Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises.

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy. Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs.

Circulating Lymphangioleiomyomatosis Tumor Cells With Loss of Heterozygosity in the TSC2 Gene Show Increased Aldehyde Dehydrogenase Activity.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a destructive metastasizing neoplasm of the lung characterized by proliferation of LAM cells in specialized lung nodules. LAM cells are characterized by expression of the prometastatic and cancer-initiating hyaluronan receptor CD44v6, and loss of heterozygosity (LOH) of TSC1 and TSC2. The circulating neoplastic LAM cells are thought to be involved in metastasis. Because LAM cells display properties of neoplastic, metastatic, and stem cell-like cancer cells, we hypothesized that elevated aldehyde dehydrogenase (ALDH) activity, characteristic of cancer and stem cells, is a property of LAM cells. METHODS: We performed an in silico search of ALDH genes in microdissected LAM lung nodules. To identify circulating LAM cells, we osmotically removed red blood cells from whole blood to obtain peripheral blood mononuclear cells, which were then sorted by fluorescence-activated cell sorting based on their level of ALDH activity. RESULTS: Microdissected LAM lung nodules possess a distinctive ALDH gene profile. The cell subpopulation with high ALDH activity, isolated from circulating cells, possessed TSC2 LOH in 8 of 14 patients with LAM. Approximately 60% of the circulating cells with high ALDH activity expressed CD44v6. Cells with TSC2 LOH from patients with LAM and LAM/TSC exhibited different properties in different body locations, but all cell types showed high ALDH activity. CONCLUSIONS: This new procedure allows for isolation of circulating LAM cells from cultured cells, blood, and chylous effusions and shows that circulating LAM cells are heterogeneous with neoplastic, metastatic, and cancer-stem cell-like properties.

PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Mature enteroendocrine cells contribute to basal and pathological stem cell dynamics in the small intestine.

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here, we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage-tracing studies identified a subset of EECs that reside at +4 position for more than 2 wk, most of which were BrdU-label-retaining cells. Under basal conditions, cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally, the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium, and in the development of EC-derived small intestinal tumors. NEW & NOTEWORTHY The current manuscript demonstrating that a subset of mature enteroendocrine cells (EECs), predominantly enterochromaffin cells, dedifferentiates to fully functional intestinal stem cells (ISCs) is novel, timely, and important. These cells dedifferentiate to ISCs not only in response to injury but also under basal homeostatic conditions. These novel findings provide a mechanism in which a specified cell can dedifferentiate and contribute to normal tissue plasticity as well as the development of EEC-derived intestinal tumors under pathologic conditions.

Masks in imaging flow cytometry.

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis.

Detection and Characterization of Rare Circulating Endothelial Cells by Imaging Flow Cytometry.

Circulating endothelial cells (CECs) are angiogenic cells that appear in increased numbers in the peripheral circulation either as a result of vascular injury or in response to angiogenic stimuli. Elevated levels of CECs have been correlated with various disease states, indicating the use of CECs as a biomarker of disease. Flow cytometry is a widely accepted method for detecting and quantitating CECs. Flow cytometry provides statistical information on large numbers of cells but no information on morphological characteristics. Imaging flow cytometry combines traditional flow cytometry and microscopy, providing a streamlined, multiparameter approach to characterize the biological properties and morphology of large numbers of cells, and is particularly amenable for rare event analysis such as CECs. This approach for identifying and characterizing CECs allows the morphological characterization of large numbers of live, nucleated, single CECs, and alleviates the need for prior enrichment.

Sickle Cell Imaging Flow Cytometry Assay (SIFCA).

Hemoglobin S polymerization under hypoxic conditions in sickle cell disorders causes characteristic shape changes to human red blood cells. Previous sickling assays used to investigate the efficacy of novel agents to treat these disorders are laborious and observer dependent. Here, we describe a partially automated, high-throughput sickling assay using imaging flow cytometry.

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells.

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

Imaging flow cytometry for the study of erythroid cell biology and pathology.

