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Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. It is unclear how monocytes contribute to fibrosis in IPF. Here, we show that levels of circulating monocytes correlated directly with the extent of fibrosis in IPF lungs, as measured by CT imaging. IPF monocytes were phenotypically distinct, displayed increased expression of CD64 (FcgR1), a type 1 IFN gene expression signature by RNA sequencing of monocytes, and an amplified type 1 IFN response ex vivo. CD64 levels correlated with levels of IFN-stimulated genes in monocytes. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytes with impaired differentiation to macrophages, and increased numbers of monocytes in lung tissue. Interrogation of single cell transcriptomic data from human IPF lung explants revealed a increased proportion of monocytes and 9transitional macrophages9 with high CCL-2 expression; and reduced mature macrophages. The transitional macrophages in IPFs also expressed higher CD64 and were enriched for type I IFN gene sets. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of chronic lung fibrosis. It provides a clear biological rationale for targeting monocytes therapeutically in IPF.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of lung fibrosis. It is progressive, and has an extremely poor outcome and limited treatment options. The disease exclusively affects the lungs, and thus less attention has been focused on blood-borne immune cells. which could be a more effective therapeutic target than lung-based cells. Here, we questioned if circulating monocytes, which has been shown to be increased in IPF, bore abnormalities that might contribute to its pathogenesis. We found that levels of circulating monocytes correlated directly with the extent of fibrosis in the lungs, and increased further during acute clinical deterioration. Monocytes in IPF were phenotypically distinct, displaying increased expression of CD64, a type 1 IFN gene expression signature and a greater magnitude of type 1 IFN response when stimulated. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytesex vivo, and increased numbers of monocytes in lung tissue. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of an aberrant repair response and fibrosis. It provides a rationale for targeting monocytes and identifies monocytic CD64 as a potential specific therapeutic target for IPF.

Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Oxidative stress from cigarette smoke and chronic inflammation is likely to induce this corticosteroid insensitivity. Quercetin is a polyphenol that has been reported to be an active oxygen scavenger as well as a functional adenosine monophosphate-activated protein kinase (AMPK) activator. The aim of this study was to investigate the effect of quercetin on corticosteroid responsiveness in COPD cells. Corticosteroid sensitivity was examined in human monocytic U937 cells exposed to cigarette smoke extract (CSE) and peripheral blood mononuclear cells (PBMC) collected frompatients with COPD. Corticosteroid sensitivity was determined as the dexamethasone concentration causing 40% inhibition of tumor necrosis factor alpha-induced CXCL8 production (Dex-IC40) in the presence orabsence of quercetin. In U937 cells, treatment with quercetin activated AMPK and induced expression of nuclear factor erythroid 2-related factor 2, and consequently reversed CSE-induced corticosteroid insensitivity. PBMC frompatients with COPD showed corticosteroid insensitivity comparedwith those from healthy volunteers, and treatment with quercetin restored corticosteroid sensitivity. In conclusion, quercetin restores corticosteroid sensitivity, and has the potential to be a novel treatment in combination with corticosteroids in COPD.

Activation of the mTOR signaling pathway by L -leucine in 5q- syndrome and other RPS14-deficient erythroblasts

Introduction and background. The protein deacetylase sirtuin-1 (SIRT1) is and anti-ageing molecule that is decreased in the lung from patients with chronic obstructive pulmonary disease (COPD). Recently, SIRT1 has been detected in the serum and is increased in the asthmatic patients. Aims and objectives. To determine serum SIRT1 levels in the patients with COPD as a potential disease biomarker. Methods. Serum SIRT1 was measured by Western blotting, and relative ratio of band density in samples against that of positive control were calculated. Results. Several SIRT1 bands with differing molecular weights, including a 75kDa truncated form and 120kDa (actual size) were observed by Western blotting. The 120kDa serum SIRT1 was significantly decreased in the patients with COPD compared to the subjects without COPD (SIRT1 ratio in healthy: 0.90±0.34, vs COPD: 0.68±0.24; p=0.014), and positively correlated with the lung function (FEV 1 /FVC; r=0.31; p=0.020) and severity of airflow obstruction represented by FEV 1 % predicted (r=0.29; p=0.029). The 120kDa serum SIRT1 also showed; 1) positive correlation with BMI (r=0.36; p=0.0078) and diffusing capacity of the lung per unit volume (KCO%; r=0.32; p=0.025); 2) the tendency to decrease in the presence of lung emphysematous change (without emphysema: 0.92±0.37 vs with emphysema: 0.71±0.24; p=0.026) and clinical history of frequent COPD exacerbations (infrequent: 0.76±0.20 vs frequent: 0.56±0.26; p=0.027). No reduction was seen in 75KDa SIRT1. Conclusions. Serum SIRT1 was decreased in the patients with COPD, and might be a potential biomarkers of COPD and particularly accelerated ageing.

