Abstract Background Cystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to chronic infection and mucus retention in the lungs, with lung function gradually deteriorating through recurrent pulmonary exacerbations (PEx). Virulence factors (VFs) of Pseudomonas aeruginosa and Staphylococcus aureus are thought to contribute to pulmonary exacerbations. Our study objective was to identify VF genes related to PEx, high Pseudomonas abundance, and high Staphylococcus abundance in persons with CF (pwCF). Methods This was an ancillary study of pwCF treated with IV antibiotics for PEx between 2016-2020 at Children’s National Hospital. Using shotgun metagenomics and ShortBRED, we identified bacterial VF genes and used DESeq2 to determine differential expression of VF genes across comparators. Results Twenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years, 41% female, 59% white, 36% Hispanic, and 45% had an F508del homozygous CFTR mutation. Minimal differences in VF gene abundance were identified across clinical state. The most differentially increased VF genes found in Pseudomonas high samples were associated with an aminotransferase (log2FC 25.9), flagellar biosynthesis (log2FC 8.3), and type VI secretion systems (log2FC 8.2). The most differentially increased VF genes found in Staphylococcus high samples were an exotoxin (log2FC 26.7), macrolide phosphotransferase (log2FC 25.8), pathogenicity island proteins (log2FC 25.2 and 24.7), and VOC family proteins (log2FC 24.8). Conclusions These findings demonstrate that specific VFs associated with immune modulation, motility secretion systems, bacterial motility, and antibiotic resistance are related to P. aeruginosa and S. aureus abundance, providing potential targets for more personalized antimicrobial interventions.

Bacterial extracellular vesicles (EVs) are important mediators of host infection. People with cystic fibrosis (CF) often suffer from chronic infection with Pseudomonas aeruginosa, an opportunistic pathogen. However, the relative abundance of P. aeruginosa is not associated with the onset of increased pulmonary symptoms, known as a pulmonary exacerbation. We hypothesized that the cargo of P. aeruginosa EVs is different at times of baseline wellness and pulmonary exacerbation onset in persons with CF. This is the first study to characterize and compare P. aeruginosa EVs at these two time points, using a novel series of steps to isolate the P. aeruginosa EVs directly from the sputum of persons with CF. Our study found a differential packaging of P. aeruginosa proteins at baseline wellness and pulmonary exacerbation, with six proteins being more frequently present in pulmonary exacerbation samples. In addition, we were able to demonstrate that the P. aeruginosa EVs isolated from the sputum of persons with CF at the time of pulmonary exacerbation induced an inflammatory response in CF human bronchial epithelial cells. These data, while preliminary, support the clinical relevance of P. aeruginosa EVs in influencing gene regulation and lung inflammation and immunity in persons with CF.

Background Respiratory syncytial virus (RSV) bronchiolitis contributes to a large morbidity and mortality burden globally. While emerging evidence suggests that airway microRNA (miRNA) is involved in the pathobiology of RSV infection, its role in the disease severity remains unclear.Methods In this multicentre prospective study of infants (aged<1 year) hospitalised for RSV bronchiolitis, we sequenced the upper airway miRNA and messenger RNA (mRNA) at hospitalisation. First, we identified differentially expressed miRNAs (DEmiRNAs) associated with higher bronchiolitis severity—defined by respiratory support (eg, positive pressure ventilation, high-flow oxygen therapy) use. We also examined the biological significance of miRNAs through pathway analysis. Second, we identified differentially expressed mRNAs (DEmRNAs) associated with bronchiolitis severity. Last, we constructed miRNA–mRNA coexpression networks and determined hub mRNAs by weighted gene coexpression network analysis (WGCNA).Results In 493 infants hospitalised with RSV bronchiolitis, 19 DEmiRNAs were associated with bronchiolitis severity (eg, miR-27a-3p, miR-26b-5p; false discovery rate<0.10). The pathway analysis using miRNA data identified 1291 bronchiolitis severity-related pathways—for example, regulation of cell adhesion mediated by integrin. Second, 1298 DEmRNAs were associated with bronchiolitis severity. Last, of these, 190 DEmRNAs were identified as targets of DEmiRNAs and negatively correlated with DEmiRNAs. By applying WGCNA to DEmRNAs, four disease modules were significantly associated with bronchiolitis severity—for example, microtubule anchoring, cell-substrate junction. The hub genes for each of these modules were also identified—for example, PCM1 for the microtubule anchoring module, LIMS1 for the cell-substrate junction module.Conclusions In infants hospitalised for RSV bronchiolitis, airway miRNA–mRNA coexpression network contributes to the pathobiology of bronchiolitis severity.

