Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

17q21 Variants Disturb Mucosal Host Defense in Childhood Asthma.

Rationale: The strongest genetic risk factor for childhood-onset asthma, the 17q21 locus, is associated with increased viral susceptibility and disease-promoting processes.Objectives: To identify biological targets underlying the escalated viral susceptibility associated with the clinical phenotype mediated by the 17q21 locus.Methods: Genome-wide transcriptome analysis of nasal brush samples from 261 children (78 healthy, 79 with wheezing at preschool age, 104 asthmatic) within the ALLIANCE (All-Age-Asthma) cohort, with a median age of 10.0 (range, 1.0-20.0) years, was conducted to explore the impact of their 17q21 genotype (SNP rs72163891). Concurrently, nasal secretions from the same patients and visits were collected, and high-sensitivity mesoscale technology was employed to measure IFN protein levels.Measurements and Main Results: This study revealed that the 17q21 risk allele induces a genotype- and asthma/wheeze phenotype-dependent enhancement of mucosal GSDMB expression as the only relevant 17q21-encoded gene in children with preschool wheeze. Increased GSDMB expression correlated with the activation of a type-1 proinflammatory, cell-lytic immune, and natural killer signature, encompassing key genes linked to an IFN type-2-signature (IFNG, CXCL9, CXCL10, KLRC1, CD8A, GZMA). Conversely, there was a reduction in IFN type 1 and type 3 expression signatures at the mRNA and protein levels.Conclusions: This study demonstrates a novel disease-driving mechanism induced by the 17q21 risk allele. Increased mucosal GSDMB expression is associated with a cell-lytic immune response coupled with compromised airway immunocompetence. These findings suggest that GSDMB-related airway cell death and perturbations in the mucosal IFN signature account for the increased vulnerability of 17q21 risk allele carriers to respiratory viral infections during early life, opening new options for future biological interventions.The All-Age-Asthma (ALLIANCE) cohort is registered at www.clinicaltrials.gov (pediatric arm, NCT02496468).

Proinflammatory pattern in the lower airways of non-asthmatic obese adolescents.

BACKGROUND: Obesity is known to be a pro-inflammatory condition affecting multiple organs. Obesity as a systemic pro-inflammatory state, might be associated with bronchial inflammation in non-smoking adolescents with a BMI ≥ 30 kg/m2 without evidence of concomitant chronic diseases. MATERIALS AND METHODS: We studied non-asthmatic obese patients (n = 20; median age 15.8 years; BMI 35.0 kg/m2) compared to age matched healthy control subjects (n = 20; median age 17.5 years; BMI 21.5 kg/m2). Induced sputum differential cell counts and sputum mRNA levels were assessed for all study subjects. Serum levels of CRP, IL-6, and IL-8 were measured. Further, IL-5, IL-6, IL-8, IL-13, IL-17, TNF-α, IFN-γ, and IP-10 protein levels were analyzed in induced sputum was. RESULTS: Serum CRP levels, sputum inflammatory cell load and sputum eosinophils differed significantly between obese and non-obese subjects, for sputum neutrophils, a correlation was shown with BMI ≥ 30 kg/m2. Differences were also observed for sputum mRNA expression of IL6, IL8, IL13, IL17, IL23, and IFN-γ, as well as the transcription factors T-bet, GATA3, and FoxP3. CONCLUSIONS: Increased bronchial inflammation, triggered by systemic or local inflammatory effects of obesity itself, may account for the higher rates of airway disease in obese adolescents.

Ex Vivo Immunomodulatory Effects of Lactobacillus-, Lacticaseibacillus-, and Bifidobacterium-Containing Synbiotics on Human Peripheral Blood Mononuclear Cells and Monocyte-Derived Dendritic Cells in the Context of Grass Pollen Allergy.

Sensing of the intestinal microbiota by the host immune system is important to induce protective immune responses. Hence, modification of the gut microbiota might be able to prevent or treat allergies, mediated by proinflammatory Th2 immune responses. The aim was to investigate the ex vivo immunomodulatory effects of the synbiotics Pollagen® and Kallergen®, containing the probiotic bacterial strains Lactobacillus, Lacticaseibacillus and Bifidobacterium, in the context of grass pollen allergy. Peripheral blood mononuclear cells (PBMCs) from grass pollen-allergic patients and healthy controls were stimulated with grass pollen extract (GPE) and synbiotics and Gata3 expression and cytokine secretion analyzed. Monocyte-derived dendritic cells (MoDCs) cells were matured in the presence of GPE and synbiotics, co-cultured with autologous naïve T cells and maturation markers and cytokine secretion analyzed. GPE stimulation of PBMCs from grass pollen-allergic patients resulted in a significant higher production of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 compared to healthy controls. Gata3+CD4+ T cell induction was independent of the allergic status. The synbiotics promoted IL-10 and IFN-γ secretion and downregulated the GPE-induced Th2-like phenotype. Co-culturing naïve T cells with MoDCs, matured in the presence of GPE and synbiotics, shifted the GPE-induced Th2 cytokine release towards Th1-Th17-promoting conditions in allergic subjects. The investigated synbiotics are effective in downregulating the GPE-induced Th2 immune response in PBMCs from grass pollen-allergic patients as well as in autologous MoDC-T cell stimulation assays. In addition to increased IL-10 release, the data indicates a shift from a Th2- to a more Th1- and Th17-like phenotype.

