Effects of cigarette smoke on barrier function and tight junction proteins in the bronchial epithelium: protective role of cathelicidin LL-37.

BACKGROUND: Airway epithelial barrier function is maintained by the formation of tight junctions (TJs) and adherens junctions (AJs). Inhalation of cigarette smoke causes airway epithelial barrier dysfunction and may contribute to the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). We assessed the effects of cigarette smoke on barrier function and expression of multiple TJ and AJ proteins in the bronchial epithelium. We also examined whether treatment with glucocorticosteroids (GCSs), long-acting ß2-agonists (LABAs), and human cathelicidin LL-37 can protect against cigarette smoke extract (CSE)-induced barrier dysfunction. METHODS: Calu-3 cells cultured at the air-liquid interface were pretreated with or without GCSs, LABAs, GCSs plus LABAs, or LL-37, and subsequently exposed to CSE. Barrier function was assessed by transepithelial electronic resistance (TEER) measurements. Gene and protein expression levels of TJ and AJ proteins were analyzed by quantitative PCR and western blotting, respectively. Immunofluorescence staining of TJ and AJ proteins was performed. RESULTS: CSE decreased TEER and increased permeability in a concentration-dependent manner. CSE suppressed gene expression of claudin-1, claudin-3, claudin-4, claudin-7, claudin-15, occludin, E-cadherin, junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) within 12 h post-CSE exposure, while suppressed protein expression levels of occludin at 12 h. CSE-treated cells exhibited discontinuous or attenuated immunostaining for claudin-1, claudin-3, claudin-4, occludin, ZO-1, and E-cadherin compared with untreated cells. GCS treatment partially restored CSE-induced TEER reduction, while LABA treatment had no effect. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction and gene suppression of TJ and AJ proteins. Human cathelicidin LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. LL-37 also attenuated CSE-induced decreases in gene and protein expression levels of occludin. CONCLUSIONS: CSE caused airway epithelial barrier dysfunction and simultaneously downregulated multiple TJ and AJ proteins. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction. LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. Use of LL-37 to counteract airway epithelial barrier dysfunction may have significant benefits for respiratory diseases such as asthma and COPD.

Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non-small-cell lung cancer.

Spliced variant isoforms of CD44 (CD44v) are a marker of cancer stem cells in solid tumors. They stabilize the xCT subunit of the transporter system xc(-) and thereby promote synthesis of the antioxidant glutathione. Salazosulfapyridine (SASP) is an inhibitor of xCT and suppresses the proliferation of CD44v-positive cancer cells. Chemotherapy-naïve patients with advanced non-squamous non-small-cell lung cancer were enrolled in a dose-escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed. The primary end-point was the percentage of patients who experience dose-limiting toxicity. Fifteen patients were enrolled in the study. Dose-limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day (elevation of aspartate and alanine aminotransferase levels, each of grade 3), two of five patients at 3 g/day (hypotension or pneumonitis, each of grade 3), and two of three patients at 4.5 g/day (anorexia of grade 3). The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression-free survival was 11.7 months, much longer than that for cisplatin-pemetrexed alone in previous studies. Exposure to SASP varied markedly among individuals according to ABCG2 and NAT2 genotypes. The serum concentration of free CD44v protein was increased after the first cycle of treatment, possibly reflecting death of cancer stem cells. Salazosulfapyridine was thus given safely in combination with cisplatin-pemetrexed, with the addition of SASP tending to prolong progression-free survival. This trial is registered in the UMIN Clinical Trials Registry as UMIN000017854.

Resection of tracheal mucosa-associated lymphoid tissue (MALT) lymphoma by bronchoscopic high-frequency electrosurgical snare: Case report.

There is no standard method of bronchoscopic local therapy for tracheal tumours. We herein present a case involving a 61-year-old woman who was diagnosed with tracheal mucosa-associated lymphoid tissue lymphoma and underwent resection by a bronchoscopic high-frequency electrosurgical snare. Few reports to date have described such use of high-frequency electrosurgical snares; however, they are effective for the treatment of tracheal tumours, especially pedunculated tumours.

