Increased ER Stress and Unfolded Protein Response Activation in Epithelial and Inflammatory Cells in Hypersensitivity Pneumonitis.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis.

Fibrotic hypersensitivity pneumonitis (FHP) is a fatal interstitial pulmonary disease with limited treatment options. Lung macrophages are a heterogeneous cell population that exhibit distinct subsets with divergent functions, playing pivotal roles in the progression of pulmonary fibrosis. However, the specific macrophage subpopulations and underlying mechanisms involved in the disease remain largely unexplored. In this study, a decision tree model showed that matrix metalloproteinase-14 (MMP14) had higher scores for important features in the up-regulated genes in macrophages from mice exposed to the Saccharopolyspora rectivirgula antigen (SR-Ag). Using single-cell RNA sequencing (scRNA-seq) analysis of hypersensitivity pneumonitis (HP) mice profiles, we identified MMP14high macrophage subcluster with a predominant M2 phenotype that exhibited higher activity in promoting fibroblast-to myofibroblast transition (FMT). We demonstrated that suppressing toll-like receptor 2 (TLR2) and nuclear factor kappa-B (NF-κB) could attenuate MMP14 expression and exosome secretion in macrophages stimulation with SR-Ag. The exosomes derived from MMP14-overexpressing macrophages were found to be more effective in regulating the transition of fibroblasts through exosomal MMP14. Importantly, it was observed that the transfer of MMP14-overexpressing macrophages into mice promoted lung inflammation and fibrosis induced by SR-Ag. NSC-405020 binding to the hemopexin domain (PEX) of MMP-14 ameliorated lung inflammation and fibrosis induced by SR-Ag in mice. Thus, MMP14-overexpressing macrophages may be an important mechanism contributing to the exacerbation of allergic reactions. Our results indicated that MMP14 in macrophages has the potential to be a therapeutic target for HP.

Macrophage P2Y6 Receptor Signaling Selectively Activates NFATC2 and Suppresses Allergic Lung Inflammation.

Innate immune responses to innocuous Ags can either prevent or facilitate adaptive type 2 allergic inflammation, but the mechanisms are incompletely understood. We now demonstrate that macrophage UDP-specific type 6 purinergic (P2Y6) receptors selectively activate NFATC2, a member of the NFAT family, to drive an innate IL-12/IFN-γ axis that prevents type 2 allergic inflammation. UDP priming potentiated IL-12p40 production in bone marrow-derived macrophages (BMMs) stimulated by the house dust mite Dermatophagoides farinae (Df) in a P2Y6-dependent manner. Inhibitions of phospholipase C, calcium increase, and calcineurin eliminated UDP-potentiated Df-induced IL-12p40 production. UDP specifically induced nuclear translocation of NFATC2, but not NFATC1 and NFATC3, in BMMs in a P2Y6-dependent manner. UDP-potentiated IL-12p40 production by BMMs and Df-induced IL-12p40 gene expression by alveolar macrophages were abrogated in cells from Nfatc2 knockout mice. Pulmonary transplantation of wild-type but not Nfatc2 knockout macrophages increased Df-induced IL-12 production and IFN-γ expression in P2ry6 fl/fl/Cre/+ recipient mice. Finally, Nfatc2 knockout mice showed significantly increased indices of type 2 immunopathology in response to Df challenge, similar to P2ry6 fl/fl/Cre/+ mice. Thus, macrophage P2Y6 receptor signaling selectively utilizes NFATC2 to potentiate an innate IL-12/IFN-γ axis, a potential mechanism that protects against inappropriate type 2 immune responses.

Contribution of Protein Kinase D1 on Acute Pulmonary Inflammation and Hypersensitivity Pneumonitis Induced by Saccharopolyspora rectivirgula.

Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula-induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula-induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula-induced HP.

ITK independent development of Th17 responses during hypersensitivity pneumonitis driven lung inflammation.

