Interstitial lung disease in children with Rubinstein-Taybi syndrome.

INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD. METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene. RESULTS: Computed tomography images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased anti-smooth muscle antibody staining, and increased staining for acetylated histone 2 and histone 3 lysine 9. DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein.

Loss of Thy-1 may reduce lung regeneration after pneumonectomy in mice.

BACKGROUND: Lung regeneration plays an important role in lung repair after injury. It is reliant upon proliferation of multiple cell types in the lung, including endothelium, epithelium, and fibroblasts, as well as remodeling of the extracellular matrix. METHODS: Lung regeneration following injury progresses via an initial inflammatory response during which macrophages clear the tissue of cellular debris. This process continues through cellular proliferation when existing cells and progenitors act to repopulate cells lost during injury, followed by tissue maturation in which newly formed cells achieve a differentiated phenotype. RESULTS: Signaling pathways critical for lung regeneration include FGF, EGF, WNT, and NOTCH. In addition, HDACs, miRNAs, ELASTIN, and MMP14 have been shown to regulate lung regeneration. Partial pneumonectomy (PNX) has been used as a therapeutic and investigational tool for several decades. Following PNX the remaining lung increases in size to compensate for loss of volume and respiratory capacity. CONCLUSIONS: Much has been learned about the triggers and mechanisms regulating pulmonary regeneration. However, the role of thymocyte differentiation antigen-1 (Thy-1) in post-PNX lung growth remains incompletely characterized. Thy-1 is a phosphatidylinositol glycoprotein with a relative molecular weight of 25000~37000 Da, which is expressed in almost all types of fibroblasts and regulates many biological functions. It not only supports the structure of fibroblasts, but also can balance cell proliferation, migration and regulate the synthesis of immune inflammatory mediators.

αvß3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis.

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvß3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvß3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvß3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvß3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Thy-1 dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFß1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFß1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by cellular phenotype alterations and deposition of extracellular matrix proteins. The alternative activation of macrophages in the lungs has been associated as a major factor promoting pulmonary fibrosis, however the mechanisms underlying this phenomenon are poorly understood. In the present study, we have defined a molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin lead to the activation of Rac2 which regulates alternative macrophage differentiation, a signaling axis within the pulmonary macrophage compartment required for bleomycin induced pulmonary fibrosis. Mice deficient in Rac2 were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with lower expression of alternatively activated profibrotic macrophage markers. We have demonstrated a macrophage autonomous process by which the injection of M2 and not M1 macrophages restored the bleomycin induced pulmonary fibrosis susceptibility in Rac2-/- mice, establishing a critical role for a macrophage Rac2 signaling axis in the regulation of macrophage differentiation and lung fibrosis in vivo. We also demonstrate that markers of alternative macrophage activation are increased in patients with IPF. Taken together, these studies define an important role for an integrin-driven Rac2 signaling axis in macrophages, and reveal that Rac2 activation is required for polarization of macrophages towards a profibrotic phenotype and progression of pulmonary fibrosis in vivo.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

Role of neprilysin in airway inflammation induced by diesel exhaust emissions.

In this study, we examined the role of neprilysin (NEP), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells' gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP or its contents because the change also occurred after exposure to urban dust (SRM 1649a), which differs in physical and chemical composition from DEP. Third, we also collected the transcriptome profiles of the concentration-effects of SRM 2975 in cultured BEAS-2B cells through a 2 X 3 factorial design. DEP exposure upregulated 151 genes and downregulated 59 genes. Cells with decreased NEP expression (accomplished by transfecting an NEP-specific small interfering RNA [siRNA]) substantially altered the expression of genes (upregulating 17 and downregulating 14) associated with DNA/protein binding, calcium channel activities, and the cascade of intracellular signaling by cytokines. Data generated from the combined RNAi and microarray approaches revealed that there is a complex molecular cascade mediated by NEP in different subcellular compartments, possibly influencing the inflammatory response. Fourth, in a controlled human exposure study, we observed significant increases in soluble NEP in sputum after acute exposure to DEE, with an average net increase of 31%. We speculate that the change in NEP activity in sputum, if confirmed in larger epidemiologic investigations at ambient exposure levels to DEE, may provide a useful endpoint and promote insight into the mechanism of DEE-induced airway alterations.

Acute changes in sputum collected from exposed human subjects in mining conditions.

