Growth Of Erythroid Progenitors Research Articles

A rat model of multicompartmental traumatic injury and hemorrhagic shock induces bone marrow dysfunction and profound anemia.

Severe trauma is associated with systemic inflammation and organ dysfunction. Preclinical rodent trauma models are the mainstay of postinjury research but have been criticized for not fully replicating severe human trauma. The aim of this study was to create a rat model of multicompartmental injury which recreates profound traumatic injury. Male Sprague-Dawley rats were subjected to unilateral lung contusion and hemorrhagic shock (LCHS), multicompartmental polytrauma (PT) (unilateral lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), or naïve controls. Weight, plasma toll-like receptor 4 (TLR4), hemoglobin, spleen to body weight ratio, bone marrow (BM) erythroid progenitor (CFU-GEMM, BFU-E, and CFU-E) growth, plasma granulocyte colony-stimulating factor (G-CSF) and right lung histologic injury were assessed on day 7, with significance defined as p values <0.05 (*). Polytrauma resulted in markedly more profound inhibition of weight gain compared to LCHS (p = 0.0002) along with elevated plasma TLR4 (p < 0.0001), lower hemoglobin (p < 0.0001), and enlarged spleen to body weight ratios (p = 0.004). Both LCHS and PT demonstrated suppression of CFU-E and BFU-E growth compared to naïve (p < 0.03, p < 0.01). Plasma G-CSF was elevated in PT compared to both naïve and LCHS (p < 0.0001, p = 0.02). LCHS and PT demonstrated significant histologic right lung injury with poor alveolar wall integrity and interstitial edema. Multicompartmental injury as described here establishes a reproducible model of multicompartmental injury with worsened anemia, splenic tissue enlargement, weight loss, and increased inflammatory activity compared to a less severe model. This may serve as a more effective model to recreate profound traumatic injury to replicate the human inflammatory response postinjury.

Posttraumatic pneumonia exacerbates bone marrow erythropoietic dysfunction.

Pneumonia is a common complication after severe trauma that is associated with worse outcomes with increased mortality. Critically ill trauma patients also have persistent inflammation and bone marrow dysfunction that manifests as persistent anemia. Terminal erythropoiesis, which occurs in bone marrow structures called erythroblastic islands (EBIs), has been shown to be impacted by trauma. Using a preclinical model of polytrauma (PT) and pneumonia, we sought to determine the effect of infection on bone marrow dysfunction and terminal erythropoiesis. Male and female Sprague-Dawley rats aged 9 to 11 weeks were subjected to either PT (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofracture) or PT with postinjury day 1 Pseudomonas pneumonia (PT-PNA) and compared with a naive cohort. Erythroblastic islands were isolated from bone marrow samples and imaged via confocal microscopy. Hemoglobin, early bone marrow erythroid progenitors, erythroid cells/EBI, and % reticulocytes/EBI were measured on day 7. Significance was defined as p < 0.05. Day 7 hemoglobin was significantly lower in both PT and PT-PNA groups compared with naive (10.8 ± 0.6 and 10.9 ± 0.7 vs. 12.1 ± 0.7 g/dL [ p < 0.05]). Growth of bone marrow early erythroid progenitors (colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte; erythroid burst-forming unit; and erythroid colony-forming unit) on day 7 was significantly reduced in PT-PNA compared with both PT and naive. Despite a peripheral reticulocytosis following PT and PT-PNA, the percentage of reticulocytes/EBI was not different between naive, PT, and PT-PNA. However, the number of erythroblasts/EBI was significantly lower in PT-PNA compared with naive (2.9 ± 1.5 [ p < 0.05] vs. 8.9 ± 1.1 cells/EBI macrophage). In addition to changes in EBI composition, EBIs were also found to have significant structural changes following PT and PT-PNA. Multicompartmental PT altered late-stage erythropoiesis, and these changes were augmented with the addition of pneumonia. To improve outcomes following trauma and pneumonia, we need to better understand how alterations in EBI structure and function impact persistent bone marrow dysfunction and anemia.