Bronchiolitis, a viral lower respiratory infection, is the leading cause of infant hospitalization, which is associated with an increased risk for developing asthma later in life. Bronchiolitis can be caused by several respiratory viruses, such as respiratory syncytial virus (RSV), rhinovirus (RV), and others. It can also be caused by a solo infection (e.g., RSV- or RV-only bronchiolitis) or co-infection with two or more viruses. Studies have shown viral etiology-related differences between RSV- and RV-only bronchiolitis in the immune response, human microRNA (miRNA) profiles, and dominance of certain airway microbiome constituents. Here, we identified bacterial small RNAs (sRNAs), the prokaryotic equivalent to eukaryotic miRNAs, that differ between infants of the 35th Multicenter Airway Research Collaboration (MARC-35) cohort with RSV- versus RV-only bronchiolitis. We first derived reference sRNA datasets from cultures of four bacteria known to be associated with bronchiolitis (i.e., Haemophilus influenzae, Moraxella catarrhalis, Moraxella nonliquefaciens, and Streptococcus pneumoniae). Using these reference sRNA datasets, we found several sRNAs associated with RSV- and RV-only bronchiolitis in our human nasal RNA-Seq MARC-35 data. We also determined potential human transcript targets of the bacterial sRNAs and compared expression of the sRNAs between RSV- and RV-only cases. sRNAs are known to downregulate their mRNA target, we found that, compared to those associated with RV-only bronchiolitis, sRNAs associated with RSV-only bronchiolitis may relatively activate the IL-6 and IL-8 pathways and relatively inhibit the IL-17A pathway. These data support that bacteria may be contributing to inflammation differences seen in RSV- and RV-only bronchiolitis, and for the first time indicate that the potential mechanism in doing so may be through bacterial sRNAs.

Background:Maternal body size, nutrition, and hyperglycemia contribute to neonatal body size and composition. There is little information on maternal-fetal transmission of messages which influence fetal growth. We analyzed adipocyte-derived small extracellular vesicular (ADsEV) microRNAs in maternal and cord blood to explore their adipogenic potential. Methods:We studied 127 mother-neonate pairs (51 lean and 76 adipose neonates, in 68 NGT and 59 GDM pregnancies). Adiposity refers to the highest tertile (T3) of sum of skinfolds in neonates of normal glucose tolerant (NGT) mothers, lean to the to lowest tertile (T1). ADsEV miRNAs from maternal and cord blood samples were profiled on Agilent 8*60K microarray. Differential expression (DE) of ADsEV miRNAs in adipose vs. lean neonates was studied before and after adjustment for maternal gestational diabetes mellitus (GDM), adiposity, and vitamin B12-folate status. Results:Multiple miRNAs were common in maternal and cord blood and positively correlated. We identified 24 maternal and 5 cord blood miRNAs differentially expressed (p ≤ 0.1) in the adipose neonate group, and 19 and 26 respectively, in the adjusted analyses. Even though DE miRNAs were different in maternal and cord blood, they targeted similar adipogenic pathways (e.g., the forkhead box O (FOXO) family of transcription factors, mitogen-activated protein kinase (MAPK) pathway, transforming growth factor beta (TGF-β) pathway). Maternal GDM and adiposity were associated with many DE ADsEV miRNAs. Conclusion:Our results suggest that the ADsEV miRNAs in mothers are potential regulators of fetal adiposity. The expression and functionality of miRNAs appears to be influenced by maternal adiposity, hyperglycemia, and micronutrient status during pregnancy.