Erythroid cell maturation and diseases affecting erythrocytes are frequently accompanied by morphologic and immunophenotypic changes to these cells. In the past, these changes have been assessed primarily through the use of manual microscopy, which substantially limits the statistical rigor, throughput, and objectivity of these studies. Imaging flow cytometry provides a technology to examine both the morphology of cells as well as to quantify the staining intensity and signal distribution of numerous fluorescent markers on a cell-by-cell basis with high throughput in a statistically robust manner, and thus is ideally suited to studying erythroid cell biology. To date imaging flow cytometry has been used to study erythrocytes in three areas: 1) erythroid cell maturation, 2) sickle cell disease, and 3) infectious diseases such as malaria. In the maturation studies, imaging flow cytometry can closely recapitulate known stages of maturation and has led to the identification of a new population of erythroid cell precursors. In sickle cell disease, imaging flow cytometry provides a robust method to quantify sickled erythrocytes and to identify cellular aggregates linked to morbidities, and in malaria, imaging flow cytometry has been used to screen for new chemotherapeutic agents. These studies have demonstrated the value of imaging flow cytometry for investigations of erythrocyte biology and pathology.

Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. ß2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Characterization of immune cells in psoriatic adipose tissue.

BACKGROUND: Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue. METHODS: Conventional and imaging flow cytometry were used to define immune cell populations in biopsy specimens of psoriatic adipose tissue (n = 30) including T cells, B cells, NK cells, NKT cells, neutrophils, and macrophages. Relationships between adipose immune cell types and body mass index were determined using Spearman regression analysis, and multivariate linear regression analysis was performed to adjust for cardiometabolic disease risk factors. RESULTS: These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use. CONCLUSIONS: Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis.

Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.

Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses.

Bone marrow mesenchymal stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 × 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system.

Sirolimus decreases circulating lymphangioleiomyomatosis cells in patients with lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM), sporadic or in women with tuberous sclerosis complex (TSC), is characterized by cystic lung destruction, lymphatic involvement (eg, chylous pleural effusions, lymphangioleiomyomas), and renal angiomyolipomas (AMLs). The multisystem manifestations of LAM appear to result from metastatic dissemination of LAM cells bearing inactivating mutations or having loss of heterozygosity (LOH) of the tumor suppressor genes TSC1 or TSC2, which leads to hyperactivation of the mammalian target of rapamycin. Sirolimus slows the decline of lung function, reduces chylous effusions, and shrinks the size of AMLs. The purpose of this study was to determine the effect of sirolimus on circulating LAM cells. METHODS: Cells from blood were isolated by a density-gradient fractionation system and from urine and chylous effusions by centrifugation. Blood cells were incubated with anti-CD45-fluorescein isothiocyanate (FITC) and anti-CD235a-R-phycoerythrin (PE) antibodies, and urine and chylous effusion cells were incubated with anti-CD44v6-FITC and anti-CD9-R-PE antibodies. Cells were sorted and analyzed for TSC2 LOH. RESULTS: LAM cells with TSC2 LOH were identified in 100% of blood specimens and 75% of urine samples from patients before therapy. Over a mean duration of 2.2 ± 0.4 years of sirolimus therapy, detection rates of LAM cells were significantly decreased to 25% in blood (P < .001) and 8% in urine (P = .003). Following therapy, a greater loss of circulating LAM cells was seen in postmenopausal patients (P = .025). CONCLUSIONS: Patients receiving sirolimus had a progressive loss of circulating LAM cells that depended on time of treatment and menopausal status.

Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

Restricted mitochondrial protein acetylation initiates mitochondrial autophagy.

Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced 'stress-resilience'. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects.

Apparent mtDNA sequence heterogeneity in single human blood CD34+ cells is markedly affected by storage and transport.

Single CD34(+) cells from adult human peripheral blood show mtDNA sequence heterogeneity. In this study, we compared mtDNA sequence variation in single CD34(+) cells from peripheral blood (PB) mononuclear cells (MNCs) from the same donors but under different conditions of storage and transport: group I, MNCs from heparinized PB that inadvertently required six days to be transported to the testing laboratory; group II, MNCs which were isolated from PB within a day of phlebotomy and frozen prior to transportation and storage. We observed more cell death for MNCs of group I than group II. Concordantly, group I CD34(+) cells had a very low potential for hematopoietic colony formation in vitro compared with group II cells. CD34(+) cells of group II showed an unexpectedly higher level of mtDNA sequence heterogeneity than was present in group I cells. These observations suggest that reduced mtDNA sequence heterogeneity in single CD34(+) cells of group I was likely due to elimination of cells harboring mutations. CD34(+) cells that survive stress ex vivo may be more enriched in quiescent primitive hematopoietic stem cells, with fewer mtDNA mutations than are present in committed progenitors. Technically, attention is required to conditions of preparation of human blood samples for single cell mtDNA analysis.