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BackgroundSepsis survivors exhibit immune dysregulation that contributes to poor long-term outcomes. Phenotypic and functional alterations within the myeloid compartment are believed to be a contributing factor. Here we dissect the cellular and transcriptional heterogeneity of splenic CD11b+Ly6Chigh myeloid cells that are expanded in mice that survive the cecal ligation and puncture (CLP) murine model of polymicrobial sepsis to better understand the basis of immune dysregulation in sepsis survivors.MethodsSham or CLP surgeries were performed on C57BL/6J and BALB/c mice. Four weeks later splenic CD11b+Ly6Chigh cells from both groups were isolated for phenotypic (flow cytometry) and functional (phagocytosis and glycolysis) characterization and RNA was obtained for single-cell RNA-seq (scRNA-seq) and subsequent analysis.ResultsCD11b+Ly6Chigh cells from sham and CLP surviving mice exhibit phenotypic and functional differences that relate to immune function,some of which are observed in both C57BL/6J and BALB/c strains and others that are not. To dissect disease-specific and strain-specific distinctions within the myeloid compartment,scRNA-seq analysis was performed on CD11b+Ly6Chigh cells from C57BL/6J and BALB/c sham and CLP mice. Uniform Manifold Approximation and Projection from both strains identified 13 distinct clusters of sorted CD11b+Ly6Chigh cells demonstrating significant transcriptional heterogeneity and expressing gene signatures corresponding to classical-monocytes,non-classical monocytes,M1- or M2-like macrophages,dendritic-like cells,monocyte-derived dendritic-like cells,and proliferating monocytic myeloid-derived suppressor cells (M-MDSCs). Frequency plots showed that the percentages of proliferating M-MDSCs (clusters 8,11 and 12) were increased in CLP mice compared to sham mice in both strains. Pathway and UCell score analysis in CLP mice revealed that cell cycle and glycolytic pathways were upregulated in proliferating M-MDSCs in both strains. Notably,granule protease genes were upregulated in M-MDSCs from CLP mice. ScRNA-seq analyses also showed that phagocytic pathways were upregulated in multiple clusters including the classical monocyte cluster,confirming the increased phagocytic capacity in CD11b+Ly6Chigh cells from CLP mice observed in ex vivo functional assays in C57BL/6J mice.ConclusionThe splenic CD11b+Ly6Chigh myeloid populations expanded in survivors of CLP sepsis correspond to proliferating cells that have an increased metabolic demand and gene signatures consistent with M-MDSCs,a population known to have immunosuppressive capacity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-024-00970-0. View Publication

IntroductionImmune resilience is the capacity of the immune system to recover from sepsis-induced organ injury and reestablish host defense. While sepsis survivors are often viewed as immunocompromised,recent studies suggest that some may acquire adaptive immune traits that enhance resistance to secondary infections.MethodsWe employed a murine cecal ligation and puncture (CLP) model to study polymicrobial sepsis and subsequent immune responses. Listeria monocytogenes was used as a model intracellular pathogen to assess immune protection. We analyzed myeloid cell phenotypes using single-cell RNA sequencing and evaluated lipid metabolic changes through quantitative RT-PCR,immunohistochemistry,serum cytokine assays,and plasma lipidomics.ResultsSepsis-surviving mice showed enhanced resistance to Listeria infection. Single-cell RNA sequencing revealed transcriptional reprogramming in splenic CD11b+Ly6Chigh myeloid cells,including downregulation of lipoprotein lipase and lipid efflux genes. CD11b+ myeloid cells exhibited increased lipid droplet accumulation,accompanied by elevated serum interferon-gamma (IFN-γ) levels. Plasma lipidomics identified systemic lipid remodeling,with increased phosphatidylserine and decreased phosphatidylinositol and phosphatidylglycerol levels.DiscussionOur findings suggest that sepsis survival induces lipid metabolic reprogramming in myeloid cells,contributing to enhanced immunity against intracellular pathogens. These insights reveal potential therapeutic targets within lipid metabolic pathways to improve host defense in sepsis survivors. View Publication