Background Infants with bronchiolitis are at high risk for developing childhood asthma. While genome-wide association studies suggest common genetic susceptibilities between these conditions, the mechanisms underlying the link remain unclear. Objective Through integrated genetics-metabolomics analysis in this high-risk population, we sought to identify genetically driven metabolites associated with asthma development and genetic loci associated with both these metabolites and asthma susceptibility. Methods In a multicenter prospective cohort study of infants hospitalized for bronchiolitis, we profiled the nasopharyngeal metabolome and genotyped the whole genome at hospitalization. We identified asthma-related metabolites from 283 measured compounds and conducted metabolite quantitative trait loci (mtQTL) analyses. We further examined the mtQTL associations by testing shared genetic loci for metabolites and asthma using colocalization analysis and the concordance between the loci and known asthma-susceptibility genes. Results In 744 infants hospitalized with bronchiolitis, 28 metabolites (e.g., docosapentaenoate [DPA], 1,2-dioleoyl-sn-glycero-3-phosphoglycerol, sphingomyelin) were associated with asthma risk. A total of 349 loci were associated with these metabolites—161 for non-Hispanic white, 120 for non-Hispanic black, and 68 for Hispanics. Of these, there was evidence for 30 shared loci between 16 metabolites and asthma risk (colocalization posterior probability ≥0.5). The significant SNPs within loci were aligned with known asthma-susceptibility genes (e.g., ADORA1 , MUC16 ). Conclusion The integrated genetics-metabolomics analysis identified genetically driven metabolites during infancy that are associated with asthma development and genetic loci associated with both these metabolites and asthma susceptibility. Identifying these metabolites and genetic loci should advance research into the functional mechanisms of the infant bronchiolitis-childhood asthma link.

Bronchiolitis is the most common lower respiratory infection in infants, yet its pathobiology remains unclear. Here we present blood DNA methylation data from 625 infants hospitalized with bronchiolitis in a 17-center prospective study, and associate them with disease severity. We investigate differentially methylated CpGs (DMCs) for disease severity. We characterize the DMCs based on their association with cell and tissues types, biological pathways, and gene expression. Lastly, we also examine the relationships of severity-related DMCs with respiratory and immune traits in independent cohorts. We identify 33 DMCs associated with severity. These DMCs are differentially methylated in blood immune cells. These DMCs are also significantly enriched in multiple tissues (e.g., lung) and cells (e.g., small airway epithelial cells), and biological pathways (e.g., interleukin-1-mediated signaling). Additionally, these DMCs are associated with respiratory and immune traits (e.g., asthma, lung function, IgE levels). Our study suggests the role of DNA methylation in bronchiolitis severity.

Background Severe bronchiolitis ( i.e. bronchiolitis requiring hospitalisation) during infancy is a major risk factor for childhood asthma. However, the exact mechanism linking these common conditions remains unclear. We examined the longitudinal relationship between nasal airway miRNAs during severe bronchiolitis and the risk of developing asthma. Methods In a 17-centre prospective cohort study of infants with severe bronchiolitis, we sequenced their nasal microRNA at hospitalisation. First, we identified differentially expressed microRNAs (DEmiRNAs) associated with the risk of developing asthma by age 6 years. Second, we characterised the DEmiRNAs based on their association with asthma-related clinical features, and expression level by tissue and cell types. Third, we conducted pathway and network analyses by integrating DEmiRNAs and their mRNA targets. Finally, we investigated the association of DEmiRNAs and nasal cytokines. Results In 575 infants (median age 3 months), we identified 23 DEmiRNAs associated with asthma development ( e.g. hsa-miR-29a-3p; false discovery rate (FDR) <0.10), particularly in infants with respiratory syncytial virus infection (FDR for the interaction <0.05). These DEmiRNAs were associated with 16 asthma-related clinical features (FDR <0.05), e.g. infant eczema and corticosteroid use during hospitalisation. In addition, these DEmiRNAs were highly expressed in lung tissue and immune cells ( e.g. T-helper cells, neutrophils). Third, DEmiRNAs were negatively correlated with their mRNA targets ( e.g. hsa-miR-324-3p/ IL13 ), which were enriched in asthma-related pathways (FDR <0.05), e.g. toll-like receptor, PI3K-Akt and FcɛR signalling pathways, and validated by cytokine data. Conclusion In a multicentre cohort of infants with severe bronchiolitis, we identified nasal miRNAs during illness that were associated with major asthma-related clinical features, immune response, and risk of asthma development.

