syndrome (pSS) patients. Methods: In this single-center, open-label pilot study, 29 patients with pSS (28 women, 1 man) received 6 mg/day orally cepharanthine for 12 months. Standard clinical assessments and stimulated salivary flow were examined at baseline and each month for 12 months in all 29 patients. In eight of the patients, inflammatory lesions in the salivary glands were histologically investigated before and after the cepharanthine treatment. We analyzed the expressions of p65, phosphorylated IκB-α, MMP-9, and type IV collagen immunohistochemically. Results: All patients completed the study without any adverse events. A significant increase in salivary flow was observed after the cepharanthine treatment compared to baseline. The serological analysis revealed that the 14 patients with an anti-Sjögren's-syndrome-related antigen A (anti-SSA/Ro) antibody value that was either negative or 64 U/ml did not. The immunohistochemical analysis demonstrated that although p65, phosphorylated IκB-α, and MMP-9 were more strongly stained in the acinar cells of the patients at baseline compared to the staining at the completion of cepharanthine treatment, the continuity of type IV collagen was observed following the cepharanthine treatment. These results indicate that cepharanthine could inhibit the phosphorylation of IκB-α, followed by the prevention of MMP-9 activation and the stabilization of type IV collagen. Conclusions: Our findings suggest that cepharanthine could be a promising agent for improving salivary secretion in pSS patients

Role of CXCL10 in pathogenesis of Sjögren's syndrome

Sjögren's syndrome (SS) is a common autoimmune disease characterized by the destruction of acinar structure by marked lymphocytic infiltrates in the salivary and lacrimal glands, resulting in sicca symptoms. Gene expression profiling of lip salivary glands (LSGs) shows that C-X-C motif chemokine 10 (CXCL10) expression is upregulated in patients with primary SS (pSS). CXCL10 and its receptor, C-X-C receptor 3 (CXCR3), contribute to the pathogenesis of SS. We investigated the clinical significance of CXCL10 and CXCR3 in the autoimmune lesions of pSS and the molecular mechanisms of CXCL10 upregulation in the salivary gland cells. CXCL10 showed particularly intense staining in LSG ductal cells from pSS patients. CXCR3 expression was detected primarily in CD163+ macrophages. The number of CXCR3+CD163+ macrophages was inversely correlated with the severity of LSG inflammatory lesions. Our in vitro experiments demonstrated that human salivary gland ductal (NS-SV-DC) cells produced higher levels of CXCL10 than acinar (NS-SV-AC) cells. Furthermore, NS-SV-DC and NS-SV-AC cells had different regulators of CXCL10 enhancement: interferon (IFN)-γ had more potential than IFN-α, tumor necrosis factor (TNF)-α, and interleukin (IL)1-β in the induction of CXCL10 production in NS-SV-DC cells, whereas TNF-α had the potential to induce CXCL10 production in NS-SV-AC cells. Our results suggest that CXCL10 overexpression in salivary glands is mainly caused by IFN-γ-stimulated salivary gland ductal cells. The enhanced production of CXCL10 by ductal cell IFN-γ results in the migration of CXCR3+ immune cells. CXCL10 plays an important role in SS pathogenesis, and CXCL10 regulation may be useful in the treatment of SS patients

Baricitinib Inhibits CXCL10 Production in Salivary Gland Cells

Sjögren's syndrome (SS) is a chronic autoimmune disease targeting salivary and lacrimal glands. C-X-C motif chemokine ligand 10 (CXCL10) expression is upregulated in lip salivary glands (LSGs) of primary SS (pSS) patients, and CXCL10 involved in SS pathogenesis via immune-cell accumulation. Moreover, interferon (IFN)-γ enhances CXCL10 production via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We investigated the effects of baricitinib, a selective JAK1/2 inhibitor, on both IFN-γ-induced CXCL10 production and immune-cell chemotaxis. We used immunohistochemical staining to determine the expression levels and localization of JAK1 and JAK2 in LSGs of SS patients (n=12) and healthy controls (n=3). We then evaluated effect of baricitinib in an immortalized normal human salivary gland ductal (NS-SV-DC) cell line. Immunohistochemical analysis of LSGs from pSS patients revealed strong JAK1 and JAK2 expression in ductal and acinar cells, respectively. Baricitinib significantly inhibited IFN-γ-induced CXCL10 expression as well as the protein levels in an immortalized human salivary gland ductal-cell clone in a dose-dependent manner. Additionally, western blot analysis showed that baricitinib suppressed the IFN-γ-induced phosphorylation of STAT1 and STAT3, with a stronger effect observed in case of STAT1. It also inhibited IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggested that baricitinib suppressed IFN-γ-induced CXCL10 expression and attenuated immune-cell chemotaxis by inhibiting JAK/STAT signaling, suggesting its potential as a therapeutic strategy for pSS

