Type I Interferon, Induced by Adenovirus or Adenoviral Vector Infection, Regulates the Cytokine Response to Lipopolysaccharide in a Macrophage Type-Specific Manner.

INTRODUCTION: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αß) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. METHODS: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-ß was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. RESULTS: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αß by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. CONCLUSION: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. METHODS: We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. RESULTS: We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. CONCLUSION: Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Genetic and geographic influence on phenotypic variation in European sarcoidosis patients.

Introduction: Sarcoidosis is a highly variable disease in terms of organ involvement, type of onset and course. Associations of genetic polymorphisms with sarcoidosis phenotypes have been observed and suggest genetic signatures. Methods: After obtaining a positive vote of the competent ethics committee we genotyped 1909 patients of the deeply phenotyped Genetic-Phenotype Relationship in Sarcoidosis (GenPhenReSa) cohort of 31 European centers in 12 countries with 116 potentially disease-relevant single-nucleotide polymorphisms (SNPs). Using a meta-analysis, we investigated the association of relevant phenotypes (acute vs. sub-acute onset, phenotypes of organ involvement, specific organ involvements, and specific symptoms) with genetic markers. Subgroups were built on the basis of geographical, clinical and hospital provision considerations. Results: In the meta-analysis of the full cohort, there was no significant genetic association with any considered phenotype after correcting for multiple testing. In the largest sub-cohort (Serbia), we confirmed the known association of acute onset with TNF and reported a new association of acute onset an HLA polymorphism. Multi-locus models with sets of three SNPs in different genes showed strong associations with the acute onset phenotype in Serbia and Lublin (Poland) demonstrating potential region-specific genetic links with clinical features, including recently described phenotypes of organ involvement. Discussion: The observed associations between genetic variants and sarcoidosis phenotypes in subgroups suggest that gene-environment-interactions may influence the clinical phenotype. In addition, we show that two different sets of genetic variants are permissive for the same phenotype of acute disease only in two geographic subcohorts pointing to interactions of genetic signatures with different local environmental factors. Our results represent an important step towards understanding the genetic architecture of sarcoidosis.

Prevention of M2 polarization and temporal limitation of differentiation in monocytes by extracellular ATP.

BACKGROUND: Elevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1ß) and Chemokine (C-C motif) ligand 18 (CCL18), respectively. RESULTS: Monocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1ß, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1ß. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1ß release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP. CONCLUSIONS: ATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.

Analysis of single nucleotide polymorphisms in chronic beryllium disease.

Sarcoidosis and chronic beryllium disease (CBD) are phenocopies, however the latter one has a clear trigger factor that is beryllium exposure. This study analyses single nucleotide polymorphisms (SNPs) in a large cohort for beryllium-exposed persons. SNPs were chosen for their relevance in sarcoidosis. Even though one of largest cohorts of beryllium-exposed persons was analysed, no statistically relevant association between any SNP and CBD could be verified. Notably, some SNPs exhibit inverse OR for beryllium sensitization and CBD with nominally statistical significance, which allows hypothesizing about pathophysiological role of genes for the disease triggering and development.

