Responses In Lung Fibroblasts Research Articles

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFβ, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.

Human Lung Fibroblasts Exhibit Induced Inflammation Memory via Increased IL6 Gene Expression and Release.

Fibroblasts of different origins are known to possess stromal memory after inflammatory episodes. However, there are no studies exploring human lung fibroblast memory which may predict a subsequent inflammatory response in chronic respiratory diseases and COVID-19. MRC-5 and HF19 human lung fibroblast cell lines were treated using different primary and secondary stimulus combinations: TNFα–WD–TNFα, Poly (I:C)–WD–TNFα, TNFα–WD–Poly (I:C), or LPS–WD–TNFα with a 24-h rest period (withdrawal period; WD) between the two 24-h stimulations. TLR3 and NF-κB inhibitors were used to determine pathways involved. The effect of SARS-Cov-2 spike protein to inflammatory response of lung fibroblasts was also investigated. mRNA expressions of genes and IL6 release were measured using qRT-PCR and ELISA, respectively. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups. Preexposure with Poly (I:C) significantly increased TNFα-induced IL6 gene expression and IL6 release in both cell lines, while it affected neither gene expressions of IL1B, IL2, IL8, and MMP8 nor fibrosis-related genes: ACTA2, COL1A1, POSTN, and TGFB1. Inhibition of TLR3 or NF-κB during primary stimulation significantly downregulated IL6 release. Simultaneous treatment of MRC-5 cells with SARS-CoV-2 spike protein further increased TNFα-induced IL6 release; however, preexposure to Poly (I:C) did not affect it. Human lung fibroblasts are capable of retaining inflammatory memory and showed an augmented response upon secondary exposure. These results may contribute to the possibility of training human lung fibroblasts to respond suitably on inflammatory episodes after viral infection.

Cellular response of lung fibroblasts and epithelial cells to particulate matter10 treatment examined via comparative transcriptome analysis.

Particulate matter (PM) can be categorized by particle size (PM10, PM2.5 and PM1.0), which is an important factor affecting the biological response. Exposure to PM in the air (dust, smoke, dirt and biological contaminants) is clearly associated with lung disease (lung cancer, pneumonia and asthma). Although PM primarily affects lung epithelial cells, the specific response of related cell types to PM remains to be elucidated. The present study performed Gene Ontology (GO) analysis programs (Clustering GO and Database for Annotation, Visualization and Integrated Discovery) on differentially expressed genes in lung epithelial cells (WI‑38 VA‑13) and fibroblasts (WI‑38) following treatment with PM10 and evaluated the cell‑specific biological responses related to cell proliferation, apoptosis, adhesion and extracellular matrix production. The results suggested that short‑ or long‑term exposure to PM may affect cell condition and may consequently be related to several human diseases, including lung cancer and cardiopulmonary disease.

Overexpression of bone morphogenetic protein receptor type 2 suppresses transforming growth factor β-induced profibrotic responses in lung fibroblasts

Purpose of the study: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a poor prognosis. As the efficacy of currently available antifibrotics is limited, development of new therapies is warranted. Transforming growth factor (TGF)-β plays a central role in the pathogenesis of IPF through mechanisms such as promoting the production of extracellular matrix by fibroblasts. Conversely, bone morphogenetic proteins (BMPs) are known to be antifibrotic and may counterbalance TGF-β signaling via BMP receptor type 2 (BMPR2). However, little is known about the expression status of BMPR2 and its function in pulmonary fibrosis, and manipulation of BMPR2 expression has never been attempted. In this study, we aimed at evaluating the effectiveness of BMPR2 upregulation for modulating the imbalance of the TGF-β/BMP axis and reduce the profibrotic changes in lung fibroblasts. Materials and Methods We investigated BMPR2 expression in pulmonary fibrosis and TGF-β/BMP signaling in lung fibroblasts. Then we evaluated the impact of BMPR2 upregulation using adenoviral transduction on TGF-β-induced Smad2/3 phosphorylation and fibronectin production in lung fibroblasts. Results BMPR2 was distributed in airway epithelium and alveolar walls in rat lungs. BMPR2 expression was decreased in fibrotic lesions in the lungs of rats with bleomycin-induced pulmonary fibrosis and in human lung fibroblasts (HLFs) stimulated with TGF-β. Although Smad2/3 phosphorylation and fibronectin production were not suppressed solely by BMPs, phosphorylated Smad2/3 was decreased in BMPR2-transduced cells even without BMP stimulation. Fibronectin was decreased only when BMPR2-transduced HLFs were stimulated with BMP7 (but not BMP4). Similar results were also observed in IPF patient HLFs and rat lung fibroblasts. Conclusions BMPR2 expression was reduced in fibrotic lungs and lung fibroblasts stimulated with TGF-β. BMPR2 transduction to lung fibroblasts reduced Smad2/3 phosphorylation, and reduced fibronectin production when treated with BMP7. Upregulation of BMPR2 may be a possible strategy for treating pulmonary fibrosis.