Bone Marrow Adipokine Expression Was Associated With Decreased Erythroid Colony Growth After Trauma.

Proinflammatory and immunomodulatory adipokines are linked to inflammation in critically ill patients but are poorly studied after injury. We hypothesized that trauma would induce systemic adipokine release and influence erythroid suppression. Blood and bone marrow (BM) were collected from trauma patients (ISS > 15, n = 90) and compared to patients undergoing elective hip replacement (n = 37). Plasma adipokine levels were measured, and BM was assayed for adipokine transcription and erythroid progenitor growth potential. Differences were detected using t-tests and correlations using simple linear regression. Trauma patients exhibited decreased adiponectin (1.8* vs 3.4mg/mL) and increased leptin (7.8* vs 4.6ng/mL) and resistin (3.1* vs 2.5ng/mL), with sex- and age-specific differences. They also showed increased BM visfatin transcription. Adipokine transcription negatively correlated with erythroid progenitor growth. Adipose tissue activity is linked to inflammatory responses after injury, with variability by age and sex. Bone marrow adipose tissue may influence erythroid recovery after trauma.

P1414: MARROW ADIPOCYTE-ENRICHED NICHE HOSTS AND INFLUENCES A FRACTION OF HEMATOPOIETIC PROGENITORS IN ADULT HIP BONE

Background: The hematopoietic process is tightly regulated by the bone marrow (BM) microenvironment. Emerging studies recognized a regulatory role of mouse BM adipose tissue (BMAT) on self-renewal and differentiation of hematopoietic stem cells (HSC) and progenitors, while results on human BMAT are still scarce. Aims: Although hip arthroplasty-derived BM serves as an available resource for non-malignant hematopoietic cell research, the BMAT component of this BM and its’ impact on hematopoietic progenitors is not yet revealed. Therefore, our aims were to explore the contribution of human BMAT to the maintenance of hematopoietic progenitors. Methods: BM samples of acetabular bones were obtained from patients undergoing hip arthroplasty (n=13, 65.7±12.5 years, Ethical approval 187/18). Centrifugation was applied to separate the fatty layer containing BMAT from the BM cell suspension. The BM fraction was used for isolation of BM mononuclear (BMC) and expansion of BM mesenchymal stromal cells (BM-MSC). Digestion of BMAT was applied to separate the non-adipocytic cells of BMAT from mature adipocytes, expand them, and to investigate their functions and transcriptional activity by RNA sequencing. Multiparametric flow cytometry was applied to explore surface marker expression. Hematopoietic activity was investigated by short-term colony assays of cells ex vivo and in vitro upon co-culture with BM-MSC or sub-cultured BMAT adipocytes. Results are presented as mean ±SD and statistical significance was estimated by paired tests. Results: We found that the total colony forming cell number in BMC (0.147±0.068) was significantly higher than in BMAT-associated cells (0.0684±0.062%, p=0.0002, n=11). The strongest reduction within BMAT was observed in colony forming unit (CFU)-macrophages and CFU-granulocyte-erythroid-macrophage/monocyte-megakaryocytes. The content of CD45+CD34+ cells was higher in BMC (0.901±1.009%) than in BMAT (0.357±0.505%), while interestingly no differences were found in CD33+ cell frequencies. Both, expanded BMAT and sub-cultured BMAT adipocytes showed stronger adipogenic potential than BM-MSC. When compared to BM-MSC, sub-cultured BMAT adipocytes showed weaker potential to support growth of committed hematopoietic progenitors within BMC ex vivo, favorizing the growth of early and late erythroid progenitors at the expense of other hematopoietic cells. Transcriptomic analyses showed that HSC niche factors (Angptl4, Wnt5b, Il7, Dkk3), but also LepR were upregulated in BM-MSC compared to expanded BMAT cells. Summary/Conclusion: Our findings indicate a yet unrevealed participation of the BMAT niche in compartmentalization of adult hip bone hematopoietic progenitors by hosting a set of hematopoietic cells and potentially, BMAT progenitors in the close proximity of BM adipocytes. Since a behavior of BM populations is dependent on local microenvironmental context and bone region, obtained results might reflect pathologic settings in arthroplasty patients. Further studies have to address the potential coupling of marrow adipogenesis and hematopoietic progenitor maintenance during aging and diseases.

