Respiratory Research

IntroductionCommunity-acquired pneumonia (CAP) is associated with high morbidity and hospitalization rate. In infectious diseases, host genetics plays a critical role in susceptibility and immune response, and the immune pathways involved are highly dependent on the microorganism and its route of entry. ObjectiveTo identify genetic risk loci for CAP using a one-stage genome-wide association study (GWAS). MethodsWe performed a GWAS on 3,765 Spanish individuals, including 257 adult patients hospitalized with CAP and 3,508 population controls. Pneumococcal CAP was documented in 30% of patients; the remaining 70% were selected among patients with unidentified microbiological etiology. Genotyping and imputation allowed testing the association of 7,6 million variants using logistic regressions. Subsequently, we prioritized genes and likely causal variants based on Bayesian fine mapping and functional evidence. Imputation and association testing of the classic HLA alleles and amino acids was also conducted. ResultsWe revealed six independent sentinel variants that were genome-wide significant (p<5x10-8), three located on chromosome 6p21.32, and one for each of the chromosomes 4q28.2, 11p12, and 20q11.22. Our analyses prioritized C4orf33 on 4q28.2, TAPBP on 6p21.32, and ZNF341 on 20q11.22. Interestingly, genetic defects of TAPBP and ZNF341 are previously known inborn errors of immunity predisposing to bacterial pneumonia, including pneumococcus and Haemophyilus influenzae. Associations were all non-significant for the classic HLA alleles. ConclusionsWe completed a GWAS of CAP and identified four novel risk loci involved in CAP susceptibility.

BackgroundChronic obstructive pulmonary disease (COPD) is the third leading cause of global mortality. Emerging evidence suggests the oral microbiome may contribute to COPD progression, though causal relationships remain elusive. MethodsUsing bidirectional Mendelian randomization (MR) on East Asian genome-wide association study (GWAS) summary data, we assessed causal links between oral microbial taxa and COPD risk. Subsequently, hub genes in COPD bulk RNA sequencing data were identified by integrating the Protein-Protein Interaction (PPI) network with machine learning, followed by target validation using single-cell RNA sequencing, immune infiltration analysis, and molecular docking. ResultsForward MR identified 43 taxa associated with COPD, primarily from genera such as Fusobacterium, Prevotella, and Streptococcus. Reverse MR detected 73 taxa affected by COPD, mainly involving Campylobacter_A, Rothia, and Streptococcus. Through the PPI network, machine learning screening, and multi-omics analysis validation, MPDZ emerged as a key hub gene, upregulated in Ciliated and Endothelial cells during early stages of COPD and linked to immune dysregulation. Molecular docking revealed six candidate drugs with strong binding affinity to MPDZ. ConclusionOur study provides insights for the development of personalized treatment strategies for COPD and offers preliminary candidate targets and drugs for future drug development.

BackgroundPatients with severe asthma may have a greater risk of dying from COVID-19 disease caused by SARS-CoV-2 virus. Angiotensin converting enzyme 2 (ACE2) receptor and enzyme proteases, transmembrane protease, serine 2 (TMPRSS2) and furin are needed for the attachment and invasion of the virus into host cells. We determined whether their expression in the airways of severe asthma patients is increased. MethodWe examined the microarray mRNA expression of ACE2, TMPRSS2 and furin in the sputum, bronchial brush and bronchial biopsies of participants in the European U-BIOPRED cohort. ResultsACE2 and furin sputum gene expression was significantly increased in severe non-smoking asthma compared to mild-moderate asthma and healthy volunteers. By contrast, TMPRSS2 expression in bronchial biopsy and bronchial brushings was increased in severe smoking and ex-smoking asthmatics, and so was furin expression in bronchial brushings. Several clinical parameters including male gender, oral steroid use and nasal polyps were positively associated with ACE2, TMPRSS2 and furin expression levels. There was a higher expression of ACE2 and furin in the sputum neutrophilic molecular phenotype with inflammasome activation compared to the eosinophilic Type2-high or paucigranulocytic phenotypes. The enrichment score of the IL-13-Type2 gene signature was positively correlated with ACE2, TMPRSS2 and furin levels. ConclusionThese key determinants of virus entry into the lungs may contribute to the poorer outcomes from COVID-19 disease in patients with severe asthma. "take home" messageIn severe asthma, gene expression of ACE, TMPRSS2 and furin are elevated compared to mild-moderate asthma and healthy volunteers, particularly in neutrophilic asthma. This might explain the increased risk of death in severe asthma afflicted with COVID19.