Anti-EJ antibody-positive interstitial pneumonia with breast cancer improved by combining immunosuppressive therapy and chemotherapy.

We present a case of a 45-year-old woman diagnosed with interstitial pneumonia (IP) during a comprehensive breast cancer evaluation. Although the patient showed no obvious clinical symptoms of polymyositis or dermatomyositis, the presence of anti-glycyl-transfer ribonucleic acid synthetase antibodies confirmed anti-synthetase syndrome. The patient began methylprednisolone for treatment of the IP. She then received preoperative chemotherapy with epirubicin and cyclophosphamide before undergoing a mastectomy. A significant improvement was seen in the patient's IP during treatment. This case emphasizes the potential advantages of personalized immunosuppressive therapy for patients who are simultaneously diagnosed with anti-synthetase syndrome and cancer.

Advanced atypical lung carcinoid tumour successfully treated with carboplatin, etoposide and atezolizumab: A case report.

Atypical carcinoid tumours are relatively rare among lung cancers. Surgery is regarded as standard treatment for localized cases, but there is little established evidence on treatment strategies for advanced cases. Moreover, the efficacy of immune checkpoint inhibitors for advanced carcinoid tumours is unclear. Here, we report a case of a patient with an atypical carcinoid tumour in whom successful disease control was achieved with the use of combined cytotoxic chemotherapy and immunotherapy.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNß or IFNλ increased PD-L1 and PD-L2, and IFNß or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNß, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Inhibition of PI3Kδ Enhances Poly I:C-Induced Antiviral Responses and Inhibits Replication of Human Metapneumovirus in Murine Lungs and Human Bronchial Epithelial Cells.

Viral infections of the airway can exacerbate respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD), and accelerate disease progression. Phosphoinositide 3-kinase (PI3K)δ, a class 1A PI3K, has been studied as a potential target for achieving anti-oncogenic and anti-inflammatory effects. However, the role of PI3Kδ in antiviral responses is poorly understood. Using a synthetic double-stranded RNA poly I:C and a selective PI3Kδ inhibitor IC87114, we investigated the role of PI3Kδ signaling in poly I:C-induced expression of the T lymphocyte-inhibitory molecule programmed death 1 ligand 1 (PD-L1), inflammatory responses and antiviral interferon (IFN) responses. C57BL/6N mice were treated with IC87114 or vehicle by intratracheal (i.t.) instillation followed by i.t. administration of poly I:C. Poly I:C increased PD-L1 expression on epithelial cells, lymphocytes, macrophages, and neutrophils in the lungs and IC87114 suppressed poly I:C-induced PD-L1 expression on epithelial cells and neutrophils possibly via inhibition of the Akt/mTOR signaling pathway. IC87114 also attenuated poly I:C-induced increases in numbers of total cells, macrophages, neutrophils and lymphocytes, as well as levels of KC, IL-6 and MIP-1ß in bronchoalveolar lavage fluid. Gene expression of IFNß, IFNλ2 and IFN-stimulated genes (ISGs) were upregulated in response to poly I:C and a further increase in gene expression was observed following IC87114 treatment. In addition, IC87114 enhanced poly I:C-induced phosphorylation of IRF3. We assessed the effects of IC87114 on human primary bronchial epithelial cells (PBECs). IC87114 decreased poly I:C-induced PD-L1 expression on PBECs and secretion of IL-6 and IL-8 into culture supernatants. IC87114 further enhanced poly I:C- induced increases in the concentrations of IFNß and IFNλ1/3 in culture supernatants as well as upregulated gene expression of ISGs in PBECs. Similar results were obtained in PBECs transfected with siRNA targeting the PIK3CD gene encoding PI3K p110δ, and stimulated with poly I:C. In human metapneumovirus (hMPV) infection of PBECs, IC87114 suppressed hMPV-induced PD-L1 expression and reduced viral replication without changing the production levels of IFNß and IFNλ1/3 in culture supernatants. These data suggest that IC87114 may promote virus elimination and clearance through PD-L1 downregulation and enhanced antiviral IFN responses, preventing prolonged lung inflammation, which exacerbates asthma and COPD.