Biology subjects

Gern, J. E.

Publications and source records attributed to Gern, J. E..

11 recordsLinked to original sources

bioRxiv2026

Rhinovirus Infection of Human Airway Epithelial Cells Stimulates Neutrophil Superoxide

Background: Neutrophils migrate to the respiratory epithelium during rhinovirus (RV) infection, but whether RV directly activates neutrophils or whether activation arises from epithelial responses remains unclear. Objective: To determine whether RV directly activates human neutrophils or indirectly activates them through RV infected airway epithelial cells. Methods: Human neutrophils were exposed to purified RV, and activation was assessed by myeloperoxidase activity, CD11b/CD66b expression, and reactive oxygen species (ROS) production. Neutrophils were co-cultured with RV-infected air liquid interface (ALI) bronchial epithelium evaluated for effects on epithelial integrity. At 48 to 72 hours post-infection, we used live-cell imaging to measure ROS responses and neutrophil-tracking assays to measure migration velocity and ROS responses to basolateral supernatants, clarified apical washes, and apical debris from RV-infected epithelial cells. Plaque assays were used to assess for RV infectivity after incubation with apical neutrophils. Results: RV did not directly activate neutrophils. Adding neutrophils to the apical surface of airway epithelial cells increased neutrophil ROS production at 48 to 72 hrs post-infection but did not alter epithelial cytotoxicity. Conversely, basolateral supernatants from infected cells enhanced neutrophil migration but not ROS, consistent with cytokine-mediated priming. Clarified apical washes had no effect on migration or ROS. By contrast, apical debris from infected cultures triggered a robust ROS response. Basolateral fractions from medium control or RV infected cultures did not induce ROS. Infectivity is not impacted by apical neutrophil presence. Conclusion: RV does not directly activate neutrophils. Instead, RV infected epithelial cells release basolateral factors that enhance neutrophil migration, while apical debris from infected cells drives ROS activation. These findings indicate that epithelial injury, not RV itself, is the dominant trigger of neutrophil activation during infection.

immunology↗

bioRxiv2026

Exclusive liquid repellency isolation (ELRi) microliter scale isolation technique unmasks T-cell migration defects to CCL21 in pediatric asthma

Exclusive Liquid Repellency Isolation (ELRi) is a novel, chip-free platform designed for efficient microscale immune cell isolation. Leveraging the inherent exclusive liquid repellency (ELR) properties of polypropylene tubes with an oil overlay, ELRi ensures that small sample volumes, as low as 8 L, have no contact with container walls, preventing cell loss. Here we describe ELRi and demonstrate magnetic bead-based isolation of diverse immune cells, including T cells, and monocytes, from whole blood. The platforms utility is highlighted in its application to pediatric asthma research, where sample volume is highly restricted. It has been reported that the T cell homing receptor CCR7 is downregulated in cells from asthmatic patients, but a direct functional link to impaired cell migration remains unconfirmed (1, 2). Using ELRi-isolated cells, we provide the first functional evidence that T cells from asthmatic children exhibit significantly impaired chemotaxis toward the CCR7 ligand, CCL21. By simplifying RBC depletion and enabling such functional assays alongside RNA sequencing from the same tiny sample, ELRi overcomes the limitations of large-volume flow cytometry or cell-specific sorting methods. Easily integrated into lab workflows and scalable for various needs, ELRi facilitates more frequent, minimally invasive, and functionally informative immune profiling of restricted volume samples.

immunology↗

bioRxiv2025

Human coronavirus HKU1 neutralization by glycan receptor mimicry

Entry of seasonal human coronavirus HKU1 (HCoV-HKU1) into host cells is facilitated by sequential binding to sialoglycans and transmembrane serine protease 2 (TMPRSS2) receptors. However, the neutralizing capacity of antibodies disrupting these receptor interactions have not been examined. Here, we describe the isolation and characterization of a human monoclonal antibody (mAb) HKU1-2 that recognizes the HCoV-HKU1 spike protein and exhibits dose-dependent neutralization of the virus. Epitope mapping and structural analysis revealed that HKU1-2 mAb targets the sialoglycan binding site in the N-terminal domain of the spike protein. A cryo-electron microscopy (cryo-EM) structure of the spike-Fab complex further demonstrated the ability of HKU1-2 to mimic sialic acid binding thereby effectively blocking sialoglycan receptor engagement. HKU1-2 binding is primarily mediated by CDRH3 recognition of NTD residues K80 and W89 that are known to be critical for sialic acid engagement. Overall, our results demonstrate antibody recognition and neutralization of HCoV-HKU1 by receptor mimicry.

