Sho Goya

Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17

Exposure to ozone, which is a major component of air pollution, induces a form of asthma that occurs in the absence of adaptive immunity. Although ozone-induced asthma is characterized by airway neutrophilia, and not eosinophilia, it is nevertheless associated with airway hyperreactivity (AHR), which is a cardinal feature of asthma. Because AHR induced by allergens requires the presence of natural killer T (NKT) cells, we asked whether ozone-induced AHR had similar requirements. We found that repeated exposure of wild-type (WT) mice to ozone induced severe AHR associated with an increase in airway NKT cells, neutrophils, and macrophages. Surprisingly, NKT cell–deficient (CD1d−/− and Jα18−/−) mice failed to develop ozone-induced AHR. Further, treatment of WT mice with an anti-CD1d mAb blocked NKT cell activation and prevented ozone-induced AHR. Moreover, ozone-induced, but not allergen-induced, AHR was associated with NKT cells producing interleukin (IL)-17, and failed to occur in IL-17−/− mice nor in WT mice treated with anti–IL-17 mAb. Thus, ozone exposure induces AHR that requires the presence of NKT cells and IL-17 production. Because NKT cells are required for the development of two very disparate forms of AHR (ozone- and allergen-induced), our results strongly suggest that NKT cells mediate a unifying pathogenic mechanism for several distinct forms of asthma, and represent a unique target for effective asthma therapy.

Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13

BACKGROUND Atopic dermatitis (AD) is characterized by local and systemic T(H)2 responses to cutaneously introduced allergens and is a risk factor for asthma. Blockade of T(H)2 cytokines has been suggested as therapy for AD. OBJECTIVES We sought to examine the effect of the absence of IL-4 and IL-13 on the T(H)17 response to epicutaneous sensitization in a murine model of allergic skin inflammation with features of AD. METHODS Wild-type, IL4 knockout (KO), IL13 KO and IL4/13 double KO (DKO) mice were subjected to epicutaneous sensitization with ovalbumin (OVA) or saline and airway challenged with OVA. Systemic immune responses to OVA, skin and airway inflammation, and airway hyperresponsiveness were examined. RESULTS OVA-sensitized DKO mice exhibited impaired T(H)2-driven responses with undetectable OVA-specific IgE levels and severely diminished eosinophil infiltration at sensitized skin sites but intact dermal infiltration with CD4(+) cells. DKO mice mounted exaggerated IL-17A but normal IFN-gamma and IL-5 systemic responses. Airway challenge of these mice with OVA caused marked upregulation of IL-17 mRNA expression in the lungs, increased neutrophilia in bronchoalveolar lavage fluid, airway inflammation characterized by mononuclear cell infiltration with no detectable eosinophils, and bronchial hyperresponsiveness to methacholine that were reversed by IL-17 blockade. IL-4, but not IL-13, was identified as the major T(H)2 cytokine that downregulates the IL-17 response in epicutaneously sensitized mice. CONCLUSION Epicutaneous sensitization in the absence of IL-4/IL-13 induces an exaggerated T(H)17 response systemically and in lungs after antigen challenge that results in airway inflammation and airway hyperresponsiveness.

Early [18F]Fluorodeoxyglucose Positron Emission Tomography at Two Days of Gefitinib Treatment Predicts Clinical Outcome in Patients with Adenocarcinoma of the Lung

Purpose: Positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) is increasingly used in early assessment of tumor response after chemotherapy. We investigated whether a change in [18F]FDG uptake at 2 days of gefitinib treatment predicts outcome in patients with lung adenocarcinoma. Experimental Design: Twenty patients were enrolled. [18F]FDG-PET/computed tomographic (CT) scan was carried out before and 2 days after gefitinib treatment. Maximum standardized uptake values (SUV) were measured, and post-gefitinib percentage changes in SUV were calculated. Early metabolic response (SUV decline < −25%) was compared with morphologic response evaluated by CT scan and with progression-free survival (PFS). Results: At 2 days of gefitinib treatment, 10 patients (50%) showed metabolic response, 8 had metabolic stable disease, and 2 had progressive metabolic disease. Percentage changes of SUV at 2 days were correlated with those of tumor size in CT at 1 month (R2 = 0.496; P = 0.0008). EGFR gene was assessable in 15 patients, and of 12 patients with EGFR mutations, 8 showed metabolic response at 2 days and 6 showed morphologic response at 1 month. None of 3 patients with wild-type EGFR showed metabolic or morphologic response. Metabolic response at 2 days was not statistically associated with PFS (P = 0.095), but when a cutoff value of −20% in SUV decline was used, metabolic responders had longer PFS (P < 0.0001). Conclusion: Early assessment of [18F]FDG tumor uptake with PET at 2 days of gefitinib treatment could be useful to predict clinical outcome earlier than conventional CT evaluation in patients with lung adenocarcinoma. Clin Cancer Res; 18(1); 220–8. ©2011 AACR.

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector‐mediated transient expression of both the human interleukin‐6 (IL‐6) and IL‐6 receptor (IL‐6R) genes, but not the IL‐6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL‐6 and IL‐6R double transgenic mice to elucidate the effects of prolonged IL‐6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue‐like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue‐like structures. These structures were distinguished from bronchus‐associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL‐6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung. Copyright

Apoptotic Cells Activate NKT Cells through T Cell Ig-Like Mucin-Like–1 Resulting in Airway Hyperreactivity

T cell Ig-like mucin-like–1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1–dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.