T helper 17 (Th17) cells develop in response to T cell receptor signals (TCR) in the presence of specific environments, and produce the inflammatory cytokine IL17A. These cells have been implicated in a number of inflammatory diseases and represent a potential target for ameliorating such diseases. The kinase ITK, a critical regulator of TCR signals, has been shown to be required for the development of Th17 cells. However, we show here that lung inflammation induced by Saccharopolyspora rectivirgula (SR) induced Hypersensitivity pneumonitis (SR-HP) results in a neutrophil independent, and ITK independent Th17 responses, although ITK signals are required for γδ T cell production of IL17A. Transcriptomic analysis of resultant ITK independent Th17 cells suggest that the SR-HP-induced extrinsic inflammatory signals may override intrinsic T cell signals downstream of ITK to rescue Th17 responses in the absence of ITK. These findings suggest that the ability to pharmaceutically target ITK to suppress Th17 responses may be dependent on the type of inflammation.

Ameliorative effects of eosinophil deficiency on immune response, endoplasmic reticulum stress, apoptosis, and autophagy in fungus-induced allergic lung inflammation.

BACKGROUND: Respiratory fungal exposure is known to be associated with various allergic pulmonary disorders. Eosinophils have been implicated in tissue homeostasis of allergic inflammation as both destructive effector cells and immune regulators. What contributions eosinophils have in Aspergillus fumigatus (Af)-induced allergic lung inflammation is worthy of investigating. METHODS: We established the Af-exposed animal asthmatic model using eosinophil-deficient mice, ∆dblGATA1 mice. Airway inflammation was assessed by histopathological examination and total cell count of bronchoalveolar lavage fluid (BALF). The protein level in BALF and lung mRNA level of type 2 cytokines IL-4, IL-5, and IL-13 were detected by ELISA and qRT-PCR. We further studied the involvement of endoplasmic reticulum (ER) stress, apoptosis, and autophagy by western blots, qRT-PCR, immunofluorescence, TUNEL, or immunohistochemistry. RNA-Seq analysis was utilized to analyze the whole transcriptome of Af-exposed ∆dblGATA1 mice. RESULTS: Hematoxylin and eosin (HE) staining and periodic acid-Schiff staining (PAS) showed that airway inflammation and mucus production were alleviated in Af-challenged ∆dblGATA1 mice compared with wild-type controls. The protein and mRNA expressions of IL-4, IL-5, and IL-13 were reduced in the BALF and lung tissues in Af-exposed ∆dblGATA1 mice. The results demonstrated that the significantly increased ER stress markers (GRP78 and CHOP) and apoptosis executioner caspase proteases (cleaved caspase-3 and cleaved caspase-7) in Af-exposed wild-type mice were all downregulated remarkably in the lungs of ∆dblGATA1 mice with Af challenge. In addition, the lung autophagy in Af-exposed ∆dblGATA1 mice was found elevated partially, manifesting as higher expression of LC3-II/LC3-I and beclin1, lower p62, and downregulated Akt/mTOR pathway compared with Af-exposed wild-type mice. Additionally, lung RNA-seq analysis of Af-exposed ∆dblGATA1 mice showed that biological processes about chemotaxis of lymphocytes, neutrophils, or eosinophils were enriched but without statistical significance. CONCLUSIONS: In summary, eosinophils play an essential role in the pathogenesis of Af-exposed allergic lung inflammation, whose deficiency may have relation to the attenuation of type 2 immune response, alleviation of ER stress and apoptosis, and increase of autophagy. These findings suggest that anti-eosinophils therapy may provide a promising direction for fungal-induced allergic pulmonary diseases.

Beneficial impact of cathelicidin on hypersensitivity pneumonitis treatment-In vivo studies.

Cathelicidin (CRAMP) is a defence peptide with a wide range of biological responses including antimicrobial, immunomodulatory and wound healing. Due to its original properties the usefulness of CRAMP in the treatment of pulmonary fibrosis was assessed in a murine model of hypersensitivity pneumonitis (HP). The studies were conducted on mouse strain C57BL/6J exposed to a saline extract of Pantoea agglomerans cells (HP inducer). Cathelicidin was administered in the form of an aerosol during and after HP development. Changes in the composition of immune cell populations (NK cells, macrophages, lymphocytes: Tc, Th, Treg, B), were monitored in lung tissue by flow cytometry. Extracellular matrix deposition (collagens, hydroxyproline), the concentration of cytokines involved in inflammatory and the fibrosis process (IFNγ, TNFα, TGFß1, IL1ß, IL4, IL5, IL10, IL12α, IL13) were examined in lung homogenates by the ELISA method. Alterations in lung tissue morphology were examined in mouse lung sections stained with haematoxylin and eosin as well as Masson trichrome dyes. The performed studies revealed that cathelicidin did not cause any negative changes in lung morphology/structure, immune cell composition or cytokines production. At the same time, CRAMP attenuated the immune reaction induced by mice chronic exposure to P. agglomerans and inhibited hydroxyproline and collagen deposition in the lung tissue of mice treated with bacteria extract. The beneficial effect of CRAMP on HP treatment was associated with restoring the balance in quantity of immune cells, cytokines production and synthesis of extracellular matrix components. The presented study suggests the usefulness of cathelicidin in preventing lung fibrosis; however, cathelicidin was not able to reverse pathological changes completely.