BackgroundPathway-based approaches may effectively dissect the polygenic architecture of complex diseases. We conducted a semi-explorative interaction study between genome-wide and pathway-specific polygenic risk scores (PRS) and occupational asthmagens (i.e., cleaning agents) on adult asthma. MethodsThis study included 2615 adults from the Personalized Environment and Genes Study (PEGS) in North Carolina, with comprehensive questionnaire-based health and exposure data and whole genome sequencing data. Occupational exposure to any cleaning agent was assessed as ever exposure to ammonia, chlorine bleach, and carbon tetrachloride over 15 minutes per week in any job. Current adult asthma was defined as doctor-diagnosed asthma with an attack in the past year. We estimated genome-wide PRS and pathway-specific PRS of oxidative stress and type 2 immune responses. We used 1) logistic regression to analyze interactions between PRS and exposure, and 2) linear principal-component regression to examine gene-based interactions. ResultsCurrent asthma prevalence was 9.56% (n=250). Occupational exposure to cleaning agents was associated with an increased asthma risk [adjusted odds ratio (aOR): 1.56-2.24]. Genome-wide PRS multiplicatively interacted with any cleaning agent or bleach (P interaction =0.029 or 0.039), while pathway-based PRS conferred asthma risk independently. Additive interactions between cleaning agents and high genetic risks (PRS>median splits) were observed, showing the highest excess asthma risk in individuals with both exposure and pathway-based risks. Notably, eight genes were identified to explain oxidative stress PRS-related interaction with cleaning agents on asthma (P interaction < 0.05/29 genes). ConclusionApplying novel and statically powerful pathway-based PRS, we found cleaning agents synergistically interacted with genetic risks of asthma, advancing mechanistic insights for precision prevention and intervention in asthma.

Dysbiosis has been linked to the pathogenesis of multiple diseases. Although dozens of publications have associated changes in the nasopharyngeal microbiota to patients susceptibility to COVID-19, results from these studies are highly variable and contradictory in many cases. Addressing the limitations in previous research responsible for that variability, this study uses 16S rRNA gene sequencing to analyse the nasopharyngeal microbiota of 395 subjects, 117 uninfected controls and 278 COVID-19 patients, of different age groups that cover the entire lifespan and across varying disease severities. Importantly, our results reveal that bacterial diversity decreases progressively throughout life but only in severely ill COVID-19 patients, in whose nasopharynx, moreover, the opportunistic pathogen bacterial genera Staphylococcus, Corynebacterium, Streptococcus, Prevotella, Acinetobacter, and Pseudomonas are overrepresented. Notably, Scardovia wiggsiae appears only in severe COVID-19 patients over 60 years of age, suggesting a potential utility of this bacterial species as a COVID-19 severity biomarker in the elderly, who are the most susceptible individuals to suffer from serious forms of the disease and the age group that presents more differences in comparison with the other age groups according to the majority of the parameters analysed in this study. Our results provide valuable insights into age-associated dynamics within nasopharyngeal microbiota during severe COVID-19, offering potential avenues for further exploration and therapeutic interventions.

