scholarly journals Erythropoietin (EPO) Regulates Bone Mass Via EPO Receptors on Myeloid and Lymphocytic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 846-846
Author(s):  
Sahar Hiram-Bab ◽  
Albert Kolomansky ◽  
Naamit Deshet-Unger ◽  
Nathalie Ben-Califa ◽  
Tamar Liron ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Bone Resorption ◽  
Bone Loss ◽  
Bone Mass ◽  
B Cell ◽  
Trabecular Bone ◽  
Conflicts Of Interest ◽  
New Findings ◽  
Equal Contribution

Abstract Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production. In response to hypoxia, EPO levels can increase 1000-fold and remain high for weeks to promote hematopoiesis. Therapeutically, the introduction of EPO and erythropoietic stimulating agents into clinical practice has revolutionized the treatment of anemia despite certain concerns regarding the safety of the therapy. Recent studies demonstrate that EPO has activities in addition to hematopoiesis, and modulates bone remodeling by increasing bone resorption and decreasing bone formation, leading to trabecular bone loss. In vitro, EPO directly inhibits murine osteoblast differentiation and mineralization at doses relatively lower than those shown to stimulate osteoclastogenesis. The aim of this study was to investigate the dose-response relationship between EPO dose, hemoglobin (Hgb) levels, and the extent of bone loss, as well as to examine the role of the monocytic and B cell EPO receptor (EPOR) in bone metabolism. Results: Treating mice for 2 weeks with escalating doses of EPO, ranging from 6-540 IU/week, led to a dose-dependent increase in Hgb accompanied by a more dramatic decrease in trabecular bone mass; regression slopes of Hgb and bone volume/total volume (BV/TV, a measure of bone density) were 0.009 vs -0.09, respectively (p<0.05). These effects were associated with a significant increase in the number of preosteoclasts (CD115+ cells) in the bone marrow (r=0.74, p<0.05). To assess whether the osteoclast lineage contributes to EPO-induced bone loss, we generated mice lacking EPOR in the monocytic lineage (LysM-cre+/+;EPORflox/flox, cKO). At steady state, these mice and their LysM+/+;EPORwt/wt controls exhibited similar levels of Hgb (16.7±0.57 and 16.8 ±0.25 g/dL, respectively) and BV/TV (2.73%± 0.73 and 3.10%±0.76, respectively). Although not completely abolished, the bone loss induced by high EPO doses (540 IU/week) was significantly attenuated in cKO compared to control mice (60%±4.7 reduction versus 40%±13.2 reduction, respectively). At the same time, the levels of osteoclast precursors (CD115+ cells) increased from 3.08%±1.12 to 4.67%±0.92 in EPO-treated control mice, although there was no change in bone marrow preosteoclasts and preosteoblasts (defined as CD11b-/ALP+) in EPO-treated cKO mice. This suggests that osteoclast EPOR is responsible, at least in part, for mediating the effect of EPO on bone mass. Adding to the complexity of EPO's osteoimmunological roles, new findings suggest that EPO also regulates bone resorption via EPOR signaling in B cells. EPO stimulated surface expression of the osteoclastogenic RANKL in B cells (MFI: 2.6%±0.1 to 3.13%±0.09 P<0.05) and we found a higher bone mass in mice with conditional EPOR KO in B cells (MB1-cre+/-;EPORflox/flox) (vBMD, 52.2±15.1 versus 40.8±8.8 mg HA/cm3 in MB1-Cre+/-;EPORwt/wt, p<0.05). Conclusions: Our data demonstrate the complexity of EPO-induced bone loss mediated at least partly by EPOR signaling in both myeloid and B cell lineages. Furthermore, since patients who require treatment with EPO are prone to osteoporosis, our data suggest that using the lowest effective EPO dose would not only decrease the risk of thromboembolic complications but also minimize adverse skeletal outcomes. SHB and AK - Equal contribution; YG and DN - Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and the Israel Science Foundation, Grant # 343/17 to DN. Disclosures No relevant conflicts of interest to declare.

