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Abstract Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single‐Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent‐lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation‐bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa‐generated tracking trajectory to improve the accuracy of pseudo‐time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. Key points SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function. The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness. The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent‐lineage differentiation. The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo. The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset. SCALeBa improves the accuracy of differentiation trajectories in monocle2‐based pseudo‐time analysis.

Ferroptosis is an iron-dependent mode of cell death characterized by intracellular lipid peroxide accumulation and a redox reaction imbalance. Compared with other modes of cell death, ferroptosis has specific biological and morphological features. The iron-dependent lipid peroxidation accumulation is manifested explicitly in the abnormal metabolism of intracellular lipid oxides catalyzed by excessive iron ions with the production of many reactive oxygen species and over-oxidization of polyunsaturated fatty acids. Recent studies have shown that various diseases, which include intestinal diseases and cancer, are associated with ferroptosis, but few studies are related to airway inflammatory diseases. This review provides a comprehensive analysis of the primary damage mechanisms of ferroptosis and summarizes the relationship between ferroptosis and airway inflammatory diseases. In addition to common acute and chronic airway inflammatory diseases, we also focus on the progress of research on COVID-19 in relation to ferroptosis. New therapeutic approaches and current issues to be addressed in the treatment of inflammatory airway diseases using ferroptosis are further proposed.

Chronic obstructive pulmonary disease (COPD) is a severely disabling chronic lung disease characterized by persistent airway inflammation, which leads to limited expiratory airflow that deteriorates over time. Isorhamnetin (Iso) is one of the most important active components in the fruit of Hippophae rhamnoides L. and leaves of Ginkgo biloba L, which is widely used in many pulmonary disease studies because of its anti-inflammatory effects. Here, we investigated the pharmacological action of Iso in CS-induced airway inflammation and dissected the anti-inflammation mechanisms of Iso in COPD mice. A mouse model of COPD was established by exposure to cigarette smoke (CS) and intratracheal inhalation of lipopolysaccharide (LPS). Our results illustrated that Iso treatment significantly reduced leukocyte recruitment and excessive secretion of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and regulated upon activation, normal T-cell expressed and secreted (RANTES) in BALF of CS-induced COPD mice in a dose-dependent manner. This improved airway collagen deposition and emphysema, and further alleviated the decline in lung functions and systemic symptoms of hypoxia and weight loss. Additionally, Iso treatment obviously improves the T lymphocyte dysregualtion in peripheral blood of COPD mice. Mechanistically, Iso may degrade Keap1 through ubiquitination of p62, thereby activating the nuclear factor erythroid 2-related factor (Nrf2) pathway to increase the expression of protective factors, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) 1, and SOD2, in lungs of CS-exposed mice, which plays an anti-inflammatory role in COPD. In conclusion, our study indicates that Iso significantly alleviates the inflammatory response in CS-induced COPD mice mainly by affecting the Nrf2/Keap1 pathway. More importantly, Iso exhibited anti-inflammatory effects comparable with Dex in COPD and we did not observe discernible side effects of Iso. The high safety profile of Iso may make it a potential drug candidate for COPD.

El sistema NADPH oxidasa de las células fagocíticas cumple una función importante durante la respuesta antimicrobiana del organismo. La activación de este sistema está precedida por la translocación de las proteínas citosólicas p67phox, p47phox y p40phox hacia la membrana para ponerse en contacto con el flavocitocromo b558, lo que induce la generación del anión superóxido, un precursor de agentes microbicidas oxidantes. El presente trabajo presenta un análisis funcional del sistema NADPH oxidasa basado en los hallazgos de polimorfismos encontrados en el gen de p67phox de individuos sanos. Para esto se generaron mutaciones en el cADN que codifica la p67phox y se expresaron en el sistema de células COSphox. Los datos obtenidos en el presente trabajo indican que los cambios Val166?Ile, Pro329?Ser y His389?Gln no generan alteraciones en el funcionamiento de la p67phox cuando su función se analizó en el sistema transgénico basado en células COS-7. Por lo tanto, estos polimorfismos no generan ningún riesgo genético de producir deficiencias en la activación del sistema NADPH oxidasa. Además, se demuestra que el modelo de células COSphox representa un nuevo sistema celular, fácilmente transfectable que permite estudiar la función del sistema NADPH oxidasa de las células fagocíticas y sus particularidades genéticas. Finalmente, los hallazgos con estos polimorfismos nos permiten avanzar en el conocimiento sobre los mecanismos moleculares involucrados en la activación del sistema NADPH oxidasa células fagocíticas.

El sistema NADPH oxidasa de las células fagocíticas tiene un papel central para la función antimicrobiana de dichas células. La activación de este sistema está presidida por la translocación de las proteínas citosólicas p67phox, p47phox y p40phox hacia la membrana para ponerse en contacto con el flavocitocromo b558, induciendo de esta manera la generación de anión superóxido, un precursor de agentes microbicidas oxidantes. En el presente trabajo presentamos una aproximación al estudio molecular del sistema NADPH oxidasa basados en los hallazgos de mutaciones en el gen de la p67phox de pacientes con EGC y algunos posibles polimorfismos encontrados en individuos sanos.

The NADPH oxidase system of the phagocytes cells has a central role for their microbicidal activity. The activation of this system is started by the translocation of cytosolic proteins p67phox, p47phox and p40phox towards the membrane to be in contact with flavocytochrome b558, inducing the superoxide anion generation. The present work presents a molecular study of NADPH oxidase system based on the findings of mutations in the p67phox gene of patients with chronic granulomatous disease (CGD) and some possible polymorphisms found in healthy humans. Mutations were generated in the p67phox cDNA by site-directed mutagenesis and then expressed in a COS-phox cells. The data collected in this work suggest that Lys19, Lys20 and Asp21 of the p67phox Nterminal are essential for the activation of the oxidase system while the C-terminal of this protein (residues 398-526) is not necessary for the O2- generation using this transgenic system. On the other hand, the changes Val166Ile, Pro329Ser and His389Gln corresponding to possible polymorphisms of normal individuals did not produce alterations in the of p67phox function, therefore these do not imply any genetic risk to produce deficiencies in the activation of NADPH oxidase system. In conclusion, the cellular model COS-phox represents a new system of complete cells, easily transfectable that serves to study the NADPH oxidase system function. Finally, the findings with the mutants of p67phox allow us to advance in the knowledge on the molecular mechanisms involved in the interaction of the cytosolic components of this system. [ABSTRACT FROM AUTHOR]