Jill R. Johnson

A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial-mesenchymal transition

Epithelial–mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1–SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1–SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.

Remodeling in asthma

Airway remodeling encompasses the structural alterations in asthmatic compared with normal airways. Airway remodeling in asthmatic patients involves a wide array of pathophysiologic features, including epithelial changes, increased smooth muscle mass, increased numbers of activated fibroblasts/myofibroblasts, subepithelial fibrosis, and vascular changes. Multiple cytokines, chemokines, and growth factors released from both inflammatory and structural cells in the airway tissue create a complex signaling environment that drives these structural changes. However, recent investigations have changed our understanding of asthma from a purely inflammatory disease to a disease in which both inflammatory and structural components are equally involved. Several reports have suggested that asthma primarily develops because of serious defects in the epithelial layer that allow environmental allergens, microorganisms, and toxins greater access to the airway tissue and that can also stimulate the release of mediators from the epithelium, thus contributing to tissue remodeling. Lung-resident fibroblasts and smooth muscle cells have also been implicated in the pathogenesis of airway remodeling. Remodeling is assumed to result in persistent airflow limitation, a decrease in lung function, and airway hyperresponsiveness. Asthmatic subjects experience an accelerated decrease in lung function compared with healthy subjects, which is proportionally related to the duration and severity of their disease.

Transforming Growth Factor-β Regulates House Dust Mite–induced Allergic Airway Inflammation but Not Airway Remodeling

RATIONALE It is now believed that both chronic airway inflammation and remodeling contribute significantly to airway dysfunction and clinical symptoms in allergic asthma. Transforming growth factor (TGF)-beta is a powerful regulator of both the tissue repair and inflammatory responses, and numerous experimental and clinical studies suggest that it may play an integral role in the pathogenesis of asthma. OBJECTIVES We investigated the role of TGF-beta in the regulation of allergic airway inflammation and remodeling using a mouse model of house dust mite (HDM)-induced chronic allergic airway disease. METHODS We have previously shown that intranasal administration of an HDM extract (5 d/wk for 5 wk) elicits robust Th2-polarized airway inflammation and remodeling that is associated with increased airway hyperreactivity. Here, Balb/c mice were similarly exposed to HDM and concurrently treated with a pan-specific TGF-beta neutralizing antibody. MEASUREMENTS AND MAIN RESULTS We observed that anti-TGF-beta treatment in the context of either continuous or intermittent HDM exposure had no effect on the development of HDM-induced airway remodeling. To further confirm these findings, we also subjected SMAD3 knockout mice to 5 weeks of HDM and observed that knockout mice developed airway remodeling to the same extent as HDM-exposed littermate controls. Notably, TGF-beta neutralization exacerbated the eosinophilic infiltrate and led to increased airway hyperreactivity. CONCLUSIONS Collectively, these data suggest that TGF-beta regulates HDM-induced chronic airway inflammation but not remodeling, and furthermore, caution against the use of therapeutic strategies aimed at interfering with TGF-beta activity in the treatment of this disease.

Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.

Induction of Vascular Remodeling in the Lung by Chronic House Dust Mite Exposure.

Structural changes to the lung are associated with chronic asthma. In addition to alterations to the airway wall, asthma is associated with vascular modifications, although this aspect of remodeling is poorly understood. We sought to evaluate the character and kinetics of vascular remodeling in response to chronic aeroallergen exposure. Because many ovalbumin-driven models used to investigate allergic airway disease do so in the absence of persistent airway inflammation, we used a protocol of chronic respiratory exposure to house dust mite extract (HDME), which has been shown to induce persistent airway inflammation consistent with that seen in humans with asthma. Mice were exposed to HDME intranasally for 7 or 20 consecutive weeks, and resolution of the inflammatory and remodeling response to allergen was investigated 4 weeks after the end of a 7-week exposure protocol. Measures of vascular remodeling, including total collagen deposition, procollagen I production, endothelial and smooth muscle cell proliferation, smooth muscle area, and presence of myofibroblasts, were investigated histologically in lung vessels of different sizes and locations. We observed an increase in total collagen content, which did not resolve upon cessation of allergen exposure. Other parameters were significantly increased after 7 and/or 20 weeks of allergen exposure but returned to baseline after allergen withdrawal. We conclude that respiratory HDME exposure induces airway remodeling and pulmonary vascular remodeling, and, in accordance with airway remodeling, some components of these structural changes may be irreversible.