Introduction and background: The phosphatidylinositol-3-kinase (PI3K) pathway is activated in chronic obstructive pulmonary disease (COPD), but the mechanisms that regulate this pathway are yet to be elucidated. Aims and objectives: We investigated the expression and role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), the major negative regulator of the PI3K pathway in COPD. Methods: The protein levels and immunopurified PTEN activities were measured in peripheral lung from patients with COPD and compared to controls. Airways epithelial BEAS-2B cells were incubated with cigarette smoke extract (CSE) in the presence or absence of L-buthionine-sulfoximine (BSO), and the effect of PTEN knock–down by RNA interference was also examined. Results: PTEN protein was significantly decreased in peripheral lung of patients with COPD compared to the subjects without COPD (PTEN/β-actin ratio in healthy: 1.30±0.62 vs COPD: 0.71±0.27; p 1 %predicted; r=0.41; p=0.014), although no difference was observed in PTEN activity. Conversely, phosphorylated Akt, as a marker of PI3K activation, showed a negative correlation with PTEN protein levels (r=-0.43; p=0.0085). In BEAS-2B cells, CSE decreased PTEN protein, mainly through the suppression of the mRNA transcription. PTEN knock-down potentiated Akt phosphorylation and enhanced production of pro-inflammatory cytokines, such as interleukin-6, and CXCL8. Conclusions: Oxidative stress reduces PTEN protein levels, which may result in increased PI3K signalling and amplification of inflammation in COPD.

Neutrophilic inflammation in acute exacerbations of asthma tends to be resistant to treatment with glucocorticoids. This may be related to decreased activity and expression of histone deacetylase-2 (HDAC2), which down-regulates expression of proinflammatory genes via recruitment to the glucocorticoid receptor complex. We assessed airway inflammation and response to steroid treatment in a novel mouse model of an acute exacerbation of chronic asthma. Systemically sensitized mice received low-level challenge with aerosolized ovalbumin for 4 weeks, followed by a single moderate-level challenge to induce enhanced inflammation in distal airways. We assessed the effects of pre-treatment with dexamethasone on the accumulation of inflammatory cells in the airways, airway responsiveness to methacholine, expression and enzymatic activity of nuclear proteins including histone acetyl transferase (HAT) and HDAC2, and levels of transcripts for neutrophil chemoattractant and survival cytokines. Dexamethasone suppressed inflammation associated with eosinophil and T-lymphocyte recruitment, but did not prevent neutrophil accumulation or development of airway hyperresponsiveness. Increased activity of HAT was suppressed by steroid treatment, but the marked diminution of HDAC2 activity and increased activity of nuclear factor-kappaB were not reversed. Correspondingly, elevated expression of mRNA for TNF-alpha, granulocyte-macrophage colony-stimulating factor, IL-8, and p21(waf) were also not suppressed by dexamethasone. Levels of lipid peroxidation and protein nitration products were elevated in the acute exacerbation model. We conclude that impaired nuclear recruitment of HDAC2 could be an important mechanism of steroid resistance of the neutrophilic inflammation in exacerbations of asthma. Oxidative stress may contribute to decreased HDAC2 activity.

