The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, ß-arrestin2, and mTORC1.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A-/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNF-α release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires ß-arrestin2 and, in ß-arrestin2-/- AMs and after intratracheal LPS challenge of ß-arrestin2-/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A-/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires ß-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, ß-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

Different responses of the oral, nasal and lung microbiomes to cigarette smoke.

To examine the role of smoking on the bacterial community composition of the upper and the lower respiratory tract, a monocentric, controlled prospective study was performed, including healthy smokers, ex-smokers and never-smokers. Smokers were further grouped according to their smoking history. Bacterial diversity was analysed using a molecular barcoding approach based on directly extracted DNA. Our study shows for the first time distinct bacterial response patterns in the upper and lower respiratory tract to cigarette smoking leading to a higher abundance of opportunistic pathogens. The clinical significance of these dysbioses for health needs to be further explored.

IgA+ memory B-cells are significantly increased in patients with asthma and small airway dysfunction.

BACKGROUND: Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS: In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS: Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS: With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

T2-high asthma phenotypes across lifespan.

RATIONALE: In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. OBJECTIVES: To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. METHODS: In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. MEASUREMENTS AND MAIN RESULTS: Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. CONCLUSIONS: Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Characteristics of the deventilation syndrome in COPD patients treated with non-invasive ventilation: an explorative study.

BACKGROUND: Non-invasive ventilation (NIV) is a recommended treatment for COPD patients suffering from chronic hypercapnic respiratory failure. Prolonged dyspnea after mask removal in the morning, often referred to as deventilation syndrome, is a common side effect but has been poorly characterized yet. This study aimed to explore the pathomechanism, identify risk factors and possible treatment strategies for the deventilation syndrome. METHODS: A prospective, controlled, non-blinded study was conducted. After a night with established NIV therapy, the patients underwent spirometry, blood gas analyses and 6-min walking tests (6MWT) directly, at 2 and 4 h after mask removal. Dyspnea was measured by the modified Borg scale. Bodyplethysmography and health-related quality of life (HRQoL) questionnaires were used. Patients suffering from deventilation syndrome (defined as dyspnea of at least three points on the Borg scale after mask removal) were treated with non-invasive pursed lip breathing ventilation (PLBV) during the second night of the study. RESULTS: Eleven of 31 patients included (35%) met the given criteria for a deventilation syndrome. They reported significantly more dyspnea on the Borg scale directly after mask removal (mean: 7.2 ± 1.0) compared to measurement after 2 h (4.8 ± 2.6; p = 0.003). Initially, mean inspiratory vital capacity was significantly reduced (VCmax: 46 ± 16%) compared to 2 h later (54 ± 15%; p = 0.002), while no changes in pulse oximetry or blood gas analysis were observed. Patients who suffered from a deventilation syndrome had a significantly higher mean airway resistance (Reff: 320 ± 88.5%) than the patients in the control group (253 ± 147%; p = 0.021). They also scored significantly lower on the Severe Respiratory Insufficiency Questionnaire (SRI; mean: 37.6 ± 10.1 vs 50.6 ± 16.7, p = 0.027). After one night of ventilation in PLBV mode, mean morning dyspnea decreased significantly to 5.6 ± 2.0 compared to 7.2 ± 1.0 after established treatment (p = 0.019) and mean inspiratory vital capacity increased from 44 ± 16.0% to 48 ± 16.3 (p = 0.040). CONCLUSIONS: The deventilation syndrome is a serious side effect of NIV in COPD patients, characterized by increase of dyspnea. It is associated with decrease in vital capacity, exercise tolerance after mask removal and lower HRQoL. Patients with high airway resistance are at greater risk of suffering from morning dyspnea. Ventilation in PLBV mode may prevent or improve the deventilation syndrome. TRIAL REGISTRATION: The study was registered in the German Clinical Trials Register (DRKS00016941) on 09 April 2019.

The alpha-melanocyte-stimulating hormone acts as a local immune homeostasis factor in experimental allergic asthma.

BACKGROUND: Originally, the neuropeptide α-melanocyte-stimulating hormone (α-MSH) has been described as a mediator of skin pigmentation. However, recent studies have shown that α-MSH is able to modulate inflammation in various tissues including the lung. So far, it is still not clear whether α-MSH also plays a role in allergic bronchial asthma. OBJECTIVE: This study aimed at investigating the role and regulatory mechanisms of α-MSH in asthma pathogenesis. METHODS: α-MSH levels were measured in bronchoalveolar lavage (BAL) fluid of asthmatic and non-asthmatic individuals as well as of healthy mice and mice with experimental asthma. Wild-type mice were sensitized to ovalbumin (OVA) and exposed to an OVA aerosol in order to induce experimental allergic asthma. α-MSH was administrated intratracheally, the α-MSH antibody intraperitoneally prior each OVA challenge. Airway inflammation, cytokine production, mucus production, airway hyperresponsiveness and receptor expression were assessed. RESULTS: α-MSH levels in BAL of asthmatic individuals and mice were significantly higher compared to healthy controls. In a mouse model of experimental asthma, α-MSH neutralization increased airway inflammation and mucus production, whereas local administration of α-MSH significantly reduced inflammation of the airways. The beneficial effects were further associated with decreased levels of eosinophilic chemoattractant factors that are released by MC5R-positive T helper 2 and airway epithelial cells. CONCLUSION AND CLINICAL RELEVANCE: α-MSH acts as a regulatory factor to maintain local immune homeostasis in allergic bronchial asthma.

