Atopic Dermatitis Complicated by Recurrent Eczema Herpeticum Is Characterized by Multiple, Concurrent Epidermal Inflammatory Endotypes.

A subgroup of patients with atopic dermatitis (AD) suffers from recurrent, disseminated herpes simplex virus skin infection, termed eczema herpeticum. To determine the transcriptional mechanisms of the skin and immune system pathobiology that underlie development of AD with eczema herpeticum (ADEH), we performed RNA-sequencing analysis of nonlesional skin (epidermis, dermis) from AD patients with and without a history of ADEH (ADEH+, n = 15; ADEH-, n = 13) along with healthy controls (n = 15). We also performed RNA sequencing on participants' plasmacytoid dendritic cells infected in vitro with herpes simplex virus 1. ADEH+ patients exhibited dysregulated gene expression, limited in the dermis (14 differentially expressed genes) and more widespread in the epidermis (129 differentially expressed genes). ADEH+-upregulated epidermal differentially expressed genes were enriched in type 2 cytokine (IL4R , CCL22, CRLF2, IL7R), interferon (CXCL10, ICAM1, IFI44, IRF7), and IL-36γ (IL36G) inflammatory gene pathways. All ADEH+ participants exhibited type 2 cytokine and inteferon endotypes, and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH- participants. ADEH+ skin also had dysregulated epidermal differentiation complex gene expression of the late-cornified envelope, S100A, and small proline-rich gene families, which are involved in skin barrier function and antimicrobial activities. Plasmacytoid dendritic cell transcriptional responses to herpes simplex virus 1 infection were unaltered by ADEH status. The study concluded that the pathobiology underlying ADEH+ risk is associated with a unique, multifaceted epidermal inflammation that accompanies dysregulation of epidermal differentiation complex genes. These findings will help direct future studies that define how these inflammatory patterns may drive risk of eczema herpeticum in AD.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

The Puerto Rican Infant Metagenomic and Epidemiologic Study of Respiratory Outcomes (PRIMERO): Design and Baseline Characteristics for a Birth Cohort Study of Early-life Viral Respiratory Illnesses and Airway Dysfunction in Puerto Rican Children.

Epidemiologic studies demonstrate an association between early-life respiratory illnesses (RIs) and the development of childhood asthma. However, it remains uncertain whether these children are predisposed to both conditions or if early-life RIs induce alterations in airway function, immune responses, or other human biology that contribute to the development of asthma. Puerto Rican children experience a disproportionate burden of early-life RIs and asthma, making them an important population for investigating this complex interplay. PRIMERO, the Puerto Rican Infant Metagenomics and Epidemiologic Study of Respiratory Outcomes , recruited pregnant women and their newborns to investigate how the airways develop in early life among infants exposed to different viral RIs, and will thus provide a critical understanding of childhood asthma development. As the first asthma birth cohort in Puerto Rico, PRIMERO will prospectively follow 2,100 term healthy infants. Collected samples include post-term maternal peripheral blood, infant cord blood, the child's peripheral blood at the year two visit, and the child's nasal airway epithelium, collected using minimally invasive nasal swabs, at birth, during RIs over the first two years of life, and at annual healthy visits until age five. Herein, we describe the study's design, population, recruitment strategy, study visits and procedures, and primary outcomes.

Human Epidemiology and RespOnse to SARS-CoV-2 (HEROS): Objectives, Design and Enrollment Results of a 12-City Remote Observational Surveillance Study of Households with Children using Direct-to-Participant Methods.

The Human Epidemiology and Response to SARS-CoV-2 (HEROS) is a prospective multi-city 6-month incidence study which was conducted from May 2020-February 2021. The objectives were to identify risk factors for SARS-CoV-2 infection and household transmission among children and people with asthma and allergic diseases, and to use the host nasal transcriptome sampled longitudinally to understand infection risk and sequelae at the molecular level. To overcome challenges of clinical study implementation due to the coronavirus pandemic, this surveillance study used direct-to-participant methods to remotely enroll and prospectively follow eligible children who are participants in other NIH-funded pediatric research studies and their household members. Households participated in weekly surveys and biweekly nasal sampling regardless of symptoms. The aim of this report is to widely share the methods and study instruments and to describe the rationale, design, execution, logistics and characteristics of a large, observational, household-based, remote cohort study of SARS-CoV-2 infection and transmission in households with children. The study enrolled a total of 5,598 individuals, including 1,913 principal participants (children), 1,913 primary caregivers, 729 secondary caregivers and 1,043 other household children. This study was successfully implemented without necessitating any in-person research visits and provides an approach for rapid execution of clinical research.

Breathing zone pollutant levels are associated with asthma exacerbations in high-risk children.

