How early life respiratory viral infections impact airway epithelial development and may lead to asthma.

Childhood asthma is a common chronic disease of the airways that results from host and environment interactions. Most risk factor studies of asthma point to the first year of life as a susceptibility window of mucosal exposure that directly impacts the airway epithelium and airway epithelial cell development. The development of the airway epithelium, which forms a competent barrier resulting from coordinated interactions of different specialized cell subsets, occurs during a critical time frame in normal postnatal development in the first year of life. Understanding the normal and aberrant developmental trajectory of airway epithelial cells is important in identifying pathways that may contribute to barrier dysfunction and asthma pathogenesis. Respiratory viruses make first contact with and infect the airway mucosa. Human rhinovirus (HRV) and respiratory syncytial virus (RSV) are mucosal pathogens that are consistently identified as asthma risk factors. Respiratory viruses represent a unique early life exposure, different from passive irritant exposures which injure the developing airway epithelium. To replicate, respiratory viruses take over the host cell transcriptional and translational processes and exploit host cell energy metabolism. This takeover impacts the development and differentiation processes of airway epithelial cells. Therefore, delineating the mechanisms through which early life respiratory viral infections alter airway epithelial cell development will allow us to understand the maturation and heterogeneity of asthma and develop tools tailored to prevent disease in specific children. This review will summarize what is understood about the impact of early life respiratory viruses on the developing airway epithelium and define critical gaps in our knowledge.

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

Although 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. SUMMARY: Nasal airway epithelial cells from children with wheeze are characterized by altered development and increased susceptibility to RSV infection.

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Glucagon-like Peptide-1 Receptor Pathway Attenuates Platelet Activation in Aspirin-Exacerbated Respiratory Disease.

Platelets are key contributors to allergic asthma and aspirin-exacerbated respiratory disease (AERD), an asthma phenotype involving platelet activation and IL-33-dependent mast cell activation. Human platelets express the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R agonists decrease lung IL-33 release and airway hyperresponsiveness in mouse asthma models. We hypothesized that GLP-1R agonists reduce platelet activation and downstream platelet-mediated airway inflammation in AERD. GLP-1R expression on murine platelets was assessed using flow cytometry. We tested the effect of the GLP-1R agonist liraglutide on lysine-aspirin (Lys-ASA)-induced changes in airway resistance, and platelet-derived mediator release in a murine AERD model. We conducted a prospective cohort study comparing the effect of pretreatment with liraglutide or vehicle on thromboxane receptor agonist-induced in vitro activation of platelets from patients with AERD and nonasthmatic controls. GLP-1R expression was higher on murine platelets than on leukocytes. A single dose of liraglutide inhibited Lys-ASA-induced increases in airway resistance and decreased markers of platelet activation and recruitment to the lung in AERD-like mice. Liraglutide attenuated thromboxane receptor agonist-induced activation as measured by CXCL7 release in plasma from patients with AERD and CD62P expression in platelets from both patients with AERD (n = 31) and nonasthmatic, healthy controls (n = 11). Liraglutide, a Food and Drug Administration-approved GLP-1R agonist for treatment of type 2 diabetes and obesity, attenuates in vivo platelet activation in an AERD murine model and in vitro activation in human platelets in patients with and without AERD. These data advance the GLP-1R axis as a new target for platelet-mediated inflammation warranting further study in asthma.

Tissue-Specific Dependence of Th1 Cells on the Amino Acid Transporter SLC38A1 in Inflammation.

Amino acid (AA) uptake is essential for T cell metabolism and function, but how tissue sites and inflammation affect CD4+ T cell subset requirements for specific AA remains uncertain. Here we tested CD4+ T cell AA demands with in vitro and multiple in vivo CRISPR screens and identify subset- and tissue-specific dependencies on the AA transporter SLC38A1 (SNAT1). While dispensable for T cell persistence and expansion over time in vitro and in vivo lung inflammation, SLC38A1 was critical for Th1 but not Th17 cell-driven Experimental Autoimmune Encephalomyelitis (EAE) and contributed to Th1 cell-driven inflammatory bowel disease. SLC38A1 deficiency reduced mTORC1 signaling and glycolytic activity in Th1 cells, in part by reducing intracellular glutamine and disrupting hexosamine biosynthesis and redox regulation. Similarly, pharmacological inhibition of SLC38 transporters delayed EAE but did not affect lung inflammation. Subset- and tissue-specific dependencies of CD4+ T cells on AA transporters may guide selective immunotherapies.