Toll-like receptor 3 blockade in rhinovirus-induced experimental asthma exacerbations: A randomized controlled study.

BACKGROUND: Human rhinoviruses (HRVs) commonly precipitate asthma exacerbations. Toll-like receptor 3, an innate pattern recognition receptor, is triggered by HRV, driving inflammation that can worsen asthma. OBJECTIVE: We sought to evaluate an inhibitory mAb to Toll-like receptor 3, CNTO3157, on experimental HRV-16 inoculation in healthy subjects and asthmatic patients. METHODS: In this double-blind, multicenter, randomized, parallel-group study in North America and Europe, healthy subjects and patients with mild-to-moderate stable asthma received single or multiple doses of CNTO3157 or placebo, respectively, and were then inoculated with HRV-16 within 72 hours. All subjects were monitored for respiratory symptoms, lung function, and nasal viral load. The primary end point was maximal decrease in FEV1 during 10 days after inoculation. RESULTS: In asthmatic patients (n = 63) CNTO3157 provided no protection against FEV1 decrease (least squares mean: CNTO3157 [n = 30] = -7.08% [SE, 8.15%]; placebo [n = 25] = -5.98% [SE, 8.56%]) or symptoms after inoculation. In healthy subjects (n = 12) CNTO3157 versus placebo significantly attenuated upper (P = .03) and lower (P = .02) airway symptom scores, with area-under-the-curve increases of 9.1 (15.1) versus 34.9 (17.6) and 13.0 (18.4) versus 50.4 (25.9) for the CNTO3157 (n = 8) and placebo (n = 4) groups, respectively, after inoculation. All of the severe and 4 of the nonserious asthma exacerbations occurred while receiving CNTO3157. CONCLUSION: In summary, CNTO3157 was ineffective in attenuating the effect of HRV-16 challenge on lung function, asthma control, and symptoms in asthmatic patients but suppressed cold symptoms in healthy subjects. Other approaches, including blockade of multiple pathways or antiviral agents, need to be sought for this high unmet medical need.

Risk for latent and active tuberculosis in Germany.

PURPOSE: Few individuals that are latently infected with M. tuberculosis latent tuberculosis infection(LTBI) progress to active disease. We investigated risk factors for LTBI and active pulmonary tuberculosis (PTB) in Germany. METHODS: Healthy household contacts (HHCs), health care workers (HCWs) exposed to M. tuberculosis and PTB patients were recruited at 18 German centres. Interferon-γ release assay (IGRA) testing was performed. LTBI risk factors were evaluated by comparing IGRA-positive with IGRA-negative contacts. Risk factors for tuberculosis were evaluated by comparing PTB patients with HHCs. RESULTS: From 2008-2014, 603 HHCs, 295 HCWs and 856 PTBs were recruited. LTBI was found in 34.5% of HHCs and in 38.9% of HCWs. In HCWs, care for coughing patients (p = 0.02) and longstanding nursing occupation (p = 0.04) were associated with LTBI. In HHCs, predictors for LTBI were a diseased partner (odds ratio 4.39), sexual contact to a diseased partner and substance dependency (all p < 0.001). PTB was associated with male sex, low body weight (p < 0.0001), alcoholism (15.0 vs 5.9%; p < 0.0001), glucocorticoid therapy (7.2 vs 2.0%; p = 0.004) and diabetes (7.8 vs. 4.0%; p = 0.04). No contact developed active tuberculosis within 2 years follow-up. CONCLUSIONS: Positive IGRA responses are frequent among exposed HHCs and HCWs in Germany and are poor predictors for the development of active tuberculosis.

Detection of transrenal DNA for the diagnosis of pulmonary tuberculosis and treatment monitoring.