M2 macrophages have unique transcriptomes but conditioned media does not promote profibrotic responses in lung fibroblasts or alveolar epithelial cells in vitro.

Macrophages are critical regulators of pulmonary fibrosis. Their plasticity, proximity, and ability to cross talk with structural cells of the lung make them a key cell type of interest in the regulation of lung fibrosis. Macrophages can express a variety of phenotypes, which have been historically represented through an "M1-like" to "M2-like" delineation. In this classification, M1-like macrophages are proinflammatory and have increased phagocytic capacity compared with alternatively activated M2-like macrophages that are profibrotic and are associated with wound healing. Extensive evidence in the field in both patients and animal models aligns pulmonary fibrosis with M2 macrophages. In this study, we performed RNA sequencing (RNAseq) to fully characterize M1- vs. M2-skewed bone marrow-derived macrophages (BMDMs) and investigated the profibrotic abilities of M2 BMDM conditioned media (CM) to promote fibroblast migration and proliferation, alveolar epithelial cell (AEC) apoptosis, and mRNA expression of key fibrotic genes in both fibroblasts and AECs. Although M2 CM-treated fibroblasts had increased migration and M2 CM-treated fibroblasts and AECs had increased expression of profibrotic proteins over M1 CM-treated cells, all differences can be attributed to M2 polarization reagents IL-4 and IL-13 also present in the CM. Collectively, these data suggest that the profibrotic effects associated with M2 macrophage CM in vitro are attributable to effects of polarization cytokines rather than additional factors secreted in response to those polarizing cytokines.

Vimentin regulation of autophagy activation in lung fibroblasts in response to lipopolysaccharide exposure in vitro.

BackgroundThe activation and assembly of the NLRP3 inflammasome is dependent on the interaction between NLRP3 and the intermediate filament protein vimentin in an acute respiratory distress syndrome (ARDS) model. We investigated the role of vimentin in this process using human fetal lung (HFL-1) fibroblasts with vimentin transfer genes or gene knockdown and lipopolysaccharide (LPS) intervention.MethodsHFL-1 cells [con-vector + LPS, vimentin-pCMV3 (VIM-pCMV3), con-siRNA, and vimentin siRNA (VIM-siRNA)] were treated with LPS. An oxidative stress damage assessment, apoptosis analysis, and quantification of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 by enzyme linked immunosorbent assay (ELISA) were performed. Immunoblotting was used to reveal the autophagy pathway.ResultsWe demonstrated that in response to LPS vimentin expression was lower in the HFL-1 cells with the vimentin gene knocked down. Specifically, an increase in oxidative stress, a decrease in mitochondrial membrane potential, or an increase in calcium ion permeability resulted in an increase in the fibroblast apoptosis rate. In addition, the inflammatory response after vimentin gene knockout was upregulated, as indicated by higher levels of TNF-a, IL-1β, IL-6, and IL-10. Importantly, the mechanism of suppression of vimentin in the lung fibroblasts was caused by a decrease in autophagy, an increase in mitochondrial membrane protein, and a decrease in mitochondrial function, which may contribute to the augmented cellular injury generated during the response to LPS.ConclusionsThis study provides insights into whether vimentin may interfere with the inflammatory cascade by activating the autophagy pathway of mitochondrial lung fibroblasts in the early stage of acute lung injury (ALI).