Tetraspanin CD9 Negatively Regulates Lipopolysaccharide-Induced Macrophage Activation and Lung Inflammation

Tetraspanins facilitate the formation of multiple molecular complexes at specialized membrane microdomains and regulate cell activation and motility. In the present study, the role of tetraspanin CD9 in LPS-induced macrophage activation and lung inflammation was investigated in vitro and in vivo. When CD9 function was ablated with mAb treatment, small interfering RNA transfection, or gene knockout in RAW264.7 cells or bone marrow-derived macrophages, these macrophages produced larger amounts of TNF-α, matrix metalloproteinase-2, and -9 upon stimulation with LPS in vitro, when compared with control cells. Sucrose gradient analysis revealed that CD9 partly colocalized with the LPS-induced signaling mediator, CD14, at low-density light membrane fractions. In CD9 knockout macrophages, CD14 expression, CD14 and TLR4 localization into the lipid raft, and their complex formation were increased whereas IκBα expression was decreased when compared with wild-type cells, suggesting that CD9 prevents the formation of LPS receptor complex. Finally, deletion of CD9 in mice enhanced macrophage infiltration and TNF-α production in the lung after intranasal administration of LPS in vivo, when compared with wild-type mice. These results suggest that macrophage CD9 negatively regulates LPS response at lipid-enriched membrane microdomains.

Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.

Involvement of Endothelial Apoptosis Underlying Chronic Obstructive Pulmonary Disease―like Phenotype in Adiponectin-null Mice: Implications for Therapy

RATIONALE Chronic obstructive pulmonary disease is frequently complicated with comorbidities, such as cardiovascular disease, osteoporosis, and body weight loss, but the causal link remains unclear. OBJECTIVES To investigate the role of adiponectin in the pathogenesis of chronic obstructive pulmonary disease and its potential use in therapy. METHODS Adiponectin localization and dynamics in the lung were analyzed in an elastase-induced emphysema model. Next, the lung of adiponectin-knockout mice, extrapulmonary effects, and the underlying mechanism were investigated. Finally, we tested whether exogenous adiponectin could ameliorate the emphysematous change in adiponectin-knockout mice. MEASUREMENTS AND MAIN RESULTS Adiponectin expression in lung vasculature and plasma concentration of adiponectin were reduced after elastase-instillation. Notably, adiponectin-knockout mice showed progressive alveolar enlargement and increased lung compliance. They further exhibited not only systemic inflammation, but also extrapulmonary phenotype, such as body weight loss, fat atrophy, and osteoporosis. Moreover, endothelial apoptosis was enhanced in the lungs of adiponectin-knockout mice, as evidenced by caspase-3 activity. Consistent with this, expressions of vascular endothelial growth factor receptor-2 and platelet endothelial cell adhesion molecule-1 on endothelial cells were decreased in the adiponectin-knockout mice. Finally, adenovirus-mediated adiponectin supplementation ameliorated the emphysematous phenotype. CONCLUSIONS Adiponectin-knockout mice develop progressive chronic obstructive pulmonary disease-like phenotype with systemic inflammation and extrapulmonary phenotypes. Hypoadiponectinemia could thus play a critical role in the progression of chronic obstructive pulmonary disease and concomitant comorbidities through endothelial dysfunction. Together, adiponectin could be a novel target for chronic obstructive pulmonary disease therapy.

Animal Models of Airway Sensitization

Asthma is a complex phenotype that involves multiple mechanisms, including adaptive and innate immunity as well as physiological and mechanical changes in the airways. A cardinal feature of asthma is airway hyperreactivity (AHR), a multifaceted reaction that can only be assessed in vivo. Mouse models of asthma replicate many of the features of human asthma, including AHR, which can be assessed using standard protocols. Examination of AHR in mice has provided important information about human asthma, primarily because the immunology of allergy is easily studied in mice, especially with the availability of reagents including genetically modified mice. In this unit we discuss the induction and measurement of AHR and the two most common methodologies: noninvasive measurement using a whole‐body plethysmograph (WBP) and invasive measurement of lung resistance and dynamic compliance. Curr. Protoc. Immunol. 79:15.18.1‐15.18.19.

Cell Surface Tetraspanin CD9 Mediates Chemoresistance in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with extremely high mortality due to the appearance of widespread metastases early in its clinical course and rapid acquisition of chemoresistance after initial therapy. A theory of cell adhesion-mediated drug resistance is thought to be a principal mechanism in which extracellular matrix proteins provide a survival advantage against cytotoxic drug-induced apoptosis. We found that the tetraspanin family member CD9 was expressed preferentially in SCLC tumors and metastases from three of seven relapsed patients, whereas chemonaïve primary tumors from 16 patients were CD9 negative with only one exception. Additionally, CD9 was highly expressed on SCLC cell lines rendered resistant to cisplatin or etoposide, and was upregulated in parental chemosensitive cells within 48 hours after exposure to either of these compounds. CD9-expressing chemoresistant SCLC cells adhered more tightly to fibronectin via β1 integrin, but they were less motile than the respective chemosensitive parental lines. Notably, treatment of the chemoresistant cells with chemokine CXCL12 downregulated CD9 and transiently restored motility. Moreover, selective targeting of CD9 by treatment with specific monoclonal antibody ALB6 or a small interfering RNA triggered apoptosis in the chemoresistant cells. Taken together, our findings implicate CD9 in the cell adhesion-mediated drug resistance mechanism, highlighting CD9 as an attractive therapeutic target to improve therapeutic outcomes in SCLC.