Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

TGFß-2 haploinsufficiency causes early death in mice with Marfan syndrome.

To assess the contribution of individual TGF-ß isoforms to aortopathy in Marfan syndrome (MFS), we quantified the survival and phenotypes of mice with a combined fibrillin1 (the gene defective in MFS) hypomorphic mutation and a TGF-ß1, 2, or 3 heterozygous null mutation. The loss of TGF-ß2, and only TGF-ß2, resulted in 80% of the double mutant animals dying earlier, by postnatal day 20, than MFS only mice. Death was not from thoracic aortic rupture, as observed in MFS mice, but was associated with hyperplastic aortic valve leaflets, aortic regurgitation, enlarged aortic root, increased heart weight, and impaired lung alveolar septation. Thus, there appears to be a relationship between loss of fibrillin1 and TGF-ß2 in the postnatal development of the heart, aorta and lungs.

Behçet's disease risk-variant HLA-B51/ERAP1-Hap10 alters human CD8 T cell immunity.

OBJECTIVES: The endoplasmic reticulum aminopeptidase (ERAP1) haplotype Hap10 encodes for a variant allotype of the endoplasmic reticulum (ER)-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity. This haplotype recessively confers the highest risk for Behçet's diseases (BD) currently known, but only in carriers of HLA-B*51, the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact. METHODS: We genotyped and immune phenotyped a cohort of 26 untreated Turkish BD subjects and 22 healthy donors, generated CRISPR-Cas9 ERAP1 KOs from HLA-B*51 + LCL, analysed the HLA class I-bound peptidome for peptide length differences and assessed immunogenicity of genome-edited cells in CD8 T cell co-culture systems. RESULTS: Allele frequencies of ERAP1-Hap10 were similar to previous studies. There were frequency shifts between antigen-experienced and naïve CD8 T cell populations of carriers and non-carriers of ERAP1-Hap10 in an HLA-B*51 background. ERAP1 KO cells showed peptidomes with longer peptides above 9mer and significant differences in their ability to stimulate alloreactive CD8 T cells compared with wild-type control cells. CONCLUSIONS: We demonstrate that hypoactive ERAP1 changes immunogenicity to CD8 T cells, mediated by an HLA class I peptidome with undertrimmed peptides. Naïve/effector CD8 T cell shifts in affected carriers provide evidence of the biological relevance of ERAP1-Hap10/HLA-B*51 at the cellular level and point to an HLA-B51-restricted process. Our findings suggest that variant ERAP1-Hap10 partakes in BD pathogenesis by generating HLA-B51-restricted peptides, causing a change in immunodominance of the ensuing CD8 T cell response.

Gpr125 is a unifying hallmark of multiple mammary progenitors coupled to tumor latency.

Gpr125 is an orphan G-protein coupled receptor, with homology to cell adhesion and axonal guidance factors, that is implicated in planar polarity and control of cell movements. By lineage tracing we demonstrate that Gpr125 is a highly specific marker of bipotent mammary stem cells in the embryo and of multiple long-lived unipotent basal mammary progenitors in perinatal and postnatal glands. Nipple-proximal Gpr125+ cells express a transcriptomic profile indicative of chemo-repulsion and cell movement, whereas Gpr125+ cells concentrated at invasive ductal tips display a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix. Gpr125 progenitors acquire bipotency in the context of transplantation and cancer and are greatly expanded and massed at the pushing margins of short latency MMTV-Wnt1 tumors. High Gpr125 expression identifies patients with particularly poor outcome within the basal breast cancer subtype highlighting its potential utility as a factor to stratify risk.

Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages.

Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3-/- mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling.

Advances in Targeted Therapy for Progressive Fibrosing Interstitial Lung Disease.

Progressive fibrosing interstitial lung disease (PF-ILD) has been redefined as a new clinical syndrome that shares similar genetics, pathophysiology, and natural history to idiopathic pulmonary fibrosis (IPF). IPF is the most common form of idiopathic interstitial pneumonias, which is progressive in nature and is associated with significant mortality. Therapies targeting an inflammatory and/or immune response have not been consistently effective or well tolerated in patients with IPF. The two antifibrotic drugs approved for IPF treatment, nintedanib and pirfenidone, have been shown to reduce lung function decline in PF-ILD. Novel uses of antifibrotic therapy are emerging due to a paucity of evidence-based treatments for multiple ILD subtypes. In this review, we describe the current body of knowledge on antifibrotic therapy and immunomodulators in PF-ILD, drawing from experience in IPF where appropriate.