Recombinant Erythropoietin and Darbepoetin Alpha Exert a Similar Dose-Dependent Osteopenic Effect Which May Advocate for "Start Low, Go Slow" Dosing Strategy in Clinical Practice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2445-2445
Author(s):  
Kolomansky Albert ◽  
Sahar Hiram-Bab ◽  
Naamit Deshet-Unger ◽  
Nathalie Ben-Califa ◽  
Dafna Gilboa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Clinical Practice ◽  
B Cells ◽  
Bone Loss ◽  
Bone Mass ◽  
B Cell ◽  
Similar Dose ◽  
Darbepoetin Alpha ◽  
Dose Dependent

Abstract Background and rationale: Identifying risk factors for bone loss is of utmost importance in the prevention of osteoporotic bone fractures. We and others have recently described the dramatic erythropoietin (EPO)-induced bone loss. Recombinant EPO and its long-acting derivative, darbepoetin alpha (DA), are frequently and interchangeably used in clinical practice for similar indications including diseases (e.g. multiple myeloma) in which bone mass may be compromised already, as a part of the disease process. Here, we examined in a murine model, the dose response to the erythroid stimulating agents (ESA) EPO and DA, in the erythroid and bone compartments. Methods:C57B6/JRcchsd 9-week old female mice were treated for two weeks with either EPO or DA using two dose intensities. EPO and DA were administered subcutaneously thrice or once weekly, respectively, for a total weekly dose of 27IU/mouse or 540IU/mouse, hereafter defined correspondingly as intermediate (ID) and high dose (HD) subgroups. Bone morphometry of the distal femoral metaphysis was assessed using microCT. Flow cytometry was used for immunophenotyping of bone marrow (BM) cells. One-way ANOVA was used to compare variables among the treatment groups. Results: First, we found that ID-DA induced bone loss to a similar extent as ID-EPO as reflected by a significant (p<0.05) reduction in the trabecular bone fraction (BV/TV, -31%), bone mineral density (BMD, -53%) and trabecular number (Tb.N, -17%). HD-EPO and HD-DA resulted in even higher degrees of bone mass reduction: -48% in BV/TV and -57% in Tb.N (p<0.01 for both). Noteworthy, despite modestly superior gain (~10%) in hemoglobin level (21 and 22.5 mg/dl p< 0.01 in HD-EPO and ID-EPO, respectively; mean hemoglobin levels in diluent-treated mice was 16.4), administration of HD-EPO resulted in substantially higher bone loss compared to ID-EPO. This was manifested in ~80% further reduction in BV/TV as well as 2.5-fold further drop in Tb.N as demonstrated by ΔBV/TV and ΔTb.N (i.e. reduction relative to the control) of 48% versus 27% and 57% versus 23%, respectively (p<0.02 for both parameters). By flow cytometry EPO, but not DA, induced a 35% reduction in alkaline phosphatase positive CD11b negative (ALPL+/CD11b-) preosteoblast population in the bone marrow (p<0.01). The percentage of CD115+ BM cells, containing osteoclast precursors, was increased by 26-30% following both EPO and DA treatments (p<0.05 for EPO/DA versus control and non-significant p>0.05 for EPO versus DA groups). In view of the established role of B cells in bone metabolism, we determined the effects of EPO and DA on BM B cell compartment. HD-EPO but not ID-EPO induced a "maturation arrest"-like state in the bone marrow B cell population, increasing the proportion of B220+/CD19-/CD43high/IgM- pre-pro-B two-fold and B220+/CD19+/CD43high/IgM- pro-B cells 2.8-fold (p<0.05) while reducing the proportion of B220+/CD19+/CD43low/IgM+immature B cells by 2.3-fold (p<0.01). Intriguingly, these alterations were not noted following DA treatment. Conclusion:Applying clinically comparable dosing regimen, both recombinant EPO and DA exert a similar, dose-dependent bone loss, not directly proportional to the erythroid response. However, DA, in contrast to EPO, spares the preosteoblast and bone marrow B cell populations. These finding suggest a different mechanism of action of these closely related ESA preparations on the skeletal and immune systems thus warranting further exploration. Finally, our findings advocate for initiating and maintaining patients with the lowest possible doses of EPO/DA to minimize untoward skeletal and other extra-erythropoietic effects, while achieving the desired correction of anemia. This study was supported by a grant from the Israel Cancer Association to DN, YG and MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals B Cell Specific Knockdown of the Erythropoietin (EPO) Receptor Attenuates EPO-Induced Bone Loss in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 939-939
Author(s):  
Albert Kolomansky ◽  
Naamit Deshet-Unger ◽  
Nathalie Ben-Califa ◽  
Zamzam Awida ◽  
Maria Ibrahim ◽  
...  
Keyword(s):  
B Cells ◽  
Bone Loss ◽  
B Cell ◽  
Trabecular Bone ◽  
Cell Lineage ◽  
Lineage Tracing ◽  
Trabecular Bone Loss ◽  
Β3 Integrin ◽  
First Time

