Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies. METHODS: We performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia). RESULTS: We identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF. CONCLUSIONS: Our findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Historical shift in pathological type of progressive massive fibrosis among coal miners in the USA.

BACKGROUND: Pneumoconiosis among coal miners in the USA has been resurgent over the past two decades, despite modern dust controls and regulatory standards. Previously published studies have suggested that respirable crystalline silica (RCS) is a contributor to this disease resurgence. However, evidence has been primarily indirect, in the form of radiographic features. METHODS: We obtained lung tissue specimens and data from the National Coal Workers' Autopsy Study. We evaluated specimens for the presence of progressive massive fibrosis (PMF) and used histopathological classifications to type these specimens into coal-type, mixed-type and silica-type PMF. Rates of each were compared by birth cohort. Logistic regression was used to assess demographic and mining characteristics associated with silica-type PMF. RESULTS: Of 322 cases found to have PMF, study pathologists characterised 138 (43%) as coal-type, 129 (40%) as mixed-type and 55 (17%) as silica-type PMF. Among earlier birth cohorts, coal-type and mixed-type PMF were more common than silica-type PMF, but their rates declined in later birth cohorts. In contrast, the rate of silica-type PMF did not decline in cases from more recent birth cohorts. More recent year of birth was significantly associated with silica-type PMF. CONCLUSIONS: Our findings demonstrate a shift in PMF types among US coal miners, from a predominance of coal- and mixed-type PMF to a more commonly encountered silica-type PMF. These results are further evidence of the prominent role of RCS in the pathogenesis of pneumoconiosis among contemporary US coal miners.

Pathology and Mineralogy of the Pneumoconioses.

Pneumoconioses represent the spectrum of lung diseases caused by inhalation of respirable particulate matter small enough (typically <5-µm diameter) to reach the terminal airways and alveoli. Pneumoconioses primarily occur in occupational settings where workers perform demanding and skilled manual labor including mining, construction, stone fabrication, farming, plumbing, electronics manufacturing, shipyards, and more. Most pneumoconioses develop after decades of exposure, though shorter latencies can occur from more intense particulate matter exposures. In this review, we summarize the industrial exposures, pathologic findings, and mineralogic features of various well-characterized pneumoconioses including silicosis, silicatosis, mixed-dust pneumoconiosis, coal workers' pneumoconiosis, asbestosis, chronic beryllium disease, aluminosis, hard metal pneumoconiosis, and some less severe pneumoconioses. We also review a general framework for the diagnostic work-up of pneumoconioses for pulmonologists including obtaining a detailed occupational and environmental exposure history. Many pneumoconioses are irreversible and develop due to excessive cumulative respirable dust inhalation. Accurate diagnosis permits interventions to minimize ongoing fibrogenic dust exposure. A consistent occupational exposure history coupled with typical chest imaging findings is usually sufficient to make a clinical diagnosis without the need for tissue sampling. Lung biopsy may be required when exposure history, imaging, and testing are inconsistent, there are unusual or new exposures, or there is a need to obtain tissue for another indication such as suspected malignancy. Close collaboration and information-sharing with the pathologist prior to biopsy is of great importance for diagnosis, as many occupational lung diseases are missed due to insufficient communication. The pathologist has a broad range of analytic techniques including bright-field microscopy, polarized light microscopy, and special histologic stains that may confirm the diagnosis. Advanced techniques for particle characterization such as scanning electron microscopy/energy dispersive spectroscopy may be available in some centers.

Rituximab and antimetabolite treatment of granulomatous and lymphocytic interstitial lung disease in common variable immunodeficiency.

BACKGROUND: Granulomatous and lymphocytic interstitial lung disease (GLILD) is a life-threatening complication in patients with common variable immunodeficiency (CVID), but the optimal treatment is unknown. OBJECTIVE: Our aim was to determine whether rituximab with azathioprine or mycophenolate mofetil improves the high-resolution computed tomography (HRCT) chest scans and/or pulmonary function test results in patients with CVID and GLILD. METHODS: A retrospective chart review of clinical and laboratory data on 39 patients with CVID and GLILD who completed immunosuppressive therapy was performed. Chest HRCT scans, performed before therapy and after the conclusion of therapy, were blinded, randomized, and scored independently by 2 radiologists. Differences between pretreatment and posttreatment HRCT scan scores, pulmonary function test results, and lymphocyte subsets were analyzed. Whole exome sequencing was performed on all patients. RESULTS: Immunosuppressive therapy improved patients' HRCT scan scores (P < .0001), forced vital capacity (P = .0017), FEV1 (P = .037), and total lung capacity (P = .013) but not their lung carbon monoxide diffusion capacity (P = .12). Nine patients relapsed and 6 completed retreatment, with 5 of 6 of these patients (83%) having improved HRCT scan scores (P = .063). Relapse was associated with an increased number of B cells (P = .016) and activated CD4 T cells (P = .016). Four patients (10%) had pneumonia while undergoing active treatment, and 2 patients (5%) died after completion of therapy. Eight patients (21%) had a damaging mutation in a gene known to predispose (TNFRSF13B [n = 3]) or cause a CVID-like primary immunodeficiency (CTLA4 [n = 2], KMT2D [n = 2], or BIRC4 [n = 1]). Immunosuppression improved the HRCT scan scores in patients with (P = .0078) and without (P < .0001) a damaging mutation. CONCLUSIONS: Immunosuppressive therapy improved the radiographic abnormalities and pulmonary function of patients with GLILD. A majority of patients had sustained remissions.