immunology↗

bioRxiv2024

Farm exposure is associated with human breast milk immune profile and microbiome

Prenatal and early life farm exposure, and breastfeeding, are associated with protection from allergic diseases. We hypothesize that farm exposure influences the human breast milk microbiome and immune proteins. The immune protein profiles and microbial communities of 152 human breast milk samples were compared among three maternal farm exposure groups (traditional agrarian, farm, and non-farm) in rural Wisconsin to identify signatures associated with farm status and atopic disease. We found significant differences between farm groups for 23 immune proteins (p-adj<0.05), microbiome diversity (p=2.2E-05), and microbiome richness (p=8.0e-06). Traditional agrarian human breast milk had the highest immune protein levels and microbiome diversity and richness, followed by farm and non-farm human breast milk. Furthermore, Gram-positive bacterial species correlated with IL-23 mediated signaling events (p-adj<1.0E-05). These data suggest that increased farm exposures promotes human breast milk that is more microbially-diverse and rich in immune-associated proteins, ultimately influencing immune development in the infant.

microbiology↗

bioRxiv2024

Single cell profiling to determine influence of wheeze and early-life viral infection on developmental programming of airway epithelium

AO_SCPLOWBSTRACTC_SCPLOWAlthough childhood asthma is in part an airway epithelial disorder, the development of the airway epithelium in asthma is not understood. We sought to characterize airway epithelial developmental phenotypes in those with and without recurrent wheeze and the impact of infant infection with respiratory syncytial virus (RSV). Nasal airway epithelial cells (NAECs) were collected at age 2-3 years from an a priori designed nested birth cohort of children from four mutually exclusive groups of wheezers/non-wheezers and RSV-infected/uninfected in the first year of life. NAECs were cultured in air-liquid interface differentiation conditions followed by a combined analysis of single cell RNA sequencing (scRNA-seq) and in vitro infection with respiratory syncytial virus (RSV). NAECs from children with a wheeze phenotype were characterized by abnormal differentiation and basal cell activation of developmental pathways, plasticity in precursor differentiation and a delayed onset of maturation. NAECs from children with wheeze also had increased diversity of currently known RSV receptors and blunted anti-viral immune responses to in vitro infection. The most dramatic changes in differentiation of cultured epithelium were observed in NAECs derived from children that had both wheeze and RSV in the first year of life. Together this suggests that airway epithelium in children with wheeze is developmentally reprogrammed and characterized by increased barrier permeability, decreased antiviral response, and increased RSV receptors, which may predispose to and amplify the effects of RSV infection in infancy and susceptibility to other asthma risk factors that interact with the airway mucosa. SUMMARYNasal airway epithelial cells from children with wheeze are characterized by altered development and increased susceptibility to RSV infection.

developmental biology↗

bioRxiv2023

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps

BackgroundThe airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. ObjectiveWe hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. MethodsEthmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. ResultsAnalysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. ConclusionsTogether, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma. KEY MESSAGESO_LIEpithelial mTORC1 activity is upregulated in CRSwNP. C_LIO_LImTOR regulates EpC cytokine generation. C_LIO_LIEpithelial metabolic reprograming correlates with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in asthma. C_LI CAPSULE SUMMARYmTORC1 mediates EpC cytokine generation in CRSwNP.

immunology↗

bioRxiv2020

Asthma-associated variants induce IL33 differential expression through a novel regulatory region

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated SNP, rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

genomics↗

bioRxiv2019

Club cell TRPV4 as a damage sensor driving lung allergic inflammation

Airway epithelium is the first body surface to contact inhaled irritants and report danger. We studied how epithelial cells recognize and respond to protease, which is a critical component of many allergens that provoke asthma. In a murine model, the aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp. elicited helper T (Th) cell-dependent lung eosinophilia. Bronchiolar club cells responded rapidly to Alp1 by coordinating the accumulation of allergic immune cells in the lung. Alp1 degraded bronchiolar cell junctions, and club cells within the bronchioles propagated this signal via calcium and calcineurin to incite inflammation. In two human cohorts, we linked fungal sensitization and asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 was also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium and calcineurin to initiate Th cell sensitization.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC=\"FIGDIR/small/773382v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (24K):\norg.highwire.dtl.DTLVardef@c03f95org.highwire.dtl.DTLVardef@18e54b6org.highwire.dtl.DTLVardef@15ba395org.highwire.dtl.DTLVardef@1509e5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2018