Phenotype modulation of airway smooth muscle in asthma

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma. These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity.

IL-22 contributes to TGF-β1-mediated epithelial-mesenchymal transition in asthmatic bronchial epithelial cells

BackgroundAllergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects.MethodsPrimary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22.ResultsPrimary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics.ConclusionThe impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.

Transient Corticosteroid Treatment Permanently Amplifies the Th2 Response in a Murine Model of Asthma

Corticosteroids (CS) remain the most efficacious pharmacotherapeutic option for the management of asthma. Although the acute anti-inflammatory effects of CS treatment have been amply documented both clinically and experimentally, recent human data intimate that exposure to CS may be associated with retrograde immune phenomena, including enhanced synthesis of IgE in vivo and elevated Th2 cytokine production in vitro. We have investigated the long-term immunologic effects of CS treatment in a murine model of allergic airway inflammation. CS treatment during initial exposure to OVA or upon long-term Ag rechallenge remarkably attenuated eosinophilic airway inflammation and airway hyperresponsiveness. Interestingly, however, Th2 cytokine production by cultured splenocytes from CS-treated mice was significantly elevated, while IFN-γ synthesis was depressed. Moreover, mice rechallenged with OVA several weeks after CS intervention during allergic sensitization not only developed airway inflammation, but also exhibited enhanced Th2 cytokine production in lymphoid tissues and OVA-specific IgE in serum. This amplification of the systemic immune response was associated with an intact APC compartment during CS-conditioned sensitization to OVA. These data indicate that immune processes underlying the allergic phenotype remain impervious to CS treatment and raise the possibility that treatment with CS during sensitization may amplify elements of the allergen-specific immune response.

Smooth muscle in tissue remodeling and hyper-reactivity: Airways and arteries.

Smooth muscle comprises a key functional component of both the airways and their supporting vasculature. Dysfunction of smooth muscle contributes to and exacerbates a host of breathing-associated pathologies such as asthma, chronic obstructive pulmonary disease and pulmonary hypertension. These diseases may be marked by airway and/or vascular smooth muscle hypertrophy, proliferation and hyper-reactivity, and related conditions such as fibrosis and extracellular matrix remodeling. This review will focus on the contribution of airway or vascular smooth dysfunction to common airway diseases.

Evaluation of allergic lung inflammation by computed tomography in a rat model in vivo

The ability of micro-computed tomography (CT) to noninvasively evaluate allergic pulmonary inflammation in an experimental model was investigated. In addition, two image segmentation methods and the value of respiratory gating were investigated in the context of this model. Brown Norway rats were exposed to one of four doses of house dust mite (HDM) extract (0, 0.15, 15 or 150 μg) delivered intratracheally every 24 h for 10 days. CT scanning was performed at baseline and after several longitudinal HDM exposures. Both thoracic- and lung-segmentation methods yielded similar results when standardisation practices were employed. While tissue histology correlated well with CT images, cell counts from bronchoalveolar lavage depicted greater inflammation than did density measures from CT images. Evidence from representative CT slices and transaxial density distribution indicated that inflammation was primarily associated with major airways and extended into the periphery from these focal points. Respiratory gating demonstrated that images of the inspiratory state provided greater contrast of inflammatory processes. Lastly, decreases in tidal volumes indicated significant mechanical respiratory changes in animals exposed to both 15 and 150 μg. In summary, CT image segmentation can extract pertinent data on in vivo allergic airway/lung inflammation. Furthermore, respiratory gating provides additional contrast and insight into these quantification practices.