Biologic treatment options have expanded considerably for severe asthma, transforming patient management, but only ~70% of patients respond to the treatment following standard criteria. This preliminary study utilized data collected in the Wessex AsThma CoHort of difficult asthma (WATCH) study. DNAm was measured in n=15 severe asthma patients at baseline, i.e., before mepolizumab (MEPO), an interleukin-5 receptor antagonist, was administered and the response to MEPO was analyzed. We aimed to assess the potential of DNA methylation as a feasible biomarker to predict responses to MEPO among severe asthma patients. Our preliminary findings suggested that pre-biologic DNA methylation as a predictor of a patients response status may outperform pre-biologic blood eosinophils cell counts.

BackgroundAsthma is a complex chronic inflammatory disease affecting approximately 339 million people worldwide. The ADAM33 gene, encoding a disintegrin and metalloproteinase, has emerged as a key susceptibility gene for asthma, with the rs2280091 (T1) polymorphism showing variable associations across different populations. This study represents the first genetic investigation of asthma in the Syrian population. MethodsA case-control study was conducted at Aleppo University Hospital from April to November 2019, including 100 participants (80 asthma patients and 20 healthy controls) aged 20-40 years. Asthma diagnosis was confirmed using spirometry and reversibility testing according to GINA guidelines. Genomic DNA was extracted from whole blood, and the rs2280091 polymorphism was genotyped using PCR-RFLP with NcoI restriction enzyme. Statistical analysis was performed using SPSS 25.0 with significance set at p[&le;]0.05. ResultsThe study population showed balanced sex distribution (50% male, 50% female) with mean ages of 26.13 years (cases) and 29.65 years (controls). Genotype frequencies were: A/A (43.0%), A/G (45.0%), and G/G (12.0%), with allele frequencies of A=0.66 and G=0.34, conforming to Hardy-Weinberg equilibrium. While no significant association was found between genotype and asthma occurrence (p=0.871), the G/G genotype showed significant association with increased asthma severity (p=0.016). ANOVA analysis revealed significantly lower FEV1 values in G/G carriers compared to A/A and A/G genotypes (p=0.001). ConclusionsThe ADAM33 rs2280091 G/G genotype is significantly associated with increased asthma severity in the Syrian population, suggesting its potential utility as a genetic marker for severe asthma phenotypes. This finding contributes to understanding asthma genetics in Middle Eastern populations and supports the role of ADAM33 in airway remodeling processes.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic and a global health emergency. The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed in nasal epithelial cells and plays a major role in cellular entry leading to infection. High expression of ACE2 has been suggested to be a potential risk factor for virus infection and disease severity. However the profile of ACE2 gene expression in diseases of the upper airways remains poorly understood. We herein investigated ACE2 gene expression in the nasal tissues of a cohort of Swedish patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) using RT-qPCR. ACE2 mRNA expression was significantly reduced in the nasal mucosa of CRSwNP patients compared to that of controls. Moreover, we observed a sex-dependant difference in nasal ACE2 expression, where significantly lower levels of the ACE2 transcript were detected in the nasal mucosa of only female CRSwNP patients. These findings indicate that CRSwNP patients with a decrease in ACE2 gene expression may thereby be less prone to be infected by SARS-CoV-2. These results enhance our understanding on the profile of ACE2 expression in the nasal mucosa of patients with upper airway diseases, and their susceptibility to infection with SARS-CoV-2.