Background: Natural killer (NK) cells are key effector lymphoid cells involved in both innate and adaptive immunity and are capable of clearing abnormally aggregated α-synuclein (αSyn). In preclinical Parkinson’s disease (PD) models,NK cell depletion worsens motor deficits and increases insoluble αSyn accumulation,suggesting a neuroprotective role. However,the therapeutic potential of NK cell transfer in modulating αSyn pathology and neurodegeneration remains unexplored. Methods: To assess the efficacy of NK cell therapy,we administered biweekly systemic injections of untouched NK cells isolated from B6C3H donor mice into 2-month-old presymptomatic homozygous M83 transgenic mice injected with human αSyn preformed fibrils. Neurological function was assessed via clasping behavior and clinical scoring. αSyn pathology and dopaminergic neurodegeneration were evaluated via immunohistochemistry. CyTOF-based immune profiling and multiplex ELISA were performed to characterize central and peripheral immune responses. Results: Adoptive NK cell transfers improved motor function and reduced αSyn pathology in a region- and dose-dependent manner,with significant reductions in phosphorylated-αSyn inclusions and tyrosine hydroxylase-positive neuronal loss in the substantia nigra. NK cell transfer modulated the CNS immune landscape by reducing CD11b+CD45high and MHCII+ activated microglial,CD4⁺ T cells,and neutrophil infiltration,while promoting CD19+ B and CD8+ T cells. Similar immunomodulatory effects were observed in the periphery,including restoration of follicular B cells and reduced neutrophil frequencies. Mechanistically,αSyn exposure downregulated activating NK ligands and upregulated inhibitory receptor ligand mQa1b,along with p21 induction in microglia,suggesting a senescence-associated,immune-evasive phenotype that may contribute to reduced therapeutic efficacy at later disease stages. Conclusions: Our study provides direct evidence of NK cells exerting neuroprotective and immunomodulatory effects in a preclinical model of synucleinopathy. These findings support NK cell transfer as a novel therapeutic strategy for PD and related neurodegenerative disorders. View Publication

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

BACKGROUND: We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study,we seek to apply Aldefluor flow cytometry-based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines,which may become a new tool that will facilitate our continued research in this field. EXPERIMENTAL DESIGN: Several established lung cancer cell lines were used,including A549 cell line expressing siRNA against aldehyde dehydrogenase class-1A1 (ALDH1A1). Western blot analysis,spectrophotometry assay,and Aldefluor staining were used to measure protein or enzyme activity in these cell lines. For the purpose of measurement of ALDH activity by Aldefluor in cells with known high ALDH levels,cells were mixed 1:10 with immortalized lung epithelial cell line (Beas-2B),which is known to lack ALDH activity. To delineate dead cells,double staining using Aldefluor and propidium iodide (PI) was done. Double staining was also used to detect changes in ALDH activity in two different cell lines after treatment with 4-hydroperoxycyclophosphamide (4-HC). RESULTS: Our results show a very good correlation between Aldefluor,Western blot,and spectrophotometry assays. Mixing experiments with Beas-2B cells allowed accurate assessment of ALDH activity in A549 cells at baseline and after siRNA expression,thus establishing an approach that facilitates the measurement of very high ALDH using the Aldefluor assay. Aldefluor staining was able to detect heterogeneity in ALDH expression among as well as within the same cell lines and better assess viability after 4-HC treatment when combined with PI. CONCLUSIONS: Aldefluor assay can be adapted successfully to measure ALDH activity in lung cancer cells and may have the advantage of providing real time changes in ALDH activity in viable cells treated with siRNA or chemotherapy. View Publication

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study,we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab,a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines,cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling,as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines,as indicated by the significant decrease in SFE and the loss of serial transplantability,following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here,we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression,thereby representing a critical survival pathway of tumor-initiating cells. View Publication

PI3K activity determines positive and negative selection of B cells,a key process for immune tolerance and B cell maturation. Activation of PI3K is balanced by phosphatase and tensin homolog (Pten),the PI3K's main antagonistic phosphatase. Yet,the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. Here,we show that PI3K control of this process is achieved post-transcriptionally by an axis composed of a transcription factor (c-Myc),a microRNA (miR17-92),and Pten. Enhancing activation of this axis through overexpression of miR17-92 reconstitutes the impaired PI3K activity for positive selection in CD19-deficient B cells and restores most of the B cell developmental impairments that are evident in CD19-deficient mice. Using a genetic approach of deletion and complementation,we show that the c-Myc/miR17-92/Pten axis critically controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death. View Publication

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro,and become more adult-like in their structure,cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al.,2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment,long-term survival,and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue,we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue,resulting in airway-like structures that were remarkably similar to the native adult human lung. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

We have established a novel cell line,designated as TF-1,from a patient with erythroleukemia,which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1,but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion,and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I,the morphological and cytochemical features,and the constitutive expression of globin genes,indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate,TF-1 entered two different pathways. Specifically,hemin and delta-aminolevulinic acid induced hemoglobin synthesis,whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary,TF-1 is a cell line of immature erythroid origin that requires GM-CSF,IL-3,or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3,GM-CSF,and EPO or the signal transduction of these hemopoietic growth factors. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice,its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that textgreater99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality,which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b,suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody,B-1 responses to phosphocholine,and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation. View Publication