Chronic Hypersensitivity Pneumonitis, an Interstitial Lung Disease with Distinct Molecular Signatures.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

Histopathologic Analysis of Surgically Resected Lungs of Patients with Non-tuberculous Mycobacterial Lung Disease: a Retrospective and Hypothesis-generating Study.

Non-tuberculous mycobacterial lung disease (NTM-LD) is most commonly due to species within the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAbC). Surgical lung resection, typically a lobectomy or segmentectomy, is occasionally undertaken for individuals with recalcitrant but localized NTM-LD. Since the growth characteristics of MAC (slow growers) and MAbC (rapid growers) as well as their drug susceptibility patterns are significantly different, the objective of this study is to characterize and compare the histopathologic features of the resected lungs due to these two major NTM groups. From 1996 to 2017, 356 patients with NTM-LD due to MAC (n=270), MAbC (n=54), or both (n=32) underwent a total of 404 lobar resections (with the lingula counted as a separate lobe) at the University of Colorado Hospital. We analyzed by microscopy the existing surgical lung tissue sections for bronchiolitis, bronchiolectasis, bronchiectasis, non-necrotizing granuloma (airway, parenchymal, and total), necrotizing granuloma (airway, parenchymal, and total), peri-airway fibrosis, fibrous pleuritis, and lymphoid follicles. There were no significant differences in the presence or absence of most of the histopathologic features of surgically removed lungs due to MAC, MAbC, or both MAC + MAbC. However, there were significantly more necrotizing granulomas (airway, parenchymal, and total) and fibrous pleuritis in MAC compared to MAbC lung diseases. Since necrotizing granulomas may be a sign of inadequate control of the infection, we posit that their presence may be an indication of increased chronicity, increased virulence of MAC compared to MAbC, and/or impaired host immunity against the NTM. Futures studies to determine the root cause of such differences in histopathologic findings in MAC versus MAbC lung disease may spawn new leads on differential pathogenic mechanisms with different NTM, with the goal of aiming for more targeted therapy against both the NTM and the lung damage induced by them.

The hallmarks of severe pulmonary arterial hypertension: the cancer hypothesis-ten years later.

Severe forms of pulmonary arterial hypertension (PAH) are most frequently the consequence of a lumen-obliterating angiopathy. One pathobiological model is that the initial pulmonary vascular endothelial cell injury and apoptosis is followed by the evolution of phenotypically altered, apoptosis-resistant, proliferating cells and an inflammatory vascular immune response. Although there may be a vasoconstrictive disease component, the increased pulmonary vascular shear stress in established PAH is caused largely by the vascular wall pathology. In this review, we revisit the "quasi-malignancy concept" of severe PAH and examine to what extent the hallmarks of PAH can be compared with the hallmarks of cancer. The cancer model of severe PAH, based on the growth of abnormal vascular and bone marrow-derived cells, may enable the emergence of novel cell-based PAH treatment strategies.

Toll-like Receptor 3 Is a Therapeutic Target for Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. TLR3 (Toll-like receptor 3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. OBJECTIVES: To study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces pulmonary hypertension in preclinical models. METHODS: Lung tissue and endothelial cells from patients with PAH were investigated by polymerase chain reaction, immunofluorescence, and apoptosis assays. TLR3-/- and TLR3+/+ mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic/polycytidylic acid (Poly[I:C]). MEASUREMENTS AND MAIN RESULTS: TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3-/- mice exhibited more severe pulmonary hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA receptors in endothelial cells. This was associated with greater susceptibility to apoptosis, a known driver of pulmonary vascular remodeling. Poly(I:C) increased TLR3 expression via IL-10 in rat endothelial cells. In vivo, high-dose Poly(I:C) reduced pulmonary hypertension in both rat models in proof-of-principle experiments. In addition, Poly(I:C) also reduced right ventricular failure in established pulmonary hypertension. CONCLUSIONS: Our work identifies a novel role for TLR3 in PAH based on the findings that reduced expression of TLR3 contributes to endothelial apoptosis and pulmonary vascular remodeling.