Neprilysin (NEP) is a key cell surface peptidase in the maintenance of airway homeostasis and the development of pulmonary disorders. However, little information is available about the effect of particulate matter (PM) on airway NEP. In this controlled human exposure study, changes in induced sputum were measured in 11 subjects at baseline, overshot (OS) mucking, and diesel exhaust (DE) exposure days. Neither OS condition nor DE exposure was found to induce significant changes in total protein, but DE induced significant increases in cell numbers of macrophages and epithelium. Moreover, significant increases in soluble NEP were observed following OS mining dust particulates (0.43 +/- 0.06 nmol/microg protein/min; p = .023) and DE exposure (0.40 +/- 0.03 nmol/microg protein/min; p = .035) when compared with the baseline control (0.30 +/- 0.04 nmol/microg protein/min), with 42% and 31% average net increase, respectively. Pearson's correlation analyses indicated that sputum NEP activity was significantly associated with personal exposure product (elemental carbon concentration [mg/m(3)] x time [min]; C x T). The data suggest that changes in NEP activity may be an early, accurate endpoint for airway epithelial injury and provide a new insight into the mechanism of airway effects following particulate exposure.

Neurogenic responses in rat lungs after nose-only exposure to diesel exhaust.

Using an in-line, real-time, in vivo exposure system, we investigated whether acute adverse effects of diesel exhaust (DE*) exposure involve neurogenic inflammation in the lungs via sensory nerve C fibers. A total of 168 female F344 rats (175 g, 8 weeks old) were randomly assigned to pretreatment with capsaicin or saline to deplete C-fiber neurotransmitters. In a 2 x 3 factorial design, groups of animals were then exposed nose-only to a low level of DE (LDE, 35.3 microg/m3), a high level of DE (HDE, 632.9 microg/m3), or side-stream cigarette smoke (CS, 0.4 mg/m3). Two control groups were exposed whole body to filtered air in the animal room (fRA) or unfiltered air in the diesel engine room (eRA), respectively. DE was taken directly from a heavy-duty Cummins N14 research engine operated at 75% throttle (California Air Resources Board [CARB] 8, mode 6). Exposure to DE or air was 4 hours/day, 5 days/week, for 3 weeks. Exposure to CS was for 4 hours/day for 7 days. Involvement of neurogenic inflammation in the response to DE or CS was assessed via comparison of plasma extravasation, a sensitive endpoint of neurogenic inflammation, between rats with and without capsaicin pretreatment. Lung injury was assessed via analysis of proinflammatory cytokines, respiratory permeability, and histopathology. Moreover, whether DE exposure affected the molecular mechanisms of neurogenic inflammation was analyzed through quantification of substance P (SP) and its primary neurokinin-1 (NK1) receptor at the gene and protein levels and through neutral endopeptidase (NEP) activity. DE and CS exposure induced dose-dependent plasma extravasation, which may play an important role in initiating the associated lung inflammation and injury. Exposure of rats to DE affected the SP signaling pathway as indicated by overexpression of the NK1 receptor or reduction of SP in the lung tissue. DE exposure consistently inactivated tissue NEP, a key factor that switches neurogenic inflammation from its physiological and protective functions to a role that increases and perpetuates lung injury. The roles of these overlapping neurokininergic mechanisms in the initiation of DE-associated lung injury are plausible, and these changes may contribute to DE-associated respiratory disorders. Capsaicin rats followed the same trends as those of saline animals when exposed to DE or CS: capsaicin rats did not have significantly different plasma extravasation in the airways or lung parenchyma compared to their corresponding controls. Histopathology evaluation likewise demonstrated the same degree of tissue changes, such as edema and alveolar macrophage collection, in capsaicin and saline rats after the same level of DE exposure. In summary, our data suggest that neurokininergic mechanisms may have been involved in DE-induced inflammatory conditions in rat lung but that C fibers did not appear to be involved under these exposure conditions. We believe that time-course or protein knockdown/knockout animal studies are required to characterize further the role of neurokininergic mechanisms in DE-induced lung injury.

Tachykinin substance P depletion by capsaicin exacerbates inflammatory response to sidestream cigarette smoke in rats.