RationaleAsthma is a chronic airway disease driven by multiple immunologic pathways that determine the clinical response to therapy. Current diagnostic methods are incapable of discriminating subtypes of asthma and guiding targeted treatment. We hypothesized that sputum cytokine profiles could help to identify immunologically-defined disease subtypes and individualize therapy in patients with severe asthma. ObjectivesDefine asthma subtypes associated with sputum alarmin and cytokine levels. MethodsCross-sectional analysis of clinical features and sputum from 200 asthmatic patients was performed. 10 cytokines belonging to alarmin, T2, and non-T2 pathways were measured. Pearson correlation was used to identify cytokine modules. Latent class analysis was used to cluster patients by cytokine expression. Measurements and Main ResultsThree modules of highly correlated cytokines were identified including a non-T2 module, the IL-1{beta}mod (IL-1{beta}, IL-6, GCSF), and two distinct T2 modules: TSLPmod (TSLP, IL-4, IL-5, IL-9) and IL-33mod (IL-33, IL-13, IL-21). The TSLPmod was associated with asthma severity, airway obstruction, eosinophilia, and elevated FeNO. Patient clustering revealed three subgroups; two different subgroups showed expression of T2 modules. ConclusionsAnalysis of sputum cytokines revealed three discrete signaling modules in patients with asthma. Unexpectedly, the inclusion of alarmins led to separation of canonical T2 cytokines into two unique modules; IL-5 grouped with TSLP, while IL-13 grouped with IL-33. In addition, patient clustering revealed two distinct endotypes associated with T2 immune signaling. These findings indicate a new layer of immunologic heterogeneity within the T2 paradigm, and suggest that sputum cytokine profiling may hold diagnostic utility for patients with asthma.

Biologic monoclonal antibody therapies for severe asthma target the Type 2 endotype through blockade of the IgE, IL-5/eosinophil, or IL-4/13 pathways, which represents at least two-thirds of patients, and have led to significant clinical benefits in severe asthma management. However, studies show that 10-20% of patients may be non-responders and require a change in therapy. There is also the emerging concept that a significant percentage of patients may enter clinical remission, with a very high level of disease control and virtually symptom-free. These clinical scenarios and heterogeneity increase the need to develop blood-based biomarkers that can predict outcome. Identifying markers of clinical remission may also have potential for expanding access to other severe asthma patients not currently identified through serum IgE, blood eosinophils, or FeNO. In this study, blood was taken prior to therapy from severe asthma patients (n=31) with a Type 2 endotype, high serum IgE, atopy, and blood eosinophilia who qualified for both Omalizumab (anti-IgE) and Mepolizumab (anti-IL-5) and were randomised to receive either treatment. White blood cells underwent single cell RNA-sequencing and patients were assessed for clinical outcomes over a 6-month period. Non-response to either Omalizumab or Mepolizumab was predicted by a gene signature expressed in antiviral plasmacytoid dendritic cells. Clinical remission was predicted by a common gene signature in rarer CD34+ blood progenitors and circulating MAIT cells with a ROC Curve AUC of 0.91 and 0.88, respectively. This discovery study identifies novel blood biomarkers that predict clinical outcome to multiple biologic therapies in severe asthma.

Epigenetic dysregulation, particularly DNA methylation variations, is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Ten-eleven translocation (TET) proteins (TET1, TET2, and TET3) regulate DNA methylation and gene transcription. Impaired TET1 expression was previously associated with airway inflammation and asthma. Here we investigated TET gene associations with COPD severity. We found that reduced TET1 expression in peripheral blood mononuclear cells was associated with higher sputum and blood neutrophil counts, decreased lung function and increased disease severity in patients. These findings support a potential protective role and warrant further mechanistic investigations into the actions of TET1 in COPD.

BackgroundChronic rhinosinusitis (CRS) is characterized by persistent nasal and paranasal sinus mucosa inflammation. It comprises two phenotypes, namely CRS with nasal polyps (CRSwNP) and without (CRSsNP). CRSwNP can be associated with asthma and hypersensitivity to non-steroidal anti-inflammatory drugs (NSAIDs) in a syndrome known as NSAID-exacerbated respiratory disease (N-ERD). Furthermore, CRS frequently intertwines with respiratory allergies. ObjectiveThis study investigated the phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) within cohorts of CRS patients, additionally examining the influence of comorbid respiratory allergies on these parameters. Methods24 participants were grouped into controls, CRSsNP, CRSwNP, and N-ERD (n=6/group), with half of the patients in each group having respiratory allergies. Levels of cytokines were quantified in nasal secretions and sera. The abundance and phenotypic features of immune cells in PBMCs were evaluated through mass cytometry and clustering methods. ResultsN-ERD patients showed heightened type 2 nasal cytokine levels. Mass cytometry analysis revealed increased activated naive B cell levels in CRSwNP and N-ERD, while resting naive B cells were higher in CRSsNP. Th2a cell levels did not differ between CRS subtypes but were significantly elevated in allergic subjects. In CRSwNP and N-ERD patients, naive B cells had a lower CXCR5 and higher CD45RA expression, while NK cells displayed reduced CD56 levels. ConclusionsThere are distinct immunological features in PBMCs of CRS phenotypes and allergy, characterized by elevated resting naive B cells in CRSsNP, increased activated naive B cells in CRSwNP and N-ERD, and higher Th2a cell levels in allergic subjects. Capsule summaryThis study examines immunological profiles in different phenotypes of CRS with and without comorbid allergy patients, highlighting immune cell intricacies in CRS subtypes and immune differences in CRS and respiratory allergy.

