Membrane type 1 matrix metalloproteinase is necessary for distal airway epithelial repair and keratinocyte growth factor receptor expression after acute injury

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a protease produced by airway epithelial cells in various diseases. Since other MMPs are involved in bronchial epithelial repair, we investigated the role of MT1-MMP in naphthalene-induced small airway injury and repair in wild-type (WT) and MT1-MMP-knockout (KO) mice. The degree of injury was similar in both strains, but the MT1-MMP KO mice were unable to reconstitute a normal, fully differentiated airway epithelium 28 days after injury. MT1-MMP was required for the proliferative response in distal airway epithelial cells, resulting in decreased cell density and airway epithelial cell differentiation in MT1-MMP KO mice. Surprisingly, EGF-mediated signaling was unaltered in MT1-MMP KO mice and therefore unrelated to the proliferative response. However, keratinocyte growth factor receptor (KGFR) expression was significantly upregulated before the proliferative response and markedly less evident in the distal airway epithelium of MT1-MMP KO mice. These results indicate MT1-MMP is involved in KGFR expression and epithelial cell proliferation after acute airway injury.     

Gender differences in pulmonary regenerative response to naphthalene-induced bronchiolar epithelial cell injury

Objectives:  Accumulating evidence suggests that gender affects the incidence and severity of several pulmonary diseases. Previous studies on mice have shown gender differences in susceptibility to naphthalene-induced lung injury, where Clara cell damage was found to occur earlier and to be more extensive in females than in males. However, very little is known about whether there are any gender differences in subsequent lung repair responses. The aim of this study was to investigate whether gender plays an important role in pulmonary regenerative response to naphthalene-induced Clara cell ablation.
Materials and methods:  Adult male and female mice were injected with a low, medium, or high dose of naphthalene, and lung tissue regeneration was examined by immunohistochemical staining for cell proliferation marker (Ki-67) and mitosis marker (phosphohistone-3).
Results:  Histopathological analysis showed that naphthalene-induced Clara cell necrosis was more prominent in the lungs of female mice as compared to male mice. Cell proliferation and mitosis in both the distal bronchiolar airway epithelium and peribronchiolar interstitium of female mice was significantly greater than that of male mice after treatment with the low and medium doses. However, after treatment with high dose of naphthalene, lung regeneration was delayed in female mice, while male mice mounted a timely regenerative response.
Conclusions:  These findings show that there are clear gender differences in naphthalene-induced lung injury and repair.     

Airway Epithelial NF-κB Activation Promotes Mycoplasma pneumoniae Clearance in Mice

Background/Objective

Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.

Methodology/Main Results

Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-_CAIKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg−) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.

Conclusion

By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.     

In vivo function of airway epithelial TLR2 in host defense against bacterial infection

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2(-/-) BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2(-/-) mice reconstituted 30-40% TLR2 expression compared with TLR2(+/+) cells. Such airway epithelial TLR2 reconstitution in TLR2(-/-) mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2(-/-) mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2(-/-) mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function.     

HIP/PAP protects against bleomycin‐induced lung injury and inflammation and subsequent fibrosis in mice

Hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein (HIP/PAP), a C‐type lectin, exerts anti‐oxidative, anti‐inflammatory, bactericidal, anti‐apoptotic, and mitogenic functions in several cell types and tissues. In this study, we explored the role of HIP/PAP in pulmonary fibrosis (PF). Expression of HIP/PAP and its murine counterpart, Reg3B, was markedly increased in fibrotic human and mouse lung tissues. Adenovirus‐mediated HIP/PAP expression markedly alleviated bleomycin (BLM)‐induced lung injury, inflammation, and fibrosis in mice. Adenovirus‐mediated HIP/PAP expression alleviated oxidative injury and lessened the decrease in pulmonary superoxide dismutase (SOD) activity in BLM‐treated mice, increased pulmonary SOD expression in normal mice, and HIP/PAP upregulated SOD expression in cultured human alveolar epithelial cells (A549) and human lung fibroblasts (HLF‐1). Moreover, in vitro experiments showed that HIP/PAP suppressed the growth of HLF‐1 and ameliorated the H₂O₂‐induced apoptosis of human alveolar epithelial cells (A549 and HPAEpiC) and human pulmonary microvascular endothelial cells (HPMVEC). In HLF‐1, A549, HPAEpiC, and HPMVEC cells, HIP/PAP did not affect the basal levels, but alleviated the TGF‐β1‐induced down‐regulation of the epithelial/endothelial markers E‐cadherin and vE‐cadherin and the over‐expression of mesenchymal markers, such as α‐SMA and vimentin. In conclusion, HIP/PAP was found to serve as a potent protective factor in lung injury, inflammation, and fibrosis by attenuating oxidative injury, promoting the regeneration of alveolar epithelial cells, and antagonizing the pro‐fibrotic actions of the TGF‐β1/Smad signaling pathway.     

Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model

Asthmatic patients are more susceptible to viral infection, and we asked whether dynamic strain on the airway wall (such as that associated with bronchoconstriction) would influence the rate of viral infection of the epithelial and subepithelial cells. To address this, we characterized the barrier function of a three-dimensional culture model of the bronchial airway wall mucosa, modified the culture conditions for optimization of ciliogenesis, and compared epithelial and subepithelial green fluorescent protein (GFP) transduction by a pWpts-GFP lentivirus, pseudotyped with VSV-G, under static vs. dynamic conditions. The model consisted of human lung fibroblasts, bronchial epithelial cells, and a type I collagen matrix, and after 21 days of culture at air liquid interface, it exhibited a pseudostratified epithelium comprised of basal cells, mucus-secreting cells, and ciliated columnar cells with beating cilia. Microparticle tracking revealed partial coordination of mucociliary transport among groups of cells. Slow dynamic compression of the airway wall model (15% strain at 0.1 Hz over 3 days) substantially enhanced GFP transduction of epithelial cells and underlying fibroblasts. Fibroblast-only controls showed a similar degree of transduction enhancement when undergoing dynamic strain, suggesting enhanced transport through the matrix. Tight junction loss in the epithelium after mechanical stress was observed by immunostaining. We conclude that dynamic compressive strain such as that associated with bronchoconstriction may promote transepithelial transport and enhance viral transgene delivery to epithelial and subepithelial cells. This finding has significance for asthma pathophysiology as well as for designing delivery strategies of viral gene therapies to the airways.     

Nix/BNIP3L-dependent mitophagy accounts for airway epithelial cell injury induced by cigarette smoke

Cigarette smoke-induced airway epithelial cell mitophagy is an important mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Mitochondrial protein Nix (also known as BNIP3L) is a selective autophagy receptor and participates in several human diseases. However, little is known about the role of Nix in airway epithelial cell injury during the development of COPD. The aim of the present study is to investigate the effects of Nix on mitophagy and mitochondrial function in airway epithelial cells exposed to cigarette smoke extract (CSE). Our present study has found that CSE could increase Nix protein expression and induce mitophagy in airway epithelial cells. And Nix siRNA significantly inhibited mitophagy and attenuated mitochondrial dysfunction and cell injury when airway epithelial cells were stimulated with 7.5% CSE. In contrast, Nix overexpression enhanced mitophagy and aggravated mitochondrial dysfunction and cell injury when airway epithelial cells were incubated with 7.5% CSE. These data suggest that Nix-dependent mitophagy promotes airway epithelial cell and mitochondria injury induced by cigarette smoke, and may be involved in the pathogenesis of COPD and other cigarette smoke-associated diseases.     

Stat3 is required for cytoprotection of the respiratory epithelium during adenoviral infection

The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3(DeltaDelta) mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3(DeltaDelta) mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3(DeltaDelta) mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3(DeltaDelta) mice were similar, TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3(DeltaDelta) mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3(DeltaDelta) mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-x(L), an E1- and E3-deleted, nonproliferative adenovirus expressing the antiapoptotic protein Bcl-x(L), protected Stat3(DeltaDelta) mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Partial SP-B deficiency perturbs lung function and causes air space abnormalities

Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60-70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content, confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged; however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement.     

Transfection of multiple pulmonary cell types following intravenous injection of PEI-DNA in normal and CFTR mutant mice

BACKGROUND: The polycationic vector polyethylenimine (PEI) has been shown to be a powerful agent for transfecting the mouse lung after injection of plasmid-based polyplexes through the tail vein. These findings raise therapeutic prospects for a number of lung conditions. For such potentials to be realised, the precise identity of the transfected cells remains to be determined; however, so far, no ultrastructural analysis has been performed on PEI-transfected lungs. The definition of which pulmonary cells are transfected is particularly critical for certain pulmonary diseases which might require transfection of defined cell types such as epithelial cells for cystic fibrosis (CF). METHODS: Here, we use a combination of light and electron microscopy to determine which cells are transfected in the lung after PEI-mediated gene delivery through the intravenous route. Furthermore, we extend the same experimental setting to a mouse model of CF to provide proof of principle that this approach can be used in genetic models of the disease. RESULTS: We show that within 18-20 h after injection through the tail vein, DNA/PEI complexes have already crossed the capillary barrier resulting in high levels of expression of reporter genes in the lungs. Transgene expression is observed in endothelial cells, in type I and type II pneumocytes, and in septal cells. Coexpression of the transgene and of the endogenous CF transmembrane conductance regulator (CFTR) gene is observed in some of the targeted epithelial cells. Levels and sites of expression are similar in normal and in CFTR-mutant mice. CONCLUSIONS: The results demonstrate that PEI-mediated gene delivery leads to transfection of epithelial cells beyond the endothelial barrier and show that this method can be used for lung gene delivery in CF fragile mutant mice.     

Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.     

TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.