Mechanisms of improved erythroid progenitor growth with removal of chronic stress after trauma

BackgroundErythropoietic dysfunction after trauma and critical illness is associated with anemia, persistent inflammation, increased hematopoietic progenitor cell mobilization from the bone marrow, and reduced erythroid progenitor growth. Yet the duration and reversibility of these postinjury bone marrow changes remain unknown. This study sought to determine whether removal of chronic postinjury stress could induce improvements in erythroid progenitor growth. MethodsSprague-Dawley rats (n = 8–11/group) were assigned to the following: naïve, lung contusion and hemorrhagic shock, lung contusion and hemorrhagic shock plus daily chronic stress for 7 days followed by 7 days of routine handling to allow recovery (lung contusion and hemorrhagic shock + chronic stress 7), or lung contusion and hemorrhagic shock plus chronic stress for 14 days (lung contusion and hemorrhagic shock + chronic stress 14). Circulating CD117+CD71+ erythroid progenitors were detected by flow cytometry. Rodents were killed on day 14, and bone marrow erythroid progenitor growth and erythroid transcription factors were assessed. Differences were assessed by analysis of variance (P < .05). ResultsCompared to lung contusion and hemorrhagic shock + chronic stress 14, lung contusion and hemorrhagic shock + chronic stress 7 rodents had improved hemoglobin (8% ± 10% increase vs 6% ± 10% decrease) with fewer mobilized erythroid progenitors (898 × vs 1,524 cells), lower granulocyte-colony stimulating factor levels (3.1 ± 1.1 × pg/mL vs 5.9 ± 1.8 pg/mL), and improved erythroid progenitor growth. Cessation of stress had no impact on erythroid transcription factors GATA-1, GATA-2, LMO2, or KLF1. ConclusionImprovements in erythroid progenitor growth and reduced hematopoietic progenitor cell mobilization were seen 7 days after cessation of chronic stress and were associated with an improvement in hemoglobin. Early bone marrow erythropoietic functional recovery may result from resolution of hematopoietic progenitor mobilization rather than upregulation of pro-erythroid transcription factors. This study suggests that postinjury anemia is reversible and has the potential to improve with the cessation of stress.

Nitric Oxide Mediation in Hydroxyurea and Nitric Oxide Metabolites' Inhibition of Erythroid Progenitor Growth.

In several systems, hydroxyurea has been shown to trigger nitric oxide (NO) release or activation of NO synthase (NOS). To elucidate this duality in its pharmacological effects, during myelosuppression, we individually examined hydroxyurea’s (NO releasing agent) and NO metabolites’ (stable NO degradation products) effects on erythroid colony growth and NOS/NO levels in mice using NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Hydroxyurea and nitrite/nitrate decreased the bone marrow cellularity that was blocked by PTIO only for the NO metabolites. Hydroxyurea inhibition of colony-forming unit-erythroid (CFU-E) formation and reticulocytes was reversed by PTIO. Moreover, hydroxyurea, through a negative feedback mechanism, reduced inducible NOS (iNOS) expressing cells in CFU-E, also prevented by PTIO. Nitrate inhibition of burst-forming units-erythroid (BFU-E) colony growth was blocked by PTIO, but not in mature CFU-E. The presented results reveal that NO release and/or production mediates the hydroxyurea inhibition of mature erythroid colony growth and the frequency of iNOS immunoreactive CFU-E.

