FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

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 FOXO1high 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 FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high 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: FOXO1high 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.

Associations Among Social Jet Lag, Sleep-Related Characteristics, and Burnout of Nurses in Tertiary Hospitals.

To investigate the status of social jet lag (SJL) through sociodemographic factors among clinical nurses and examine the correlation with burnout. There has been relatively little research on the possible factors resulting in SJL among nurses in China and its role in burnout. A multicenter cross-sectional study recruited 596 nurses from 7 Chinese hospitals. Online questionnaires were delivered to assess sociodemographics, shift work, SJL, chronotypes, and the burnout of nurses. Nurses had severe levels of SJL. The number of children, forms of employment, specialty area, length of professional service, and chronotypes were the main predictors of SJL. Moreover, SJL affected burnout (emotional exhaustion and deindividuation), and reducing the nurses' SJL could relieve their burnout. Additional evidence-based interventions indicate that reducing the SJL is essential as the nurses are suffering severe job burnout.

Metabotropic glutamate receptor 5 promotes blood-brain barrier recovery after traumatic brain injury.

Blood-brain barrier (BBB) impairment and glutamate release are two pathophysiological features of traumatic brain injury (TBI), contributing to secondary brain damage and neuroinflammation. However, our knowledge of BBB integrity damage and dysfunction are still limited due to the diverse and fluctuating expression of glutamate receptors after trauma. Here, we confirmed the downregulation of metabotropic glutamate receptor 5 (mGluR5) on microvascular endothelial cell within the acute phase of TBI, and the recovered mGluR5 levels on BBB was positively associated with blood perfusion and neurological recovery. In whole body mGluR5-knockout mice, BBB dysfunction and neurological deficiency were exacerbated after TBI compared with wild type mice. In terms of mechanism, the amino acid sequence 201-259 of cytoskeletal protein Alpha-actinin-1 (ACTN1) interacted with mGluR5, facilitating mGluR5 translocation from cytoplasmic compartment to plasma membrane in endothelial cells. Activation of plasma membrane mGluR5 triggers the PLC/PKCµ/c-Jun signaling pathway, leading to increased expression of the tight junction-actin cytoskeleton connecting protein zonula occludens-1 (ZO-1). Our findings uncover a novel mechanism mediated by membrane and cytoplasmic mGluR5 in endothelial cell integrity maintenance and repair, providing the potential therapeutic target for TBI treatment targeting at mGluR5 and mGluR5/ACTN1 complex in BBB.

Independent and Interactive Roles of Immunity and Metabolism in Aortic Dissection.

Aortic dissection (AD) is a cardiovascular disease that seriously endangers the lives of patients. The mortality rate of this disease is high, and the incidence is increasing annually, but the pathogenesis of AD is complicated. In recent years, an increasing number of studies have shown that immune cell infiltration in the media and adventitia of the aorta is a novel hallmark of AD. These cells contribute to changes in the immune microenvironment, which can affect their own metabolism and that of parenchymal cells in the aortic wall, which are essential factors that induce degeneration and remodeling of the vascular wall and play important roles in the formation and development of AD. Accordingly, this review focuses on the independent and interactive roles of immunity and metabolism in AD to provide further insights into the pathogenesis, novel ideas for diagnosis and new strategies for treatment or early prevention of AD.

Change and challenge: An online course in Medical Biochemistry and Molecular Biology.

The development of information technology and portable devices has sparked a revolution in the field of education, facilitating access to diverse educational resources and lifelong learning. In particular, the COVID-19 pandemic has accelerated the transition from face-to-face to distance teaching, which requires online education to be provided worldwide. Biochemistry and Molecular Biology are key basic medical courses in laboratory-based science that cover complicated theories and applications. The balance between traditional and online courses, and the effectiveness of online courses, are fundamental to the teaching quality of Biochemistry and Molecular Biology. In this study, we explored the concepts, designs, and practices of a new blended online course and identified potential challenges. We hope that our experiences will provide new ideas for online teaching and promote teaching reform and the development of Medical Biochemistry and Molecular Biology education.

MiR-216a-3p inhibits the proliferation and invasion of fibroblast-like synoviocytes by targeting dual-specificity phosphatase 5.