To evaluate the role of substance P (SP)-containing C-fiber nerves in the development of the inflammatory responses to sidestream cigarette smoke (SSCS), female Fischer 344 rats were randomly assigned into vehicle and capsaicin groups, respectively. Then, half the number in each group (N = 24) was nose-only exposed to air or 0.4 mg/m3 total particulate matter of SSCS for 4 h/day for 7 days. Exposure of the vehicle rats to SSCS induced obvious pulmonary neurogenic inflammation as indicated by elevations in plasma extravasation and proinflammatory cytokine secretions [interieukin (IL)-1beta and IL-12]. In addition, except for SP release, SSCS exposure significantly induced the tachykininergic toxicities at the gene level: upregulation of beta-preprotachykinin-I (beta-PPT-I) mRNA. However, neither SSCS exposure nor capsaicin pretreatment affects the immunolabeling density of neurokinin-1 receptor (NK-1R) in airway epithelium. SSCS also significantly inactivated pulmonary neutral endopeptidase (NEP) in lung tissue. Moreover, pretreatment with capsaicin significantly exacerbated the SSCS-induced inflammatory responses mentioned above as well as the release of plasma protein. Considering that capsaicin did not affect the normal control baselines of these parameters except for a decrease in NK-1R mRNA, we conclude that the degree of SSCS-induced inflammatory response was exacerbated because of the depletion of stored SP and/or inactivation of capsaicin-sensitive C-fiber nerves. Our data suggest the loss of afferent tachykinin SP signaling may lead to dysfunction of the sensory C-fiber nerve reflexes during exposure to SSCS, suggesting that SP serves a protective role.

Substance P and neutral endopeptidase in development of acute respiratory distress syndrome following fire smoke inhalation.

To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.

Dose-dependent transcriptome changes by metal ores on a human acute lymphoblastic leukemia cell line.

The increased morbidity of childhood leukemia in Fallon, Nevada and Sierra Vista, Arizona has prompted great health concern. The main characteristic that these two towns share is the environmental pollution attributed to metal ore from abandoned mining operations. Consequently, we have investigated the transcriptome effects of metal ores from these endemic areas using a human T-cell acute lymphoblastic leukemia cell line (T-ALL). Metal ore from Fallon significantly increased cell growth after 24, 48 and 72 h of incubation at 1.5 microg/mL concentration, as measured by trypan-blue. Sierra Vista ore significantly increased cell growth with 0.15 and 1.5 microg/mL following 72 h of incubation. From human cDNA microarray, results indicate that in total, eight genes, mostly metallothionein (MT) genes, were up-regulated and 10 genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 microg/mL of metal ores at 72 h, in comparison with untreated cells. Twenty-eight metals of both ores were quantified and their presence may be associated with the cell growth rate and dose-dependent activation of transcriptomes in immature T-cells.

Tissue-specific patterns of neurokinin-1 receptor (NK-1R) gene expression in mice exposed to sidestream cigarette smoke.

Neurokinin-1 receptor (NK-1R), a high-affinity plasma membrane-bound receptor for neurokinin substance P, plays important roles in the onset of the pathophysiological responses. To test whether the transcript levels of gene encoding NK-1R in organs are affected by sidestream cigarette smoke (SSCS) exposure, the C57BL/6 mice were randomly assigned to five groups (six/group) in a study of the dose-effect relationship. The mice were exposed to 0 (filtered room air), 2, 4, 8 and 16 mg total particulate matter (TPM) of SSCS/exposure/day, respectively, for seven days through a nose-only exposure chamber (IN-TOX, Albuquerque, NM, USA). The levels of NK-1R mRNA in the lung, heart, liver, kidney and spleen tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) techniques and normalized against GAPDH expression. NK-1R mRNA in heart tissue showed SSCS-induced dose-dependent downregulation, with minimum expression at a dose of 8.0 mg TPM. Whereas, the levels of NK-1R mRNA in the liver were upregulated to 2.86 and 5.13-fold after exposure to 2.0 and 4.0 mg TPM of SSCS respectively, then returned to 4.19 and 3.93-fold at the exposure doses of 8.0 and 16.0 mg TPM, respectively, when compared to that of the control. In the kidney, SSCS exposure at a dose of 2.0 TPM, but not higher than that level, induced significant elevation of NK-1R mRNA expression. These findings suggest that there are the paracrine and/or autocrine signalling mechanisms through receptor-ligand interactions. No alteration of NK-1R gene expression was observed in the lungs and spleen tissues in this study. The tissue-specific patterns by which SSCS affect NK-1R gene expression in these organs may partially explain dissimilarity of NK-1R activation and the associated toxicity caused by environmental tobacco smoke.