Aging is a risk factor for many non-communicable diseases such as cardiovascular and neurodegenerative diseases. Extracellular vesicles and particles (EVP) carry microRNAs that may play a role in age-related diseases and may induce oxidative stress. We hypothesized that aging could impact EVP miRNA and impair redox homeostasis, contributing to chronic age-related diseases. Our aims were to investigate the microRNA profiles of circulating total EVPs from aged and young adult animals and to evaluate the pro- and antioxidant machinery in circulating total EVPs. Plasma from 3- and 21-month-old male Wistar rats were collected, and total EVPs were isolated. MicroRNA isolation and microarray expression analysis were performed on EVPs to determine the predicted regulation of targeted mRNAs. Thirty-one mature microRNAs in circulating EVPs were impacted by age and were predicted to target molecules in canonical pathways directly related to cardiovascular diseases and oxidative status. Circulating total EVPs from aged rats had significantly higher NADPH oxidase levels and myeloperoxidase activity, whereas catalase activity was significantly reduced in EVPs from aged animals. Our data shows that circulating total EVP cargo-specifically microRNAs and oxidative enzymes-are involved in redox imbalance in the aging process and can potentially drive cardiovascular aging and, consequently, cardiac disease.

Background Maternal body size, nutrition, and hyperglycemia contribute to neonatal body size and composition. There is little information on maternal-fetal transmission of messages which influence fetal growth. We analyzed adipocyte-derived small extracellular vesicular (ADsEV) microRNAs in maternal and cord blood to explore their adipogenic potential.Methods We studied 127 mother-neonate pairs (51 lean and 76 adipose neonates, in 68 NGT and 59 GDM pregnancies). Adiposity refers to highest tertile (T3) of sum of skinfolds in neonates of normal glucose tolerant (NGT) mothers, lean to lowest tertile (T1). ADsEV miRNAs from maternal and cord blood samples were profiled on Agilent 8*60K microarray. Differential expression (DE) of ADsEV miRNAs in adipose vs. lean neonates was studied before and after adjustment for maternal gestational diabetes mellitus (GDM), adiposity, and vitamin B12-folate status.Results Multiple miRNAs were common in maternal and cord blood and positively correlated. We identified 24 maternal and 5 cord blood miRNAs differentially expressed (p≤0.1) in the adipose neonate group, and 19 and 26 respectively, in the adjusted analyses. Even though DE miRNAs were different in maternal and cord blood, they targeted similar adipogenic pathways (e.g., the forkhead box O (FOXO) family of transcription factors, mitogen-activated protein kinase (MAPK) pathway, transforming growth factor beta (TGF-β) pathway). Maternal GDM and adiposity were associated with many DE ADsEV miRNAs.Conclusion Our results suggest that the DE ADsEV miRNAs in mothers of adipose neonates are potential regulators of fetal adiposity. Further, the composition and functionality of miRNAs may be influenced by maternal hyperglycemia, adiposity, and micronutrient status during pregnancy.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe study was funded by InDiaGDM grant of the Department of Biotechnology, New Delhi, India (BT/IN/Denmark/02/CSY/2014) and National Institute of Health (NIH), the U.S government (R21HD094127-01).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics committee of King Edward Memorial Hospital Research Centre, Pune, India gave ethical approval for this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.* PMNS : Pune Maternal Nutrition Study PCS : Pune Children Study SSF : Sum of skin folds EV : Extracellular vesicles ADsEV : Adipocyte derived small extracellular vesicles GDM : Gestational diabetes mellitus NGT : Normal glucose tolerant FABP4 : Fatty acid binding protein 4 DE : miRNA Differentially expressed microRNA