MMP-9 Inhibition Suppresses Interferon-γ-Induced CXCL10 Production in Human Salivary Gland Ductal Cells

Gene expression profiling of lip salivary gland (LSG) has shown that C-X-C motif chemokine 10 (CXCL10) and matrix metalloproteinase 9 (MMP9) expression is up-regulated in primary Sjögren's syndrome (pSS) patients. Although CXCL10 and MMP-9 are both associated with pSS pathogenesis, the potential relationship between these two factors has not been investigated. In this study, we used LSG sections from pSS patients and human salivary gland cell lines to investigate the relationship between CXCL10 and MMP-9. Immunofluorescence analyses revealed that CXCL10 and MMP-9 were co-expressed in the LSG of pSS patients, particularly in expanded ductal cells. Furthermore, RT-qPCR analyses on human salivary gland ductal NS-SV-DC cells confirmed that CXCL10 expression was induced by interferon (IFN)-γ, whereas that of MMP9 was stimulated by IFN-α, tumor necrosis factor-α, and interleukin 1β. Remarkably, MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells significantly decreased CXCL10 mRNA and secreted protein levels. Further analyses established that MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells decreased STAT1 phosphorylation and hence suppressed IFN-γ signaling. Collectively, these results suggest that in addition to its reported role in the destruction of acinar structures, MMP-9 is involved in the IFN-γ-induced production of CXCL10 in pSS lesions. We believe that our findings open the door to the development of novel treatments for pSS, based on the modulation of MMP-9 activity

Clinical Significance of Carbapenem-Tolerant Pseudomonas aeruginosa Isolated in the Respiratory Tract

We often come across difficult to treat infections—even after administering appropriate antibiotics according to the minimal inhibitory concentration of the causative bacteria. Antibiotic tolerance has recently started to garner attention as a crucial mechanism of refractory infections. However, few studies have reported the correlation between clinical outcomes and antibiotic tolerance. This study aims to clarify the effect of antibiotic tolerance on clinical outcomes of respiratory tract infection caused by Pseudomonas aeuginosa (P. aeruginosa). We examined a total of 63 strains isolated from sputum samples of different patients and conducted a retrospective survey with the medical records of 37 patients with imipenem-sensitive P. aeruginosa infections. Among them, we selected 15 patients with respiratory infections, and they were divided into high-tolerance minimal bactericidal concentration for adherent bacteria (MBCAD)/minimal inhibitory concentration for adherent bacteria (MICAD) ≥ 32 (n = 9) group and low-tolerance MBCAD/MICAD ≤ 16 (n = 6) group for further investigations. The findings indicated that the high-tolerance group consisted of many cases requiring hospitalization. Chest computed tomography findings showed that the disease was more extensive in the high-tolerance group compared to the low-tolerance group. Regarding the bacterial phenotypic characterization, the high-tolerance group significantly upregulated the production of the virulence factors compared to the low-tolerance group. Our study provided evidence that carbapenem tolerance level is a potent prognostic marker of P. aeruginosa infections, and carbapenem tolerance could be a potential target for new antimicrobial agents to inhibit the progression of persistent P. aeruginosa infections

Management of tooth extraction in a patient with ELANE gene mutation-induced cyclic neutropenia

