Systemic inflammatory response syndrome (SIRS) is an overwhelming whole body inflammation caused by infectious diseases or sterile insults. Neutrophils are the dominant participants during inflammation, and their survival and death determine the initiation as well as resolution of SIRS. Apoptosis and autophagy are two fundamental cellular processes that modulating cell fate, but their correlation and regulators in neutrophils under SIRS condition have not been elucidated. In this study, we demonstrated that high dose of LPS induced both apoptosis and autophagy of neutrophils in a mouse SIRS model and LPS-stimulated neutrophils in vitro. Moreover, we found that the adenosine 2A receptor (A2AR), a known anti-inflammatory G protein-coupled receptor (GPCR), could inhibit LPS-induced neutrophil apoptosis by suppressing the LPS-induced autophagy. Activation of A2AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting GPCR βϒ subunit–AKT signaling. The A2AR-inhibited autophagy suppressed apoptosis of neutrophils by blocking caspase8, caspase3 and PARP signaling. These findings not only increase our understandings of neutrophils’ fate and function in response to systemic inflammation, but also identify a novel anti-inflammatory role of A2AR in modulating neutrophils’ survival during inflammation.

Background: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. Methods: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. Results: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1 ^high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1 ^high neutrophils. In the chronic stage, the “FOXO1-transferrin receptor (TFRC)” mechanism contributes to FOXO1 ^high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. Conclusions: FOXO1 ^high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Neutrophils from skull bone marrow (N_skull) are activated under some brain stresses, but their effects on traumatic brain injury (TBI) are lacking. Here, we find N_skull infiltrates brain tissue quickly and persistently after TBI, which is distinguished by highly and specifically expressed osteocalcin (OCN) from blood-derived neutrophils (N_blood). Reprogramming of glucose metabolism by reducing glycolysis-related enzyme glyceraldehyde 3-phosphate dehydrogenase expression is involved in the antiapoptotic and proliferative abilities of OCN-expressing N_skull. The transcription factor Fos-like 1 governs the specific gene profile of N_skull including C-C motif chemokine receptor-like 2 (CCRL2), arginase 1 (Arg1), and brain-derived neurotrophic factor (BDNF) in addition to OCN. Selective knockout of CCRL2 in N_skull demonstrates that CCRL2 mediates its recruitment, whereas high Arg1 expression is consistent with its immunosuppressive effects on N_blood, and the secretion of BDNF facilitating dendritic growth contributes to its neuroprotection. Thus, our findings provide insight into the roles of N_skull in TBI.

Background: High‐grade intraepithelial neoplasia (HIN) is the precursor of oesophageal squamous cell carcinoma. The molecular and functional properties of HIN are determined by intrinsic origin cells and the extrinsic microenvironment. Yet, these factors are poorly understood.Methods: We performed single‐cell RNA sequencing of cells from HINs and adjacent tissues from the human oesophagus. We analysed the heterogeneity of basal layer cells and confirmed it using immunostaining. Aneuploid cells in HIN were studied using primary cell culture combined with karyotype analysis. We reconstructed the lineage relationship between tumour and normal populations based on transcriptome similarity. Integration analysis was applied to our epithelial data and published invasive cancer data, and results were confirmed by immunostaining and 3D organoid functional experiments. We also analysed the tumour microenvironment of HIN.Results: The basal layer contained two cell populations: KRT15^highSTMN1^low and KRT15^highSTMN1^high cells, which were located mainly in the interpapillary and papillary zones, respectively. The KRT15^highSTMN1^low population more closely resembled stem cells and transcriptome similarity revealed that HIN probably originated from these slow‐cycling KRT15^highSTMN1^low cells. 3D Organoid experiments and RNA‐sequencing showed that basal‐cell features and the differentiation ability of the normal epithelium were largely retained in HIN, but may change dramatically in tumour invasion stage. Moreover, the tumour microenvironment of HIN was characterised by both inflammation and immunosuppression.Conclusions: Our study provides a comprehensive single‐cell transcriptome landscape of human oesophageal HIN. Our findings on the origin cells and unique microenvironment of HIN will allow for the development of strategies to block tumour progression and even prevent cancer initiation.

Clarifying the locations, molecular markers, functions and roles of bladder interstitial cells is crucial for comprehending the pathophysiology of the bladder. This research utilized human, rat and mouse bladder single‐cell sequencing, bioinformatics analysis and experimental validation. The main cell types found in human, rat and mouse bladder tissues include epithelial cells, smooth muscle cells, endothelial cells, fibroblasts, myofibroblasts, neurons and various immune cells. Our study identified two significant types of interstitial cells (PTN⁺IGFBP6⁺PI16 (CD364)⁺ CD34⁺) and myofibroblasts (STC1⁺PLAT⁺TNC⁺). These two types of interstitial cells are mainly located in the subepithelial lamina propria, between muscles and between muscle bundles. In the CYP (cyclophosphamide)‐induced bladder injury mouse model, the interaction types and signals (MK, MIF, GDF and CXCL) of fibroblasts and myofibroblasts significantly increased compared with the normal group. However, in the aging mouse model, the signals CD34, LAMININ, GALECTIN, MK, SELPLG, ncWNT, HSPG, ICAM and ITGAL‐ITGB2 of fibroblasts and myofibroblasts disappeared, but the signals PTN and SEMA3 significantly increased. Our findings identified two crucial types of interstitial cells in bladder tissue, fibroblasts and myofibroblasts, which play a significant role in normal bladder physiology, CYP‐induced bladder injury and aging bladder development.