Abstract Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis‐resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin‐β5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial‐to‐mesenchymal transition (EnMT). We found that elevated integrin‐β5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin‐β5 and its heterodimerization partner integrin‐αν and showed accelerated barrier formation. Inhibition of integrin‐ανβ5 in vitro partially blocked transforming growth factor (TGF)‐β1‐induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin‐ανβ5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF‐β1. In vivo, blocking of integrin‐ανβ5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin‐ανβ5 in lung endothelial survival and migration, but also a partial contribution to TGF‐β1‐induced EnMT gene expression. Our results suggest that integrin‐ανβ5 is required for physiologic function of ECs and lung vascular homeostasis.

Aging is associated with adipose tissue dysfunction and is recognized as a risk factor for shortened life span. Considering that in vitro findings have shown the involvement of microRNA in extracellular vesicles and particles (EVPs) on senescence, we hypothesized that circulating EVPs derived from adipocytes can be involved in the aging process via their microRNA cargo. We aimed to determine the microRNA profiles of circulating EVPs derived from adipocytes (FABP4+) from aged and young adult animals and to perform in silico prediction of their downstream signaling effects. Plasma was obtained from Wistar rats (3 and 21 months old), and adipocyte-derived EVPs were isolated using the commercially available kit. Fatty acid-binding protein 4 (FABP4) was used for adipocyte-derived EVPs isolation; microRNA isolation and microarray expression analysis were performed. The analysis revealed 728 miRNAs, 32 were differentially between groups (p < 0.05; fold change ≥ |1.1|), of which 15 miRNAs were upregulated and 17 were downregulated in circulating EVPs from aged animals compared to young adults. A conservative filter was applied, and 18 microRNAs had experimentally validated and highly conserved predicted mRNA targets, with a total of 2,228 mRNAs. Canonical pathways, disease and functions, and upstream regulator analyses were performed using IPA-QIAGEN, allowing a global and interconnected evaluation. IPA categories impacted negatively were cell cycle, cellular development, cellular growth and proliferation, and tissue development, while those impacted positively were “digestive system cancer” and “endocrine gland tumor.” Interestingly, the upregulated miR-15-5p targets several cyclins, such as CCND1 and CCND2, and miR-24-3p seems to target CDK4 (cyclin-dependent kinase 4); then potentially inhibiting their expression, both miRNAs can induce a negative regulation of cell cycle progression. In contrast, silencing of negative cell cycle checkpoint regulators, such as p21 and p16, can be predicted, which can induce impairment in response to genotoxic stressors. In addition, predicted targets, such as SMAD family members, seem to be involved in the positive control of digestive and endocrine tumors. Taken together, this exploratory study indicates that miRNA signature in circulating adipocyte-derived EVPs may be involved with the double-edged sword of cellular senescence, including irreversible proliferation arrest and tissue-dependent cancer, and seems to be suitable for further validation and confirmatory studies.

Respiratory syncytial virus (RSV) bronchiolitis is not only the leading cause of hospitalization in U.S. infants, but also a major risk factor for asthma development. While emerging evidence suggests clinical heterogeneity within RSV bronchiolitis, little is known about its biologically-distinct endotypes. Here, we integrated clinical, virus, airway microbiome (species-level), transcriptome, and metabolome data of 221 infants hospitalized with RSV bronchiolitis in a multicentre prospective cohort study. We identified four biologically- and clinically-meaningful endotypes: A) clinical classic microbiome M. nonliquefaciens inflammation IFN-intermediate , B) clinical atopic microbiome S. pneumoniae / M. catarrhalis inflammation IFN-high , C) clinical severe microbiome mixed inflammation IFN-low , and D) clinical non-atopic microbiome M.catarrhalis inflammation IL-6 . Particularly, compared with endotype A infants, endotype B infants—who are characterized by a high proportion of IgE sensitization and rhinovirus coinfection, S. pneumoniae/M. catarrhalis codominance, and high IFN-α and -γ response—had a significantly higher risk for developing asthma (9% vs. 38%; OR, 6.00: 95%CI, 2.08–21.9; P = 0.002). Our findings provide an evidence base for the early identification of high-risk children during a critical period of airway development.