Background: Indoor and outdoor air pollution levels are associated with poor asthma outcomes in children. However, few studies have evaluated whether breathing zone pollutant levels associate with asthma outcomes. Objective: Determine breathing zone exposure levels of NO 2 , O 3 , total PM 10 and PM 10 constituents among children with exacerbation-prone asthma, and examine correspondence with in-home and community measurements and associations with outcomes. Methods: We assessed children's personal breathing zone exposures using wearable monitors. Personal exposures were compared to in-home and community measurements and tested for association with lung function, asthma control, and asthma exacerbations. Results: 81 children completed 219 monitoring sessions. Correlations between personal and community levels of PM 10 , NO 2 , and O 3 were poor, whereas personal PM 10 and NO 2 levels correlated with in-home measurements. However, in-home monitoring underdetected brown carbon (Personal:79%, Home:36.8%) and ETS (Personal:83.7%, Home:4.1%) personal exposures, and detected black carbon in participants without these personal exposures (Personal: 26.5%, Home: 96%). Personal exposures were not associated with lung function or asthma control. Children experiencing an asthma exacerbation within 60 days of personal exposure monitoring had 1.98, 2.21 and 2.04 times higher brown carbon (p<0.001), ETS (p=0.007), and endotoxin (p=0.012), respectively. These outcomes were not associated with community or in-home exposure levels. Conclusions: Monitoring pollutant levels in the breathing zone is essential to understand how exposures influence asthma outcomes, as agreement between personal and in-home monitors is limited. Inhaled exposure to PM 10 constituents modifies asthma exacerbation risk, suggesting efforts to limit these exposures among high-risk children may decrease their asthma burden. CLINICAL IMPLICATIONS: In-home and community monitoring of environmental pollutants may underestimate personal exposures. Levels of inhaled exposure to PM 10 constituents appear to strongly influence asthma exacerbation risk. Therefore, efforts should be made to mitigate these exposures. CAPSULE SUMMARY: Leveraging wearable, breathing-zone monitors, we show exposures to inhaled pollutants are poorly proxied by in-home and community monitors, among children with exacerbation-prone asthma. Inhaled exposure to multiple PM 10 constituents is associated with asthma exacerbation risk.

Epigenomic response to albuterol treatment in asthma-relevant airway epithelial cells.

BACKGROUND: Albuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables. RESULTS: We followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10-8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10-5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10-14 ≤ p ≤ 6.60 × 10-5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05). CONCLUSIONS: This study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.

Atopic dermatitis complicated by recurrent eczema herpeticum is characterized by multiple, concurrent epidermal inflammatory endotypes.

BACKGROUND: A subgroup of atopic dermatitis (AD) patients suffer from recurrent, disseminated herpes simplex virus (HSV) skin infections, termed eczema herpeticum (EH), which can be life-threatening and contribute to AD morbidity. The pathobiology underlying ADEH is unknown. OBJECTIVE: To determine transcriptional mechanisms of skin and immune system pathobiology that underlie ADEH disease. METHODS: We performed whole transcriptome RNA-sequencing of non-lesional skin samples (epidermis, dermis) of AD patients with (ADEH + , n=15) and without (ADEH - , n=13) recurrent EH history, and healthy controls (HC, n=15). We also performed RNA-sequencing on plasmacytoid dendritic cells (pDCs) collected from these participants and infected in vitro with HSV-1. Differential expression, gene set enrichment, and endotyping analyses were performed. RESULTS: ADEH + disease was characterized by dysregulation in skin gene expression, which was limited in dermis (differentially expressed genes [DEGs]=14) and widespread in epidermis (DEGs=129). ADEH + -upregulated epidermal DEGs were enriched in type 2 cytokine (T2) ( IL4R, CCL22, CRLF2, IL7R ), interferon ( CXCL10, ICAM1, IFI44 , and IRF7) , and IL-36γ ( IL36G ) inflammatory pathway genes. At a person-level, all ADEH + participants exhibited T2 and interferon endotypes and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH - participants. ADEH + patient skin also exhibited dysregulation in epidermal differentiation complex (EDC) genes within the LCE, S100 , and SPRR families, which are involved in skin barrier function, inflammation, and antimicrobial activities. pDC transcriptional responses to HSV-1 infection were not altered by ADEH status. CONCLUSIONS: ADEH + pathobiology is characterized by a unique, multi-faceted epidermal inflammation that accompanies dysregulation in the expression of EDC genes. Key Messages: AD patients with a history of recurrent EH exhibit molecular skin pathobiology that is similar in form, but more severe in degree, than in AD patients without this complication. Non-lesional skin of ADEH + patients concurrently exhibits excessive type 2 cytokine, interferon, and IL-36γ-driven epidermal inflammation. Expression of these inflammatory skin endotypes among ADEH + patients is associated with dysregulation in expression of epidermal differentiation complex genes involved in barrier function, inflammation, and antimicrobial activity. Capsule Summary: AD patients with a history of recurrent disseminated HSV-1 skin infections form a unique molecular skin endotype group that concurrently exhibits type 2 cytokine, interferon, and IL-36γ-driven skin inflammation, accompanied by dysregulation in expression of epidermal differentiation complex genes involved in barrier function, inflammation, and antimicrobial activity.