The role of osteosarcoma cell-derived exosomes in the promotion of lung metastasis

Abstract Osteosarcoma (OS) is the most common primary tumor of bone and typically occurs in children and young adults. 30–40% of OS patients develop lung metastases after initial diagnosis, with only 20% of these patients surviving 4 years post-relapse. For over 30 years, there have been no improvements in the treatment of OS, and more importantly, no diagnostic tools that help identify those patients at high risk for lung metastases. Recent data has shown that in other tumor types, tumor-secreted extracellular vesicles (EVs) deliver biological cargo to distant sites, resulting in priming of non-malignant host cells for promotion of metastasis. The aim of this project is to understand how OS EVs influence the response of lung fibroblasts (LFs) and alveolar macrophages (AMs), and if their uptake of EVs results in pre-metastatic priming that can be detected via AM transcriptomic changes. Hypothesis LFs are primary targets of OS EVs, which will increase LF IL-6 production and cause paracrine STAT3 activation of AM’s into an M2 tumor-promoting phenotype. Results Flow cytometry and confocal microscopy demonstrated in vitro uptake of OS EVs by LF cells. In vitro ‘education’ of LFs with OS EVs induced significant production of IL-6, IL-8, and CCL2, with the greatest mean difference between EV-educated and naïve LFs observed with IL-6 secretion. Conclusion This data suggest that OS EVs efficiently prime LFs to induce IL-6 secretion, and this response may be associated with promotion of OS lung metastasis. Further data is being collected to determine the role of IL-6 in OS cell survival and lung metastatic colonization. RNA sequencing of AMs will be used to characterize phenotype which could serve as an effective cellular biomarker to predict lung metastasis.

Paracrine signalling during ZEB1-mediated epithelial\u2013mesenchymal transition augments local myofibroblast differentiation in lung fibrosis

The contribution of epithelial–mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR–RAS–ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial–mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

Manning the Barricades: Lung Fibroblasts and CD4+ T Cells as the Last Line of Defense against Bacterial Invasion?

Fibroblasts are a major structural cell in the human lung, being responsible for the production of extracellular matrix components that provide the intricate structure necessary for correct lung function. Generally located in the submucosa, fibroblasts do not usually directly interact with the commensal microbes that we now know are resident in the airways. However, during situations in which alveolar macrophages and epithelial cells are impaired, for example, during severe viral infections leading to pneumonia, bacteria can invade the lung mesenchyme. In these circumstances, fibroblasts may represent another immunological barrier to bacterial invasion, not just as innate immune effectors, but also by interacting with migrating and tissue-resident adaptive immune cell populations, such as CD4+ T cells. The cytokines produced by CD4+ T helper cells are integral in directing appropriate innate and adaptive immune responses against bacteria, but the nature of fibroblast-CD4+ cell interaction, unlike the CD8+ T-cell interaction, is not clearly established. Here, we review the responses of lung fibroblasts to bacteria and discuss emerging data indicating a key role for these cells in directly presenting bacterial antigens to CD4+ T cells.

Real-time imaging of mechanically and chemically induced ATP release in human lung fibroblasts

Extracellular adenosine 5′-triphosphate (ATP) acts as an inflammatory mediator of pulmonary fibrosis. We investigated the effects of mechanical and chemical stimuli on ATP release from primary normal human lung fibroblasts. We visualized the ATP release from fibroblasts in real time using a luminescence imaging system while acquiring differential interference contrast cell images with infrared optics. Immediately following a single uniaxial stretch for 1s, ATP was released from a certain population of cells and spread to surrounding spaces. Hypotonic stress, which causes plasma membrane stretching, also induced the ATP release. Compared with the effects of mechanical stretch, ATP-induced release sites were homogeneously distributed. In contrast to the effects of mechanical stimuli, application of platelet-derived growth factor caused ATP release from small numbers of the cells. Our real-time ATP imaging demonstrates that there is a heterogeneous nature of ATP release from lung fibroblasts in response to mechanical and chemical stimuli.

Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response

Radiation-induced lung fibrosis, the most serious effect of lung cancer radiotherapy on normal tissue, remains a major technical obstacle to the broader application of radiotherapy to patients with lung cancer. This study describes the use of an image-guided irradiation system in mice mimicking stereotactic body radiotherapy (SBRT) to examine the molecular features of chronic fibrotic response after radiation injury. MicroRNA (miR) array analysis of injured pulmonary tissue identified a set of miRs whose expression was significantly increased in damaged lung tissue. In particular, miR-21 expression was increased at the radiation injury site, concurrent with collagen deposition. Although the inhibition of miR-21 by its specific inhibitor anti-miR-21 only marginally affected endothelial-mesenchymal transition (EndMT) in lung endothelial cells, this inhibition significantly reduced collagen synthesis in lung fibroblasts. Furthermore, ectopic expression of miR-21 was sufficient to promote a fibrotic response in lung fibroblasts, enhancing Smad2 phosphorylation concurrent with Smad7 downregulation. These findings indicate that the induction of miR-21 expression is responsible for fibrotic responses observed in mesenchymal cells at the injury site through the potentiation of TGF-β signaling. Local targeting of miR-21 at the injured area could have potential therapeutic utility in mitigating radiation-induced lung fibrosis.