Identification of Autophagy-related Proteins in Lungs From Hypersensitivity Pneumonitis Patients.

Autophagy has been involved in the pathogenesis of various lung diseases. However, it is not yet known whether autophagy plays a role in hypersensitivity pneumonitis (HP). HP is an interstitial lung disease resulting from exposure to a wide variety of antigens that provoke an exaggerated immune response in susceptible individuals. The aim of this study was to explore the localization of autophagy key proteins in lungs from HP patients and controls by immunohistochemistry and analyze their expression levels by immunoblot. Macrophages and epithelial cells were strongly positive for the autophagosome biomarker LC3B (microtubule-associated protein light chain 3 beta) in HP lungs compared with controls. A similar pattern was found for the autophagy receptor p62 and the enzyme ATG4B. Unexpectedly, nuclear p62 signal was also noticed in macrophages from HP lungs. Regarding ATG5 and ATG7 localization, we observed positive staining in neutrophils, vascular smooth muscle cells, and endothelial cells. Our findings provide for the first time evidence that proteins from the autophagy machinery are highly expressed in the lungs of HP patients and describe the specific cellular and subcellular localization of LC3B, p62, ATG4B, ATG5, and ATG7 in HP lungs.

Decreased expression of transmembrane TNFR2 in lung leukocytes subpopulations of patients with non-fibrotic hypersensitivity pneumonitis compared with the fibrotic disease.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease, characterized by lung inflammation (non-fibrotic HP) that may often progresses to fibrosis (Fibrotic HP). The tumor necrosis factor (TNF) and its receptors (TNFR1 and TNFR2) can be found as soluble (sol) and transmembrane (tm) forms, playing pro-inflammatory functions but also has been related to immune regulatory functions. Bronchioalveolar lavage from fibrotic and non-fibrotic HP patients was obtained, and immune cells were characterized by flow cytometry, whereas soluble proteins were analyzed by ELISA. Compare to fibrotic HP patients, HP patients with non-fibrotic disease have accumulation of pro-inflammatory CD3+ myeloid cells, cell subpopulations that have decreased tmTNFR2 expression, and low frequency of regulatory-T cells. Whereas solTNF, solTNFR2, and IL-8 are increased. These findings suggest that the TNF pathway may explain, at least partially, the differences between both HP clinical forms. The evaluation of the TNF family molecules may help to develop new therapeutic approaches.

Evaluation of Renin and Soluble (Pro)renin Receptor in Patients with IPF. A Comparison with Hypersensitivity Pneumonitis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an unclear pathogenic mechanism. Components of the renin-angiotensin system (RAS) have a role in the pathogenesis of IPF, specifically, the aspartyl protease renin acts as a profibrotic factor in the lung. However, the concentration of the RAS components renin and soluble (pro)renin receptor (sPRR) have not been previously evaluated neither in serum nor in bronchoalveolar lavage fluid (BAL) of patients with IPF or chronic Hypersensitivity pneumonitis (cHP), a disease which may be confused with IPF. METHODS: The serum levels of renin [IPF patients (n = 70), cHP patients (n = 83), and controls (n = 26)] and sPRR [IPF (n = 28), cHP (37), and controls (n = 20)] were measured by ELISA. Renin was also quantified in BALs of IPF patients and controls by Western blot. RESULTS: We found that the levels of renin were higher in serum samples from IPF patients when compared with cHP patients and controls. Furthermore, BALs from IPF patients had more renin than BALs from controls. Unlike renin, the serum levels of sPRR were lower in IPF and cHP patients than in control individuals. CONCLUSIONS: The high levels of renin in sera and BALs of IPF patients suggest that renin might play a major role in the pathogenesis of IPF. Results from BAL confirm that renin is produced locally in the lung. Serum levels of renin could be used to differentiate IPF from cHP.