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p&lt;0.05, Figure 1). Remarkably, this effect was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment (21.1±0.1 mg/dL vs 20.4±0.2 mg/dL in the cKD vs control mice, p&lt;0.05). An EPO-induced increase in CD115+ Pro-B cells was observed in EPO-treated control mice but was absent in the cKD mice. The latter finding correlates with the observed bone loss and indicates that the increased number of MCSF-R-expressing pro-B cells is dependent on B cell EPO-R. Supporting the osteoclastic potential of this specific B cell subpopulation is the fact that most of the CD115+ Pro-B cells also express β3 integrin (CD61) which is essential for osteoclast differentiation and function. Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice

2018 ◽  
Vol 238 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Thomas Funck-Brentano ◽  
Karin H Nilsson ◽  
Robert Brommage ◽  
Petra Henning ◽  
Ulf H Lerner ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
Bone Mass ◽  
Trabecular Bone ◽  
Cortical Bone ◽  
Bone Strength ◽  
Bending Test ◽  
Bone Homeostasis ◽  
Mineral Density

WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.

Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4943-4943
Author(s):  
Charles Repetti ◽  
Hsueh-Hua Chen ◽  
Yongbao Wang ◽  
Vanessa A Jones ◽  
Albert K Ho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Sequence Variant ◽  
Low Grade ◽  
Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results  Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15).  Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 79-79
Author(s):  
Zev J. Greenberg ◽  
Darlene A. Monlish ◽  
Rachel L. Bartnett ◽  
Jeffrey J. Bednarski ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Development ◽  
B Cell Development ◽  
Cellular Migration ◽  
Physical Interaction ◽  
Hematopoietic Stem ◽  
Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p&lt;0.005), splenic (35% fewer, p&lt;0.05), lymphatic (65% fewer, p&lt;0.0001), and peripheral (30% fewer, p&lt;0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p&lt;0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p&lt;0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

scholarly journals Demethylase Activity of Aid during Germinal Center B Cell Maturation Could Contribute to Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 59-59 ◽  
Author(s):  
Maria Del Pilar Dominguez ◽  
Matt Teater ◽  
Nyasha Chambwe ◽  
David Redmond ◽  
Bao Vuong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cytosine Methylation ◽  
Conflicts Of Interest ◽  
Retroviral Vectors ◽  
Dna Hypomethylation ◽  
Demethylase Activity

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors that arise from germinal center B cells (GCBs) and post-GCBs and are noted for their heterogeneity and variable clinical outcomes. Epigenetic modifications like DNA methylation of cytosine nucleotides have emerged as important mechanisms of gene regulation and have been implicated in carcinogenesis. Our previous genome-wide studies in primary samples revealed profound alterations in the cytosine methylation patterning of DLBCLs. We also found that expression of activation-induced deaminase (AID) was significantly associated with the loss of methylation in DLBCL patients and was predominantly identified within computationally predicted AID-binding RGYW motifs. AID is a cytidine deaminase required for class switch recombination and somatic hypermutation (SHM) of immunoglobulin genes in GCBs. The enzymatic machinery that mediates these processes is error-prone and may introduce point-mutations and changes in DNA methylation, resulting in genomic and epigenomic instability. Since AID can also function as a demethylase during embryonic development, we asked whether AID has demethylase activity during transit of B cells through the GCs and if its overexpression can contribute to lymphomagenesis through disrupting DNA methylation. To address this question, we studied the epigenetic function of AID in GCBs and GC-derived lymphomas. We characterized the methylome of naïve B cells (NBs) and GCBs isolated from human tonsils and spleens of immunized mice by enhanced Reduced Representation Bisulfite Sequencing (eRRBS). We observed that the transition from NBs to GCBs was characterized by DNA hypomethylation, with 60,000 and 8,000 differentially methylated CpGs (DMCs) that were hypomethylated in GCBs compared to NBs, in human and mouse respectively. We also found that hypomethylated regions were enriched for the putative AID binding site RGYW (Wilcoxon P <.001). Furthermore, AID knockdown in lymphoma cells (RAMOS) resulted in preferential hypermethylation at AID-binding sites (Chi square P ~ 0). We then isolated DNA from splenic NBs and GCBs from Aicda-/- (AID-deficient) and Aicda+/+ (wild type) mice and performed eRRBS analysis, obtaining single nucleotide resolution for 2.5-3 million represented CpGs. We observed that most of the 8,000 hypoDMCs identified between GCBs and NBs in Aicda+/+ mice were absent in Aicda-/- mice (800 hypoDMCs between GCBs and NBs Aicda-/- cells), implying that AID is a regulator of DNA methylation in GCBs. In addition, those AID-dependent hypoDMCs were predominantly localized in introns (35%), and also in promoters (10%) and exons (10%). We then defined differentially methylated regions (DMRs) based on the following criteria: ≥ 5 DMCs and methylation difference ≥10%, with >250bp between DMRs. We identified DMRs that get hypomethylated in GCBs in the Aicda+/+ mice, but are not hypomethylated in Aicda-/- GCBs, corresponding to >200 genes that represent AID epigenetic targets. These genes include factors involved in B cell function and differentiation like PAX5, BCL2L1, IRF8 and others. Not unexpectedly, many of epigenetic targets are also known targets for SHM, but some are novel targets that only demonstrate evidence of epigenetic deregulation. We also analyzed the transcriptome of NBs and GCBs from Aicda-/- and Aicda+/+ mice by RNA-seq and detected an increase in DNMT1 expression in Aicda-/- cells compared to Aicda+/+ cells. There were no significant changes in expression of other factors involved in modification of cytosine methylation, such as DNMT3a/3b, TET1/2/3, UNG or MSH2/6. Finally, we performed bone marrow transplantation experiments using VavP-Bcl2 mice, which are known to develop GC-derived lymphomas. We transplanted VavP-Bcl2 bone marrow cells infected with AID-expressing retroviral vectors into C57BL/6 mice and monitored the progression of the resulting BCL2-driven lymphomas. Our preliminary results indicate that high AID expression is correlated with a more aggressive phenotype of the disease. We are currently analyzing the epigenetic targets of AID in both normal GCBs and tumors, in order to find genes that could be epigenetically deregulated and contribute to the formation of lymphomas. Our results demonstrate for the first time that AID functions as a demethylase in GCBs in vivo and suggest that the epigenetic role of AID could contribute to lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

