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.

scRNA-seq profiling of human granulocytes reveals expansion of developmentally flexible neutrophil precursors with mixed neutrophil and eosinophil properties in asthma.

Neutrophils and eosinophils share common hematopoietic precursors and usually diverge into distinct lineages with unique markers before being released from their hematopoietic site, which is the bone marrow (BM). However, previous studies identified an immature Ly6g(+) Il-5Rα(+) neutrophil population in mouse BM, expressing both neutrophil and eosinophil markers suggesting hematopoietic flexibility. Moreover, others have reported neutrophil populations expressing eosinophil-specific cell surface markers in tissues and altered disease states, confusing the field regarding eosinophil origins, function, and classification. Despite these reports, it is still unclear whether hematopoietic flexibility exists in human granulocytes. To answer this, we utilized single-cell RNA sequencing (scRNA-seq) and CITE-seq to profile human BM and circulating neutrophils and eosinophils at different stages of differentiation and determine whether neutrophil plasticity plays role in asthmatic inflammation. We show that immature metamyelocyte neutrophils in humans expand during severe asthmatic inflammation and express both neutrophil and eosinophil markers. We also show an increase in tri-lobed eosinophils with mixed neutrophil and eosinophil markers in allergic asthma and that IL-5 promotes differentiation of immature blood neutrophils into tri-lobed eosinophilic phenotypes suggesting a mechanism of emergency granulopoiesis to promote myeloid inflammatory or remodeling response in patients with chronic asthma. By providing insights into unexpectedly flexible granulocyte biology and demonstrating emergency hematopoiesis in asthma, our results highlight the importance of granulocyte plasticity in eosinophil development and allergic diseases.

Eosinophil biology from the standpoint of metabolism: implications for metabolic disorders and asthma.

Eosinophils, recognized for their immune and remodeling functions and participation in allergic inflammation, have recently garnered attention due to their impact on host metabolism, especially in the regulation of adipose tissue. Eosinophils are now known for their role in adipocyte beiging, adipokine secretion, and adipose tissue inflammation. This intricate interaction involves complex immune and metabolic processes, carrying significant implications for systemic metabolic health. Importantly, the interplay between eosinophils and adipocytes is bidirectional, revealing the dynamic nature of the immune-metabolic axis in adipose tissue. While the homeostatic regulatory role of eosinophils in the adipose tissue is appreciated, this relationship in the context of obesity or allergic inflammation is much less understood. Mechanistic details of eosinophil-adipose interactions, especially the direct regulation of adipocytes by eosinophils, are also lacking. Another poorly understood aspect is the metabolism of the eosinophils themselves, encompassing metabolic shifts during eosinophil subset transitions in different tissue microenvironments, along with potential effects of host metabolism on the programming of eosinophil hematopoiesis and the resulting plasticity. This review consolidates recent research in this emerging and fascinating frontier of eosinophil investigation, identifying unexplored areas and presenting innovative perspectives on eosinophil biology in the context of metabolic disorders and associated health conditions, including asthma.

Sex differences in a murine model of asthma are time and tissue compartment dependent.

CONCLUSION: Sexual dimorphism in lung inflammation is both time and tissue compartment dependent. Spatiotemporal variability in sex differences in a murine model of asthma must be accounted for when planning experiments to model the sex bias in allergic inflammation.

Association of citrulline concentration at birth with lower respiratory tract infection in infancy: Findings from a multi-site birth cohort study.

Assessing the association of the newborn metabolic state with severity of subsequent respiratory tract infection may provide important insights on infection pathogenesis. In this multi-site birth cohort study, we identified newborn metabolites associated with lower respiratory tract infection (LRTI) in the first year of life in a discovery cohort and assessed for replication in two independent cohorts. Increased citrulline concentration was associated with decreased odds of LRTI (discovery cohort: aOR 0.83 [95% CI 0.70-0.99], p = 0.04; replication cohorts: aOR 0.58 [95% CI 0.28-1.22], p = 0.15). While our findings require further replication and investigation of mechanisms of action, they identify a novel target for LRTI prevention and treatment.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs.