Anionic Pulmonary Surfactant Lipid Treatment Inhibits Rhinovirus A Infection of the Human Airway Epithelium.

Rhinoviruses (RVs) are major instigators of acute exacerbations of asthma, COPD, and other respiratory diseases. RVs are categorized into three species (RV-A, RV-B, and RV-C), which comprise more than 160 serotypes, making it difficult to develop an effective vaccine. Currently, no effective treatment for RV infection is available. Pulmonary surfactant is an extracellular complex of lipids and proteins that plays a central role in regulating innate immunity in the lung. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), are potent regulators of inflammatory processes and exert antiviral activity against respiratory syncytial virus (RSV) and influenza A viruses (IAV). In the current study, we examined the potencies of POPG and PI against rhinovirus A16 (RV-A16) in primary human airway epithelial cells (AECs) differentiated at an air-liquid interface (ALI). After AECs were infected with RV-A16, PI reduced the viral RNA copy number by 70% and downregulated (55-75%) the expression of antiviral (MDA5, IRF7, and IFN-lambda) and CXCL11 chemokine genes. In contrast, POPG only slightly decreased MDA5 (24%) and IRF7 (11%) gene expression but did not inhibit IFN-lambda gene expression or RV-A16 replication in AECs. However, both POPG and PI inhibited (50-80%) IL6 gene expression and protein secretion and CXCL11 protein secretion. PI treatment dramatically attenuated global gene expression changes induced by RV-A16 infection alone in AECs. The observed inhibitory effects were indirect and resulted mainly from the inhibition of virus replication. Cell-type enrichment analysis of viral-regulated genes opposed by PI treatment revealed the PI-inhibited viral induction of goblet cell metaplasia and the virus-induced downregulation of ciliated, club, and ionocyte cell types. Notably, the PI treatment also altered the ability of RV-A16 to regulate the expression of some phosphatidylinositol 4-kinase (PI4K); acyl-CoA-binding, domain-containing (ACBD); and low-density lipoprotein receptor (LDLR) genes that play critical roles in the formation and functioning of replication organelles (ROs) required for RV replication in host cells. These data suggest PI can be used as a potent, non-toxic, antiviral agent for RV infection prophylaxis and treatment.

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Human Epidemiology and RespOnse to SARS-CoV-2 (HEROS): Objectives, Design and Enrollment Results of a 12-City Remote Observational Surveillance Study of Households with Children using Direct-to-Participant Methods.

The Human Epidemiology and Response to SARS-CoV-2 (HEROS) is a prospective multi-city 6-month incidence study which was conducted from May 2020-February 2021. The objectives were to identify risk factors for SARS-CoV-2 infection and household transmission among children and people with asthma and allergic diseases, and to use the host nasal transcriptome sampled longitudinally to understand infection risk and sequelae at the molecular level. To overcome challenges of clinical study implementation due to the coronavirus pandemic, this surveillance study used direct-to-participant methods to remotely enroll and prospectively follow eligible children who are participants in other NIH-funded pediatric research studies and their household members. Households participated in weekly surveys and biweekly nasal sampling regardless of symptoms. The aim of this report is to widely share the methods and study instruments and to describe the rationale, design, execution, logistics and characteristics of a large, observational, household-based, remote cohort study of SARS-CoV-2 infection and transmission in households with children. The study enrolled a total of 5,598 individuals, including 1,913 principal participants (children), 1,913 primary caregivers, 729 secondary caregivers and 1,043 other household children. This study was successfully implemented without necessitating any in-person research visits and provides an approach for rapid execution of clinical research.

Risk factors for SARS-CoV-2 infection and transmission in households with children with asthma and allergy: A prospective surveillance study.