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells (ECs) contribute to severe pulmonary arterial hypertension (PAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). The remodelled pulmonary arteries of PAH patients harboured CD117+ ECs. Rat lung CD117+ ECs underwent four generations of clonal expansion to enrich hyperproliferative ECs. The resulting clonally enriched ECs behaved like ECs, as measured by in vitro and in vivo angiogenesis assays. The same primitive ECs showed a limited ability for mesenchymal lineage differentiation. Endothelial differentiation and function were enhanced by blocking TGF-β signalling, promoting bone morphogenic protein (BMP) signalling. The transplantation of the EC clones caused arterio-occlusive PH in rats exposed to chronic hypoxia. These EC clones engrafted in the pulmonary arteries. Yet cessation of chronic hypoxia promoted lung cell apoptosis and resolution of vascular lesions. In conclusion, this is to the best of our knowledge, the first report that clonally enriched primitive ECs promote occlusive pulmonary arteriopathy and severe PH. These primitive EC clones further give rise to cells of endothelial and mesenchymal lineage as directed by BMP and TGF-β signaling.

Background Epigenetic changes in visceral adipose tissue (VAT) with obesity and their effects on gene expression are poorly understood, especially during emergent obesity in youth. The current study tested the hypothesis that methylation and gene expression profiles of key growth factor and inflammatory pathways are altered in VAT from obese compared to non-obese youth. Methods VAT samples from adolescent females grouped as Lean (L; n = 15; age = 15 ± 3 years, BMI = 21.9 ± 3.0 kg/m 2 ) or Obese (Ob; n = 15, age = 16 ± 2 years, BMI = 45.8 ± 9.8 kg/m 2 ) were collected. Global methylation (n = 20) and gene expression (N = 30) patterns were profiled via microarray and interrogated for differences between groups by ANCOVA (p < 0.05), followed by biological pathway analyses. Results Overlapping differences in methylation and gene expression in 317 genes were found in VAT from obese compared to lean groups. PI3K/AKT Signaling (p = 1.83 × 10 −6 ; 11/121 molecules in dataset/pathway) was significantly overrepresented in Ob VAT according to pathway analysis. Upregulations in the PI3K/AKT signaling pathway mRNAs TFAM (p = 0.03; fold change = 1.8) and PPP2R5C (p = 0.03, FC = 2.6) were confirmed via qRT-PCR. Conclusion Our analyses show obesity-related differences in DNA methylation and gene expression in visceral adipose tissue of adolescent females. Specifically, we identified methylation site/gene expression pairs differentially regulated and mapped these differences to pathways including PI3K/AKT signaling, suggesting that PI3K/AKT signaling pathway dysfunction in obesity may be driven in part by changes in DNA methylation.