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR−/−) and wild-type (AhR+/+) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR−/− cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR−/− compared to AhR+/+ cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR+/+ fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR+/+ lung fibroblasts in response to serum, corresponding to a decrease in p27KIP1 protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27KIP1 in AhR−/− fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR.

Anti-inflammation and anti-fiborosis actions of resveratrol on in vitro pneumoconiosis model

Pneumoconiosis is the most severe one in occupational diseases, especially in mainland China. Persistent inflammation and subsequent fibrosis in the lung constitute two main pathological processes of pneumoconiosis. Previous studies showed a promising potential of resveratrol to intervene the inflammatory and fibrotic processes in pulmonary disease. Present study investigated the potential therapeutic effects and underlying mechanisms of resveratrol on silica-stimulated inflammatory response in lung macrophages and TGF-β-induced fibrotic response in lung fibroblasts.

Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts

BackgroundIn vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes.ObjectivesWe investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response.MethodsPrimary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 μg/mL equivalent to 0.37-6.0 μg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms.ResultsMWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1β, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used.ConclusionsTaken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.

Abstract 9843: Proton Pump Inhibitors Inhibit DDAH and Processes Underlying Idiopathic Pulmonary Fibrosis

Rationale: The cardiovascular enzyme dimethylarginine dimethylaminohydrolase (DDAH) has recently been implicated in the progression of idiopathic pulmonary fibrosis (IPF). By degrading asymmetric dimethylarginine (ADMA), an inhibitor of inducible nitric oxide (NO) synthase (iNOS), DDAH releases the brake on a pathway implicated in lung injury and fibrosis. In addition, DDAH promotes the pro-fibrotic activity of the TGFβ pathway. Objectives: We embarked on studies to discover DDAH inhibitors, and to assess the effect of such inhibitors on processes involved in IPF. Methods: We performed high throughput screening (HTS) of over 130,000 small molecules and discovered that the proton pump inhibitors (PPIs) modestly inhibit DDAH. We used cellular and molecular assays to study the effect of the PPIs on DDAH activity by measuring the concentration of ADMA. In addition, we assessed the effect of PPIs on collagen synthesis by IPF lung fibroblasts, and on the survival and proliferation of lung alveolar epithelial cells. Finally, a retrospective analysis of the Stanford University interstitial lung disease (ILD) database was performed to assess the effect of PPIs on transplant-free survival. Results: We found that the PPIs inhibit DDAH activity in biochemical assays, cell culture and in vivo. The resulting increase in ADMA level was mirrored by a reduction in NO production. In addition, the PPIs reduced the production of soluble collagen by primary IPF lung fibroblasts in response to TGFβ and regulated both proliferation and survival of lung alveolar epithelial cells. Retrospective review of the ILD database of 131 IPF patients (of who 85 were on PPIs for > 1 year) revealed a survival advantage (i.e. time to transplant or death when calculated from the time of diagnosis) of the patients on PPIs, including those without symptoms of Gastroesophageal disease (GERD); an indication for which the PPIs are primarily prescribed for, suggesting the involvement of a mechanism other than acid-suppression. Conclusions: Our data reveal a plausible biological explanation for the clinical benefit of the PPIs in IPF. The reported reduction in radiologic fibrosis score and significant survival advantage of IPF patients on PPIs may be due to modulation of the DDAH/NOS pathway.

IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts.