C-C Motif Chemokine Ligand 15 May Be a Useful Biomarker for Predicting the Prognosis of Patients with Chronic Hypersensitivity Pneumonitis.

BACKGROUND: Chronic hypersensitivity pneumonitis (CHP) is characterized by lymphocytic inflammation and progressive fibrosis of the lung caused by a variety of inhaled antigens. Due to the difficulty of accurately diagnosing CHP, and the poor prognosis associated with the condition, a novel clinical biomarker is urgently needed. OBJECTIVE: To investigate the usefulness of C-C motif chemokine ligand 15 (CCL15), which had been demonstrated to highly express in the lungs of CHP patients, as a clinical biomarker for CHP. METHOD: Immunohistochemical investigations were performed on lung tissue from CHP patients, and CCL15 levels in serum and bronchoalveolar lavage fluid (BALF) were measured via the enzyme-linked immunosorbent assay. RESULTS: Immunohistochemistry investigations revealed high CCL15 expression in the lungs of CHP patients. Serum CCL15 levels in CHP patients (29.1 ± 2.1 µg/mL) were significantly higher than those of idiopathic pulmonary fibrosis patients (19.7 ± 1.3 µg/mL, p = 0.01) and healthy subjects (19.5 ± 1.7 µg/mL, p = 0.003). When BALF CCL15 level was divided by BALF albumin (Alb) level (BALF CCL15/Alb), it was significantly inversely correlated with forced vital capacity (ß = -0.47, p = 0.0006), percentage of predicted carbon monoxide diffusion capacity of the lung (ß = -0.41, p = 0.0048), and BALF lymphocyte count (ß = -0.34, p = 0.01) in CHP patients. Multivariate Cox proportional hazards analysis revealed that high BALF CCL15/Alb and poor prognosis were statistically significantly independently correlated in CHP patients (HR 1.1, 95% CI 1.03-1.18, p = 0.004). CONCLUSION: The results of the current study suggest that CCL15 may be a useful prognostic biomarker for CHP. CCL15 was highly expressed in the lung tissue of CHP patients, and BALF CCL15/Alb was significantly associated with CHP prognosis.

Elevated Levels of Intelectin-1, a Pathogen-binding Lectin, in the BAL Fluid of Patients with Chronic Eosinophilic Pneumonia and Hypersensitivity Pneumonitis.

Objective Human intelectin-1 (hITLN-1) binds to galactofuranosyl residues, which are present in the microbial cell wall, but which are absent in mammalian tissues, and has been suggested to play an immunological role against microorganisms. However, the involvement of hITLN-1 in the pathogenesis of diffuse pulmonary diseases remains unknown. The aim of this study was to compare the hITLN-1 concentrations in the bronchoalveolar lavage (BAL) fluid of patients with diffuse pulmonary diseases. Methods The cell components and concentrations of hITLN-1 were analyzed in the BAL fluid of 8 patients with idiopathic chronic eosinophilic pneumonia (ICEP), 3 patients with drug-induced eosinophilic pneumonia, 4 patients with hypersensitivity pneumonitis (HP), 11 patients with sarcoidosis, 9 patients with cryptogenic organizing pneumonia, and 5 patients with idiopathic fibrosing interstitial pneumonia (fibrosing nonspecific interstitial pneumonia or usual interstitial pneumonia). Results The hITLN-1 concentrations in the BAL fluid of patients with ICEP and HP were higher than in those with other diseases. In the ICEP group, no significant difference was observed in the hITLN-1 concentrations of patients with or without a history of bronchial asthma. Conclusion The results of the present study suggest that hITLN-1 may be involved in the pathogenesis of ICEP and HP, and that an increase in the hITLN-1 concentration in the BAL fluid may represent a new biomarker for these diseases.

Increased levels of interleukin 27 in patients with early clinical stages of non-small cell lung cancer.

