miR-146a regulates emphysema formation and abnormal inflammation in the lungs of two mouse models.

miR-146a, a microRNA (miRNA) that regulates inflammatory responses, plays an important role in many inflammatory diseases. Although an in vitro study had suggested that miR-146a is involved in abnormal inflammatory response, being a critical factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), in vivo evidence of its pathogenic role in COPD remains limited. Eight-week-old male B6(FVB)-Mir146tm1.1Bal/J [miR-146a knockout (KO)] and C57BL/6J mice were intratracheally administered elastase and evaluated after 28 days or exposed to cigarette smoke (CS) and evaluated after 5 mo. miR-146a expression was significantly increased in C57BL/6J mouse lungs due to elastase administration (P = 0.027) or CS exposure (P = 0.019) compared with that in the control group. Compared with C57BL/6J mice, elastase-administered miR-146a-KO mice had lower average computed tomography (CT) values (P = 0.017) and increased lung volume-to-weight ratio (P = 0.016), mean linear intercept (P < 0.001), and destructive index (P < 0.001). Moreover, total cell (P = 0.006), macrophage (P = 0.001), neutrophil (P = 0.026), chemokine (C-X-C motif) ligand 2/macrophage inflammatory protein-2 [P = 0.045; in bronchoalveolar lavage fluid (BALF)], cyclooxygenase-2, and matrix metalloproteinase-2 levels were all increased (in the lungs). Following long-term CS exposure, miR-146a-KO mice showed a greater degree of emphysema formation in their lungs and inflammatory response in the BALF and lungs than C57BL/6J mice. Collectively, miR-146a protected against emphysema formation and the associated abnormal inflammatory response in two murine models.NEW & NOTEWORTHY This study demonstrates that miR-146a expression is upregulated in mouse lungs because of elastase- and CS-induced emphysema and that the inflammatory response by elastase or CS is enhanced in the lungs of miR-146a-KO mice than in those of control mice, resulting in the promotion of emphysema. This is the first study to evaluate the protective role of miR-146a in emphysema formation and the associated abnormal inflammatory response in different in vivo models.

Exposure to the heated tobacco product IQOS generates apoptosis-mediated pulmonary emphysema in murine lungs.

Pulmonary emphysema is predominantly caused by chronic exposure to cigarette smoke (CS). Novel tobacco substitutes, such as heated tobacco products (HTPs), have emerged as healthier alternatives to cigarettes. IQOS, the most popular HTP in Japan, is advertised as harmless compared with conventional cigarettes. Although some studies have reported its toxicity, few in vivo studies have been conducted. Here, 12-wk-old C57BL6/J male mice were divided into three groups and exposed to air (as control), IQOS aerosol, or CS for 6 mo. After exposure, the weight gain was significantly suppressed in the IQOS and CS groups compared with the control (-4.93 g; IQOS vs. air and -5.504 g; CS vs. air). The serum cotinine level was significantly higher in the IQOS group than in the control group. The neutrophils and lymphocyte count increased in the bronchoalveolar lavage fluid of the IQOS and CS groups compared with those in the control group. Chronic IQOS exposure induced pulmonary emphysema similar to that observed in the CS group. Furthermore, expression levels of the genes involved in the apoptosis-related pathways were significantly upregulated in the lungs of the IQOS-exposed mice. Cytochrome c, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase-1 were overexpressed in the IQOS group compared with the control. Single-stranded DNA and TdT-mediated dUTP nick-end labeling-positive alveolar septal cell count significantly increased in the IQOS group compared with the control. In conclusion, chronic exposure to IQOS aerosol induces pulmonary emphysema predominantly via apoptosis-related pathways. This suggests that HTPs are not completely safe tobacco products.

Regional heterogeneity in response of airway epithelial cells to cigarette smoke.

BACKGROUND: Cigarette smoke (CS) exposure causes an abnormal inflammatory response, which can result in chronic obstructive pulmonary disease (COPD). Previous studies show that this disorder predominantly occurs in peripheral or small-airway areas, whereas the same condition has not been identified in the larger airways during the course of COPD. However, the different biochemical and genetic alterations occurring in response to CS exposure among airway epithelial cells from different sites in the lungs have not been fully investigated. METHODS: Human small airway epithelial cells (SAECs) and normal human bronchial epithelial cells (NHBEs) were exposed to CS extract (CSE), and microarray analysis was used to determine gene- and protein-expression profiles and identify alterations following CSE exposure in both cell types. An in vivo smoking experiment was also performed to confirm differential responses to CS between sites in the lung. RESULTS: Microarray analysis of SAECs and NHBEs following 24 h of CSE exposure showed that inflammatory related pathways and terms, including the tumor necrosis factor-signaling pathway, were overrepresented, especially in SAECs. Clustering analysis highlighted prostaglandin-endoperoxide synthase-2 [also known as cyclooxygenase (COX)-2] as a gene specifically upregulated in SAECs, with COX-2 mRNA and protein expression significantly elevated by CSE exposure in SAECs (3.1- and 3.1-fold, respectively), but not in NHBEs. Furthermore, time-course analysis of COX-2 expression revealed earlier increases in SAECs compared with NHBEs following CS exposure. Short-term exposure of mouse lungs to CS was found to predominantly induce COX-2 expression in the small airway. CONCLUSIONS: The small airway is more susceptible to CSE than the large airway and could be the initial site of development of CS-related respiratory diseases, such as COPD.

Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice.

Chronic obstructive pulmonary disease (COPD) is predominantly a cigarette smoke (CS)-triggered disease with features of chronic systemic inflammation. Oxidants derived from CS can induce DNA damage and stress-induced premature cellular senescence in the respiratory system, which play significant roles in COPD. Therefore, antioxidants should provide benefits for the treatment of COPD; however, their therapeutic potential remains limited owing to the complexity of this disease. Recently, molecular hydrogen (H2) has been reported as a preventive and therapeutic antioxidant. Molecular H2 can selectively reduce hydroxyl radical accumulation with no known side effects, showing potential applications in managing oxidative stress, inflammation, apoptosis, and lipid metabolism. However, there have been no reports on the efficacy of molecular H2 in COPD patients. In the present study, we used a mouse model of COPD to investigate whether CS-induced histological damage in the lungs could be attenuated by administration of molecular H2. We administered H2-rich pure water to senescence marker protein 30 knockout (SMP30-KO) mice exposed to CS for 8 weeks. Administration of H2-rich water attenuated the CS-induced lung damage in the SMP30-KO mice and reduced the mean linear intercept and destructive index of the lungs. Moreover, H2-rich water significantly restored the static lung compliance in the CS-exposed mice compared with that in the CS-exposed H2-untreated mice. Moreover, treatment with H2-rich water decreased the levels of oxidative DNA damage markers such as phosphorylated histone H2AX and 8-hydroxy-2'-deoxyguanosine, and senescence markers such as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1, and ß-galactosidase in the CS-exposed mice. These results demonstrated that H2-rich pure water attenuated CS-induced emphysema in SMP30-KO mice by reducing CS-induced oxidative DNA damage and premature cell senescence in the lungs. Our study suggests that administration of molecular H2 may be a novel preventive and therapeutic strategy for COPD.

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung's structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.