Genome-Wide Gene Expression Analysis Reveals Unique Genes Signatures of Epithelial Reorganization in Primary Airway Epithelium Induced by Type-I, -II and -III Interferons.

Biosensors such as toll-like receptors (TLR) induce the expression of interferons (IFNs) after viral infection that are critical to the first step in cell-intrinsic host defense mechanisms. Their differential influence on epithelial integrity genes, however, remains elusive. A genome-wide gene expression biosensor chip for gene expression sensing was used to examine the effects of type-I, -II, and -III IFN stimulation on the epithelial expression profiles of primary organotypic 3D air-liquid interface airway cultures. All types of IFNs induced similar interferon-stimulated genes (ISGs): OAS1, OAS2, and IFIT2. However, they differentially induced transcription factors, epithelial modulators, and pro-inflammatory genes. Type-I IFN-induced genes were associated with cell-cell adhesion and tight junctions, while type-III IFNs promoted genes important for transepithelial transport. In contrast, type-II IFN stimulated proliferation-triggering genes associated and enhanced pro-inflammatory mediator secretion. In conclusion, with our microarray system, we provide evidence that the three IFN types exceed their antiviral ISG-response by inducing distinct remodeling processes, thereby likely strengthening the epithelial airway barrier by enhancing cross-cell-integrity (I), transepithelial transport (III) and finally reconstruction through proliferation (II).

Role of microRNAs in type 2 diseases and allergen-specific immunotherapy.

MicroRNAs (miRs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases as well as their potential as biomarkers in allergen-specific treatment options. Their function as post-transcriptional regulators, controlling various cellular processes, is of high importance since any single miR can target multiple mRNAs, often within the same signalling pathway. MiRs can alter dysregulated expression of certain cellular responses and contribute to or cause, but in some cases prevent or repress, the development of various diseases. In this review article, we describe current research on the role of specific miRs in regulating immune responses in epithelial cells and specialized immune cells in response to various stimuli, in allergic diseases, and regulation in the therapeutic approach of allergen-specific immunotherapy (AIT). Despite the fact that AIT has been used successfully as a causative treatment option since more than a century, very little is known about the mechanisms of regulation and its connections with microRNAs. In order to fill this gap, this review aims to provide an overview of the current knowledge.

Thermosensitive PLGA-PEG-PLGA Hydrogel as Depot Matrix for Allergen-Specific Immunotherapy.

Allergen-specific immunotherapy (AIT) is the only currently available curative treatment option for allergic diseases. AIT often includes depot-forming and immunostimulatory adjuvants, to prolong allergen presentation and to improve therapeutic efficacy. The use of aluminium salts in AIT, which are commonly used as depot-forming adjuvants, is controversially discussed, due to health concerns and Th2-promoting activity. Therefore, there is the need for novel delivery systems in AIT with similar therapeutic efficacy compared to classical AIT strategies. In this study, a triblock copolymer (hydrogel) was assessed as a delivery system for AIT in a murine model of allergic asthma. We show that the hydrogel combines the advantages of both depot function and biodegradability at the same time. We further demonstrate the suitability of hydrogel to release different bioactive compounds in vitro and in vivo. AIT delivered with hydrogel reduces key parameters of allergic inflammation, such as inflammatory cell infiltration, mucus hypersecretion, and allergen-specific IgE, in a comparable manner to standard AIT treatment. Additionally, hydrogel-based AIT is superior in inducing allergen-specific IgG antibodies with potentially protective functions. Taken together, hydrogel represents a promising delivery system for AIT that is able to combine therapeutic allergen administration with the prolonged release of immunomodulators at the same time.

Lung Epithelial CYP1 Activity Regulates Aryl Hydrocarbon Receptor Dependent Allergic Airway Inflammation.

The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR-/- and CYP1B1-/- but not CYP1A1-/- or CYP1A2-/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.

Role of Respiratory Epithelial Cells in Allergic Diseases.