BACKGROUND: Whether children and people with asthma and allergic diseases are at increased risk for severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection is unknown. OBJECTIVE: Our aims were to determine the incidence of SARS-CoV-2 infection in households with children and to also determine whether self-reported asthma and/or other allergic diseases are associated with infection and household transmission. METHODS: For 6 months, biweekly nasal swabs and weekly surveys were conducted within 1394 households (N = 4142 participants) to identify incident SARS-CoV-2 infections from May 2020 to February 2021, which was the pandemic period largely before a vaccine and before the emergence of SARS-CoV-2 variants. Participant and household infection and household transmission probabilities were calculated by using time-to-event analyses, and factors associated with infection and transmission risk were determined by using regression analyses. RESULTS: In all, 147 households (261 participants) tested positive for SARS-CoV-2. The household SARS-CoV-2 infection probability was 25.8%; the participant infection probability was similar for children (14.0% [95% CI = 8.0%-19.6%]), teenagers (12.1% [95% CI = 8.2%-15.9%]), and adults (14.0% [95% CI = 9.5%-18.4%]). Infections were symptomatic in 24.5% of children, 41.2% of teenagers, and 62.5% of adults. Self-reported doctor-diagnosed asthma was not a risk factor for infection (adjusted hazard ratio [aHR] = 1.04 [95% CI = 0.73-1.46]), nor was upper respiratory allergy or eczema. Self-reported doctor-diagnosed food allergy was associated with lower infection risk (aHR = 0.50 [95% CI = 0.32-0.81]); higher body mass index was associated with increased infection risk (aHR per 10-point increase = 1.09 [95% CI = 1.03-1.15]). The household secondary attack rate was 57.7%. Asthma was not associated with household transmission, but transmission was lower in households with food allergy (adjusted odds ratio = 0.43 [95% CI = 0.19-0.96]; P = .04). CONCLUSION: Asthma does not increase the risk of SARS-CoV-2 infection. Food allergy is associated with lower infection risk, whereas body mass index is associated with increased infection risk. Understanding how these factors modify infection risk may offer new avenues for preventing infection.

Nasal airway transcriptome-wide association study of asthma reveals genetically driven mucus pathobiology.

To identify genetic determinants of airway dysfunction, we performed a transcriptome-wide association study for asthma by combining RNA-seq data from the nasal airway epithelium of 681 children, with UK Biobank genetic association data. Our airway analysis identified 95 asthma genes, 58 of which were not identified by transcriptome-wide association analyses using other asthma-relevant tissues. Among these genes were MUC5AC, an airway mucin, and FOXA3, a transcriptional driver of mucus metaplasia. Muco-ciliary epithelial cultures from genotyped donors revealed that the MUC5AC risk variant increases MUC5AC protein secretion and mucus secretory cell frequency. Airway transcriptome-wide association analyses for mucus production and chronic cough also identified MUC5AC. These cis-expression variants were associated with trans effects on expression; the MUC5AC variant was associated with upregulation of non-inflammatory mucus secretory network genes, while the FOXA3 variant was associated with upregulation of type-2 inflammation-induced mucus-metaplasia pathway genes. Our results reveal genetic mechanisms of airway mucus pathobiology.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Influenza virus infection increases ACE2 expression and shedding in human small airway epithelial cells.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV), suggesting pathogenic interactions. METHODS: We investigated how IAV may increase the risk of COVID-19 lung disease, focusing on the receptor angiotensin-converting enzyme (ACE)2 and the protease TMPRSS2, which cooperate in the intracellular uptake of SARS-CoV-2. RESULTS: We found, using single-cell RNA sequencing of distal human nondiseased lung homogenates, that at baseline, ACE2 is minimally expressed in basal, goblet, ciliated and secretory epithelial cells populating small airways. We focused on human small airway epithelial cells (SAECs), central to the pathogenesis of lung injury following viral infections. Primary SAECs from nondiseased donor lungs apically infected (at the air-liquid interface) with IAV (up to 3×105 pfu; ∼1 multiplicity of infection) markedly (eight-fold) boosted the expression of ACE2, paralleling that of STAT1, a transcription factor activated by viruses. IAV increased the apparent electrophoretic mobility of intracellular ACE2 and generated an ACE2 fragment (90 kDa) in apical secretions, suggesting cleavage of this receptor. In addition, IAV increased the expression of two proteases known to cleave ACE2, sheddase ADAM17 (TACE) and TMPRSS2 and increased the TMPRSS2 zymogen and its mature fragments, implicating proteolytic autoactivation. CONCLUSION: These results indicate that IAV amplifies the expression of molecules necessary for SARS-CoV-2 infection of the distal lung. Furthermore, post-translational changes in ACE2 by IAV may increase vulnerability to lung injury such as acute respiratory distress syndrome during viral co-infections. These findings support efforts in the prevention and treatment of influenza infections during the COVID-19 pandemic.

Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection.

The high prevalence of Johne's disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

Type 2 and interferon inflammation regulate SARS-CoV-2 entry factor expression in the airway epithelium.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

Single-Cell and Population Transcriptomics Reveal Pan-epithelial Remodeling in Type 2-High Asthma.

The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.

Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium.

Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium.

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.