Insights into immunometabolism: A dataset correlating the 18FDG PET/CT maximum standard uptake value of the primary tumor with the CCL18 serum level in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the western world [1]. Despite multiple therapeutic and diagnostic advances, the overall survival is low and recurrence of NSCLC is a common problem with different treatment regimens. The inclusion of 18fluorine fluorodeoxyglucose (18FDG) positron emission tomography (PET) in combination with computed tomography (CT) in clinical practice was revolutionary for the staging of NSCLC [2]. 18FDG-PET/CT provides morphological, functional, and metabolic information about the tumor, which is usually highly, metabolically active. Due to the increased glucose uptake, 18FDG is actively accumulatedin the tumor tissues, resulting in an increased standardized uptake value (SUV). The tumor tissue itself consists of neoplastic cells, extracellular matrix, fibroblasts, and various immune cells. These immune cells include tumor-infiltrating lymphocytes, regulatory T cells, and macrophages. Macrophages have different activation patterns and play an essential role in inflammation and cancer. In particular, tumor-associated macrophages (TAMs) are a specialized group of alternatively activated or M2 macrophages. TAMs release several chemokines that are different from those released by classically activated macrophages found in an inflammatory environment. One of the most important chemokines released by TAMs is CC-chemokine ligand 18 (CCL18). Although CCL18 is present in healthy subjects, its levels are significantly elevated in the serum of patients with NSCLC. It correlates with overall survival and tumor stage in several malignant diseases [3,4]. A recurring problem is that increased glucose metabolism can be found in the inflammatory tissue, which can also lead to an increased SUV in 18FDG PET/CT, lowering its oncological specificity [5]. In a previous study, we demonstrated that serum CCL18 levels can be used to differentiate between patients with NSCLC and healthy subjects [3]. Hence, we investigated the correlation between serum CCL18 levels and the maximum standardized uptake value (SUVmax) of the primary tumor using 18FDG-PET/CT. We found a significant correlation between the SUVmax of the primary tumor and the serum CCL18 level. The data are important because they can be used to draw conclusions about immunometabolism. Furthermore, they can serve as basis for future prospective clinical studies.

Surveillance Bronchoscopy for the Care of Lung Transplant Recipients: A Retrospective Single Center Analysis.

INTRODUCTION: Lung transplantation is often the only treatment for end-stage lung disease. Following lung transplantation, infections and transplant rejections are major obstacles to short- and long-term success. Therefore, close monitoring for these complications is required after lung transplantation. The role of prescheduled surveillance bronchoscopies after lung transplantation is controversial. Thus, we aimed to retrospectively analyze the therapeutic implications of surveillance bronchoscopies in 110 consecutive lung transplant recipients. MATERIALS AND METHODS: Results of 400 prescheduled surveillance bronchoscopies of 110 consecutive lung transplant recipients were analyzed. Positive results (pathologic histology, microbiology, or virology) were further investigated for their effect on clinical decision making. Additionally, cellular composition of bronchoalveolar lavage (BAL) was analyzed. RESULTS: Two hundred five surveillance bronchoscopies showed pathologic findings. In 81 cases clinical treatment was changed based on the results. That is, 20% of all prescheduled bronchoscopies directly influenced clinical decision making. Furthermore, analyses of BAL indicate that increased alveolar eosinophils are associated with an increased risk of transplant rejection. CONCLUSIONS: Prescheduled surveillance bronchoscopies identify clinically unsuspected but therapeutically relevant pathologic findings in approximately 20% of cases. BAL cell composition may confer additional information, especially in cases when biopsy is not possible.

A Cluster of Beryllium Sensitization Traced to the Presence of Beryllium in Concrete Dust.

BACKGROUND: Chronic beryllium disease (CBD), a granulomatous disease with similarities to sarcoidosis, arises only in individuals exposed to beryllium. Inhaled beryllium can elicit a T-cell-dominated alveolitis leading nonnecrotizing granulomata. CBD can be distinguished from sarcoidosis by demonstrating beryllium sensitization in a lymphocyte proliferation test. RESEARCH QUESTION: Beryllium exposure usually occurs in an occupational setting. Because of the diagnosis of CBD in a patient without evident beryllium exposure, we performed a beryllium-lymphocyte proliferation test (BeLPT) among his work colleagues. STUDY DESIGN AND METHODS: This field study investigated a cohort of work colleagues without obvious beryllium exposure. Twenty-one of 30 individuals were assessed in our outpatient clinic for beryllium sensitization. Therefore, BeLPT was performed with freshly collected peripheral blood mononuclear cells. Data were extracted from clinical charts, including geographical data. Beryllium content in dust samples collected at the workplace was measured by graphite-furnace atomic absorption spectroscopy and was compared with samples from different areas of Germany. RESULTS: For the initial patient, the diagnosis of sarcoidosis was reclassified as CBD based on two positive BeLPT results. Assessment of his workplace did not identify a source of beryllium. However, BeLPTs performed on his workmates demonstrated beryllium sensitization in 5 of 21 individuals, suggesting a local beryllium source. Concrete dust obtained from the building yard, the workplace of the index patient, contained high amounts of beryllium (1138 ± 162 µg/kg), whereas dust from other localities (control samples) showed much lower beryllium content (range, 147 ± 18-452 ± 206 µg/kg). Notably, the control dust collected from different places all over Germany exhibit different beryllium concentrations. INTERPRETATION: We describe a cluster of beryllium-sensitized workers from an industry not related to beryllium caused by environmental exposure to beryllium-containing concrete dust, which exhibited markedly elevated beryllium content. Importantly, analyses of dust samples collected from different localities showed that they contain markedly different amounts of beryllium. Thus, besides workplace-related exposure, environmental factors also are capable of eliciting a beryllium sensitization.