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

High Mobility Group Box 1 Protein (HMGB1) Mediates Anemia of Inflammation through Lineage Skewing and Direct Inhibition of EPO Signaling

Erythropoiesis is sensitive to inflammation and its associated mediators. We previously identified the damage-associated molecular pattern, high mobility group box 1 protein (HMGB1), as a causative factor involved in the development of anemia using a murine model of chronic inflammation, and recently, showed that HMGB1 negatively affects human erythropoiesis, in part, through altered erythropoietin (EPO) signaling and hematopoietic stem and progenitor cell (HSPC) lineage commitment. Despite these findings, a mechanistic understanding of erythroid dysfunction in the presence of HMGB1 is lacking.We first documented a dose-dependent logarithmic difference in erythroid cell growth at Days 7 and 11 of culture of CD34+ HSPCs during erythroid differentiation as well as a relatively smaller loss of cells at Day 14 between HMGB1-treated and vehicle-treated cultures. These results suggest that HMGB1 exerts its effects at distinct stages of erythroid differentiation: at both the progenitor and the precursor stages. Indeed, flow analysis of erythroblast differentiation showed that HMGB1 caused an accumulation of precursors at the basophilic erythroblast stage. The loss of poly- and orthochromatic erythroblasts resulted from increased apoptosis at these stages of erythroid differentiation as assessed by annexin-V staining. Induction of apoptosis was confirmed by a dose-dependent increase in apoptosis and 3-fold increase in cleaved caspase-3 in HUDEP-2 cells, an erythroid cell line. Furthermore, a mutant form of HMGB1 (3S-HMGB1) in which critical cysteine residues were mutated to abolish the activity of the protein failed to alter erythroid proliferation and survival.HMGB1 appears to deprive differentiating erythroid cells of growth and survival signals through attenuated phosphorylation of downstream EPO effectors including pJAK2 and pSTAT5, independent of SHP1/2 activity. As a consequence of perturbed EPO signaling, erythroid cells contained reduced levels of nuclear HSP70 and GATA1, which ultimately led to apoptosis. Importantly, EPO co-administrated with either tumor necrosis factor (TNF), interleukin-1β (IL-1β), or IL-6 led to phosphorylation levels of JAK2-STAT5 comparable to controls, suggesting that direct inhibition of EPO signaling is specific to HMGB1.To document the effect of HMGB1 on EPO-dependent and independent progenitors, erythroid progenitor growth and differentiation were monitored at 24hr intervals in culture. CFU-E but not BFU-E numbers were decreased following treatment with HMGB1. Moreover, HMGB1-treated cells expressed high levels of GATA2 indicating deregulation of GATA transcription factor switching. Myelo-erythroid culture systems demonstrated that HMGB1 strongly inhibited erythroid commitment of CD34+ HSPCs while it favored the generation of increased numbers of CD11b+ and CD13+ expressing myeloid cells. The induced switch in lineage commitment is consistent with increased GATA2 expression and decreased GATA1 expression. Further, we observed that erythroid and hematopoietic progenitor cultures became more acidic in the presence of HMGB1 suggesting that HMGB1 may also influence progenitor metabolic processes.Our data demonstrate that HMGB1 negatively affects erythropoiesis at EPO-dependent and -independent stages of erythroid differentiation through decreased CFU-E numbers and apoptosis of erythroblasts, and also favor increased myeloid differentiation of HSPCs. Contrary to classical inflammatory mediators, HMGB1 relies on a novel mechanism, involving downstream EPO effectors, to mediate erythroid dysfunction. Ongoing pharmacologic and genetic screens of EPO signaling axes, putative HMGB1 receptor(s), and progenitor metabolic profiling are currently underway to better understand how HMGB1 causes erythropoietin refractory anemia in the setting of inflammation. DisclosuresNo relevant conflicts of interest to declare.

Transcriptomic Changes Within Human Bone Marrow After Severe Trauma.