PURPOSE: Molecular diagnostics of patients with MTB tuberculosis from urine samples. METHODS: We developed a new molecular assay based on the detection of M. tuberculosis-specific transrenal DNA (trDNA) and tested it for the diagnosis of active tuberculosis at the initiation of anti-tuberculosis therapy and during treatment follow-up. RESULTS: The overall sensitivity of trDNA was 96 and 100% when smear-microscopy and trDNA was combined. In a subset of TB treatment naïve patients (n = 11) sensitivity and specificity of trDNA was 64 and 100%, respectively. For this subset of patients the sensitivity was 91% when smear-microscopy and trDNA diagnosis were combined. After treatment initiation, trDNA showed a significant reduction in concentration over time reaching undetectable trDNA values at week 12 in 9 of 11 accessible patients (82%). Kinetics in treatment-naïve patients showed low base-line trDNA levels, which increased to maximal trDNA levels within one week indicating bactericidal activity of anti-tuberculosis drugs after the initiation of effective therapy. Maximal trDNA levels correlated positively with a radiological score, suggesting that the process of DNA excretion may reflect the extent of pulmonary disease. Matched samples showed an inverse correlation between the time to positivity of solid culture with maximum trDNA levels as well as the expected positive correlation between smear grade and maximum trDNA values. CONCLUSION: The detection of M. tuberculosis trDNA from urine specimen is a promising method for the diagnosis tuberculosis. The assay may be a candidate diagnostic tool for patients with paucibacillary and extrapulmonary disease, as method to assess treatment responses and could be helpful to diagnose tuberculosis in children.

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RATIONALE: The development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis. OBJECTIVES: To identify T-cell-dependent mechanisms that are protective in tuberculosis. METHODS: Multicolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood. MEASUREMENTS AND MAIN RESULTS: At two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%). CONCLUSIONS: Our data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis.

Personalized Medicine for Chronic Respiratory Infectious Diseases: Tuberculosis, Nontuberculous Mycobacterial Pulmonary Diseases, and Chronic Pulmonary Aspergillosis.

Chronic respiratory infectious diseases are causing high rates of morbidity and mortality worldwide. Tuberculosis, a major cause of chronic pulmonary infection, is currently responsible for approximately 1.5 million deaths per year. Although important advances in the fight against tuberculosis have been made, the progress towards eradication of this disease is being challenged by the dramatic increase in multidrug-resistant bacilli. Nontuberculous mycobacteria causing pulmonary disease and chronic pulmonary aspergillosis are emerging infectious diseases. In contrast to other infectious diseases, chronic respiratory infections share the trait of having highly variable treatment outcomes despite longstanding antimicrobial therapy. Recent scientific progress indicates that medicine is presently at a transition stage from programmatic to personalized management. We explain current state-of-the-art management concepts of chronic pulmonary infectious diseases as well as the underlying methods for therapeutic decisions and their implications for personalized medicine. Furthermore, we describe promising biomarkers and techniques with the potential to serve future individual treatment concepts in this field of difficult-to-treat patients. These include candidate markers to improve individual risk assessment for disease development, the design of tailor-made drug therapy regimens, and individualized biomarker-guided therapy duration to achieve relapse-free cure. In addition, the use of therapeutic drug monitoring to reach optimal drug dosing with the smallest rate of adverse events as well as candidate agents for future host-directed therapies are described. Taken together, personalized medicine will provide opportunities to substantially improve the management and treatment outcome of difficult-to-treat patients with chronic respiratory infections.

Increased frequencies of pulmonary regulatory T-cells in latent Mycobacterium tuberculosis infection.

Regulation of specific immune responses following exposure to Mycobacterium tuberculosis in humans and the role of regulatory T (Treg) cells in the immune control of latent infection with M. tuberculosis are incompletely understood. Latent infection was assayed by an interferon-γ release assay (IGRA) in healthcare workers regularly exposed to tuberculosis (TB) patients and in household TB contacts in Germany. Immunophenotypes of bronchoalveolar lavage (BAL) mononuclear cells and peripheral blood mononuclear cells (PBMCs) were analysed by fluorescence-activated cell sorting. All TB contacts with latent infection (n=15) had increased (p<0.0001) frequencies of CD4+ CD25+ CD127- Treg cells (median 2.12%, interquartile range (IQR) 1.63-3.01%) among BAL mononuclear cells compared with contacts (n=25) with negative IGRA results (median 0.68%, IQR 0.32-0.96%) No differences were seen when PBMC immunophenotypes of IGRA+ and IGRA- TB contacts were compared (IGRA+ median 9.6%, IQR 5.9-10.1%; IGRA- median 7.7%, IQR 4.6-11.3%; p=0.47). Five out of 25 contacts with negative blood IGRAs showed a positive IGRA from BAL cells, possibly indicating a limited local immune response. In Germany, latent infection with M. tuberculosis, as defined by a positive M. tuberculosis-specific IGRA response on cells from the peripheral blood, is characterised by an increased frequency of Treg cells in the BAL.

Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva.

BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

Correction: Non-invasive ventilation with pursed lips breathing mode for patients with COPD and hypercapnic respiratory failure: A retrospective analysis.

[This corrects the article DOI: 10.1371/journal.pone.0238619.].