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.

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.

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.