Host Tumor Suppressor p18INK4c Functions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis.

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

K-homology splicing regulatory protein (KSRP) promotes post-transcriptional destabilization of Spry4 transcripts in non-small cell lung cancer.

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3'-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis indicated that elevated KSRP expression was correlated with poor overall survival of lung cancer patients. Furthermore, cigarette smoke, a leading risk factor for lung cancer, was also identified to be an important contributor to increased KSRP expression. Remarkably, silencing of KSRP decreased cell proliferation, reversed anchorage-independent growth, and reduced migration/invasion, suggesting an oncogenic role for KSRP in lung cancer. Finally, we provide mechanistic evidence that KSRP promotes the down-regulation of Spry4 by a previously unidentified mechanism, i.e. post-transcriptional mRNA regulation.

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis.

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20-2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

IL-32 promotes angiogenesis.

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVß3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-ß levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVß3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVß3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Relationship between gene expression and lung function in Idiopathic Interstitial Pneumonias.

BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.

Glutaredoxin-1 attenuates S-glutathionylation of the death receptor fas and decreases resolution of Pseudomonas aeruginosa pneumonia.

RATIONALE: The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection. OBJECTIVES: Fas ligand (FasL)-induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection. METHODS: Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1(-/-)), Glrx1(-/-) mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa. MEASUREMENTS AND MAIN RESULTS: Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1(-/-) cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1(-/-) mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals. CONCLUSIONS: These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.

Nuclear factor κB inhibition reduces lung vascular lumen obliteration in severe pulmonary hypertension in rats.

NF-κB and IL-6, a NF-κB downstream mediator, play a central role in the inflammatory response of tissues. We aimed to determine the role of the classical NF-κB pathway in severe pulmonary arterial hypertension (PAH) induced by SU5416 and chronic hypoxia (SuHx) in rats. Tissue samples from patients with idiopathic PAH (iPAH) and control subjects were investigated. SuHx rats were treated from Days 1 to 3, 1 to 21, and 29 to 42 with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and/or from Days 1 to 21 with anti-IL-6 antibody. Nuclear staining for NF-κB, an indicator of the activation of the classical NF-κB pathway, was detected in pulmonary arterial lesions of patients with iPAH and SuHx rats. NF-κB inhibition with PDTC prevented and reduced pulmonary arterial obliteration without reducing muscularization. However, the elevated lung levels of IL-6 were not reduced in PDTC-treated SuHx animals. PDTC treatment prevented or reduced apoptosis of pulmonary artery wall cells and pulmonary arterial obliteration. IL-6 inhibition had only a partial effect on apoptosis and obliteration. Pulmonary arterial media wall thickness was not affected by any of these treatments. Preventive and therapeutic PDTC treatment promoted immune regulation by increasing the number of perivascular CD4(+) T cells, in particular regulatory T cells (early treatment), and by reducing the number of perivascular CD8(+) T lymphocytes and CD45RA(+) B lymphocytes. Therapeutic PDTC treatment further preserved right ventricular function in SuHx animals. Inhibition of NF-κB may represent a therapeutic option for pulmonary arterial obliteration via reduced vessel wall cell apoptosis and improved regulation of the immune system.

Endothelial cell adhesion molecule CD146: implications for its role in the pathogenesis of COPD.

CD146 is an adhesion molecule localized at endothelial cell junctions and facilitates cell-cell interactions. The circulating soluble form (sCD146) lacks both the intracellular and the transmembrane domains. In this study we show that CD146 expression was significantly decreased in the lung tissue of smokers with chronic obstructive pulmonary disease (COPD) and also in rats exposed to second-hand smoke (SHS). Concurrently, levels of sCD146 were increased in both the plasma and bronchoalveolar lavage fluid (BALF) of COPD patients as well as in BALF from rats exposed to SHS. Decreased or abolished CD146 protein expression in rat pulmonary micro- and macrovascular endothelial cells was found after treatment with cigarette smoke extract (CSE), proinflammatory cytokine interleukin 18 (IL-18) or after silencing CD146 expression with siRNA. The decrease in CD146 protein was accompanied by increased endothelial monolayer permeability and enhanced macrophage infiltration in vitro. In CD146 knockout (KO) mice, distinct perivascular oedema was seen and increased numbers of inflammatory cells, along with increased protein levels in BALF. Increased sCD146 was found in BALF and plasma from patients with COPD. The circulating plasma levels of sCD146 correlated positively with the presence of anti-endothelial cell antibodies (AECAs). sCD146 in combination with AECAs may be useful markers for early detection of COPD. Our study indicates that loss of CD146 function damages pulmonary endothelial integrity. This damage may represent part of the pathophysiological processes that are involved in the basic aetiology of COPD/emphysema.