Safety and efficacy of abatacept in patients with treatment-resistant SARCoidosis (ABASARC) - protocol for a multi-center, single-arm phase IIa trial.

INTRODUCTION: Sarcoidosis is a granulomatous systemic disease that becomes chronic in approximately one third of affected patients resulting in quality of life and functional impairment. Immunosuppressive drugs other than steroids represent alternative therapeutic options, but side effects like liver and bone marrow toxicity or increased susceptibility to infections limit their use. Pathophysiological studies in sarcoidosis patients demonstrate altered regulatory T-cell functions with a reduced expression of CTLA-4 (CD152) and prolonged inflammation. Therefore, interfering with CTLA-4 using abatacept might be a therapeutic option in sarcoidosis similar to rheumatoid arthritis therapy. METHODS/DESIGN: This is a multicenter prospective open-labeled single arm phase II study addressing the safety of abatacept in sarcoidosis patients. 30 patients with chronic sarcoidosis requiring immunosuppressive therapy beyond 5 mg prednisolone equivalent will be treated with abatacept in combination with corticosteroids for one year in two centers.The primary endpoint is the number and characterization of severe infectious complications under treatment with abatacept.Secondary endpoints are the rate of all infections, patient-related outcomes (assessed by questionnaires), lung function and immunological parameters including alveolar inflammation assessed by bronchoaveolar lavage. DISCUSSION: This is the first trial of abatacept in patients with sarcoidosis. It is hypothesized that administration of abatacept is safe in patients with chronic sarcoidosis and can limit ongoing inflammation. Patients' wellbeing is assessed by established questionnaires. Immunological work-up will highlight the effect of abatacept on inflammatory pathways in sarcoidosis. TRIAL REGISTRATION: The trial has been registered at the German Clinical Trial Registry (Deutsches Register Klinischer Studien, DRKS) with the identity number DRKS00011660.

Interaction Between CCL18 and GPR30 Differs from the Interaction Between Estradiol and GPR30.