The airway epithelium provides the first line of defense to the surrounding environment. However, dysfunctions of this physical barrier are frequently observed in allergic diseases, which are tightly connected with pro- or anti-inflammatory processes. When the epithelial cells are confronted with allergens or pathogens, specific response mechanisms are set in motion, which in homeostasis, lead to the elimination of the invaders and leave permanent traces on the respiratory epithelium. However, allergens can also cause damage in the sensitized organism, which can be ascribed to the excessive immune reactions. The tight interaction of epithelial cells of the upper and lower airways with local and systemic immune cells can leave an imprint that may mirror the pathophysiology. The interaction with effector T cells, along with the macrophages, play an important role in this response, as reflected in the gene expression profiles (transcriptomes) of the epithelial cells, as well as in the secretory pattern (secretomes). Further, the storage of information from past exposures as memories within discrete cell types may allow a tissue to inform and fundamentally alter its future responses. Recently, several lines of evidence have highlighted the contributions from myeloid cells, lymphoid cells, stromal cells, mast cells, and epithelial cells to the emerging concepts of inflammatory memory and trained immunity.

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.

Allergen Content of Therapeutic Preparations for Allergen-Specific Immunotherapy of European Paper Wasp Venom Allergy.

Allergy to Polistes dominula (European paper wasp) venom is of particular relevance in Southern Europe, potentially becoming a threat in other regions in the near future, and can be effectively cured by venom immunotherapy (VIT). As allergen content in extracts may vary and have an impact on diagnostic and therapeutic approaches, the aim was to compare five therapeutic preparations for VIT of P. dominula venom allergy available in Spain. Products from five different suppliers were analyzed by SDS-PAGE and LC-MS/MS and compared with a reference venom sample. Three products with P. dominula venom and one product with a venom mixture of American Polistes species showed a comparable band pattern in SDS-PAGE as the reference sample and the bands of the major allergens phospholipase A1 and antigen 5 were assignable. The other product, which consists of a mixture of American Polistes species, exhibited the typical band pattern in one, but not in another sample from a second batch. All annotated P. dominula allergens were detected at comparable levels in LC-MS/MS analysis of products containing P. dominula venom. Due to a lack of genomic information on the American Polistes species, the remaining products were not analyzed by this method. The major Polistes allergens were present in comparable amounts in the majority, but not in all investigated samples of venom preparations for VIT of P. dominula venom allergy.

Early reduction of SARS-CoV-2-replication in bronchial epithelium by kinin B2 receptor antagonism.

SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B2 receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B2R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B2R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein-coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B2R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R-antagonists, like icatibant, in the treatment of early-stage COVID-19. KEY MESSAGES: Induction of kinin B2 receptor in the nose of SARS-CoV-2-positive patients. Treatment with B2R-antagonist protects airway epithelial cells from SARS-CoV-2. B2R-antagonist reduces ACE2 levels in vivo and ex vivo. Protection by B2R-antagonist is mediated by inhibiting viral replication and spread.

Bronchial inflammation biomarker patterns link humoral immunodeficiency with bronchiectasis-related small airway dysfunction.

BACKGROUND: The progression of chronic destructive lung disease in patients with humoral immunodeficiency (ID) and concomitant development of bronchiectasis is difficult to prevent. Lung function tests in these patients typically show bronchial obstruction of the small airways in combination with increased air trapping in the distal airways, which is consistent with small airway dysfunction. OBJECTIVE: The objective was to assess the grade of chronic lower airway inflammation and small airway dysfunction from induced sputum and the corresponding local pro-inflammatory mediator pattern to discriminate patients affected by bronchiectasis-related Small Airway Dysfunction (SAD). METHODS: In a prospective design, 22 patients with ID (14 CVID, 3 XLA, 3 hyper-IgM syndrome, 1 hyper-IgE syndrome and low IgG levels due to treatment with rituximab and 1 SCID after BMT and persistent humoral defect) and 21 healthy controls were examined. Lung function, Fraction Expiratory Nitric Oxide (FeNO) and pro-inflammatory cytokine levels were compared in subsets of patients with (ID + BE) and without bronchiectasis (ID) pre-stratified using high-resolution computed tomography (HRCT) scans and control subjects. RESULTS: Analysis of induced sputum showed significantly increased total cell counts and severe neutrophilic inflammation in ID. The concomitant SAD revealed higher total cell numbers compared to ID. Bronchial inflammation in ID is clearly mirrored by pro-inflammatory mediators IL-1ß, IL-6 and CXCL-8, whilst TNF-α revealed a correlation with lung function parameters altered in the context of bronchiectasis-related Small Airway Dysfunction. CONCLUSIONS: In spite of immunoglobulin substitution, bronchial inflammation was dominated by neutrophils and was highly increased in patients with ID + BE. Notably, the pro-inflammatory cytokines in patients with ID were significantly increased in induced sputum. The context-dependent cytokine pattern in relation to the presence of concomitant bronchiectasis associated with SAD in ID patients could be helpful in delimiting ID patient subgroups and individualizing therapeutic approaches.