Building Career Paths for Ph.D., Basic and Translational Scientists in Clinical Departments in the United States: An Official American Thoracic Society Workshop Report.

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO 2 ), we developed a model of repetitive exposure to SO 2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO 2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO 2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

Low Gut Microbial Diversity Augments Estrogen-Driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease.

Although profibrotic cytokines, such as IL-17A and TGF-ß1, have been implicated in the pathogenesis of interstitial lung disease (ILD), the interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as the phosphorylation of STAT3, have not been defined. Here, through chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells, we show that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. The genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after the repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. An assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota in relation to lung fibrosis severity. An analysis of female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-ß1 levels in CD4+ T cells compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis.

Cystic Fibrosis Reprograms Airway Epithelial IL-33 Release and Licenses IL-33-Dependent Inflammation.

Rationale: Type 2 inflammation has been described in people with cystic fibrosis (CF). Whether loss of CFTR (cystic fibrosis transmembrane conductance regulator) function contributes directly to a type 2 inflammatory response has not been fully defined. Objectives: The potent alarmin IL-33 has emerged as a critical regulator of type 2 inflammation. We tested the hypothesis that CFTR deficiency increases IL-33 expression and/or release and deletion of IL-33 reduces allergen-induced inflammation in the CF lung. Methods: Human airway epithelial cells (AECs) grown from non-CF and CF cell lines and Cftr+/+ and Cftr-/- mice were used in this study. Pulmonary inflammation in Cftr+/+ and Cftr-/- mice with and without IL-33 or ST2 (IL-1 receptor-like 1) germline deletion was determined by histological analysis, BAL, and cytokine analysis. Measurements and Main Results: After allergen challenge, both CF human AECs and Cftr-/- mice had increased IL-33 expression compared with control AECs and Cftr+/+ mice, respectively. DUOX1 (dual oxidase 1) expression was increased in CF human AECs and Cftr-/- mouse lungs compared with control AECs and lungs from Cftr+/+ mice and was necessary for the increased IL-33 release in Cftr-/- mice compared with Cftr+/+ mice. IL-33 stimulation of Cftr-/- CD4+ T cells resulted in increased type 2 cytokine production compared with Cftr+/+ CD4+ T cells. Deletion of IL-33 or ST2 decreased both type 2 inflammation and neutrophil recruitment in Cftr-/- mice compared with Cftr+/+ mice. Conclusions: Absence of CFTR reprograms airway epithelial IL-33 release and licenses IL-33-dependent inflammation. Modulation of the IL-33/ST2 axis represents a novel therapeutic target in CF type 2-high and neutrophilic inflammation.

Low Gut Microbial Diversity Augments Estrogen-driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease.

Although profibrotic cytokines such as IL-17A and TGF-ß1 have been implicated in interstitial lung disease (ILD) pathogenesis, interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as phosphorylation of STAT3, have not been defined. Here we show by chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. Genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. Assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota on lung fibrosis severity. Analysis in female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-ß1 levels in CD4+ T cells, compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis.

Effect of Infant RSV Infection on Memory T Cell Responses at Age 2-3 Years.

Background: It is unknown whether RSV infection in infancy alters subsequent RSV immune responses. Methods: In a nested cohort of healthy, term children, peripheral blood mononuclear cells (PBMCs) were collected at ages 2-3 years to examine RSV memory T cell responses among children previously RSV infected during infancy (first year of life) compared to those RSV-uninfected during infancy. The presence vs. absence of infant RSV infection was determined through a combination of RSV molecular and serologic testing. Memory responses were measured in RSV stimulated PBMCs. Results: Compared to children not infected with RSV during the first year of life, children infected with RSV during infancy had lower memory T cell responses at ages 2-3 years to in vitro stimulation with RSV for most tested type-1 and type-17 markers for a number of memory T cell subsets. Conclusions: RSV infection in infancy has long-term effects on memory T cell responses. This is the first study to show the potential for RSV infection in infancy to have long-term effects on the immune memory irrespective of the severity of the infection. Our results suggest a possible mechanism through which infant RSV infection may result in greater risk of subsequent childhood respiratory viral morbidity, findings also relevant to vaccine development.