INTRODUCTION    Interleukin 27 (IL­27) is a cytokine secreted mostly by antigen­presenting cells. It is important for the immune polarization of T helper­1 (Th1) cells, and its role in interstitial lung diseases (ILDs) and lung cancer has been investigated. OBJECTIVES    We assessed IL­27 expression in the lower airways of patients with selected ILDs and early­stage non-small cell lung cancer (NSCLC). PATIENTS AND METHODS    IL­27 concentrations were examined by an enzyme­linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants collected from patients with pulmonary sarcoidosis (PS; n = 30), extrinsic allergic alveolitis (EAA; n = 14), idiopathic pulmonary fibrosis (IPF; n = 12), nonspecific interstitial pneumonia (NSIP; n = 14), and NSCLC stages I to IIa (n = 16) with peripheral localization, and in controls (n = 14). The major lymphocyte subsets in BAL fluid were phenotyped, and intracellular IL­27 expression was evaluated by flow cytometry.  RESULTS    IL­27 concentrations in BAL fluid supernatants were significantly increased in Th1­mediated conditions such as EAA and PS, but not in IPF or NSIP. The highest IL­27 levels (median [SEM], 16.9 [17.5] pg/ml) were reported for NCSLC, and the lowest-for controls (median [SEM], 0.4 [0.2] pg/ml). IL­27 was undetectable in corticosteroid­treated patients with PS. Both CD4+ and CD8+ lymphocytes were positive for IL­27; they were a possible local source of IL­27 because the cytokine levels were positivelysignificantly correlated with the total number of lymphocytes, including CD4+ cells. CONCLUSIONS    Our results support the Th1­linked activity of IL­27in ILDs. Early­stageNSCLC is characterizedby high IL­27expression in the lower airways. IL­27 is produced by a high percentage of CD4+ and CD8+ cells in BAL fluid, both in patients and controls.

Cathelicidin related antimicrobial peptide, laminin, Toll-like receptors and chemokines levels in experimental hypersensitivity pneumonitis in mice.

INTRODUCTION: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by unresolved inflammation and tissue repair pathologies triggered by repeated organic dust exposure. The aim of the study was to investigate changes in levels of the cathelicidin related antimicrobial peptide (CRAMP), laminin (LAM-A1), selected Toll-like receptors (TLR) and chemokines in experimental HP in mice. MATERIALS AND METHODS: Three and 18-month-old female C57BL/6J mice underwent inhalations of the saline extract of Pantoea agglomerans cells, Gram-negative bacterium common in organic dust and known for its pathogenic impact. The inhalations were repeated daily (28 days). ELISA was used for measuring in lung tissue homogenates concentration of CRAMP, LAM-A1, TLR2, TLR4, TLR8, CXCL9 (chemokine [C-X-C motif] ligand) and CXCL10. RESULTS: Levels of TLR2, TLR4 and CXCL9 were significantly higher in both young and old mice lungs already after 7 days of inhalations, while significant increase of LAM-A1 and CXCL10 was noted after 28 days, compared to untreated samples. TLR8 level was significantly augmented only in young mice. Only CRAMP level significantly declined. Significantly higher TLR8 and CXCL9 concentration in untreated samples were noted in old animals compared to young ones. CONCLUSION: Significant alterations of the examined factors levels indicate their role in HP pathogenesis.

Fibrocytes contribute to inflammation and fibrosis in chronic hypersensitivity pneumonitis through paracrine effects.

RATIONALE: Hypersensitivity pneumonitis (HP) represents a lung inflammation provoked by exposure to a variety of antigens. Chronic HP may evolve to lung fibrosis. Bone marrow-derived fibrocytes migrate to injured tissues and contribute to fibrogenesis, but their role in HP is unknown. OBJECTIVES: To assess the possible participation of fibrocytes in chronic HP. METHODS: CD45(+)/CXCR4(+)/Col-I(+) circulating fibrocytes were evaluated by flow cytometry, and the presence of fibrocytes in HP and normal lungs by confocal microscopy. The concentration of CXCL12 in plasma and bronchoalveolar lavage fluids was quantified by ELISA. The effect of fibrocytes on lung fibroblasts and T lymphocytes was examined in co-cultures. MEASUREMENTS AND MAIN RESULTS: The percentage of circulating fibrocytes was significantly increased in patients with HP compared with healthy individuals (5.3 ± 3.4% vs. 0.8 ± 0.7%; P = 0.00004). Numerous fibrocytes were found infiltrating the HP lungs near fibroblasts and lymphocytes. Plasma CXCL12 concentration was significantly increased in patients with HP (2,303.3 ± 813.7 vs. 1,385.6 ± 318.5 pg/ml; P = 0.00003), and similar results were found in bronchoalveolar lavage fluids. The chemokine was primarily expressed by epithelial cells. In co-cultures, fibrocytes induced on lung fibroblasts a significant increase in the expression of α1 type I collagen, matrix metalloprotease-1, and platelet-derived growth factor-ß. Likewise, fibrocytes induced the up-regulation of CCL2 in HP lymphocytes and fibroblasts. CONCLUSIONS: These findings demonstrate that high levels of fibrocytes are present in the peripheral blood of patients with chronic HP and that these cells infiltrate the HP lungs. Fibrocytes may participate in the pathogenesis of HP, amplifying the inflammatory and fibrotic response by paracrine signaling inducing the secretion of a variety of proinflammatory and profibrotic molecules.

