A novel function of MUC18: amplification of lung inflammation during bacterial infection.

Bacterial infection plays a critical role in exacerbations of various lung diseases, including chronic pulmonary obstructive disease (COPD) and asthma. Excessive lung inflammation is a prominent feature in disease exacerbations, but the underlying mechanisms remain poorly understood. Cell surface glycoprotein MUC18 (alias CD146 or melanoma cell adhesion molecule) has been shown to promote metastasis in several tumors, including melanoma. We explored the function of MUC18 in lung inflammatory responses to bacteria (eg, Mycoplasma pneumoniae) involved in lung disease exacerbations. MUC18 expression was increased in alveolar macrophages from lungs of COPD and asthma patients, compared with normal healthy human subjects. Mouse alveolar macrophages also express MUC18. After M. pneumoniae lung infection, Muc18(-/-) mice exhibited lower levels of the lung proinflammatory cytokines KC and TNF-α and less neutrophil recruitment than Muc18(+/+) mice. Alveolar macrophages from Muc18(-/-) mice produced less KC than those from Muc18(+/+) mice. In Muc18(-/-) mouse alveolar macrophages, adenovirus-mediated MUC18 gene transfer increased KC production. MUC18 amplified proinflammatory responses in alveolar macrophages, in part through enhancing the activation of nuclear factor-κB (NF-κB). Our results demonstrate, for the first time, that MUC18 exerts a proinflammatory function during lung bacterial infection. Up-regulated MUC18 expression in lungs (eg, in alveolar macrophages) of COPD and asthma patients may contribute to excessive inflammation during disease exacerbations.

MicroRNA-21 inhibits toll-like receptor 2 agonist-induced lung inflammation in mice.

Impaired airway innate immunity (e.g., suppressed Toll-like receptor 2 [TLR2] signaling) has been reported in allergic lungs with bacterial infection. Recently, an allergic mouse lung milieu including the T-helper type 2 (Th2) cytokine interleukin-13 (IL-13) has been shown to up-regulate lung microRNA-21 (miR-21) expression. Whether miR-21 modulates in vivo TLR2 signaling is unknown. The goal of this study was to determine if in vivo, miR-21 regulates a TLR2 agonist-induced lung inflammatory response. Balb/c mice were intranasally pretreated with a locked nucleic acid (LNA) in vivo inhibitor probe for mouse miR-21 or a control probe, followed by intranasal instillation of a TLR2 agonist Pam3CSK4, or saline (control). Four and/or 24 hours later, mice treated with the miR-21 inhibitor probe, as compared to the control probe, significantly increased lung leukocytes, including neutrophils and the keratinocyte-derived chemokine (KC). IL-13 treatment for 72 hours increased (P < .05) miR-21 in cultured primary normal human airway epithelial cells. These results, for the first time, suggest an in vivo role of miR-21 in suppressing TLR2 signaling, and further support that IL-13 can up-regulate miR-21 in human airway epithelial cells. Functional studies on miR-21 likely provide novel approaches to modulate TLR2 signaling in Th2 cytokine-exposed airways.

SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice.

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is highly expressed in normal airways, but is dramatically decreased in allergic and cigarette smoke exposure settings. We have previously demonstrated SPLUNC1 in vitro antibacterial property against Mycoplasma pneumoniae (Mp). However, its in vivo biological functions remain unclear. The objectives of this study were to determine the in vivo functions of SPLUNC1 following bacterial (eg, Mp) infection, and to examine the underlying mechanisms. We generated SPLUNC1-deficient mice and utilized transgenic mice overexpressing human SPLUNC1 exclusively within the airway epithelium. These mice were infected with Mp and, twenty-four hours post infection, their host defense responses were compared to littermate controls. Mp levels and inflammatory cells increased in the lungs of SPLUNC1(-/-) mice as compared to wild type controls. SPLUNC1 deficiency was shown to contribute to impaired neutrophil activation. In contrast, mice overexpressing hSPLUNC1 exclusively in airway epithelial cells demonstrated lower Mp levels. Furthermore, neutrophil elastase activity was significantly increased in mice overexpressing hSPLUNC1. Lastly, we demonstrated that SPLUNC1 enhanced Mp-induced human neutrophil elastase (HNE) activity, and HNE directly inhibited the growth of Mp. Our findings demonstrate a critical in vivo role of SPLUNC1 in host defense against bacterial infection, and likely provide a novel therapeutic approach to restore impaired lung innate immune responses to bacteria in patients with chronic lung diseases.

Roflumilast increases Clara cell secretory protein in cigarette smoke-exposed mice.

Decreased Clara cell secretory protein (CCSP) levels have been found in smokers and chronic obstructive pulmonary disease (COPD) patients, which may be related to the development of COPD. A phosphodiesterase-4 (PDE4) inhibitor, roflumilast, appears to have therapeutic value for COPD. However, its effect on CCSP in cigarette smoke (CS)-exposed lungs has not been investigated. AKR/J mice were treated as follows: air control, CS, roflumilast plus CS, and roflumilast. Mice underwent four weeks of air or CS exposure. Roflumilast was administrated at 5mg/kg via gavage once daily for the duration of the study. CCSP levels in bronchoalveolar lavage (BAL) fluid and ERK1/2 activation in lungs were examined. CS exposure tended to decrease CCSP levels in BAL fluid compared to air controls. Treatment with roflumilast significantly reversed CS-induced downward trend of CCSP in BAL fluid. Roflumilast significantly inhibited CS-induced upward trend of ERK1/2 activation in lungs, and the levels of activated ERK1/2 in lungs negatively correlated with CCSP levels of BAL fluid in CS, and CS plus roflumilast groups. Our results demonstrate that one of the therapeutic mechanisms of roflumilast is to reverse CS-induced downward trend in CCSP levels of BAL fluid, which may be mediated by down-regulating ERK1/2 activity.

Impact of cigarette smoke exposure on innate immunity: a Caenorhabditis elegans model.

BACKGROUND: Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture). CONCLUSIONS: Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure.

Cigarette smoke decreases MARCO expression in macrophages: implication in Mycoplasma pneumoniae infection.

Bacterial infections including Mycoplasma pneumoniae (Mp) are a major cause of exacerbations in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is the leading cause of COPD, and affects the function of alveolar macrophages that act as the first line of defense against the invading respiratory pathogens. Macrophages express a transmembrane receptor called macrophage receptor with collagenous structure (MARCO) that is involved in the clearance of microorganisms. Whether CS down-regulates MARCO and eventually decreases the clearance of Mp has not been investigated. We utilized human monocytic cell line (THP-1)-derived macrophages to examine the effects of CS extract (CSE) on MARCO expression and Mp growth. Specifically, macrophages were pre-exposed to CSE for 6 h, and then infected with or without Mp for 2 h. MARCO was examined at both mRNA and protein levels by using real-time PCR and immunofluorescent staining, respectively. Mp in the supernatants was quantified by quantitative culture. In addition, a neutralizing MARCO antibody was added to macrophages to test if blockade of MARCO impaired Mp clearance. We found that CSE significantly decreased MARCO expression in a dose-dependant manner at 6 h post-CSE. Mp levels in CSE-treated cells were higher than those in non-CSE-treated cells, indicating a decreased pathogen clearance. Additionally, neutralizing MARCO in macrophages markedly increased Mp levels. Our results indicate that cigarette smoke exposure down-regulates MARCO expression in macrophages, which may be in part responsible for impaired bacterial (e.g., Mp) clearance.