Single-cell Transcriptomics Reveal The Chronic Inflammatory Mechanism Of Allergic Asthma

BackgroundAsthma is one of the most common respiratory diseases,which imposes a heavy burden on healthcare systems.Asthma is a chronic airway inflammatory disease and immune memory is a key mechanism that regulate airway inflammation.Neutrophilic asthma is associated with severe asthma and glucocorticoid resistance and is the main clinical phenotype contributing to the medical burden of asthma,but its mechanisms remain to be further investigated.Methods and ResultsTo investigate the key cell types that promote airway inflammation in asthma,this study used single-cell transcriptome sequencing to detect changes in cell subpopulations in the lung tissue of a chronic allergic asthma model induced by ovalbumin.It was found that neutrophils were significantly increased in asthma,and there were four different subgroups,including FcyRIIb+neutrophils and Cd177+neutrophils.Functional and cell interaction analyses suggested that Cd177+ neutrophils were degranulation-associated neutrophils that could secrete neutrophil extracellular traps to promote airway inflammation and promote airway remodeling by secreting Oncostatin M(Osm).Cell trajectory and transcription factor enrichment analysis found that transcription factors such as C/EBPβ and PU.1 may regulate neutrophil phenotypic switching-mediated neutrophil heterogeneity.FcyRIIb+neutrophil subpopulation was exclusively increased in asthma and associated with the IL-4 receptor signaling.Functional analysis suggested that FcyRIIb+neutrophils may be memory neutrophils with molecular characteristics of innate immune memory,mainly manifested by transcription factor activation-mediated epigenetic modifications and Toll-like receptor pathway activation,as well as upregulation of the inflammation factor secretion pathway.To clarify whether FcyRIIb+neutrophils are memory neutrophils,chronic and acute asthma models were constructed,and the key feature molecules of innate immune memory were validated by qPCR and Western-blot analysis.It was found that these molecules were highly expressed in FcyRIIb+neutrophils sorted by flow cytometry,including activation of immune memory-related transcription factors(C/EBPβ,Run×1),histone modification pathways(Tet2,β-catenin),Toll-like receptor pathway(Tlr2,Cd3001f),cAMP pathway(PDE4B),as well as upregulation of the secretion pathways of inflammation factors IL-1β and IL-6.Finally,it was inferred that FcyRIIb+neutrophils were a key cell subgroup that promoted airway inflammation.In addition,FcyRIIb+neutrophils were closely related to neutrophilic asthma induced by house dust mite with lipopolysaccharide,suggesting that memory neutrophils may be commonly present in neutrophilic asthma.To investigate the shared inflammatory mechanisms between asthma and atherosclerosis,we found that YB1-mediated mRNA stability may play an important role through co-target network analysis.We constructed mouse cells with YB1 phosphorylation site missense mutations through lentivirus transfection and performed transcriptome sequencing.The results showed that YB1 phosphorylation could affect the expression of inflammatory factors such as CCL2 and the glucocorticoid receptor(GR)response pathway.Western-blot and qPCR validation showed that the GR-related complex(GR,PNRC2,DCP1 A,UPF1,and HRSP12)can bind to CCL2 mRNA to regulate its mRNA stability.ConclusionThis study elucidates the heterogeneity of neutrophils in asthmatic lung tissue,in which the FcyRIIb+neutrophil subgroup is a memory neutrophil and a key cell subset that promotes asthma,while Cd177+neutrophils promote asthma through the secretion of neutrophil extracellular traps and Osm.This study revealed that YB1 as a shared target involved in the inflammatory mechanisms of asthma and atherosclerosis.