Lung fibrotic tenascin-C upregulation is associated with other extracellular matrix proteins and induced by TGFß1.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lung disease of unknown aetiology and poor prognosis, characterized by altered tissue repair and fibrosis. The extracellular matrix (ECM) is a critical component in regulating cellular homeostasis and appropriate wound healing. The aim of our study was to determine the expression profile of highlighted ECM proteins in IPF lungs. METHODS: ECM gene and protein expression was analyzed by cDNA microarrays, rt-PCR, immunohistochemistry and western-blot in lungs from idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP), categorized as chronic (cHP) and subacute (saHP), and healthy lung tissue. Primary fibroblast cultures from normal subjects and fibrotic patients were studied to evaluate tenascin-C (TNC) synthesis. RESULTS: A total of 20 ECM proteins were upregulated and 6 proteins downregulated in IPF. TNC was almost undetected in normal lungs and significantly upregulated in fibrotic lungs (IPF and cHP) compared to saHP. Furthermore, it was located specifically in the fibroblastic foci areas of the fibrotic lung with a subepithelial gradient pattern. TNC levels were correlated with fibroblastic foci content in cHP lungs. Versican and fibronectin glycoproteins were associated with TNC, mainly in fibroblastic foci of fibrotic lungs. Fibroblasts from IPF patients constitutively synthesized higher levels of TNC than normal fibroblasts. TNC and α-sma was induced by TGF-ß1 in both fibrotic and normal fibroblasts. TNC treatment of normal and fibrotic fibroblasts induced a non-significant increased α-sma mRNA. CONCLUSIONS: The difference in ECM glycoprotein content in interstitial lung diseases could contribute to the development of lung fibrosis. The increase of TNC in interstitial areas of fibrotic activity could play a key role in the altered wound healing.

Exposure to low doses of formaldehyde during pregnancy suppresses the development of allergic lung inflammation in offspring.

Formaldehyde (FA) is an environmental and occupational pollutant, and its toxic effects on the immune system have been shown. Nevertheless, no data are available regarding the programming mechanisms after FA exposure and its repercussions for the immune systems of offspring. In this study, our objective was to investigate the effects of low-dose exposure of FA on pregnant rats and its repercussion for the development of allergic lung inflammation in offspring. Pregnant Wistar rats were assigned in 3 groups: P (rats exposed to FA (0.75 ppm, 1 h/day, 5 days/week, for 21 days)), C (rats exposed to vehicle of FA (distillated water)) and B (rats non-manipulated). After 30 days of age, the offspring was sensitised with ovalbumin (OVA)-alum and challenged with aerosolized OVA (1%, 15 min, 3 days). After 24 h the OVA challenge the parameters were evaluated. Our data showed that low-dose exposure to FA during pregnancy induced low birth weight and suppressed the development of allergic lung inflammation and tracheal hyperresponsiveness in offspring by mechanisms mediated by reduced anaphylactic antibodies synthesis, IL-6 and TNF-alpha secretion. Elevated levels of IL-10 were found. Any systemic alteration was detected in the exposed pregnant rats, although oxidative stress in the uterine environment was evident at the moment of the delivery based on elevated COX-1 expression and reduced cNOS and SOD-2 in the uterus. Therefore, we show the putative programming mechanisms induced by FA on the immune system for the first time and the mechanisms involved may be related to oxidative stress in the foetal microenvironment.

Stromal cells can be cultured and characterized from diagnostic bronchoalveolar fluid samples obtained from patients with various types of interstitial lung diseases.