Dual-specificity phosphatase 5 (DUSP5) is a novel anti-inflammatory modulator in many inflammatory diseases. However, the role of DUSP5 in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) remains unknown. In this study, we aimed to explore the biological function and regulation of DUSP5 in FLS. We found that lower DUSP5 expression level was detected in collagen-induced arthritis (CIA) and synoviocyte MH7A. Overexpression of DUSP5 markedly decreased the proliferation, migration, and invasion of MH7A, which correlated with suppressing the phosphorylation of extracellular signal-regulated kinase (ERK). Moreover, DUSP5 was identified as a novel target gene of miR-216a-3p, which was upregulated in FLS. Therefore, DUSP5 expression was negatively regulated by miR-216a-3p, and the effect of DUSP5 overexpression on FLS was reversed by miR-216a-3p mimics. Overall, our study demonstrates that DUSP5 is a miR-216a-3p target gene and its anti-inflammatory function in FLS via inactivation of ERK. These results revealed that the miR-216a-3p/DUSP5 pathway may play a crucial role in the malignant behavior of FLS, which may serve as a new target for the treatment of RA.

Single-cell profiling reveals various types of interstitial cells in the bladder.

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.

Histones of Neutrophil Extracellular Traps Induce CD11b Expression in Brain Pericytes Via Dectin-1 after Traumatic Brain Injury.

The brain pericyte is a unique and indispensable part of the blood-brain barrier (BBB), and contributes to several pathological processes in traumatic brain injury (TBI). However, the cellular and molecular mechanisms by which pericytes are regulated in the damaged brain are largely unknown. Here, we show that the formation of neutrophil extracellular traps (NETs) induces the appearance of CD11b+ pericytes after TBI. These CD11b+ pericyte subsets are characterized by increased permeability and pro-inflammatory profiles compared to CD11b- pericytes. Moreover, histones from NETs by Dectin-1 facilitate CD11b induction in brain pericytes in PKC-c-Jun dependent manner, resulting in neuroinflammation and BBB dysfunction after TBI. These data indicate that neutrophil-NET-pericyte and histone-Dectin-1-CD11b are possible mechanisms for the activation and dysfunction of pericytes. Targeting NETs formation and Dectin-1 are promising means of treating TBI.

Single-cell transcriptomics provides insights into the origin and microenvironment of human oesophageal high-grade intraepithelial neoplasia.

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: KRT15high STMN1low and KRT15high STMN1high cells, which were located mainly in the interpapillary and papillary zones, respectively. The KRT15high STMN1low population more closely resembled stem cells and transcriptome similarity revealed that HIN probably originated from these slow-cycling KRT15high STMN1low 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.

Activation of CXCL13/CXCR5 axis aggravates experimental autoimmune cystitis and interstitial cystitis/bladder pain syndrome.

The abnormal CXCL13/CXCR5 axis is involved in many inflammatory diseases and its selective inhibitor, TAK-799 has exhibited strong anti-inflammatory potency. The sequencing of clinical specimens from interstitial cystitis/bladder pain syndrome (IC/BPS) has shown that CXCL13 and CXCR5 are highly expressed, but the role of CXCL13/CXCR5 axis in IC/BPS has not been rarely reported. Therefore, in this study, we analyzed the GSE11783 sequencing data of IC/BPS patients and investigate the role and mechanism of CXCL13/CXCR5 axis and TAK-779 in the mouse model of experimental autoimmune cystitis (EAC). We verified that CXCL13 and CXCR5 were significantly up-regulated in EAC model. EAC mice exhibited increased bladder inflammatory factors (IL-6, TNF-α, IL-1ß), apoptosis-related proteins (Bax, Caspase-3, Caspase-8), frequency of voiding. Using TAK779 to block CXCL13/CXCR5 axis significantly attenuated these inflammatory damages and efficiently improved bladder function (significant reduction in micturition frequency, significant prolongation of inter-contraction interval). Further investigation showed that inhibiton of JNK and NF-kappaB activation, the bioinformatics analysis-indicated downstream signaling of CXCL13/CXCR5 axis, is responsible for the protective effect of TAK779. Taken together, we demonstrate that activation of the CXCL13/CXCR5 axis is involved in the pathophysiology of IC/BPS and EAC. Blocking CXCL13/CXCR5 axis activation by TAK-779 reduces bladder inflammation and improves bladder function in EAC mice.

Glutamate blunts cell-killing effects of neutrophils in tumor microenvironment.