The Hedgehog target Gli1 is not required for bleomycin-induced lung fibrosis.

Sonic Hedgehog (SHH) signaling, a developmental pathway promoting lung mesenchymal expansion and differentiation during embryogenesis, has been increasingly recognized as a profibrotic factor in mature lung, where it might contribute to the pathogenesis of lung fibrosis. Pathway inhibition at the level of the downstream Gli transcription factors Gli1 and Gli2 (by GANT61) ameliorates lung fibrosis in the bleomycin model, whereas inhibition proximally at the level of HH ligand (by anti Hh antibody 5E1) or Smo (by GDC-0449) of the canonical pathway does not, implicating Gli1 and/or Gli2 as a key target. The fact that both the Gli1-labelled cell lineage and Gli1 expressing cells expand during fibrosis formation and contribute significantly to the pool of myofibroblasts in the fibrosis scars suggests a fibrogenic role for Gli1. Therefore to further dissect the roles of Gli1 and Gli2 in lung fibrosis we evaluated Gli1 KO and control mice in the bleomycin model. Monitoring of Gli1+/+ (n = 12), Gli1lZ/+ (n = 37) and Gli1lZ/lZ (n = 18) mice did not reveal differences in weight loss or survival. Lung evaluation at the 21-day endpoint did not show differences in lung fibrosis formation (as judged by morphology and trichrome staining), Ashcroft score, lung collagen content, lung weight, BAL protein content or BAL cell differential count. Our data suggest that Gli1 is not required for bleomycin-induced lung fibrosis.

Sonic Hedgehog Signaling Regulates Myofibroblast Function during Alveolar Septum Formation in Murine Postnatal Lung.

Sonic Hedgehog (Shh) signaling regulates mesenchymal proliferation and differentiation during embryonic lung development. In the adult lung, Shh signaling maintains mesenchymal quiescence and is dysregulated in diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease. Our previous data implicated a role for Shh in postnatal lung development. Here, we report a detailed analysis of Shh signaling during murine postnatal lung development. We show that Shh pathway expression and activity during alveolarization (postnatal day [P] 0-P14) are distinct from those during maturation (P14-P24). This biphasic pattern is paralleled by the transient presence of Gli1+;α-smooth muscle actin (α-SMA)+ myofibroblasts in the growing alveolar septal tips. Carefully timed inhibition of Hedgehog (Hh) signaling during alveolarization defined mechanisms by which Shh influences the mesenchymal compartment. First, interruption of Hh signaling at earlier time points results in increased lung compliance and wall structure defects of increasing severity, ranging from moderately enlarged alveolar airspaces to markedly enlarged airspaces and fewer secondary septa. Second, Shh signaling is required for myofibroblast differentiation: Hh inhibition during early alveolarization almost completely eliminates Gli1+;α-SMA+ cells at the septal tips, and Gli1-lineage tracing revealed that Gli1+ cells do not undergo apoptosis after Hh inhibition but remain in the alveolar septa and are unable to express α-SMA. Third, Shh signaling is vital to mesenchymal proliferation during alveolarization, as Hh inhibition decreased proliferation of Gli1+ cells and their progeny. Our study establishes Shh as a new alveolarization-promoting factor that might be affected in perinatal lung diseases that are associated with impaired alveolarization.

Sonic hedgehog signaling in the lung. From development to disease.