Introduction: Cyclic neutropenia (CyN) is a rare hematological disease, and patients with CyN often experience an early onset of severe periodontitis and are forced to undergo tooth extraction. Here, we report a case of a patient with CyN who showed different periodicity and oscillations of neutrophil count compared with her mother, despite sharing the same novel genetic mutation. Patient concerns: A 17-year-old Japanese girl who had been diagnosed with CyN shortly after birth presented to our hospital with a complaint of mobility of her teeth and gingivitis. Upon presentation, an intraoral examination was performed and revealed redness and swelling of the marginal and attached gingiva. Radiographs revealed extreme resorption of the alveolar bone and apical lesions in her mandibular lateral incisors. The patient's hematologic data demonstrated a lack of blood neutrophils (0/μL). The patient had no history of dental extraction, and her mother also had a history of CyN. Diagnoses: The patient was diagnosed with severe periodontitis that was associated with CyN. Gene testing showed a novel heterozygous mutation in exon 4 of the ELANE gene (c.538delC, p.Leu180Ser fsX11). Interventions: Based on the clinical findings, we planned to extract the patient's mandibular lateral incisors. Although the tooth extraction was scheduled considering the cyclic variation in neutrophil count, the patient's neutrophil count was 0/μL on the day before the planned extraction. Therefore, granulocyte-colony stimulating factor (G-CSF) was administered to increase the patient's neutrophil count. On the day of the patient's admission for the tooth extraction, she presented with fever (body temperature, 38.5°C), tonsillitis, and stomatitis. The extraction was subsequently delayed, and the patient was administered antibiotics and G-CSF for 4 days. At this time, the neutrophil count increased to 750/μL, and the tooth extraction was carried out safely. Outcomes: The postoperative course was uneventful, and the healing process at the extraction site was excellent. Conclusion: There is a possibility that the periodicity and oscillations of neutrophil count may change with growth in patients with CyN. Therefore, it is important to frequently examine and treat patients with fluctuating neutrophil levels for the management of invasive dental treatment in patients with CyN

Cepharanthine Inhibits IFN-γ-Induced CXCL10

Cepharanthine, a biscolaurine alkaloid isolated from the plant Stephania cephalantha Hayata, has been reported to have potent anti-inflammatory properties. Here we investigated the effects of cepharanthine on the expression of CXCL10 (a CXC chemokine induced by interferon-gamma [IFN-γ] that has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions) in IFN-γ-treated human salivary gland cell lines. We observed that IFN-γ induced CXCL10 production in NS-SV-DC cells (a human salivary gland ductal cell line), but not in NS-SV-AC cells (a human salivary gland acinar cell line). Cepharanthine inhibited the IFN-γ-induced CXCL10 production in NS-SV-DC cells. A Western blot analysis showed that cepharanthine prevented the phosphorylation of JAK2 and STAT1, but did not interfere with the NF-κB pathway. Moreover, cepharanthine inhibited the IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggest that cepharanthine suppresses IFN-γ-induced CXCL10 production via the inhibition of the JAK2/STAT1 signaling pathway in human salivary gland ductal cells. Our findings also indicate that cepharanthine could inhibit the chemotaxis of Jurkat T cells by reducing CXCL10 production

Resident Macrophages in SS

Macrophages (MΦs) are critical regulators of immune response and serve as a link between innate and acquired immunity. The precise mechanism of involvement of tissue-resident MΦs in the pathogenesis of autoimmune diseases is not clear. Here, using a murine model for Sjögren’s syndrome (SS), we investigated the role of tissue-resident MΦs in the onset and development of autoimmunity. Two unique populations of CD11bhigh and CD11blow resident MΦs were observed in the target tissue of the SS model. Comprehensive gene expression analysis of chemokines revealed effective production of CCL22 by the CD11bhigh MΦs. CCL22 upregulated the migratory activity of CD4+ T cells by increasing CCR4, a receptor of CCL22, on T cells in the SS model. In addition, CCL22 enhanced IFN-γ production of T cells of the SS model, thereby suggesting that CCL22 may impair the local immune tolerance in the target organ of the SS model. Moreover, administration of anti-CCL22 antibody suppressed autoimmune lesions in the SS model. Finally, histopathological analysis revealed numerous CCL22-producing MΦs in the minor salivary gland tissue specimens of the SS patients. CCL22-producing tissue-resident MΦs may control autoimmune lesions by enhancing T cell response in the SS model. These results suggest that specific chemokines and their receptors may serve as novel therapeutic or diagnostic targets for SS