Neutrophils are the first defenders of the innate system for injury and infection. They have gradually been recognized as important participants in tumor initiation and development due to their heterogeneity and plasticity. In the tumor microenvironment (TME), neutrophils can exert antitumor and protumor functions, depending on the surroundings. Tumor cells systemically alter intracellular amino acid (AA) metabolism and extracellular AA distribution to meet their proliferation need, leading to metabolic reprogramming and TME reshaping. However, the underlying mechanisms that determine how altered AAs affect neutrophils in TME are less-explored. Here, we identified that abundant glutamate releasing from tumor cells blunted neutrophils' cell-killing effects toward tumor cells in vitro and in vivo. Mass spectrometric detection, flow cytometry, and western blot experiments proved that increased levels of pSTAT3/RAB10/ARF4, mediated by glutamate, were accompanied with immunosuppressive phenotypes of neutrophils in TME. We also discovered that riluzole, an FDA-approved glutamate release inhibitor, significantly inhibited tumor growth by restoring neutrophils' cell-killing effects and decreasing glutamate secretion from tumor cells. These findings highlight the importance of tumor-released glutamate on neutrophil transformation in TME, providing new possible cancer treatments targeting altered glutamate metabolism.

Targeting metabotropic glutamate receptor 4 for cancer immunotherapy.

In this study, we report a novel role of metabotropic glutamate receptor 4 (GRM4) in suppressing antitumor immunity. We revealed in three murine syngeneic tumor models (B16, MC38, and 3LL) that either genetic knockout (Grm4−/−) or pharmacological inhibition led to significant delay in tumor growth. Mechanistically, perturbation of GRM4 resulted in a strong antitumor immunity by promoting natural killer (NK), CD4+, and CD8+ T cells toward an activated, proliferative, and functional phenotype. Single-cell RNA sequencing and T cell receptor profiling further defined the clonal expansion and immune landscape changes in CD8+ T cells. We further showed that Grm4−/− intrinsically activated interferon-γ production in CD8+ T cells through cyclic adenosine 3',5'-monophosphate (cAMP)/cAMP response element binding protein­mediated pathway. Our study appears to be of clinical significance as a signature of NKhigh-GRM4low and CD8high-GRM4low correlated with improved survival in patients with melanoma. Targeting GRM4 represents a new approach for cancer immunotherapy.

The emerging role of neutrophils in autoimmune-associated disorders: effector, predictor, and therapeutic targets.

Neutrophils are essential components of the immune system and have vital roles in the pathogenesis of autoimmune disorders. As effector cells, neutrophils promote autoimmune disease by releasing cytokines and chemokines cascades that accompany inflammation, neutrophil extracellular traps (NETs) regulating immune responses through cell-cell interactions. More recent evidence has extended functions of neutrophils. Accumulating evidence implicated neutrophils contribute to tissue damage during a broad range of disorders, involving rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), primary sjögren's syndrome (pSS), multiple sclerosis (MS), crohn's disease (CD), and gout. A variety of studies have reported on the functional role of neutrophils as therapeutic targets in autoimmune diseases. However, challenges and controversies in the field remain. Enhancing our understanding of neutrophils' role in autoimmune disorders may further advance the development of new therapeutic approaches.

Transplantation with mGluR5 deficiency bone marrow displays antidepressant-like effect in C57BL/6J mice.

Antidepressant-like effects of metabotropic glutamate receptor 5 (mGluR5) have been verified by specific antagonists or whole body knock-out (KO) mice. Previous experiments indicate that blocking mGluR5 exerts antidepressant-like effects through neuronal mechanisms, like modulating NMDA receptor activity or 5-HT system. Here we found that transplanting bone marrow from mGluR5 KO mice to WT mice could also show antidepressant-like effects, which were confirmed by sucrose preference test and tail suspension test. Furthermore, mGluR5 deficiency dramatically inhibits cytokines release from bone marrow cells, such as IL-1ß, TNF-α and IL-6, alleviating proinflammatory responses in LPS-induced depression model. In addition, inhibited cytokines could decrease the activation of brain endothelial cells in ERK-dependent manner. These data provide the evidence that blocking mGluR5 could improve depression through inhibiting peripheral immune responses, confirming the causal relationship between peripheral immune phenotype and brain behavior.

Neutrophils in traumatic brain injury (TBI): friend or foe?

Our knowledge of the pathophysiology about traumatic brain injury (TBI) is still limited. Neutrophils, as the most abundant leukocytes in circulation and the first-line transmigrated immune cells at the sites of injury, are highly involved in the initiation, development, and recovery of TBI. Nonetheless, our understanding about neutrophils in TBI is obsolete, and mounting evidences from recent studies have challenged the conventional views. This review summarizes what is known about the relationships between neutrophils and pathophysiology of TBI. In addition, discussions are made on the complex roles as well as the controversial views of neutrophils in TBI.