Abstract 171 The 5q- syndrome is the most distinct of all myelodysplastic syndromes with a clear genotype/phenotype relationship. Haploinsufficiency of the ribosomal protein gene RPS14 underlies the erythroid defect found in the 5q- syndrome. The 5q- syndrome is a disorder of aberrant ribosome biogenesis and we have recently demonstrated that erythrocytes obtained from patients with the 5q- syndrome show impaired translation. This defect in translation may represent a potential therapeutic target in the 5q- syndrome and other ribosomopathies, and there are some indications that the use of the translation enhancer L-leucine may have some efficacy. L-leucine is a branched chain amino acid that has been shown to improve haemoglobin levels and transfusion independence in patients with the ribosomopathy Diamond Blackfan Anemia (DBA). Moreover, the treatment of zebrafish models of DBA and the 5q- syndrome with L-leucine has recently been shown by others to result in partial reversal of the anemia. To model the RPS14 haploinsufficiency observed in the 5q- syndrome, we used lentivirally delivered shRNA sequences to reduce the expression of RPS14 in human bone marrow CD34+ cells from healthy controls. We have recently shown that treatment of cultured human erythroid cells derived from CD34+ cells of healthy controls with RPS14 knockdown and cultured erythroid cells derived from the CD34+ cells of patients with the 5q- syndrome with L-leucine results in an increase in cell proliferation, erythroid differentiation and mRNA translation. There is evidence to suggest that L-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls many cellular processes including cell growth and mRNA translation. In order to investigate the mechanism of action of L-leucine, we have studied the phosphorylation levels of S6K1 and 4EBP1, the key downstream targets of mTORC1 (mTOR Complex 1), by sandwich ELISA, Human Phospho-kinase Antibody Array and flow cytometry in RPS14-deficient human erythroblasts. We have shown a significant increase in the levels of phospho-S6K1 and phospho-4EBP1 following L-leucine treatment of cultured erythroid cells derived from CD34+ cells of healthy controls with RPS14 knockdown. The effects of L-leucine on phospho-S6K1 and phospho-4EBP1 were abrogated by rapamycin (an mTOR inhibitor), suggesting that L-leucine has a specific action on the mTOR signaling pathway. Consistent to the results observed in the RPS14 knockdown model, treatment with L-leucine also increased the level of phospho-S6K1 in cultured erythroid cells derived from the bone marrow cells of 5q- MDS patients. These data suggest that L-leucine activates the mTOR pathway in RPS14-deficient human erythroblasts. Our studies support the evaluation of L-leucine as a potential therapeutic agent in the treatment of the 5q- syndrome, and provide evidence on its mode of action through activation of the mTOR signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Patients with the 5q- syndrome and Diamond-Blackfan anemia (DBA) suffer from a severe macrocytic anemia. The 5q- syndrome and DBA are disorders of aberrant ribosome biogenesis (ribosomopathies) and haploinsufficiency of the ribosomal protein genes RPS14 and RPS19, respectively, underlies the anemia found in these disorders. Erythroblasts obtained from patients with the 5q- syndrome and DBA show impaired mRNA translation and this defect in translation may represent a potential therapeutic target in these ribosomopathies. There are some indications that the amino acid l-leucine, a translation enhancer, may have some efficacy in this group of disorders. Recent studies have shown that l-leucine treatment of zebrafish and murine models of the 5q- syndrome and DBA results in a marked improvement in the anemia. l-leucine treatment of RPS14-deficient and RPS19-deficient erythroblasts and erythroblasts from patients with the 5q- syndrome has been shown to result in an increase in cell proliferation, erythroid differentiation and mRNA translation in culture. l-leucine has been shown to improve hemoglobin levels and transfusion independence in a patient with DBA. l-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls cell growth and mRNA translation. There is evidence to suggest that the promotion of translation via the mTOR pathway by l-leucine is the mechanism that underlies the enhanced erythroid progenitor cell growth and differentiation observed in animal and cellular models of the 5q- syndrome and DBA treated with this amino acid. These data support the rationale for clinical trials of l-leucine as a therapeutic agent for the 5q- syndrome and DBA.

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.

Histone deacetylases (HDACs) are key molecules involved in epigenetic regulation of gene expression. We have previously demonstrated that oxidative stress caused a reduction in HDAC2, resulting in amplified inflammation and reduced corticosteroid responsiveness. Here we showed nitrative/oxidative stress reduced HDAC2 expression via nitration of distinct tyrosine residues. Peroxynitrite, hydrogen peroxide and cigarette smoke-conditioned medium reduced HDAC2 expression in A549 epithelial cells in vitro. This reduction was due to increased proteasomal degradation following ubiquitination rather than reduction of mRNA expression or stability. HDAC2 was nitrated under nitrative/oxidative stress and in the peripheral lung tissues of smokers and patients with chronic obstructive pulmonary disease. Mutagenesis studies replacing tyrosine (Y) residues with alanine revealed that Y253 is at least partly responsible for the proteasomal degradation of HDAC2 under nitrative stress. Thus, nitration of distinct tyrosine residues modifies both the expression and activity of HDAC2, having an impact on epigenetic regulation.