BACKGROUND/AIM: C-C motif chemokine ligand 18 (CCL18) is overexpressed in the microenvironment of tumors, promotes invasion and metastasis and is thus important for the therapeutic outcome of many tumor entities. The Gs-coupled seven-transmembrane receptor GPR30 is known as both a CCL18 and an estrogen receptor; its activation by estradiol leads to a transactivation of membrane-tethered pro-heparin-binding EGF-like growth factor and the MAPK/ERK pathway. We examined whether this signaling pathway remains the same under CCL18 stimulation, as opposed to estradiol stimulation. MATERIALS AND METHODS: We investigated the effects of CCL18 on the lung cancer cell line A549, that show low GPR30 expression and the breast cancer cell lines MCF-7, that has high GPR30 expression and MDA-MB-231. These cells were stimulated in different media with CCL18 and then analyzed by qPCR, In-Cell Western®, western blot and ELISA. RESULTS: Many similarities on the effect of CCL18 on the already known estradiol-activated signaling pathway via the G protein-coupled estrogen receptor GPR30 were identified. GPR30 is involved in the expression of matrix metalloproteinases (MMPs), which may play a role in the transactivation of ERK-1/-2 via the cleavage of membrane-bound HB-EGF, via Src-related tyrosine kinases and Gßγ-subunits. With increasing CCL18 concentration, the expression of MMP7 decreased in A549 cells. With decreasing estrogen content of the medium, there was an increasing effect of CCL18 on the inhibition of the relative expression of MMP7. Inhibition of GPR30 with G15 also resulted in a decrease in the relative expression of MMP7, irrespective of the subsequent stimulation with CCL18. This is a rather unexpected result, because the estrogen estradiol and CCL18 both activate GPR30. MCF-7 cells which express more GPR30 did not show any dependence of the relative MMP7 expression on CCL18 except in estrogen-free FCS medium. CCL18 induced an increased relative ERK activation in In-Cell western (ICW) at A549 cells. Stimulation with CCL18 caused decreased ERK activation with simultaneous inhibition of adenylate cyclase in MCF-7. However, stimulation with CCL18 and simultaneous inhibition of cyclooxygenase in MCF-7 resulted in increased ERK activation. In A549, stimulation with CCL18 and co-incubation with dbcAMP resulted in decreased ERK activation in both ICW and Western blot. CONCLUSION: In summary, the Gs-coupled receptor GPR30 plays an important role in the signaling pathway of CCL18. CCL18 and estradiol may not lead to the same signaling pathway after activating GPR30.

Bronchoalveolar Lavage Fluid Reflects a TH1-CD21low B-Cell Interaction in CVID-Related Interstitial Lung Disease.

Background: About 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool. Objective: To gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD). Methods: Sixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology. Results: Both sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (TFH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This TFH-like cell subset is clearly skewed toward TH1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17. Conclusion: Unlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of TFH- and TPH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described TH1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

Kinetics of Torque Teno Virus-DNA Plasma Load Predict Rejection in Lung Transplant Recipients.

BACKGROUND: Lung transplantation is the only therapeutic option in end-stage lung diseases; however, survival after transplantation is limited by acute and chronic rejection or infectious events being results of inappropriate immunosuppression. Torque Teno Viruses (TTVs) are ubiquitous DNA viruses in humans but not found to be causative for any disease. However, some reports suggest that TTV-DNA levels reflect the grade of immunosuppression with higher levels being found in more immunosuppressed individuals. METHODS: We investigated the TTV-DNA levels in 34 lung transplant recipients within their first year after transplantation by quantitative real-time polymerase chain reaction. Clinical data were extracted from charts. RESULTS: In accordance with previous results TTV-DNA levels increase after lung transplantation reaching a steady state after 3 months. The TTV-DNA levels were not correlated with immunosuppressive trough levels and a selective increase was not observed with other DNA viruses. In steady state TTV-DNA levels were significantly higher in patients with infectious complications compared to the group of patients without. Additionally, TTV-DNA levels decreased significantly before biopsy-proven rejection. Sensitivity of a 10-fold decrease in TTV-DNA levels for a subsequent rejection episode was 0.74 with a specificity of 0.99. CONCLUSIONS: In summary, TTV-DNA might be used as an additional tool to monitor immunosuppression in lung transplant recipients. Higher TTV-DNA levels reflect more intense immunosuppression, whereas the TTV-DNA kinetic (ie, decrease of TTV-DNA levels) indicate rejection.

Human alveolar epithelial cells type II are capable of TGFß-dependent epithelial-mesenchymal-transition and collagen-synthesis.