Increased proliferation of stromal cells is a typical feature encountered in several lung diseases. The objective of this study was to evaluate the success of standardized process for culturing stromal cells from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples collected from various patients and to characterize the cultured cells. Small volumes (average 15 mL) of BAL fluid samples were collected from 98 patients who underwent bronchoscopy and BAL for diagnostic purposes. The cells were cultured in vitro and characterized by immunohistochemistry, electron microscopy, flow cytometry and differentiation tests. Cells could be cultured from 62% of samples with the success rate varying with the disease (p = 0.003). Cultures from samples of the patients with idiopathic pulmonary fibrosis, non-specific interstitial pneumonia, connective tissue disorder associated interstitial lung disease and allergic alveolitis had a higher success rate than samples derived from control lung (p < 0.001, 0.03, 0.03 and 0.044, respectively). Smokers had a higher success rate compared with non-smokers (p = 0.035). The cultured cells were fibroblasts or myofibroblasts, but shared also similarities with progenitor-type cells. The study shows that mesenchymal cells can be cultured and studied from small volumes of diagnostic BAL fluid samples from patients with several different types of lung diseases.

Variability and consistency in lung inflammatory responses to particles with a geogenic origin.

BACKGROUND AND OBJECTIVE: Particulate matter <10 µm (PM10 ) is well recognized as being an important driver of respiratory health; however, the impact of PM10 of geogenic origin on inflammatory responses in the lung is poorly understood. This study aimed to assess the lung inflammatory response to community sampled geogenic PM10 . METHODS: This was achieved by collecting earth material from two regional communities in Western Australia (Kalgoorlie-Boulder and Newman), extracting the PM10 fraction and exposing mice by intranasal instillation to these particles. The physicochemical characteristics of the particles were assessed and lung inflammatory responses were compared to control particles. The primary outcomes were cellular influx and cytokine production in the lungs of the exposed mice. RESULTS: The physical and chemical characteristics of the PM10 from Kalgoorlie and Newman differed with the latter having a higher concentration of Fe and a larger median diameter. Control particles (2.5 µm polystyrene) caused a significant influx of inflammatory cells (neutrophils) with little production of proinflammatory cytokines. In contrast, the geogenic particles induced the production of MIP-2, IL-6 and a significant influx of neutrophils. Qualitatively, the response following exposure to particles from Kalgoorlie and Newman were consistent; however, the magnitude of the response was substantially higher in the mice exposed to particles from Newman. CONCLUSIONS: The unique physicochemical characteristics of geogenic particles induced a proinflammatory response in the lung. These data suggest that particle composition should be considered when setting community standards for PM exposure, particularly in areas exposed to high geogenic particulate loads.

Angiogenic axis angiopoietin-1 and angiopoietin-2/Tie-2 in non-small cell lung cancer: a bronchoalveolar lavage and serum study.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands for the Tie-2 receptor expressed on endothelial cells, play a critical role in angiogenesis, in concert with vascular endothelial growth factor (VEGF). Angiogenesis is important for tumor growth and development and also is implicated in the pathogenesis of interstitial lung diseases. The aim of this study was to evaluate the concentration of Ang-1, Ang-2, Tie-2, interleukin-18 (IL-18), transforming growth factor beta-1 (TGF ß1), and VEGF domain in both serum and bronchoalveolar lavage fluid (BALF) of lung cancer patients before chemotherapy. We studied 45 non-small cell lung cancer (NSCLC) patients (M/F; 38/7; mean age 62 ± 4 years). The age-matched control groups consisted of 15 sarcoidosis (BBS), 15 hypersensivity pneumonitis (HP), and 15 healthy subjects. The patients with NSCLC had a significantly higher level of Ang-1 compared with the BBS and healthy subjects, and a higher level of Ang-2 compared with the healthy subjects in both serum and BALF. BALF level of IL-18 was lower in the NSCLC than that in the HP group, but higher than that in the BBS patients. Serum level of IL-18 was higher in the NSCLC than in the healthy subjects. The NSCLC group had lower VEGF in BALF than that in healthy subjects. Receiver-operating characteristics (ROC) curves were applied to find the cut-off the serum levels of Ang-1 and Ang-2 levels in BALF. We did not find any correlation between the levels of Ang-1, Ang-2, Tie-2, and the stage of tumor or treatment response (prospectively). We conclude that the angiogenic axis Ang-1 and Ang-2/Tie-2 may play an important role in lung cancer development and their concentrations may be a useful marker at the time of initial diagnosis of lung cancer.