IntroductionCoronavirus disease 2019 (COVID-19) is a respiratory infection caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This virus uses the angiotensin converting enzyme II (ACE-2) as the cellular entry receptor to infect the lower respiratory tract. Because individuals with chronic obstructive pulmonary disease (COPD) are at increased risk of severe COVID-19, we determined whether ACE-2 expression in the lower airways was related to COPD and cigarette smoking. MethodsUsing RNA-seq, we determined gene expression levels in bronchial epithelia obtained from cytologic brushings of 6th to 8th generation airways in individuals with and without COPD. We eternally validated these results from two additional independent cohorts, which used microarray technologies to measure gene expression levels from 6th to 12th generation airways. ResultsIn the discovery cohort (n=42 participants), we found that ACE-2 expression levels were increased by 48% in the airways of COPD compared with non-COPD subjects (COPD=2.52{+/-}0.66 log2 counts per million reads (CPM) versus non-COPD= 1.70{+/-}0.51 CPM, p=7.62x10-4). There was a significant inverse relationship between ACE-2 gene expression and FEV1% of predicted (r=-0.24; p=0.035). Current smoking also significantly increased ACE-2 expression levels compared with never smokers (never current smokers=2.77{+/-}0.91 CPM versus smokers=1.78{+/-}0.39 CPM, p=0.024). These findings were replicated in the two eternal cohorts. ConclusionsACE-2 expression in lower airways is increased in patients with COPD and with current smoking. These data suggest that these two subgroups are at increased risk of serious COVID-19 infection and highlight the importance of smoking cessation in reducing the risk.

BackgroundAsthma has been associated with impaired interferon responses. Multiple cell types have been implicated in these impaired responses and may be responsible for increased exacerbations and immunopathology of asthma. ObjectiveCharacterize the single-cell response to Poly I:C of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA). MethodsTwo complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA and healthy controls (HC). Poly I:C and unstimulated cells were analyzed in this study. ResultsPBMCs (n=9,414) from five SA (n=6,099) and three HC (n=3,315) were profiled using scRNA-Seq. Six main cell subsets, including CD4+ T cells, CD8+ T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4+ T cells were the main cell type and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression in unstimulated cells. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. Additional analyses to identify core regulators of the enhanced interferon response in SA identified IRF1, STAT1, IRF7, STAT2, and IRF9. CyTOF profiling of Poly I:C and unstimulated PBMCs (n=120,000) from the same individuals (SA=4; HC=2) demonstrated higher numbers of CD8+ effector cells and Th1 CD4+ T cells in unstimulated conditions, followed by a decrease of these two cell subsets after poly I:C stimulation. ConclusionSingle-cell profiling of PBMCs with scRNA-seq and CyTOF in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8+ effector cells and Th1 cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in severe asthma. Key Messages- Single-cell RNA sequencing identified a pro-inflammatory status in unstimulated PBMCs of severe asthmatics. - Mass cytometry identified quantitative differences in CD8+ effector cells and Th1 cells of severe asthmatics. - The response to Poly I:C stimulation, an interferon agonist, was not impaired in a subgroup of patients with severe asthma. Capsule summarySingle-cell profiling of PBMCs in severe asthmatics characterized gene expression responses to an interferon agonist and quantitative differences in distinct cell populations. Comprehensive single-cell immune may help identify key cell features responsible for asthma heterogeneity.