BACKGROUND: The origin of collagen-producing cells in lung fibrosis is unclear. The involvement of embryonic signaling pathways has been acknowledged and trans-differentiation of epithelial cells is discussed critically. The work presented here investigates the role of TGFB in cytoskeleton remodeling and the expression of Epithelial-Mesenchymal-Transition markers by Alveolar Epithelial Cells Type II and tests the hypothesis if human alveolar epithelial cells are capable of trans-differentiation and production of pro-fibrotic collagen. METHODS: Primary human alveolar epithelial cells type II were extracted from donor tissues and stimulated with TGFß and a TGFß-inhibitor. Transcriptome and pathway analyses as well as validation of results on protein level were conducted. RESULTS: A TGFß-responsive fingerprint was found and investigated for mutual interactions. Interaction modules exhibited enrichment of genes that favor actin cytoskeleton remodeling, differentiation processes and collagen metabolism. Cross-validation of the TGFß-responsive fingerprint in an independent IPF dataset revealed overlap of genes and supported the direction of regulated genes and TGFß-specificity. CONCLUSIONS: Primary human alveolar epithelial cells type II seem undergo a TGFß-dependent phenotypic change, exhibit differential expression of EMT markers in vitro and acquire the potential to produce collagen.

Correction: Is serum level of CC chemokine ligand 18 a biomarker for the prediction of radiation induced lung toxicity (RILT)?

[This corrects the article DOI: 10.1371/journal.pone.0185350.].

Atopobium and Fusobacterium as novel candidates for sarcoidosis-associated microbiota.

Sarcoidosis is a granulomatous disease that mainly affects the lung. A role of microbial factors in disease pathogenesis is assumed, but has not been investigated systematically in a large cohort.This cross-sectional study compared the lung microbiota of 71 patients with sarcoidosis, 15 patients with idiopathic pulmonary fibrosis (non-infectious controls) and 10 healthy controls (HCs). Next-generation sequencing of 16S DNA was used on bronchoalveolar lavage samples to characterise the microbial composition, which was analysed for diversity and indicator species. Host genotypes for 13 known sarcoidosis risk variants were determined and correlated with microbial parameters.The microbial composition differed significantly between sarcoidosis and HC samples (redundancy analysis ANOVA, p=0.025) and between radiographic Scadding types. Atopobium spp. was detected in 68% of sarcoidosis samples, but not in HC samples. Fusobacterium spp. was significantly more abundant in sarcoidosis samples compared with those from HCs. Mycobacteria were found in two of 71 sarcoidosis samples. Host-genotype analysis revealed an association of the rs2076530 (BTNL2) risk allele with a decrease in bacterial burden (p=0.002).Our results indicate Scadding type-dependent microbiota in sarcoidosis BAL samples. Atopobium spp. and Fusobacterium spp. were identified as sarcoidosis-associated bacteria, which may enable new insights into the pathogenesis and treatment of the disease.

Is serum level of CC chemokine ligand 18 a biomarker for the prediction of radiation induced lung toxicity (RILT)?

The CC chemokine ligand 18 (CCL18) is produced by alveolar macrophages in patients with fibrosing lung disease and its concentration is increased in various fibrotic lung diseases. Furthermore CCL18 is elevated in several malignancies as it is produced by tumor associated macrophages. In this study we aimed to analyze the role of CCL18 as a prognostic biomarker for the development of early radiation induced lung toxicity (RILT), i.e. radiation pneumonitis after thoracic irradiation and its significance in the course of the disease. Sixty seven patients were enrolled prospectively in the study. Patients were treated with irradiation for several thoracic malignancies (lung cancer, esophageal cancer, thymoma), either with conventionally fractionated or hypo-fractionated radiotherapy. The CCL18 serum levels were quantified with ELISA (enzyme-linked immunosorbent assay) at predefined time points: before, during and at the end of treatment as well as in the first and second follow-up. Treatment parameters and functional tests were also correlated with the development of RILT.Fifty three patients were evaluable for this study. Twenty one patients (39%) developed radiologic signs of RILT Grade >1 but only three of them (5.6%) developed clinical symptoms (Grade 2). We could not find any association between the different CCL18 concentrations and a higher incidence of RILT. Statistical significant factors were the planning target volume (odds ratio OR: 1.003, p = 0.010), the volume of the lung receiving > 20 Gy (OR: 1.132 p = 0.004) and age (OR: 0.917, p = 0.008). There was no association between serial CCL18 concentrations with tumor response and overall survival.In our study the dosimetric parameters remained the most potent predictors of RILT. Further studies are needed in order to estimate the role of CCL18 in the development of early RILT.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5'-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.