Maria Velegraki

Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production

Excessive pro-inflammatory cytokine production in the bone marrow has been associated with the pathogenesis of myelodysplastic syndromes. We herein investigated the involvement of toll-like receptors and their endogenous ligands in the induction/maintenance of the inflammatory process in the marrow of patients with myelodysplastic syndromes. We evaluated the expression of toll-like receptors in marrow monocytes of patients (n=27) and healthy controls (n=25) by flow-cytometry and also assessed the activation of the respective signaling using a real-time polymerase chain reaction-based array. We measured the high mobility group box-1 protein, a toll-like receptor-4 ligand, in marrow plasma and long-term bone marrow culture supernatants by an enzyme-linked immunosorbent assay and we performed cross-over experiments using marrow plasma from patients and controls in the presence/absence of a toll-like receptor-4 inhibitor to evaluate the pro-inflammatory cytokine production by chemiluminescence. We assessed the apoptotic cell clearance capacity of patients’ macrophages using a fluorescence microscopy-based assay. We found over-expression of toll-like receptor-4 in patients’ marrow monocytes compared to that in controls; this over-expression was associated with up-modulation of 53 genes related to the respective signaling. Incubation of patients’ monocytes with autologous, but not with normal, marrow plasma resulted in over-production of pro-inflammatory cytokines, an effect that was abrogated by the toll-like receptor-4 inhibitor suggesting that the pro-inflammatory cytokine production in myelodysplastic syndromes is largely mediated through toll-like receptor-4. The levels of high mobility group box-1 protein were increased in patients’ marrow plasma and culture supernatants compared to the levels in controls. Patients’ macrophages displayed an impaired capacity to engulf apoptotic cells and this defect was associated with excessive release of high mobility group box-1 protein by dying cells. A primary apoptotic cell clearance defect of marrow macrophages in myelodysplastic syndromes may contribute to the induction/maintenance of the inflammatory process through aberrant release of molecules inducing toll-like receptor-4 such as high mobility group box-1 protein.

Increased Levels of the High Mobility Group Box 1 Protein Sustain the Inflammatory Bone Marrow Microenvironment in Patients with Chronic Idiopathic Neutropenia via Activation of Toll-Like Receptor 4

PurposeChronic idiopathic neutropenia (CIN) is a granulocytic disorder characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells and an inflammatory BM microenvironment. The aim of this study was to investigate the possible involvement of Toll-like receptors (TLRs) in the production of pro-inflammatory mediators in CIN BM.MethodsWe evaluated the expression of TLRs in patient BM cell subsets and adherent cells of long-term BM cultures (LTBMCs) using flow cytometry. We also examined the activation of TLR-mediated signaling using real-time PCR arrays and explored for potential endogenous TLR-specific ligands in CIN BM.ResultsCIN patients (n = 30) displayed significantly increased expression of surface TLR4 in monocytes of BM and LTBMC adherent cells compared to controls (n = 27). The TLR signaling gene array study in purified BM CD14+ cells showed that numerous TLR-related genes displayed at least two-fold increase in patients compared to controls. Among the over-expressed genes were genes related to the MyD88-dependent and MyD88-independent pathway suggesting a TLR4-mediated signaling. BM plasma from CIN patients induced the production of pro-inflammatory mediators including interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and IL-8 by autologous BM monocytes, and this effect was abrogated by a specific TLR4 inhibitor. The levels of the high mobility group box 1 protein (HMGB1), representing a TLR4 ligand, were significantly increased in patient LTBMC supernatants compared to controls.ConclusionThese data demonstrate a significant role of BM monocytes in the pathophysiology of CIN through the production of pro-inflammatory cytokines in a TLR4-mediated mechanism under the influence of endogenous ligands such as HMGB1.

Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia

Background Chronic idiopathic neutropenia is characterized by immune-mediated suppression of neutrophil production. Because patients with immune-mediated bone marrow failure syndromes display age-inappropriate telomere shortening in leukocytes, we investigated telomere lengths in peripheral blood mononuclear cells and granulocytes of patients with chronic idiopathic neutropenia. Design and Methods We studied 37 patients with chronic idiopathic neutropenia and 68 age- and sex-matched healthy controls. Relative telomere length and telomerase reverse transcriptase expression were assessed by a quantitative real time polymerase chain reaction. Telomerase activity was determined by a polymerase chain reaction-based immunoassay. Results The mean relative telomere values of peripheral blood mononuclear cells and granulocytes were significantly lower in patients compared to controls, and significantly lower than expected on the basis of the age-adjusted healthy control distribution. The difference in the relative telomere lengths between patients and controls in both peripheral blood mononuclear cells and granulocytes was prominent in those under the age of 50 years. Contrary to the peripheral blood mononuclear cells, in which an inverse correlation was observed between relative telomere values and age, no significant correlation was noted between granulocyte telomere values and patient age. A significant correlation was observed between individual relative telomere values and absolute neutrophil counts. There was no difference in expression of telomerase reverse transcriptase in peripheral blood mononuclear cells between patients and controls but telomerase activity was identified at a significantly higher frequency in controls than in patients. No correlation was found between telomerase activity or telomerase reverse transcriptase expression and relative telomere lengths of peripheral blood mononuclear cells. Conclusions Patients with chronic idiopathic neutropenia display age-inappropriate telomere shortening of peripheral blood cells and low telomerase activity in peripheral blood mononuclear cells. A compensatory increased proliferation of bone marrow hematopoietic progenitor cells in association with lymphocyte replicative exhaustion probably account for these abnormalities.

The −509C/T polymorphism of transforming growth factor-β1 is associated with increased risk for development of chronic idiopathic neutropenia

Objective: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro‐inflammatory and pro‐apoptotic mediators, such as tumor necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β1, and Fas‐Ligand (Fas‐L). In this study, we evaluated the frequency of TNF‐α, TGF‐β1 and Fas‐L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. Methods: The TNF‐α−308G/A, TGF‐β1 −509C/T, +869T/C, +915G/C, and Fas‐L −844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well‐defined area with genetically homogeneous population, using a polymerase chain reaction‐based restriction fragment length polymorphism assay. Results: The mutant genotype C/T or T/T of TGF‐β1 −509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild‐type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18–27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF‐β1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF‐α−308G/A, TGF‐β1 +869T/C and +915G/C and Fas‐L ‐844T/C polymorphisms. Conclusions: The TGF‐β1 −509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF‐β1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.

Clonal patterns of X-chromosome inactivation in peripheral blood cells of female patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by prolonged, unexplained reduction in peripheral blood (PB) neutrophils due to accelerated apoptosis of the bone marrow (BM) granulocytic progenitors.[1][1] There has been growing evidence to suggest that

Transforming growth factor‐beta1 affects interleukin‐10 production in the bone marrow of patients with chronic idiopathic neutropenia

Background:  Chronic idiopathic neutropenia (CIN) is a bone marrow (BM) failure syndrome characterized by accelerated apoptosis of myeloid progenitor cells because of a local imbalance between pro‐inflammatory and anti‐inflammatory cytokines. In this study, we investigated the interplay among transforming growth factor‐beta1 (TGF‐β1), interleukin‐10 (IL‐10), and soluble flt‐3 ligand (sFL) within the BM of CIN patients and probed the role of these cytokines in the pathophysiology of CIN.

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T‐lymphocytes containing T‐cell receptor excision circles

Objectives:  Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T‐lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T‐cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T‐cell lymphopenia in CIN.

Bone marrow-derived mesenchymal stem/stromal cells from patients with splenic marginal zone lymphoma are intrinsically impaired and influence the malignant B-cells

Previous studies have shown that bone marrow mesenchymal stem/stromal cells (BM-MSCs) are involved in the pathogenesis and disease progression of some B-cell lymphomas, including chronic lymphocyti...

Interactions among myeloid regulatory cells in cancer

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.