In Vitro Study Of The Mechanisms Involved In The Bone Marrow Mesenchymal Stromal Cell Modulatory Effect On B Cell Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1053-1053
Author(s):  
Eliana Amati ◽  
Giulio Bassi ◽  
Mariano Di Trapani ◽  
Francesco Liotta ◽  
Francesco Annunziato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Cpg Oligodeoxynucleotide ◽  
Ifn Γ ◽  
Cell Inhibition

Abstract Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches. Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism. Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation. Disclosures: No relevant conflicts of interest to declare.

The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1465-1465
Author(s):  
Jason Mullenix ◽  
Kimi Y Kong ◽  
Kristin Severns Owens ◽  
Jason Rogers ◽  
Shannon FitzPatrick ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mature Mirnas ◽  
Cell Development ◽  
B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

Demylinating Peripheral Neuropathy and Lymphoplasmacytic Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5090-5090
Author(s):  
Carol Ann Huff ◽  
Vinay Chaudhry ◽  
Charlotte Sumner ◽  
David Cornblath
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Neuropathy ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Demyelinating Polyneuropathy ◽  
Lymphoplasmacytic Lymphoma ◽  
Modest Improvement ◽  
Bone Marrow Biopsies

Abstract Abstract 5090 Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and plasma cells, which does not fulfill criteria for any of the other small B-cell lymphoid neoplasms (WHO 2008). Neuropathy has been described in association with Waldenstrom's macroglobulinemia, but less so with LPL. We present 7 cases of neuropathy and LPL to highlight the variable presentations. 1) A 56 year old man developed sensory ataxia with an IgM kappa non-MAG paraprotein. CSF protein was 97. Stabilized on MMF, he slowly worsened. Plasma exchange (PE) was given with improvement. Bone marrow (BM) 4 years into his course revealed LPL. Rituximab was given, and his PE reduced. 2) A 49 year old woman developed progressive weakness with rapid decline in January 2010. NCS showed demyelinating polyneuropathy, and CSF protein was 179. An IgM kappa non-MAG paraprotein was found. BM was normal. PE was given with improvement but later prednisone, IVIg and rituximab twice did not help. Repeat BM revealed 2% clonal CD20+ CD5negCD10neg B cells by flow cytometry. PE was given with modest improvement. Cyclophosphamide 1 gm/m2 monthly was given with improvement. 3) A 53 year old man noted imbalance and distal weakness. NCS showed absent SAPs and prolonged distal and F wave latencies. An anti-MAG positive, IgM kappa paraprotein was found. In 2002 BM was normal. He received rituximab weekly × 4 doses. 7 years later, he developed anemia and worsening neuropathy. Repeat BM showed 0.5% CD20+CD5negCD10neg kappa-restricted B cells by flow cytometry and weekly rituximab was reinitiated. 4) A 62 year old man developed weakness and areflexia. NCS showed asymmetric demyelinating polyneuropathy. Biclonal gammopathy of IgM kappa and IgG kappa was found. BM showed LPL by histopathology. Prednisone was given with improvement. Later two courses of weekly rituximab were given. 5) A 55 year old woman developed asymmetric weakness. NCS showed asymmetric demyelinating polyneuropathy. MRI showed enlargement, abnormal signal intensity, and abnormal enhancement of bilateral radial, median, and ulnar nerves. She was found to have an IgG kappa paraprotein and LPL on BM biopsy. She was treated with rituximab, cyclophosphamide, fludarabine, and PE. In each case, the primary feature driving the need for therapy was the neuropathy and not the underlying hematologic process. Further, worsening neuropathy in 3 cases led to repeat bone marrow biopsies revealing a clonal B cell process and a diagnosis of lymphoplasmacytic lymphoma. Thus, in the presence of an IgM monoclonal gammopathy and peripheral neuropathy, we suggest bone marrow examination for LPL and then consideration of therapy directed toward the abnormal B cell clone. Disclosures: No relevant conflicts of interest to declare.