To the Editor, Bronchiolitis is the leading cause of hospitalizations in U.S. infants. In addition to the acute morbidity, cohort studies have also shown that 30%‐40% of infants hospitalized for bronchiolitis (severe bronchiolitis) develop childhood asthma. Particularly, early life infection with rhinovirus (RV)—the second most common pathogen of bronchiolitis—is associated with an increased risk of childhood asthma. Yet, the mechanism through which RV raises asthma risk in infants is largely unknown. Experimental models and human (cross‐sectional and retrospective) studies have reported that RV infection may induce type 2 cytokines (eg, interleukin [IL]‐4, IL‐5, IL‐13, thymic stromal lymphopoietin [TSLP]) and that the levels of these cytokines are elevated in the asthmatic airway. However, no prospective study has investigated the longitudinal relation of type 2 airway inflammation in children—let alone infants with bronchiolitis—to the development of childhood asthma. To address this knowledge gap, we prospectively examined the association of nasopharyngeal cytokines in infants with RV bronchiolitis with the risk of developing childhood asthma, by using data from a multicenter cohort of infants with severe bronchiolitis. Details of the study design, samples, measurement, and analysis may be found in the Methods S1). Briefly, this multicenter prospective cohort study, the 35th Multicenter Airway Research Collaboration (MARC‐35), enrolled 1016 infants (aged < 12 months) hospitalized for bronchiolitis at 17 sites across 14 US states (Table S1) during the 2011‐2014 winter seasons. Bronchiolitis was defined by the American Academy of Pediatrics guidelines. In addition to the phenotypic data measurement via structured interview and medical record review, nasopharyngeal airway samples were collected within 24 hours of hospitalization using a standardized protocol. Levels of 10 nasopharyngeal cytokines (IL‐4, IL‐5, IL‐10, IL‐12, IL‐13, IL‐25, IL‐33, interferon [IFN]‐β, macrophage inflammatory protein [MIP]‐1α, and TSLP) were quantified using electrochemiluminescence immunoassays. Respiratory viruses were tested using real‐time PCR assays for RV and respiratory syncytial virus (RSV). To quantify the relative RV genomic load, cycle threshold (CT) values—the number of amplification cycles needed for a positive PCR test result—were used. The primary outcome was asthma at age 4 years based on a commonly used epidemiologic definition—that is, physician diagnosis of asthma plus either asthma medication use or asthma‐related symptoms in the past year. For the current study, we analyzed 132 infants with RV bronchiolitis who underwent nasopharyngeal cytokine measurement. To examine the association of exposures (cytokine levels and RV genomic load) with asthma, we used generalized linear mixed‐effects models, adjusting for potential confounders (age, sex, parental history of asthma, breathing problems prior to enrollment, IgE sensitization [aeroallergens or food], and virology [solo RV, RV/RSV coinfection]) and hospital‐level clustering. As the models indicated statistically significant virology‐exposure interactions, we then stratified the analysis by virology. Of 132 infants with severe RV bronchiolitis, the median age was 4 (IQR 2‐6) months, 64% were male, 40% were non‐Hispanic white, and 55% had RSV coinfection. Asthma was observed in 30% of children at age 4 years (Tables 1 and S2). There were statistically significant interactions between virology and six cytokine levels (IL‐4, IL‐5, IL‐13, IFN‐β, MIP‐1α, and TSLP) on the asthma risk (Pinteraction < 0.05), indicating that exposure‐asthma associations differ between solo RV infection and RV/RSV coinfection. Indeed, in infants with solo RV infection, the cytokine levels (IL‐4, IL‐5, and TSLP) and RV genomic load significantly differed between those with and without asthma (unadjusted P < 0.05; Table S3) while there were no differences in those with RV/RSV (unadjusted P > 0.05). Heatmap (Figure S1) also showed that distributions of these type 2 cytokines differed by virology and outcome. In the adjusted analysis (Figures 1 and S2), only infants with solo RV bronchiolitis had significant associations of higher type 2 cytokine levels (IL‐4, IL‐5, IL‐13, and TSLP) with an increased risk of asthma (adjusted P < 0.05). In the sensitivity analysis using normalized cytokine levels and the subgroup analysis excluding infants with a breathing problem prior to enrollment, the results were similar (Figures S3 and S4). These findings are concordant with previous cross‐sectional and retrospective studies that suggested potential interrelations between RV infection, type 2 cytokines (eg, IL‐4, IL‐5, IL‐13, TSLP), and asthmatic airway inflammation. The current prospective study builds on these earlier reports and extends them by demonstrating the prospective association between type 2 cytokine levels in the airway of infants with solo RV bronchiolitis and the risk of developing asthma. The mechanisms underlying these findings warrant further investigation. It is possible that severe RV infection is an early marker of TH2 bias in predisposed infants, which leads to augmented RV infection and replication. Yet, our data also showed no significant correlations between serum total IgE levels and nasopharyngeal cytokine levels (Table S4). Alternatively, the association may be DOI: 10.1111/all.13723