Severe trauma is associated with severe systemic inflammation and neuroendocrine activation that is associated with erythroid progenitor growth suppression and refractory anemia. Although distinct transcriptional profiles have been detected in numerous tissue types after trauma, no study has yet characterized this within the bone marrow. This study sought to identify a unique bone marrow transcriptomic response following trauma. In a prospective observational cohort study, bone marrow was obtained from severely injured trauma patients with a hip or femur fracture (n = 52), elective hip replacement patients (n = 33), and healthy controls (n = 11). RNA was isolated from bone marrow using a Purelink RNA mini kit. Direct quantification of mRNA copies was performed by NanoString Technologies on a custom gene panel. Trauma patients displayed an upregulation of genes encoding receptors known to have inhibitory downstream effects on erythropoiesis, including ferroportin, interleukin-6 (IL-6) receptor, transforming growth factor-beta (TGF-β) receptor, and IL-10, as well as genes involved in innate immunity including toll-like receptor 4 (TLR4)-mediated signaling factors. In contrast, hip replacement patients had downregulated transcription of IL-1β, IL-6, TGF-β, tumor necrosis factor alpha, and the HAMP gene with no change in TLR4-mediated signaling factors. A unique transcriptomic response within the bone marrow was identified following severe trauma compared to elective hip replacement. These transcriptomic differences were related to the innate immune response as well as known inhibitors of erythropoiesis. Although confined to just one time point, this differential transcriptional response may be linked to refractory anemia and inflammation after injury.

P249 What’s the role of anti-TNF α in correcting anemia in Crohn’s disease?

Abstract Background Anemia is the most common extra intestinal complication in Inflamatory Bowel Diseases (IBD) affecting the quality of life of patients. Iron deficiency and inflammation are the two most common mechanisms. It has been suggested that controlling disease activity is sufficient to correct well-tolerated anemia. Anti-TNF α by their powerful anti-inflammatory action and their role in increasing the growth of erythroid progenitors can lead to correction of anemia. The aim of this work was to determine the effect of anti-TNFα on the course of anemia in Crohn’s disease (CD). Methods This is a single-center retrospective study including patients followed for CD, between 2011 and 2017, on anti-TNF. Anemia was tested in these patients before the initiation of anti-TNF. After six months of treatment, a correlation between the achievement of therapeutic efficacy and the correction of the anemia was sought. The judgment criteria adopted were: clinical response (CDAI score &amp;lt;150 points), mucosal healing at endoscopy (absence of ulcerations), and normalization of the C-reactive protein (CRP &amp;lt;5mg / l). Results 120 patients were included, 60% of whom are female. The mean age at diagnosis was 29.8 years [12–56 years]. The mean duration of the disease was 7.42 ± 4.8 years. The disease phenotype was penetrating in 55% of cases and structuring in 45% of patients. Anoperineal manifestations (PAD) were noted in 20% of patients. 50% of patients were on Adalimumab, 50% on Infliximab and 45% of cases were on combination therapy. The indications for Biotherapy were: the presence of anoperineal manifestations (20%), failure of immunosuppressants (25%), postoperative recurrence (20%), intolerance to immunosuppressants (20%), and extradigestive manifestations in particular articular (15%). Pre-therapy, anemia was noted in 84 patients (70%), most of whom had chronic microcytic hypochromic anemia. The presence of anemia was well correlated with disease activity (p = 0.038). During the control, correction of anemia was obtained in 72 patients (60%), with a statistically significant association with the therapeutic efficacy criteria (clinical response: p &amp;lt;10–3, mucosal healing: p = 0.009, normalization of CRP: p &amp;lt;10–3). Conclusion Our study showed the effectiveness of anti-TNF alpha agents in correcting anemia in Crohn’s disease. Larger scale studies are needed to confirm our results.

P460 Evaluation of potential mechanisms underlying the safety observations of filgotinib in clinical studies in rheumatoid arthritis