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.

Immunological effects of adjuvanted low-dose allergoid allergen-specific immunotherapy in experimental murine house dust mite allergy.

BACKGROUND: Native allergen extracts or chemically modified allergoids are routinely used to induce allergen tolerance in allergen-specific immunotherapy (AIT), although mechanistic side-by-side studies are rare. It is paramount to balance optimal dose and allergenicity to achieve efficacy warranting safety. AIT safety and efficacy could be addressed by allergen dose reduction and/or use of allergoids and immunostimulatory adjuvants, respectively. In this study, immunological effects of experimental house dust mite (HDM) AIT were investigated applying high-dose HDM extract and low-dose HDM allergoids with and without the adjuvants microcrystalline tyrosine (MCT) and monophosphoryl lipid A (MPL) in a murine model of HDM allergy. METHODS: Cellular, humoral, and clinical effects of the different AIT strategies were assessed applying a new experimental AIT model of murine allergic asthma based on physiological, adjuvant-free intranasal sensitization followed by subcutaneous AIT. RESULTS: While low-dose allergoid and high-dose extract AIT demonstrated comparable potency to suppress allergic airway inflammation and Th2-type cytokine secretion of lung-resident lymphocytes and draining lymph node cells, low-dose allergoid AIT was less effective in inducing a potentially protective IgG1 response. Combining low-dose allergoid AIT with MCT or MCT and dose-adjusted MPL promoted Th1-inducing mechanisms and robust B-cell activation counterbalancing the allergic Th2 immune response. CONCLUSION: Low allergen doses induce cellular and humoral mechanisms counteracting Th2-driven inflammation by using allergoids and dose-adjusted adjuvants. In light of safety and efficacy improvement, future therapeutic approaches may use low-dose allergoid strategies to drive cellular tolerance and adjuvants to modulate humoral responses.

Secretoglobins in the big picture of immunoregulation in airway diseases.

The proteins of the secretoglobin (SCGB) family are expressed by secretory tissues of barrier organs. They are embedded in immunoregulatory and anti-inflammatory processes of airway diseases. This review particularly illustrates the immune regulation of SCGBs by cytokines and their implication in the pathophysiology of airway diseases. The biology of SCGBs is a complex topic of increasing importance, as they are highly abundant in the respiratory tract and can also be detected in malignant tissues and as elements of immune control. In addition, SCGBs react to cytokines, they are embedded in Th1 and Th2 immune responses, and they are expressed in a manner dependent on cell maturation. The big picture of the SCGB family identifies these factors as critical elements of innate immune control at the epithelial barriers and highlights their potential for diagnostic assessment of epithelial activity. Some members of the SCGB family have so far only been superficially examined, but have high potential for translational research.

IL-37 regulates allergic inflammation by counterbalancing pro-inflammatory IL-1 and IL-33.

BACKGROUND: Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation. METHODS: IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1ß, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1ß, IL-33, and IL-37 levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy. RESULTS: IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37 led to differential expression of >90 genes induced by IL-1ß and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1ß- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37 had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1ß and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls. CONCLUSION: IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1ß and IL-33.

MEK1/2 regulates APOBEC3B and polymerase iota-induced mutagenesis in head and neck cancer cells.

Resistance to chemotherapy provides a major challenge in treatment of metastatic cancer. Prolonged exposure to almost any drug regimen leads to the formation of resistant subclones in almost all advanced solid tumors. Tumor heterogeneity because of intrinsic genetic instability is seen as one of the major contributing factors. In this work, we present evidence that genetic instability measured by mutation frequency is induced by treatment with the EGFR inhibitor afatinib or cisplatin in head and neck squamous cancer cells. We find that APOBEC3B and polymerase iota are upregulated, and inhibition of MEK1/2 by U0126 leads to downregulation on the protein level. Costimulation of afatnib and cisplatin with U0126 leads to a significantly lower mutation frequency. These findings may represent a molecular mechanism for dynamically controlling genetic instability during chemotherapy in head and neck squamous cell carcinoma (HNSCC) cancer cells.

Sputum microRNA-screening reveals Prostaglandin EP3 receptor as selective target in allergen-specific immunotherapy.

BACKGROUND: Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). METHODS: Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2  levels were measured using ELISA. RESULTS: Two hundred and fifty nine miRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21 miRs were downregulated, only 4 miRs were upregulated in asthmatics upon AIT. Of these 4 miRs, miR-3935 stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2  levels correlated with ILC2 frequencies, secreted sputum IL-13 levels, inflammatory cell load, sputum eosinophils and symptom burden. CONCLUSIONS: While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.