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

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.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

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.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2-3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Eosinophils in Health and Disease: A State-of-the-Art Review.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Comparative Study of SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-229E and Influenza Host Gene Expression in Asthma: Importance of Sex, Disease Severity, and Epithelial Heterogeneity.

Epithelial characteristics underlying the differential susceptibility of chronic asthma to SARS-CoV-2 (COVID-19) and other viral infections are currently unclear. By revisiting transcriptomic data from patients with Th2 low versus Th2 high asthma, as well as mild, moderate, and severe asthmatics, we characterized the changes in expression of human coronavirus and influenza viral entry genes relative to sex, airway location, and disease endotype. We found sexual dimorphism in the expression of SARS-CoV-2-related genes ACE2, TMPRSS2, TMPRSS4, and SLC6A19. ACE2 receptor downregulation occurred specifically in females in Th2 high asthma, while proteases broadly assisting coronavirus and influenza viral entry, TMPRSS2, and TMPRSS4, were highly upregulated in both sexes. Overall, changes in SARS-CoV-2-related gene expression were specific to the Th2 high molecular endotype of asthma and different by asthma severity and airway location. The downregulation of ACE2 (COVID-19, SARS) and ANPEP (HCoV-229E) viral receptors wascorrelated with loss of club and ciliated cells in Th2 high asthma. Meanwhile, the increase in DPP4 (MERS-CoV), ST3GAL4, and ST6GAL1 (influenza) was associated with increased goblet and basal activated cells. Overall, this study elucidates sex, airway location, disease endotype, and changes in epithelial heterogeneity as potential factors underlying asthmatic susceptibility, or lack thereof, to SARS-CoV-2.

Metabolism of Epithelial Cells in Health and Allergic Disease: Collegium Internationale Allergologicum Update 2021.

Concomitant dramatic increase in prevalence of allergic and metabolic diseases is part of a modern epidemic afflicting technologically advanced societies. While clinical evidence points to clear associations between various metabolic factors and atopic disease, there is still a very limited understanding of the mechanisms that link the two. Dysregulation of central metabolism in metabolic syndrome, obesity, diabetes, and dyslipidemia has a systemic impact on multiple tissues and organs, including cells of the epithelial barrier. While much of epithelial research in allergy has focused on the immune-driven processes, a growing number of recent studies have begun to elucidate the role of metabolic components of disease. This review will revisit clinical evidence for the relationship between metabolic and allergic diseases, as well as discuss potential mechanisms driving metabolic dysfunction of the epithelial barrier. Among them, novel studies highlight links between dysregulation of the insulin pathway, glucose metabolism, and loss of epithelial differentiation in asthma. Studies of mitochondrial structure and bioenergetics in lean and obese asthmatic phenotypes recently came to light to provide a novel framework linking changes in tricarboxylic acid cycle and oxidative phosphorylation with arginine metabolism and nitric oxide bioavailability. New research established connections between arachidonate metabolism, autophagy, and airway disease, as well as systemic dyslipidemia in atopic dermatitis and ceramide changes in the epidermis. Taken together, studies of metabolism have a great potential to open doors to a new class of therapeutic strategies, better characterization of disease endotypes, as well as enable a systems biology approach to mechanisms of allergic disease.

The twilight zone: plasticity and mixed ontogeny of neutrophil and eosinophil granulocyte subsets.