Metabotropic glutamate receptor 5 deficiency inhibits neutrophil infiltration after traumatic brain injury in mice.

Both brain native inflammatory cells and infiltrated peripheral white blood cells (WBCs) are primary participants in the brain inflammatory damage post-TBI. Metabotropic glutamate receptor 5 (mGluR5) has been reported to regulate microglias and astrocytes to affect inflammation after TBI, but its effect on modulating infiltrated peripheral WBCs remains unclear. In a mouse moderate TBI model, we found that mGluR5 knockout (KO) significantly reduced neutrophil infiltration and inflammatory cytokine expression in the brain at 24 hours post TBI, which was accompanied by improved neurological dysfunction. Further investigation indicated that mGluR5 KO reduced the permeability of blood-brain barrier (BBB), the entrance for neutrophils to enter brain, and markedly decreased the mRNA levels of neutrophil-associated chemokines in brain tissue, including CXCL1, CXCL2, CCL2, CCL4 and CCL5. Using brain microvascular endothelial cells (BMECs), neutrophils and a BBB model in vitro, we confirmed the inhibitory effect of mGluR5 deficiency on neutrophil infiltration and demonstrated that blockade of protein kinase C (PKC) signaling was involved in it. These results provide insight into the role of mGluR5 in the regulation of inflammation in the acute phase of TBI, which may provide novel clues for TBI therapy.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600­13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome.

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 ßϒ 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.

The mutual regulation between miR-214 and A2AR signaling plays an important role in inflammatory response.

Inflammation is a pathological course involved in several diseases. Both adenosine A2A receptor (A2AR) and miR-214 play important roles in regulation of inflammation. However, the internal link between them and their synergic modulation in inflammatory response has not been elucidated. In this study, we found that miR-214 and A2AR activation could downregulate the expressions of each other in murine macrophages. Comparing with the well known anti-inflammatory role of A2AR, miR-214 promoted the release of inflammatory cytokines TNF-α and IL-6. Further investigation demonstrated that miR-214 downregulated A2AR expression by directly targeting the 3'-untranslated region of A2AR mRNA. Instead of directly interacting with miR-214, A2AR activation repressed miR-214 expression by stimulating PKA signaling to suppress the nuclear translocation of NF-κB which could enhance the transcript activity of miR-214 gene promoter. Then using an LPS-induced ALI mouse model, in which inflammation is a hallmark, we confirmed their negative relationship and demonstrated that combination of miR-214 antagomir and A2AR agonist CGS21680 exerts more anti-inflammatory effect including alleviating the pathological changes, suppressing the neutrophil infiltration and the expression of inflammatory cytokines than using one of them alone. These findings for the first time uncovered a mutual suppression feedback loop between A2AR signaling and miR-214 in inflammation, which may provide new insight of inflammatory regulation and potential therapeutic significance for some inflammation-associated diseases.

A feedback loop in PPARγ-adenosine A2A receptor signaling inhibits inflammation and attenuates lung damages in a mouse model of LPS-induced acute lung injury.

Although peroxisome proliferator-activated receptor-γ (PPARγ) and adenosine A2A receptor (A2AR) are reported to be anti-inflammatory factors in acute lung injury (ALI), their internal link and synergic or antagonistic effect after activation are poorly understood. Here, we found that PPARγ and A2AR could upregulate the mRNA and protein expressions of each other in lung tissues of LPS-induced mouse ALI model and murine macrophages. Further investigation demonstrated that PPARγ upregulated A2AR expression by directly binding to a DR10 response element (-218 to -197) within A2AR gene promoter region. Instead of directly interacting with PPARγ, A2AR stimulated PPARγ expression via protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling by provoking the binding of CREB to a cAMP responsive element (CRE)-like site in PPARγ gene promoter region. In addition, combination of PPARγ and A2AR agonists was found to exert obviously better effect on suppressing neutrophil infiltration and inflammatory cytokine expressions, attenuating lung edema, pathological changes and improving lung function of blood gas exchange than their single application. These findings reveal a novel functional positive feedback loop between PPARγ and A2AR signaling to potentialize their effect on inhibiting inflammation and attenuating lung damages in ALI. It suggests that targeting this PPARγ-A2AR signaling rather than PPARγ or A2AR alone may be a more attractive and efficient potential therapeutic strategy for ALI.