Antiviral CD8+ T-cell immune responses are impaired by cigarette smoke and in COPD.

BACKGROUND: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells. METHODS: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology. RESULTS: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics. CONCLUSION: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

Prospective evaluation of immunological, molecular-genetic, image-based and microbial analyses to characterize tumor response and control in patients with unresectable stage III NSCLC treated with concurrent chemoradiotherapy followed by consolidation therapy with durvalumab (PRECISION): protocol for a prospective longitudinal biomarker study.

Background: Concurrent platinum-based chemoradiotherapy (CRT) followed by durvalumab maintenance treatment represents the new standard of care in unresectable stage III non-small cell lung cancer (NSCLC). In this prospective hypothesis-generating single-center study, we aim to identify a framework of prognostic and predictive biomarkers by longitudinal characterization of tumor- and patient (host)-related parameters over all phases of multimodal treatment. Methods: This study will enroll 40 patients (≥18 years, Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2, with a diagnosis of PD-L1 positive (≥1%), inoperable stage III NSCLC) with an indication for CRT followed by maintenance treatment with durvalumab according to European Medicines Agency (EMA) approval. Comprehensive analysis will include peripheral blood cellular and humoral immunophenotyping and circulating tumor DNA as well as gut/saliva microbiota analyses. Additional morphological analysis with 18F-FDG-PET/computed tomography (CT) before, 6 weeks, 6 and 12 months after the end of CRT is included. Statistical analysis using multiple testing will be used to examine the impact of different parameters on progression-free survival (PFS) and overall survival (OS) as well as tumor response and response duration. Discussion: This protocol describes the methodology of a comprehensive biomarker study in order to identify a framework of prognostic and predictive markers for unresectable stage III NSCLC in a real-world setting. Trial Registration: ClinicalTrials.gov identifier (NCT05027165), data registered on August 2021.

Combining HDAC and MEK Inhibitors with Radiation against Glioblastoma-Derived Spheres.

Glioblastoma stem-like cells (GSLCs) in glioblastoma limit effective treatment and promote therapeutic resistance and tumor recurrence. Using a combined radiation and drug-screening platform, we tested the combination of a histone deacetylase inhibitor (HDACi) and MAPK/ERK kinase inhibitor (MEKi) with radiation to predict the efficacy against GSLCs. To mimic a stem-like phenotype, glioblastoma-derived spheres were used and treated with a combination of HDACi (MS-275) and MEKi (TAK-733 or trametinib) with 4 Gy irradiation. The sphere-forming ability after the combined radiochemotherapy was investigated using a sphere formation assay, while the expression levels of the GSLC markers (CD44, Nestin and SOX2) after treatment were analyzed using Western blotting and flow cytometry. The combined radiochemotherapy treatment inhibited the sphere formation in both glioblastoma-derived spheres, decreased the expression of the GSLC markers in a cell-line dependent manner and increased the dead cell population. Finally, we showed that the combined treatment with radiation was more effective at reducing the GSLC markers compared to the standard treatment of temozolomide and radiation. These results suggest that combining HDAC and MEK inhibition with radiation may offer a new strategy to improve the treatment of glioblastoma.

Gender specific airway gene expression in COPD sub-phenotypes supports a role of mitochondria and of different types of leukocytes.

Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term "response to bacterium" was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term "response to stress" was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females.

Interleukin-10 counteracts T-helper type 1 responses in B-cell lymphoma and is a target for tumor immunotherapy.

To establish strategies for immunotherapy of B-cell lymphoma, it is mandatory to gain deeper insights into the mechanisms of tumor immune escape. In a mouse model of endogenously arising lymphoma, we investigated the impact of IL-10 on the regulation of antitumor responses. Despite progressive functional impairment of NK cells and lack of IFN-γ in the tumor milieu, we found an augmented fraction of T helper type 1 (Th1) cells, which continued to express IFN-γ but also upregulated IL-10 during disease development. Using a lymphoma microenvironment in vitro, we showed that Th1 cells were converted to Foxp3-negative T regulatory type 1 (Tr1) cells, which coexpressed IFN-γ and IL-10 and upregulated PD-1. This differentiation required pre-existing IL-10, which was primarily provided by malignant B cells and dendritic cells. IFN-γ only declined in cells with the uppermost PD-1 levels. Importantly, antibody-mediated IL-10 ablation in vivo improved effector cell functions and significantly suppressed tumor development. While the contribution of IL-10 to cancer immune escape has been controversially discussed in the past, we show that IL-10 suppresses ongoing, potentially protective immune responses in lymphoma and might be a target for immunotherapy.

