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Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here,we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs,which are the most potent subsets,it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol,please refer to Liang et al. (2020) and Qiu et al. (2021). View Publication

BACKGROUND Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far,the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD,and compare them to blood neutrophils. METHODS Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n??=??32) and a control group of patients with non-PCD,non-cystic fibrosis bronchiectasis (n??=??5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization,production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production,actin polymerization and the expulsion of NETs. In addition,PCD sputum displayed high activity of neutrophil elastase,and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD. View Publication

BACKGROUND There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However,there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further,it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. RESULTS We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell,CD4 T,and CD8 T na{\{i}}ve and memory cells states. In the process of na{\"{i}}ve to memory activation across the three lineages we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further in central to effector memory differentiation our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. CONCLUSIONS Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes." View Publication

Asthmatic women tend to develop severe airway disease in their reproductive years,and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells,or ILC2,are readily detected in allergic conditions,such as rhinosinusitis,in individuals that develop nasal polyps do to allergen exposures,and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33,an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in na{\{i}}ve and ovalbumin (OVA) challenged female BALB/c mice in comparison to male mice following stimulation with IL-33. Here we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-$\kappa$B signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated and had reduced NF-$\kappa$B activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2 and Nfkb1 was found among top-ranked regulatory interactions. Together these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases." View Publication

Neutrophils are central players in the innate immune system. To protect against invading pathogens,neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense,they also have deleterious effects,and dysregulation of NETs formation has been implicated in autoimmune diseases,atherosclerosis and thrombotic conditions,cancer progression and dissemination,and acute respiratory distress syndrome. Here,we report that selinexor,a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma,markedly suppressed the release of NETs in vitro. Furthermore,we demonstrate a significant inhibitory effect of selinexor on NETs formation,but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase,myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers,including PMA,TGF-$\beta$,TNF-$\alpha$ and IL-8. Maximal inhibition of NETs formation was observed using TGF-$\beta$,for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs. View Publication

BACKGROUND Suppression of cardiac iinflammasome,which can be inhibited by Farnesoid X receptor (FXR) agonist,can ameliorate cardiac inflammation and fibrosis. Increased cardiac inflammasome decrease the abundance of regulatory T (Treg) cells and exacerbate cardiac dysfunction. Interaction between cardiomyocytes and Treg cells is involved in the development of nonalcoholic steatohepatitis (NASH)-related cardiac dysfunction. AIMS This study evaluates whether the FXR agonist obeticholic acid (OCA) treatment improves NASH-associated cardiac dysfunction. METHODS The in vivo and in vitro mechanisms and effects of two weeks of OCA treatment on inflammasome and Treg dysregulation-related cardiac dysfunction in NASH mice (NASH-OCA) at systemic,tissue and cellular levels were investigated. RESULTS The OCA treatment suppressed the serum and cardiac inflammasome levels,reduced the cardiac infiltrated CD3+ T cells,increased the cardiac Treg-represented anti-inflammatory cytokines (IL-10/IL-10R) and improved cardiac inflammation,fibrosis and function [decreased left ventricle (LV) mass and increased fractional shortening (FS)] in NASH-OCA mice. The percentages of OCA-decreased cardiac fibrosis and OCA-increased FS were positively correlated with the percentage of OCA-increased levels of cardiac FXR and IL-10/IL-10R. In the Treg cells from NASH-OCA mice spleen,in comparison with the Treg cells of the NASH group,higher intracellular FXR but lower inflammasome levels,and more proliferative/active and less apoptotic cells were observed. Incubation of H9c2 cardiomyoblasts with Treg-NASHcm [supernatant of Treg from NASH mice as condition medium (cm)],increased inflammasome levels,decreased the proliferative/active cells,suppressed the intracellular FXR,and downregulated differentiation/contraction marker. The Treg-NASHcm-induced hypocontractility of H9c2 can be attenuated by co-incubation with OCA,and the OCA-related effects were abolished by siIL-10R pretreatment. CONCLUSIONS Chronic FXR activation with OCA is a potential strategy for activating IL-10/IL-10R signalling,reversing cardiac regulatory T cell dysfunction,and improving inflammasome-mediated NASH-related cardiac dysfunction. View Publication

INTRODUCTION Based on the immunologic effects of anti-cancer treatment and their therapeutic implications,we evaluated radiotherapy (RT)-induced dynamic alterations in programmed death-1 (PD-1)/PD ligand-1 (PD-L1) expression profiles. METHODS Local RT with 2 Gy ?— 5 or 7.5 Gy ?— 1 was administered to the CT26 mouse model. Thereafter,tumors were resected and evaluated at the following predefined timepoints according to radiation response status: baseline,early (immediately after RT),middle (beginning of tumor shrinkage),late (stable status with RT effect),and progression (tumor regrowth). PD-1/PD-L1 activity and related immune cell profiles were quantitatively assessed. RESULTS RT upregulated PD-L1 expression in tumor cells from the middle to late phase; however,the levels subsequently decreased to levels comparable to baseline in the progression phase. RT with 2 Gy ?— 5 induced a higher frequency of PD-L1+ myeloid-derived suppressor cells,with a lesser degree of tumor regression,compared to 7.5 Gy. The proportion of PD-1+ and interferon (IFN)-$\gamma$+CD8$\alpha$ T cells continued to increase. The frequency of splenic PD-1+CD8+ T cells was markedly elevated,and was sustained longer with 2 Gy ?— 5. Based on the transcriptomic data,RT stimulated the transcription of immune-related genes,leading to sequentially altered patterns. DISCUSSION The dynamic alterations in PD-1/PD-L1 expression level were observed according to the time phases of tumor regression. This study suggests the influence of tumor cell killing and radiation dosing strategy on the tumor immune microenvironment. View Publication

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here,we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample,and 10?— Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein,we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol,please refer to Nozais et al. (2021). View Publication

Regulatory T cells (Tregs) are critically involved in neovascularization,an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174),which is a regulator of Treg function and development,in neovascularization remains elusive. Here,we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs,thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically,GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via G$\alpha$s/cAMP/PKA signal pathway activation. Collectively,these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases. View Publication

Previous studies demonstrated that CD4+ T cells can uptake tumor antigen-pulsed dendritic cell-derived exosomes (DEXO),which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However,in preliminary study,CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity,indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore,LEX-based anti-leukemia vaccines need to be optimized. In this study,we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs,which harbor upregulated CD80 and CD86,and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells,and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover,CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules,CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore,the animal study revealed that the CD4+ TLEX-CD8086 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice than other formulations did in both protective and therapeutic models. In conclusion,this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone,suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy. View Publication

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however,the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously,we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover,the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here,we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways,which are recognized as drivers of resistance. Mechanistically,this drug combination attenuated both interacting signaling networks,leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently,leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK,JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation,viability and the stress response. Collectively,these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment. View Publication