Megakaryocyte progenitors are the main APCs inducing Th17 response to lupus autoantigens and foreign antigens.
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ABSTRACT:
SUBMITTER: Kang HK
PROVIDER: S-EPMC3370101 | biostudies-literature | 2012 Jun
REPOSITORIES: biostudies-literature
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Project description:Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.
| S-EPMC4476694 | biostudies-literature
Project description:In helper T cells, IL-13 is traditionally considered a Th2-type cytokine that is coexpressed with IL-4. Using mouse models of immunization and autoimmunity, we demonstrate that IL-13 is frequently uncoupled from IL-4, and that it can be produced by both IFN-?(+) Th1 cells and IL-17(+) Th17 cells. We report that these IL-13-producing Th1 and Th17 cells are distinct from classical IL-4(+) Th2 cells and that they are relatively common, appearing in the context of both protective and pathogenic T-cell responses. We also demonstrate that IL-13 and Th2-type cytokines can have important consequences in Th1- and Th17-dominated settings, such as lymphopenia-induced autoimmune disease, where they can be either pro- or anti-inflammatory, depending on whether they act on innate or adaptive immune cells. Taken together, our studies indicate that IL-13 production is more widespread than previously appreciated and that blocking this cytokine may have therapeutic benefits even in settings where traditional IL-4-driven Th2-type responses are not evident.
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Project description:BACKGROUND:Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by "bulk" assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. RESULTS:To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: "Pre-MEP," enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; "E-MEP," strongly biased towards erythroid differentiation; and "MK-MEP," a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. CONCLUSIONS:Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.
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Project description:The Sigirr gene (also known as Tir8) encodes for an orphan receptor of the Toll-like receptor (TLR)/interleukin 1 receptor family that inhibits TLR-mediated pathogen recognition in dendritic cells. Here, we show that Sigirr also inhibits the activation of dendritic cells and B cells upon exposure to RNA and DNA lupus autoantigens. To evaluate the functional role of Sigirr in the pathogenesis of systemic lupus erythematosus (SLE), we generated Sigirr-deficient C57BL/6-lpr/lpr mice. These mice developed a progressive lymphoproliferative syndrome followed by severe autoimmune lung disease and lupus nephritis within 6 mo of age as compared with the minor abnormalities observed in C57BL/6-lpr/lpr mice. Lack of Sigirr was associated with enhanced activation of dendritic cells and increased expression of multiple proinflammatory and antiapoptotic mediators. In the absence of Sigirr, CD4 T cell numbers were increased and CD4(+)CD25(+) T cell numbers were reduced. Furthermore, lack of Sigirr enhanced the activation and proliferation of B cells, including the production of autoantibodies against multiple nuclear lupus autoantigens. These data identify Sigirr as a novel SLE susceptibility gene in mice.
| S-EPMC2525585 | biostudies-literature
Project description:Chemokine-directed leukocyte migration is a critical component of all innate and adaptive immune responses. The atypical chemokine receptor ACKR2 is expressed by lymphatic endothelial cells and scavenges pro-inflammatory CC chemokines to indirectly subdue leukocyte migration. This contributes to the resolution of acute inflammatory responses in vivo. ACKR2 is also universally expressed by innate-like B cells, suppressing their responsiveness to the non-ACKR2 ligand CXCL13, and controlling their distribution in vivo. The role of ACKR2 in autoimmunity remains relatively unexplored, although Ackr2 deficiency reportedly lessens the clinical symptoms of experimental autoimmune encephalomyelitis induced by immunization with encephalogenic peptide (MOG(35-55)). This was attributed to poor T-cell priming stemming from the defective departure of dendritic cells from the site of immunization. However, we report here that Ackr2-deficient mice, on two separate genetic backgrounds, are not less susceptible to autoimmunity induced by immunization, and in some cases develop enhanced clinical symptoms. Moreover, ACKR2 deficiency does not suppress T-cell priming in response to encephalogenic peptide (MOG(35-55)), and responses to protein antigen (collagen or MOG(1-125)) are characterized by elevated interleukin-17 production. Interestingly, after immunization with protein, but not peptide, antigen, Ackr2 deficiency was also associated with an increase in lymph node B cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances T helper type 17 (Th17) cell development and survival. Thus, Ackr2 deficiency does not suppress autoreactive T-cell priming and autoimmune pathology, but can enhance T-cell polarization toward Th17 cells and increase the abundance of GM-CSF(+) B cells in lymph nodes draining the site of immunization.
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Project description:Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells.
| S-EPMC2688789 | biostudies-literature
Project description:Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16 s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.
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Project description:Platelets have a key role in atherogenesis and its complications. Both hypercholesterolemia and increased platelet production promote atherothrombosis; however, a potential link between altered cholesterol homeostasis and platelet production has not been explored. Here we show that transplantation of bone marrow deficient in ABCG4, a transporter of unknown function, into Ldlr(-/-) mice resulted in thrombocytosis, accelerated thrombosis and atherosclerosis. Although not detected in atherosclerotic lesions, Abcg4 was highly expressed in bone marrow megakaryocyte progenitors (MkPs). Abcg4(-/-) MkPs had defective cholesterol efflux to high-density lipoprotein (HDL), increased cell surface expression of the thrombopoietin (TPO) receptor (c-MPL) and enhanced proliferation. These consequences of ABCG4 deficiency seemed to reflect disruption of negative feedback regulation of c-MPL signaling by the E3 ligase c-CBL and the cholesterol-sensing LYN kinase. HDL infusion reduced platelet counts in Ldlr(-/-) mice and in a mouse model of myeloproliferative neoplasm in an ABCG4-dependent fashion. HDL infusions may offer a new approach to reducing atherothrombotic events associated with increased platelet production.
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Project description:Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin(-)CD34(+)CD38(mid)CD45RA(-)FLT3(-)MPL(+)CD36(-)CD41(-) population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.
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Project description:Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.
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