Abstract Background Inhibitors of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway have demonstrated efficacy in the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Differences in selectivity of JAK inhibitors for JAK1, JAK2, JAK3 and TYK2 may influence their respective safety profiles, and the mechanisms responsible are not currently known. Filgotinib (FIL), a JAK1 inhibitor, did not negatively impact haemoglobin, LDL:HDL ratios or natural killer (NK) cell counts in clinical trials. Here, we compare the in vitro mechanistic profiles of four JAK inhibitors at clinically relevant doses. Methods JAK inhibitors (FIL, FIL metabolite [GS-829845], baricitinib [BARI], tofacitinib [TOFA], and upadacitinib [UPA]) were evaluated in vitro in human-cell-based assays. Growth of erythroid progenitors from human cord blood CD34+ cells was assessed using a HemaTox™ liquid expansion assay, NK cell proliferation was induced by IL-15 and LXR agonist-induced cholesteryl ester transfer protein (CETP) expression was assessed in the hepatic cell line, HepG2. Using assay-generated IC50 values and the reported human plasma concentrations from clinical studies, we calculated the target coverage for each JAK inhibitor at clinically relevant doses. The activity of FIL in humans was based on PK/PD modelling of FIL + GS-829845. Results Inhibition of cellular activity was calculated for each JAK inhibitor based on in vitro dose-response data, human exposure data and modelled PK/PD relationships. At clinically relevant doses, FIL resulted in lower calculated inhibition of NK cell proliferation compared with other JAK inhibitors. FIL 100 mg and 200 mg also reduced CETP expression, whereas other JAK inhibitors had no effect. There was no difference in the effect of FIL vs. other JAK inhibitors on erythroid progenitor cell differentiation or maturation. Conclusion FIL, a JAK1 inhibitor, resulted in less inhibition of NK cell proliferation compared with BARI, TOFA, and UPA. FIL also reduced LXR agonist-induced CETP expression, while the other inhibitors did not alter these levels. These results provide a potential mechanistic link between the observed reduction of CETP concentration following FIL treatment and the previously observed reduction in the LDL:HDL ratio in RA patients.

Overexpression or Deficiency of RUNX3 Results in Defective Normal Human Hematopoietic Development Similar to RUNX1-ETO Expression in AML

A common abnormality in AML (observed at a frequency of ~12%) is the t(8;21)(q22;q22) encoding the RUNX1-ETO fusion protein. Previously we showed that RUNX1-ETO can block the differentiation of normal human primary CD34+ hematopoietic stem progenitor cells (HSPC) and promote their self-renewal (consistent with its putative initiating role; Tonks et al 2003, 2004). To identify the genes that may mediate this phenotype, transcriptome analysis was performed (Tonks et al 2007). We now report that RUNX1-ETO significantly downregulates the expression of RUNX3 (2.1±0.6 fold in CD34+ HSPC; p&lt;0.05). Furthermore, we also show that RUNX3 mRNA is significantly downregulated in t(8;21) patients compared to normal human bone marrow by 4 fold (p&lt;0.001). Interestingly, in non-t(8;21) AML, RUNX3 expression is overexpressed compared with normal HSC, suggesting that both down and upregulation of RUNX3 may contribute to the pathogenesis of AML. To investigate this further, we examined the impact of RUNX3 expression on normal human hematopoiesis. Initially, we investigated the role of RUNX3 in erythropoiesis (which is severely disrupted by RUNX1-ETO). We mimicked the effects of RUNX1-ETO through shRNA-mediated knockdown (KD) of RUNX3 in CD34+ HSPC which co-expressed GFP. Following infection, GFP+ CD13low cells were isolated by FACS to yield a population of cells enriched for erythroid progenitors. Under clonal conditions, RUNX3-KD cells formed significantly less erythroid colonies than control (1.9±0.7 fold, p&lt;0.05), matching the impairment seen in erythroid progenitors expressing RUNX1-ETO. To examine this phenotype in more detail, we studied the effect of RUNX3-KD on the EPO-independent and EPO-dependent stages of erythroid development. RUNX3-KD significantly inhibited growth during the EPO-independent development (suppressed by 1.7±0.1 fold; p&lt;0.001) and showed impaired differentiation with reduced CD36 expression (1.5±0.3 fold, p&lt;0.01), retention of HLA-DR (1.2±0.1 fold, p&lt;0.01) and CD34 (1.2±0.1 fold, p&lt;0.05). These cells showed partial response to EPO but again growth response was suppressed (2±0.8 fold compared to control). Developmentally, RUNX3-KD cells failed to upregulate GlyA to the same levels as controls (suppressed by 1.3±0.1 fold p&lt;0.05), suggesting that the inhibition of differentiation persisted in the EPO-dependent phase of development. In summary, KD of RUNX3 in HSPC leads to an impairment in erythropoiesis similar to the phenotype induced by RUNX1-ETO, which suggests that suppression of RUNX3 by this fusion protein contributes to the RUNX1-ETO phenotype. We next assessed the effect of RUNX3 overexpression on erythroid development as above, this time using retrovirus co-expressing RUNX3 and DsRed. Interestingly, RUNX3 overexpression also suppressed erythroid colony formation by 1.7±0.7 fold (p&lt;0.01) compared to control, however, in a serial replating strategy, RUNX3 cells were able to form 2.7±0.6 fold more erythroid colonies than control, which suggests that RUNX3 promotes self-renewal. In the EPO-independent phase of development RUNX3 overexpression significantly inhibited the growth of erythroid progenitors by 2.7±1.3 fold (p&lt;0.05), possibly arising from reduced erythroid commitment (CD36+CD13neg reduced by 1.5±0.2 fold, p&lt;0.01). Erythroid committed cells showed a greater proliferative response to EPO (4.6±2.8 fold greater than controls) and showed phenotypic evidence of inhibition of development: decreased expression of GlyA (suppressed by 1.3±0.1 fold, p&lt;0.01), retention of CD36 (1.4±0.2 fold, p&lt;0.05) and 20% increased cell size (p&lt;0.05) compared to controls. Morphologically, these cells appeared to be blocked in an early/intermediate stage of development. These data imply that RUNX3 overexpression impairs erythroid differentiation and blocks terminal differentiation. Overall, these results show that both the overexpression and KD of RUNX3 in normal HSPC leads to a block in erythroid differentiation. Further, our preliminary data suggests that ectopic expression of RUNX3 in CD34+ HSPC selectively inhibits granulocytic development. These experimental models indicate that the level of RUNX3 expression is an important regulator of normal human hematopoietic development. We are currently determining the underlying mechanisms by which RUNX3 perturbs normal hematopoiesis, which ultimately may lead to new therapies for AML patients. Disclosures Knapper: Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Tolero: Membership on an entity's Board of Directors or advisory committees.