It is now becoming clear that neutrophils and eosinophils are heterogeneous cells with potentially multiple subsets in health and disease. With greater marker coverage by multi-color flow cytometry and single-cell level sequencing of granulocyte populations, novel phenotypes of these cells began to emerge. Intriguingly, many newly described subsets blend distinctions between classical myeloid lineage phenotypes, which are especially true for tissue resident or recruited cells in contexts of inflammation and disease. This includes reports of neutrophils with features of eosinophils, monocytes and dendritic cells, and eosinophil subsets expressing neutrophil markers. Moreover, novel studies show the ability of immature neutrophils to transdifferentiate into mature cells belonging to other myeloid lineages (eosinophils, monocytes/macrophages). In this review, we summarize novel findings in this exciting research frontier and shed light on potential processes driving the plasticity and heterogeneity of granulocyte subsets. Specifically, we discuss the hematopoietic flexibility of granulocyte precursors in bone marrow and the adaptation of myeloid cells to local tissue microenvironments. The understanding of such intermediate and developmental phenotypes is very important, as it can teach us about origins of functionally distinct myeloid cells during inflammation, and explain reasons for successes and failures of biologics targeting terminally differentiated granulocytes.

Beyond Il-5: Metabolic Reprogramming and Stromal Support Are Prerequisite for Generation and Survival of Long-Lived Eosinophil.

Eosinophils play surprisingly diverse roles in health and disease. Accordingly, we have now begun to appreciate the scope of the functional and phenotypic heterogeneity and plasticity of these cells. Along with tissue-recruited subsets during inflammation, there are tissue resident eosinophil phenotypes with potentially longer life spans and less dependency on IL-5 for survival. Current models to study murine eosinophils ex vivo rely on IL-5-sustained expansion of eosinophils from bone marrow hematopoietic progenitors. Although it does generate eosinophils (bmEos) in high purity, such systems are short-lived (14 days on average) and depend on IL-5. In this report, we present a novel method of differentiating large numbers of pure bone marrow-derived eosinophils with a long-lived phenotype (llEos) (40 days on average) that require IL-5 for initial differentiation, but not for subsequent survival. We identified two key factors in the development of llEos: metabolic adaptation and reprogramming induced by suppressed nutrient intake during active differentiation (from Day 7 of culture), and interaction with IL-5-primed stromal cells for the remainder of the protocol. This regimen results in a higher yield and viability of mature eosinophils. Phenotypically, llEos develop as Siglec-F(+)Ly6G(+) cells transitioning to Siglec-F(+) only, and exhibit typical eosinophil features with red eosin granular staining, as well as the ability to chemotax to eotaxin Ccl11 and process fibrinogen. This culture system requires less reagent input and allows us to study eosinophils long-term, which is a significant improvement over IL-5-driven differentiation protocols. Moreover, it provides important insights into factors governing eosinophil plasticity and the ability to assume long-lived IL-5-independent phenotypes.

Hypercapnia selectively modulates LPS-induced changes in innate immune and DNA replication-related gene transcription in the macrophage.

Hypercapnia, the elevation of CO2 in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases and is associated with increased risk of death. Recent studies have shown that hypercapnia inhibits expression of select innate immune genes and suppresses host defence against bacterial and viral pneumonia in mice. In the current study, we evaluated the effect of culture under conditions of hypercapnia (20% CO2) versus normocapnia (5% CO2), both with normoxia, on global gene transcription in human THP-1 and mouse RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We found that hypercapnia selectively downregulated transcription of LPS-induced genes associated with innate immunity, antiviral response, type I interferon signalling, cytokine signalling and other inflammatory pathways in both human and mouse macrophages. Simultaneously, hypercapnia increased expression of LPS-downregulated genes associated with mitosis, DNA replication and DNA repair. These CO2-induced changes in macrophage gene expression help explain hypercapnic suppression of antibacterial and antiviral host defence in mice and reveal a mechanism that may underlie, at least in part, the high mortality of patients with severe lung disease and hypercapnia.

Estrogen regulates the expression of SARS-CoV-2 receptor ACE2 in differentiated airway epithelial cells.

There is marked sexual dimorphism in the current coronavirus disease 2019 (COVID-19) pandemic. Here we report that estrogen can regulate the expression of angiotensin-converting enzyme 2 (ACE2), a key component for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry, in differentiated airway epithelial cells. Further studies are required to elucidate the mechanisms by which sex steroids regulate SARS-CoV-2 infectivity.