Activation of the innate immune system plays a key role in exacerbations of chronic lung disease, yet the potential role of lung fibroblasts in innate immunity and the identity of epithelial danger signals (alarmins) that may contribute to this process are unclear. The objective of the study was to identify lung epithelial-derived alarmins released during endoplasmic reticulum stress (ER stress) and oxidative stress and evaluate their potential to induce innate immune responses in lung fibroblasts. We found that treatment of primary human lung fibroblasts (PHLFs) with conditioned media from damaged lung epithelial cells significantly upregulated interleukin IL-6, IL-8, monocyte chemotactic protein-1, and granulocyte macrophage colony-stimulating factor expression (P<0.05). This effect was reduced with anti-IL-1α or IL-1Ra but not anti-IL-1β antibody. Costimulation with a Toll-like receptor 3 ligand, polyinosinic–polycytidylic acid (poly I:C), significantly accentuated the IL-1α-induced inflammatory phenotype in PHLFs, and this effect was blocked with inhibitor of nuclear factor kappa-B kinase subunit beta and TGFβ-activated kinase-1 inhibitors. Finally, Il1r1−/− and Il1a−/− mice exhibit reduced bronchoalveolar lavage (BAL) neutrophilia and collagen deposition in response to bleomycin treatment. We conclude that IL-1α plays a pivotal role in triggering proinflammatory responses in fibroblasts and this process is accentuated in the presence of double-stranded RNA. This mechanism may be important in the repeated cycles of injury and exacerbation in chronic lung disease.

Antifibrotic Effects of Focal Adhesion Kinase Inhibitor in Bleomycin-Induced Pulmonary Fibrosis in Mice

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor β to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of α-smooth muscle actin induced by transforming growth factor β, indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

Assessment of Brd4 Inhibition in Idiopathic Pulmonary Fibrosis Lung Fibroblasts and in Vivo Models of Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of high unmet medical need. Although bromodomain (Brd) and extra terminal domain isoforms have recently been implicated in mediating inflammatory and oncologic indications, their roles in lung fibrosis have not been comprehensively assessed. We investigated the role of Brd on the profibrotic responses of lung fibroblasts (LFs) in patients with rapidly progressing IPF and a mouse bleomycin model of lung fibrosis. The enhanced migration, proliferation, and IL-6 release observed in LFs from patients with rapidly progressing IPF are attenuated by pharmacologic inhibition of Brd4. These changes are accompanied by enhanced histone H4 lysine5 acetylation and association of Brd4 with genes involved in the profibrotic responses in IPF LFs as demonstrated using chromatin immunoprecipitation and quantitative PCR. Oral administration of 200 mg/kg per day Brd4 inhibitor JQ1 in a therapeutic dosing regimen substantially attenuated lung fibrosis induced by bleomycin in C57BL/6 mice. In conclusion, this study shows that the Brd4 inhibitor JQ1, administered in a therapeutic dosage, is capable of inhibiting the profibrotic effects of IPF LFs and attenuates bleomycin-induced lung fibrosis in mice. These results suggest that Brd4 inhibitors mayrepresent a novel therapy for the treatment of rapidly progressing IPF.

Differential analysis of gene regulation at transcript resolution with RNA-seq

Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.

Regulation of lung fibroblast activation by annexin A1

Annexin-A1 (AnxA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The contribution of AnxA1 to human cell biology is not well understood. We investigated the contribution of AnxA1 and its receptor, formyl-peptide receptor 2 (FPR2), to the regulation of inflammatory responses in human normal lung fibroblasts (NLF). Silencing constitutive AnxA1 expression in NLF using small interfering RNA (siRNA) was associated with moderate but significant increases in tumor necrosis factor (TNF)-induced proliferation and interleukin (IL)-6 production, accompanied by reduction of ERK and NF-κB activity. AnxA1 regulation of ERK and NF-κB activation was associated with effects on proliferation. Blocking FPR2 using the specific antagonist WRW4 mimicked the effects of AnxA1 silencing on TNF-induced proliferation, IL-6, ERK, and NF-κB activation. AnxA1 silencing also impaired inhibitory effects of glucocorticoid on IL-6 production and on the expression of glucocorticoid-induced leucine zipper (GILZ), but blocking FPR2 failed to mimic these effects of AnxA1 silencing. These data suggest that AnxA1 regulates TNF-induced proliferation and inflammatory responses in lung fibroblasts, via effects on the ERK and NF-κB pathways, which depend on FPR2. AnxA1 also mediates effects of glucocorticoids and GILZ expression, but these effects appear independent of FPR2. These findings suggest that mimicking AnxA1 actions might have therapeutic potential in chronic inflammatory lung diseases.