Systemic Regulation of Bone Marrow Stromal Cytokines After Severe Trauma

BackgroundTraumatic injury generates a prolonged hypercatecholamine state that is associated with reduced growth of bone marrow erythroid progenitors mediated by the bone marrow stroma. The bone marrow stroma is made up of many cells including fibroblasts, which respond to inflammatory stimuli and alter the cytokine profile. We hypothesized that trauma plasma would increase bone marrow stromal fibroblast expression of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells and correlate with injury severity and anemia. Materials and methodsPlasma from 15 trauma patients was cultured with bone marrow fibroblast cells and compared with that from healthy volunteers. At 6, 24, and 48 h, the expression of IL-6, G-CSF, EPO, SCF, and the activation of nuclear factor kappa-light-chain-enhancer of activated B cells were measured using quantitative polymerase chain reaction. The influence of trauma plasma on cytokine expression was further stratified by injury severity score (ISS). ResultsThe average hemoglobin significantly decreased from admission to discharge (10.7 ± 2.5 to 9.2 ± 1.1 g/dL, P < 0.04). The discharge hemoglobin significantly decreased by 14% from the admission hemoglobin. After 48 h, trauma plasma significantly increased IL-6, G-CSF, and EPO bone marrow fibroblast expression when compared with normal plasma. When stratified by ISS, IL-6, G-CSF, and EPO, bone marrow fibroblast expression was highest in the trauma plasma ISS 27-41 group and was significantly elevated compared with normal plasma. When SCF expression was stratified by ISS, there was a significant increase in expression in ISS 27-41. Higher ISS was also associated with a larger decrease in hemoglobin despite no difference in total blood transfusions. ConclusionsSevere trauma can systemically increase IL-6, G-CSF, and EPO expression in bone marrow stroma. Increased hematopoietic cytokine expression after traumatic injury correlated with a hypercatecholamine state, anemia, and injury severity.