Androgen receptor signaling promotes Treg suppressive function during allergic airway inflammation.

Women have higher prevalence of asthma compared with men. In asthma, allergic airway inflammation is initiated by IL-33 signaling through ST2, leading to increased IL-4, IL-5, and IL-13 production and eosinophil infiltration. Foxp3+ Tregs suppress and ST2+ Tregs promote allergic airway inflammation. Clinical studies showed that the androgen dehydroepiandrosterone (DHEA) reduced asthma symptoms in patients, and mouse studies showed that androgen receptor (AR) signaling decreased allergic airway inflammation. Yet the impact of AR signaling on lung Tregs remains unclear. Using AR-deficient and Foxp3 fate-mapping mice, we determined that AR signaling increased Treg suppression during Alternaria extract (Alt Ext; allergen) challenge by stabilizing Foxp3+ Tregs and limiting the number of ST2+ ex-Tregs and IL-13+ Th2 cells and ex-Tregs. AR signaling also decreased Alt Ext-induced ST2+ Tregs in mice by limiting expression of Gata2, a transcription factor for ST2, and by decreasing Alt Ext-induced IL-33 production from murine airway epithelial cells. We confirmed our findings in human cells where 5α-dihydrotestosterone (DHT), an androgen, decreased IL-33-induced ST2 expression in lung Tregs and decreased Alt Ext-induced IL-33 secretion in human bronchial epithelial cells. Our findings showed that AR signaling stabilized Treg suppressive function, providing a mechanism for the sex difference in asthma.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Cellular and systemic energy metabolic dysregulation in asthma development-a hypothesis-generating approach.

BACKGROUND: The roles of systemic and airway-specific epithelial energy metabolism in altering the developmental programming of airway epithelial cells (AECs) in early life are poorly understood. OBJECTIVE: Our aim was to assess carbohydrate metabolism in developing AECs among children with and without wheeze and test the association of infant plasma energy biomarkers with subsequent recurrent wheeze and asthma outcomes. METHODS: We measured cellular carbohydrate metabolism in live nasal AECs collected at age 2 years from 15 male subjects with and without a history of wheeze and performed a principal component analysis to visually assess clustering of data on AEC metabolism of glycolitic metabolites and simple sugars. Among 237 children with available year 1 plasma samples, we tested the associations of year 1 plasma energy biomarkers and recurrent wheeze and asthma by using generalized estimating equations and logistic regression. RESULTS: Children with a history of wheeze had lower utilization of glucose in their nasal AECs than did children with no wheeze. Systemically, a higher plasma glucose concentration at year 1 (within the normal range) was associated with decreased odds of asthma at age 5 years (adjusted odds ratio = 0.56; 95% CI = 0.35-0.90). Insulin concentration, glucose-to-insulin ratio, C-peptide concentration, and leptin concentration at year 1 were associated with recurrent wheeze from age 2 years to age 5 years. CONCLUSION: These results suggest that there is significant energy metabolism dysregulation in early life, which likely affects AEC development. These pertubations of epithelial cell metabolism in infancy may have lasting effects on lung development that could render the airway more susceptible to allergic sensitization.

Sex and gender in asthma.

Asthma is a heterogenous disease, and its prevalence and severity are different in males versus females through various ages. As children, boys have an increased prevalence of asthma. As adults, women have an increased prevalence and severity of asthma. Sex hormones, genetic and epigenetic variations, social and environmental factors, and responses to asthma therapeutics are important factors in the sex differences observed in asthma incidence, prevalence and severity. For women, fluctuations in sex hormone levels during puberty, the menstrual cycle and pregnancy are associated with asthma pathogenesis. Further, sex differences in gene expression and epigenetic modifications and responses to environmental factors, including SARS-CoV-2 infections, are associated with differences in asthma incidence, prevalence and symptoms. We review the role of sex hormones, genetics and epigenetics, and their interactions with the environment in the clinical manifestations and therapeutic response of asthma.