Iterative Germinal Center Re-Entries of Memory B-Cells with t(14;18) Translocation and Early Steps of Follicular Lymphoma Progression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 150-150
Author(s):  
Sandrine Roulland ◽  
Jocelyne Stephanie Sungalee ◽  
Ester Morgado ◽  
Emilie Mamessier ◽  
Emilie Gregoire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Lymph Nodes ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Systemic Disease ◽  
Memory B Cells ◽  
Lymphoid Organs ◽  
Healthy Humans

Abstract Abstract 150 The recent demonstration that memory B-cells can re-enter germinal centers (GCs) and participate to new rounds of GC reactions has opened the possibility that multi-hit B-cell lymphomagenesis could be a much more dynamic process than initially anticipated, gradually progressing throughout the successive passages of memory B-cells in GCs during a lifetime of successive immunological challenges. Here, we provide evidence for this scenario in follicular lymphoma (FL), a GC derived B-cell malignancy initiated in the bone marrow by the hallmark t(14;18) BCL2/IGH translocation. To address this issue, we engineered an original sporadic BCL2tracer mouse model mimicking the rare occurrence of t(14;18) translocation in humans through V(D)J recombination errors (1 in a million B-cells) allowing to track the resultant BCL2-expressing clones; and underwent a molecular/immunofluorescent tracking of t(14;18)+ clones vs. normal memory B-cells in paired lymphoid tissue samples (spleen, lymph nodes, bone marrow) from healthy individuals. We first show that contrary to the current dogma, ectopic BCL2 expression is not sufficient to provoke the FL's characteristic differentiation arrest of activated B-cells as GC B-cells, thereby suggesting that differentiated BCL2+ memory B-cells must return to the GC to acquire additional oncogenic hits and “fix” in situ growth. Strikingly, we further find that in a small fraction of “healthy” humans, such differentiation arrest already operated, and that a clonally expanded population of t(14;18)+ cells with FL-like features have widely disseminated in blood and in multiple lymphoid organs (spleen, lymph nodes, bone marrow), with unprecedentedly reported frequencies (from 1/million to 1/500 cells in some individuals), shaping the systemic disease presentation observed in FL patients. Using molecular/immunofluorescent backtracking of such clones in various paired and remote lymphoid organs, we further demonstrate that t(14;18)+ clones systematically display an extensive history of AID (activation-induced cytidine deaminase)-mediated events compatible with iterative rounds of GC co-opting, in sharp contrast to single memory B-cell clones from the same individuals. We thus show that BCL2-expressing memory B-cells require multiple GC transits to acquire the distinctive FL-like maturation arrest as centrocyte/centroblasts and to progress to advanced FL precursor stages. Altogether, our data argue for a model of lymphomagenesis, in which progression from precursor stages to FL occurs asymptomatically over an extended period of time by subverting the dynamic and plastic attributes of memory B-cells. This understanding of the pre-clinical phases driving FL development in asymptomatic patients should help rationalize prospective approaches designed to identify biomarkers of risk, and innovative therapeutic targets present in early, potentially more curable phases of the disease. Disclosures: No relevant conflicts of interest to declare.

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