BackgroundMetagenomic sequencing of respiratory microbial communities for etiologic pathogen identification in pneumonia may help overcome the limitations of current culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore sequencing of respiratory samples for severe pneumonia diagnosis. Methods and FindingsWe conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirate samples (ETAs) and applied a microbial DNA enrichment method prior to performing metagenomic sequencing with the Oxford Nanopore MinION device. We compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. In nine culture-positive cases, Nanopore revealed communities with low alpha diversity and high abundance of the bacterial (n=8) or fungal (n=1) species isolated by clinical cultures. In four culture-positive cases with resistant organisms, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance identified by clinical antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and airway colonization by Candida species in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in the three other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. ConclusionWe demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures and clinically actionable information offered from the sequencing profiles of culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

The prevalence, inter-relationships, and longitudinal behavior of type 1 (T1) and type 2 (T2) immune responses in asthma are uncertain, as is the role of viruses as determinants of these responses. Here, we performed whole transcriptome network analysis on sputum cells collected from Severe Asthma Research Program (SARP)-3 patients before and after treatment with intramuscular corticosteroid and again at 1 and 3-year follow-up visits. We used network analysis to analyze whole-transcriptome gene expression and metagenomic analysis of these RNA-seq data to detect viruses. We identified T1 and T2 airway networks, the expression of which showed that 26% and 44% of patients had T1-high and T2-high asthma at baseline, respectively. Asthma severity outcomes were worse in T2-high asthma than in T1-high asthma and most severe in the subgroup of patients (14%) with combined T1- and T2-high disease. Corticosteroid treatment suppressed T2 but not T1 gene expression, and corticosteroid-associated improvements in FEV1 occurred only in patients with T1-L/T2-H disease and not in T1-H/T2-H patients. Although T1 and T2 inflammation at baseline was a significant predictor of T1 and T2 inflammation at follow-up visits, most patients had variable rather than persistent expression of T1 and T2 network genes. Viral metagenomic analyses uncovered that 24% of asthma sputum samples tested positive for a virus and high viral carriage was associated with an 11-fold increased risk of T1-high disease. Together our results uncover a relatively high burden of T1-high and T1/T2-high disease subtypes in severe asthma, which are corticosteroid-resistant and manifest with sub-clinical viral infection.

The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). Here we report a novel disease association of a SNP rs9984273, which is situated in the interferon alpha/beta receptor (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, lower IFN-gamma and IL-6 levels and less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database, and better outcome in interferon (IFN) beta treated patients with ARDS. Thus, the distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signalling and glucocorticoids. One-Sentence SummarySingle nucleotide polymorphism in interferon receptor contributes to corticosteroid response and outcome in ARDS and COVID-19