Over the past two decades, the secreted protein sonic hedgehog (SHH) has emerged as a critical morphogen during embryonic lung development, regulating the interaction between epithelial and mesenchymal cell populations in the airway and alveolar compartments. There is increasing evidence that the SHH pathway is active in adult lung diseases such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and lung cancer, which raises two questions: (1) What role does SHH signaling play in these diseases? and (2) Is it a primary driver of the disease or a response (perhaps beneficial) to the primary disturbance? In this review we aim to fill the gap between the well-studied period of embryonic lung development and the adult diseased lung by reviewing the hedgehog (HH) pathway during the postnatal period and in adult uninjured and injured lungs. We elucidate the similarities and differences in the epithelial-mesenchymal interplay during the fibrosis response to injury in lung compared with other organs and present a critical appraisal of tools and agents available to evaluate HH signaling.

Hedgehog signaling in neonatal and adult lung.

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Integrin alpha3beta1-dependent beta-catenin phosphorylation links epithelial Smad signaling to cell contacts.

Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, alpha3beta1, were found to have a markedly blunted EMT response to TGF-beta1. A mechanism for this defect was explored in alpha3-null cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-beta1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-beta1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-beta-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2-pY654-beta-catenin complexes do not form in the absence of alpha3 or when alpha3beta1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.

Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis.

Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-beta1. As the ECM critically regulates AEC responses to TGF-beta1, we explored the role of the prominent epithelial integrin alpha3beta1 in experimental fibrosis by generating mice with lung epithelial cell-specific loss of alpha3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through beta-catenin has been implicated in EMT; we found that in primary AECs, alpha3 integrin was required for beta-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-beta-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-beta-catenin/pSmad2 complexes and showed accumulation of pY654-beta-catenin in myofibroblasts. These findings demonstrate epithelial integrin-dependent profibrotic crosstalk between beta-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.

Urokinase receptors are required for alpha 5 beta 1 integrin-mediated signaling in tumor cells.

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha3beta1 (D262A) or alpha5beta1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha5beta1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha5beta1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor.alpha5beta1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha5beta1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha5beta1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.

Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.

Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing beta-galactosidase (beta-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-beta1. beta-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-beta1 expression. The increase in vimentin-positive cells within injured lungs was nearly all beta-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-beta1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-beta1 pathway and, if exposed to active TGF-beta1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.

Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Isolation and characterization of Rac1 pseudogenes (psi1Rac1-psi4Rac1) in the human genome.

Ras-related C3 toxin substrate 1 (Rac1) is a small Rho-GTPase with important functions in fundamental cellular processes such as cytoskeleton rearrangements, signal transduction, cell cycle progression and malignant transformation. Using Rac1 primer, we identified a 5.5-kb DNA sequence on chromosome 4 (Chr. 4) in the human genome, containing the intronless protein coding sequence of Rac1. Sequence analysis revealed features of a processed pseudogene, which we named psi1Rac1, that could be detected by Southern blot and polymerase chain reaction (PCR) on genomic DNA. A psi1Rac1 pseudogene transcript was not detected by reverse transcription-polymerase chain reaction (RT-PCR), nor had the psi1Rac1 promoter any transcriptional activity. In addition, three other intronless pseudogenes of Rac1 on chromosomes 4, 13 and X were identified (psi1Rac1-psi4Rac1) sharing an 86-96% sequence similarity with Rac1. Neither RT-PCR with pseudogene specific restriction enzymes, nor the sequencing of 130 cDNA clones from benign and malignant breast tissue and cell lines, detected the transcription of any of the Rac1 pseudogenes (psi2Rac1-psi4Rac1). Existence of Rac1 pseudogenes should be taken into consideration when analyzing genomic alterations of the human Rac1 gene.

Urokinase receptor and integrin interactions.

Urokinase receptors (uPAR) were initially thought to function simply as a mechanism to concentrate the urokinase/plasmin system toward the cell surface. However, extensive evidence has accumulated that this glycolipid-anchored receptor also functions in both the adhesive and signaling pathways of many migratory cells. Mechanisms by which uPAR exercises these functions involve complexing with other membrane proteins for signal transduction. One set of functional partners for uPAR on the cell surface are integrins. Recent studies point to important structural features of uPAR:integrin interactions, indicating uPAR to be a cis-acting integrin ligand. In vivo data reveal altered integrin function and cell migration when uPAR:integrin interactions are impaired. Together these observations support the idea that uPAR:integrin interactions may be a focal point of intervention in pathobiology where integrin function is crucial, such as tumor metastasis.