Bacterial competition mediated by siderophore production among the human nasal microbiota

Resources available in the human nasal cavity are limited. Therefore, to successfully colonize the nasal cavity, bacteria must compete for scarce nutrients. Competition may occur directly through interference (e.g., antibiotics) or indirectly by nutrient sequestration. To investigate the nature of nasal bacterial competition, we performed co-culture inhibition assays between nasal Actinobacteria and Staphylococcus spp. We found that Staphylococcus epidermidis isolates were sensitive to growth inhibition by Actinobacteria but Staphylococcus aureus isolates were resistant to inhibition. Among Actinobacteria, we observed that Corynebacterium spp. were variable in their ability to inhibit S. epidermidis. We sequenced the genomes of ten Corynebacterium spp. isolates, including three Corynebacterium propinquum that strongly inhibited S. epidermidis and seven other Corynebacterium spp. isolates that only weakly inhibited S. epidermidis. Using a comparative genomics approach, we found that the C. propinquum genomes were enriched in genes for iron acquisition and encoded a biosynthetic gene cluster (BGC) for siderophore production, absent in the non-inhibitory Corynebacterium spp. genomes. Using a chromeazurol S assay, we confirmed that C. propinquum produced siderophores. We demonstrated that iron supplementation rescued S. epidermidis from inhibition by C. propinquum, suggesting that inhibition was due to iron restriction through siderophore production. Using comparative metabolomics, we identified the siderophore produced by C. propinquum as dehydroxynocardamine. Finally, we confirmed that the dehydroxynocardamine BGC is expressed in vivo by analyzing human nasal metatranscriptomes from the NIH Human Microbiome Project.\n\nTogether, our results suggest that bacteria produce siderophores to compete for limited available iron in the nasal cavity and improve their fitness.\n\nIMPORTANCEWithin the nasal cavity, interference competition through antimicrobial production is prevalent. For instance, nasal Staphylococcus spp. strains can inhibit the growth of other bacteria through the production of nonribosomal peptides and ribosomally synthesized and post-translationally modified peptides. In contrast, bacteria engaging in exploitation competition modify the external environment to prevent competitors from growing, usually by depleting access to essential nutrients. As the nasal cavity is a nutrient limited environment, we hypothesized that exploitation competition occurs in this system. We determined that Corynebacterium propinquum produces an iron-chelating siderophore and is able to use this molecule to sequester iron and inhibit the growth of Staphylococcus epidermidis. Further, we found that the genes required for siderophore production are expressed in vivo. Thus, though siderophore production by bacteria is often considered a virulence trait, our work indicates that bacteria may produce siderophores to compete for limited iron in the human nasal cavity.

microbiology↗

bioRxiv2017

Non-parametric mixture models identify trajectories of childhood immune development relevant to asthma and allergy

Events in early life contribute to subsequent risk of asthma; however, the causes and trajectories of childhood wheeze are heterogeneous and do not always result in asthma. Similarly, not all atopic individuals develop wheeze, and vice versa. The reasons for these differences are unclear. Using unsupervised model-based cluster analysis, we identified latent clusters within a prospective birth cohort with deep immunological and respiratory phenotyping. We characterised each cluster in terms of immunological profile and disease risk, and replicated our results in external cohorts from the UK and USA. We discovered three distinct trajectories, one of which is a high-risk \"atopic\" cluster with increased propensity for allergic diseases throughout childhood. Atopy contributes varyingly to later wheeze depending on cluster membership. Our findings demonstrate the utility of unsupervised analysis in elucidating heterogeneity in asthma pathogenesis and provide a foundation for improving management and prevention of childhood asthma.

systems biology↗

bioRxiv2017

Dynamics of the upper airway microbiome in the pathogenesis of asthma-associated persistent wheeze in preschool children

Repeated cycles of infection-associated lower airway inflammation drives the pathogenesis of persistent wheezing disease in children. Tracking these events across a birth cohort during their first five years, we demonstrate that >80% of infectious events indeed involve viral pathogens, but are accompanied by a shift in the nasopharyngeal microbiome (NPM) towards dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change in NPM frequently precedes the appearance of viral pathogens and acute symptoms. In non-sensitized children these events are associated only with \"transient wheeze\" that resolves after age three. In contrast, in children developing early allergic sensitization, they are associated with ensuing development of persistent wheeze, which is the hallmark of the asthma phenotype. This suggests underlying pathogenic interactions between allergic sensitization and antibacterial mechanisms.

genetics↗