Subsets of CD1c+ DCs: Dendritic Cell Versus Monocyte Lineage.

Currently three bona fide dendritic cell (DC) types are distinguished in human blood. Herein we focus on type 2 DCs (DC2s) and compare the three defining markers CD1c, CD172, and CD301. When using CD1c to define DC2s, a CD14+ and a CD14- subset can be detected. The CD14+ subset shares features with monocytes, and this includes substantially higher expression levels for CD64, CD115, CD163, and S100A8/9. We review the current knowledge of these CD1c+CD14+ cells as compared to the CD1c+CD14- cells with respect to phenotype, function, transcriptomics, and ontogeny. Here, we discuss informative mutations, which suggest that two populations have different developmental requirements. In addition, we cover subsets of CD11c+CD8- DC2s in the mouse, where CLEC12A+ESAMlow cells, as compared to the CLEC12A-ESAMhigh subset, also express higher levels of monocyte-associated markers CD14, CD3, and CD115. Finally, we summarize, for both man and mouse, the data on lower antigen presentation and higher cytokine production in the monocyte-marker expressing DC2 subset, which demonstrate that the DC2 subsets are also functionally distinct.

Epigenetics in non-classical monocytes support their pro-inflammatory gene expression.

Non-classical human monocytes are characterized by high-level expression of cytokines like TNF, but the mechanisms involved are elusive. We have identified miRNAs and CpG-methylation sites that are unique to non-classical monocytes, defined via CD14 and CD16 expression levels. For down-regulated miRNAs that are linked to up-regulated mRNAs the dominant gene ontology term was intracellular signal transduction. This included down-regulated miRNA-20a-5p and miRNA-106b-5p, which both are linked to increased mRNA for the TRIM8 signaling molecule. Methylation analysis revealed 16 hypo-methylated CpG sites upstream of 14 differentially increased mRNAs including 2 sites upstream of TRIM8. Consistent with a positive role in signal transduction, high TRIM8 levels went along with high basal TNF mRNA levels in non-classical monocytes. Since cytokine expression levels in monocytes strongly increase after stimulation with toll-like-receptor ligands, we have analyzed non-classical monocytes (defined via slan expression) after stimulation with lipopolysaccharide (LPS). LPS-stimulated cells continued to have low miRNA-20a and miRNA-106b and high TRIM8 mRNA levels and they showed a 10-fold increase in TNF mRNA. These data suggest that decreased miRNAs and CpG hypo-methylation is linked to enhanced expression of TRIM8 and that this can contribute to the increased TNF levels in non-classical human monocytes.

6-Sulfo LacNAc (Slan) as a Marker for Non-classical Monocytes.

Monocytes are subdivided into three subsets, which have different phenotypic and functional characteristics and different roles in inflammation and malignancy. When in man CD14 and CD16 monoclonal antibodies are used to define these subsets, then the distinction of non-classical CD14low and intermediate CD14high monocytes requires setting a gate in what is a gradually changing level of CD14 expression. In the search for an additional marker to better dissect the two subsets we have explored the marker 6-sulfo LacNAc (slan). Slan is a carbohydrate residue originally described to be expressed on the cell surface of a type of dendritic cell in human blood. We elaborate herein that the features of slan+ cells are congruent with the features of CD16+ non-classical monocytes and that slan is a candidate marker for definition of non-classical monocytes. The use of this marker may help in studying the role of non-classical monocytes in health and in diagnosis and monitoring of disease.

Increased Extracellular Vesicles Mediate WNT5A Signaling in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-ß in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome.

BACKGROUND: Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited. METHODS: Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons. RESULTS: We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype. CONCLUSION: Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.

Emphysema- and airway-dominant COPD phenotypes defined by standardised quantitative computed tomography.

EvA (Emphysema versus Airway disease) is a multicentre project to study mechanisms and identify biomarkers of emphysema and airway disease in chronic obstructive pulmonary disease (COPD). The objective of this study was to delineate objectively imaging-based emphysema-dominant and airway disease-dominant phenotypes using quantitative computed tomography (QCT) indices, standardised with a novel phantom-based approach.441 subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1-3) were assessed in terms of clinical and physiological measurements, laboratory testing and standardised QCT indices of emphysema and airway wall geometry.QCT indices were influenced by scanner non-conformity, but standardisation significantly reduced variability (p<0.001) and led to more robust phenotypes. Four imaging-derived phenotypes were identified, reflecting "emphysema-dominant", "airway disease-dominant", "mixed" disease and "mild" disease. The emphysema-dominant group had significantly higher lung volumes, lower gas transfer coefficient, lower oxygen (PO2 ) and carbon dioxide (PCO2 ) tensions, higher haemoglobin and higher blood leukocyte numbers than the airway disease-dominant group.The utility of QCT for phenotyping in the setting of an international multicentre study is improved by standardisation. QCT indices of emphysema and airway disease can delineate within a population of patients with COPD, phenotypic groups that have typical clinical features known to be associated with emphysema-dominant and airway-dominant disease.

slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation.

Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.

Decreased expression of HLA-DQ and HLA-DR on cells of the monocytic lineage in cystic fibrosis.

UNLABELLED: We studied HLA class II molecules on blood monocyte subsets, blood dendritic cells, sputum macrophages, and monocyte-derived macrophages at the protein (flow cytometry) and mRNA level (RT-PCR) in adult patients with cystic fibrosis (CF) and healthy control subjects as putative contributors to the CF phenotype. In healthy donors, we found a high average HLA-DQ expression of 4.35 mean specific fluorescence intensity units (ΔMnI) on classical blood monocytes. In F508del homozygous CF patients, the average ΔMnI was low (1.80). Patients were divided into two groups, in which 14 of these patients had HLA-DQ expression above 2 ΔMnI (average 3.25 ΔMnI, CF-DQ(group1)) and 36 below (average 1.24 ΔMnI, CF-DQ(group2)). Also, the CD16-positive monocyte subset and blood dendritic cells showed much lower levels of HLA-DQ for the CF-DQ(group2) patients compared with healthy controls. In macrophages from sputum and derived from monocytes, in vitro HLA-DQ expression was dramatically decreased to background levels in CF-DQ(group2). MHC class II transcripts were reduced in CF with a sevenfold decrease in HLA-DQß1 for CF-DQ(group2) patients. Higher levels of the inflammation marker CRP were associated with low HLA-DQ protein expression, and in vitro treatment with the inflammatory molecule lipopolysaccharide reduced HLA-DQ expression. Interferon γ (IFNγ) could overcome this effect in healthy donor cells while, in CF, the IFNγ-induced activation was impaired. Our data demonstrate a pronounced reduction of HLA-DQ expression in CF, which is associated with inflammation and a reduced response to IFNγ. KEY MESSAGE: • CF patients show a reduced expression of MHCII molecules in monocytes and macrophages. • HLA-DQ and HLA-DR transcript levels are also reduced in CF patients. • CF patient C-reactive protein levels correlate with low HLA-DQ expression. • Reduced expression of MHC class II molecules appears to be linked to inflammation. • CF patients exhibit an impaired response to IFNgamma.

Toward a refined definition of monocyte subsets.

In a nomenclature proposal published in 2010 monocytes were subdivided into classical and non-classical cells and in addition an intermediate monocyte subset was proposed. Over the last couple of years many studies have analyzed these intermediate cells, their characteristics have been described, and their expansion has been documented in many clinical settings. While these cells appear to be in transition from classical to non-classical monocytes and hence may not form a distinct cell population in a strict sense, their separate analysis and enumeration is warranted in health and disease.

A defect of CD16-positive monocytes can occur without disease.

The CD16-positive monocytes have been first described in 1988 but to date no selective defect in the number of these cells in blood has been reported. We now describe a family in which three of four siblings lack both CD16-positive monocyte subsets, i.e. the nonclassical and the intermediate monocytes. All three had CD16-positive monocytes of 2 cells/µl or less as compared to 52±18 cells/µl in healthy controls. The index case was affected by recurrent pleural effusion and infections and had evidence of an auto-inflammatory condition but no mutation of any of the relevant candidate genes. The other two siblings without CD16-positive monocytes were apparently healthy. There was no defect in serum M-CSF levels and no mutation in the M-CSF and M-CSFR genes. The data indicate that the absence of CD16-positive monocytes in blood does not lead to disease.

Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man.

Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 µg LPS to the airways and 5 µg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 µg LPS targeted to the alveoli caused significantly stronger effects compared to 5 µg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.

Transcript profiling of CD16-positive monocytes reveals a unique molecular fingerprint.