BackgroundFunctional or compositional perturbations of the microbiome can occur at different sites of the body and this dysbiosis has been linked to various diseases. Changes in the nasopharyngeal microbiome are associated to patients susceptibility to multiple viral infections, including COVID-19, supporting the idea that the nasopharynx may be playing an important role in health and disease. Most studies on the nasopharyngeal microbiome have focused on a specific component in the lifespan, such as infanthood or the elderly, or have other limitations such as low sample sizes. Therefore, detailed studies analyzing the age- and sex-associated changes in the nasopharyngeal microbiome of healthy people across their whole life are essential to understand the relevance of the nasopharynx in the pathogenesis of multiple diseases, particularly viral infections such as COVID-19. Results120 nasopharyngeal samples from healthy subjects of all ages and both sexes were analyzed by 16s rRNA sequencing. Nasopharyngeal bacterial alpha diversity did not vary in any case between age or sex groups. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant phyla in all the age groups, with several sex-associated differences probably due to the different levels of sex hormones between both sexes. Acinetobacter, Brevundimonas, Dolosigranulum, Finegoldia, Haemophilus, Leptotrichia, Moraxella, Peptoniphilus, Pseudomonas, Rothia, and Staphylococcus were the only 11 bacterial genera that presented significant age-associated differences. Other bacterial genera such as Anaerococcus, Burkholderia, Campylobacter, Delftia, Prevotella, Neisseria, Propionibacterium, Streptococcus, Ralstonia, Sphingomonas, and Corynebacterium appeared in the population with a very high frequency, suggesting that their presence might be biologically relevant. ConclusionsIn contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains very stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated changes in taxonomic composition were observed at phylum, family, and genus levels, as well as several sex-associated changes probably due to the different levels of sex hormones present in both sexes at certain ages. Our results provide a complete and valuable dataset that will be useful for future research aiming for studying the relationship between changes in the nasopharyngeal microbiome and susceptibility to or severity of multiple diseases, including COVID-19.

Asthma-COPD overlap (ACO) is a common clinical syndrome, yet there is no single objective definition. We hypothesized that Immunoglobulin E measurements could be used to refine the definition of ACO. In baseline plasma samples from 2870 subjects in the COPDGene Study, we measured total IgE levels and specific IgE levels to six common allergens. Compared to usual COPD, subjects with ACO had higher total IgE levels (median 67.0 vs 42.2 IU/ml) and more frequently had at least one positive specific IgE (43.5 vs 24.5%). We previously used a strict definition of ACO in subjects with COPD, based on self-report of a doctors diagnosis of asthma before the age of 40. This strict ACO definition was refined by the presence of atopy, determined by total IgE >100 IU/ml or at least one positive specific IgE, as was a broader definition of ACO based on any asthma history. Subjects will all three ACO definitions were younger (mean age 60.0-61.3), were more commonly African American (36.8-44.2%), had a higher exacerbation frequency (1.0-1.2 in the past year), and had more airway wall thickening on quantitative analysis of chest CT scans. Among subjects with clinical ACO, 37-46% did not have atopy; these subjects had more emphysema on chest CT scan. Based on associations with exacerbations and CT airway disease, IgE did not clearly improve the clinical definition of ACO. However, IgE measurements could be used to subdivide subjects with atopic and non-atopic ACO, who might have different biologic mechanisms and potential treatments.

To investigate whether the 21q21 region may contain a quantitative trait locus (QTL) with pleiotropic effect on % predicted FEV1 (forced expiratory volume in 1 second) and SPTQ (number of positive skin test responses to 11 allergens), in 295 EGEA families ascertained through asthmatic probands, we conducted a bivariate linkage analysis using two approaches: (1) a bivariate variance components (VC) analysis and (2) A combined principal components (CPC) analysis, with 13 microsatellites covering the whole chromosome 21. To identify the genetic variants associated with these traits, we performed family-based association analysis (FBAT) for the second principal component (PC2) using two microsatellites and 27 SNPs belonging to three candidate genes, located in the vicinity of the linkage peak. Univariate linkage analyses showed suggestive evidence of linkage to %FEV1 and SPTQ at two positions. Bivariate VC linkage analysis of these phenotypes led to an increase in linkage signals as compared to univariate analysis at the same positions. The peaks obtained by the CPC led to similar results as the full bivariate VC approach; evidence for linkage mainly coming from PC2. The strongest association signal, using single marker analysis for PC2, was obtained with D21S1252 (p=0.003 for global test; p=0.004 for allele 2 and p=0.001 for allele 11) and rs2834213 of IFNGR2 (p=0.003), these two loci being 3 Mb apart. Multi-marker analysis further strengthened this finding. These results indicate that at least two genetic factors may be involved in SPTQ and %FEV1 variability but further genotyping is needed to better understand these findings.