CD16-positive (CD14(++) CD16(+) and CD14(+) CD16(++) ) monocytes have unique features with respect to phenotype and function. We have used transcriptional profiling for comparison of CD16-positive monocytes and classical monocytes. We show herein that 187 genes are greater than fivefold differentially expressed, including 90 genes relevant to immune response and inflammation. Hierarchical clustering of data for monocyte subsets and CD1c(+) myeloid blood dendritic cells (DCs) demonstrate that CD16-positive cells are more closely related to classical monocytes than to DCs. Reverse transcriptase polymerase chain reaction for ten genes with the strongest differential expression confirmed the pattern including a lower messenger RNA level for CD14, CD163, and versican in CD16-positive monocytes. The pattern was similar for CD16-positive monocytes at rest and after exercise mobilization from the marginal pool. By contrast, alveolar macrophages, small sputum macrophages, breast milk macrophages, and synovial macrophages all showed a different pattern. When monocyte-derived macrophages (MDMs) were generated from CD16-positive monocytes by culture with macrophage colony-stimulating factor in vitro, then the MDMs maintained properties of their progeny with lower expression of CD14, CD163, and versican compared with CD14(++) CD16(-) MDMs. Furthermore, CD16-positive MDMs showed a higher phagocytosis for opsonized Escherichia coli. The data demonstrate that CD16-positive monocytes form a distinct type of cell, which gives rise to a distinct macrophage phenotype.

All-trans retinoic acid up-regulates Prostaglandin-E Synthase expression in human macrophages.

All-trans retinoic acid (ATRA) is a potent retinoid, which has been used successfully in different clinical settings as a potential drug to treat COPD and emphysema. In the present study, we analyzed genes modulated by ATRA by performing mRNA expression array analysis on alveolar macrophages after treatment with ATRA. Here we observed a 375-fold up-regulation of Prostaglandin-E Synthase (microsomal PGES-1, NM_004878 PTGES) which mediates the conversion of prostaglandin H(2) (PGH(2)) to Prostaglandin E(2) (PGE(2)). We furthermore studied the expression of PTGES after treatment with ATRA in human monocyte-derived macrophages (MDMs) and bronchoalveolar lavage (BAL) cells. ATRA up-regulated PTGES mRNA expression in MDMs generated with M-CSF by 2500-fold whereas in M-CSF+IL-13 macrophages the up-regulation was only 20-fold. Similarly, ATRA up-regulated PTGES mRNA expression by factor 1524 in BAL cells. The up-regulation of PTGES mRNA expression by ATRA is both time and dose dependent. IL-13 suppressed the ATRA induced PTGES expression at both mRNA and protein level in MDM and BAL cells. We also observed that LPS acts synergistically with ATRA in MDMs and strongly induces PTGES expression. ATRA had little impact on cyclooxygenase-1 and -2 (COX-1 and -2) expression as compared to PTGES expression under the same experimental conditions. Furthermore, we observed an induction of PGE(2) levels by ATRA in BAL cells. These data indicate that ATRA is a potent inducer of PTGES expression in human macrophages but not in alternatively activated macrophages and suggest that the eicosanoid pathway is important for ATRA action in macrophages.

Chemokine expression by small sputum macrophages in COPD.

Small sputum macrophages represent highly active cells that increase in the airways of patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It has been reported often that levels of cytokines, chemokines and pro-teases are increased in sputum supernatants of these patients. In COPD, the small sputum macrophages may contribute to these supernatant proteins and recruit additional cells via specific chemokine expression patterns. We therefore investigated the expression profile of chemokines in sputum macrophages obtained from COPD patients in comparison to cells from healthy donors and cells isolated after inhalation of lipopolysaccharide (LPS). We used the minimally invasive procedure of sputum induction and have purified macrophages with the RosetteSep technology. Using macrophage purification and flow cytometry we show that in COPD small sputum macrophages account for 85.9% ± 8.3% compared with 12.9% ± 7.1% of total macrophages in control donors. When looking at chemokine expression we found, for the small macrophages in COPD, increased transcript and protein levels for CCL2, CCL7, CCL13 and CCL22 with a more than 100-fold increase for CCL13 mRNA (P < 0.001). Looking at active smokers without COPD, there is a substantial increase of small macrophages to 60% ± 15% and, here, chemokine expression is increased as well. In a model of airway inflammation healthy volunteers inhaled 20 µg of lipopolysaccharide (LPS), which resulted in an increase of small sputum macrophages from 18% ± 19% to 64% ± 25%. The pattern of chemokine expression was, however, different with an upregulation for CCL2 and CCL7, while CCL13 was downregulated three-fold in the LPS-induced small macrophages. These data demonstrate that sputum macrophages in COPD show induction of a specific set of CCL chemokines, which is distinct from what can be induced by LPS.