Stimulation of the Proliferation of Mouse and Human Definitive Erythroid Progenitors By Activation of the Vitamin D Receptor Transcription Factor

How the proliferation of erythroid progenitors is regulated is still now well understood. We found, using a computational analysis, that the vitamin D receptor(Vdr) nuclear hormone receptor transcription factor gene is expressed in the fetal and adult (definitive) but not the embryonic (primitive) stage of mouse erythroid ontogeny. Vdr is transcribed in definitive erythroid (EryD) progenitors and was downregulated during their maturation. VDR transcription factor activation by its ligand vitamin D3 (1,25(OH)2D3) results in conformational changes that stabilize the protein and induce its translocation into the nucleus, where it recruits co-regulatory complexes. The VDR signaling pathway has been studied primarily in the biology of bone but has been largely unexplored in erythropoiesis, where the limited published studies were performed almost exclusively in leukemic cell lines and not in normal primary cells. Activation of Vdr signaling by the vitamin D3 agonist calcitriol increased the outgrowth of EryD colonies from fetal liver and adult bone marrow, maintained progenitor potential, and delayed terminal erythroid maturation, as revealed by clonogenic assays, suspension culture studies, cell surface phenotype, and gene expression analyses. The stimulation in growth of erythroid progenitors resulted in a large increase in the numbers of mature red blood cells. The early (cKit+CD71lo/neg) but not the late (cKit+CD71hi) EryD progenitor subset of Linneg cKit+ cells was responsive both to calcitriol and to calcipotriol (which is 1-to-200 fold less potent in its calcemic effects than calcitriol). Therefore, the increase in progenitor numbers in response to calcitriol is mediated through activation of VDR rather than by effects on calcium flux. In preliminary studies of human Lineageneg cells, we find that the vitamin D agonists calcitriol and calcipotriol increase the numbers of CFU-E colony numbers from peripheral blood or BM. These results are similar to our findings for mouse. The glucocorticoid receptor (Gr), like Vdr, is a member of the nuclear hormone receptor transcription factor family and has been shown to stimulate the proliferation of cKit+CD71lo/neg cells. To determine whether the Vdr and Gr signaling pathways can cooperate to modulate erythroid progenitor growth, cKit+CD71lo/negcells were cultured with or without calcitriol, dexamethasone, or the two ligands in combination. Culture of cKit+CD71lo/neg progenitors in the presence of both calcitriol and dexamethasone resulted in an increase in proliferation that was at least additive, compared to either ligand alone, suggesting a role in stress erythropoiesis. This possibility is supported by our recent finding that an erythroid specific deletion in Vdr that interferes with DNA binding results in a reticulocytosis that occurs earlier and is more pronounced than in control animals, in response to phenylhydrazine (PHZ)-induced anemia. In addition, this deletion in Vdr blocked the increase in early erythroid progenitors from fetal liver seen for wild type mice. Lentivirus shRNA-mediated knockdown of Vdr expression abrogated the stimulation of early erythroid progenitor growth by calcitriol. These findings suggest that Vdr has a cell-intrinsic function in early erythroid progenitors. Activation of Vdr by calcitriol blocked the up regulation of the erythroid transcription factor genes Gata1, Fog1 and Klf1. In contrast, expression of genes known to regulate erythroid progenitors (Gata2, Zfp36l2, Bmi1, and Hopx) was not affected by Vdr signaling. Therefore, other genes must be involved in the Vdr signaling pathway in erythroid progenitors. Targeting of downstream components of the VDR signaling pathway could lead to new approaches for expansion of erythroid progenitors ex vivo. (This work was supported by grants to MHB from the NIH, R01 DK102945 and HL62248.) DisclosuresNo relevant conflicts of interest to declare.

The Postinjury Inflammatory State and the Bone Marrow Response to Anemia.

The pathophysiology of persistent injury-associated anemia is incompletely understood, and human data are sparse. To characterize persistent injury-associated anemia among critically ill trauma patients with the hypothesis that severe trauma would be associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, and decreased erythropoiesis. A translational prospective observational cohort study comparing severely injured, blunt trauma patients who had operative fixation of a hip or femur fracture (n = 17) with elective hip repair patients (n = 22). Bone marrow and plasma obtained at the index operation were assessed for circulating catecholamines, systemic inflammation, erythropoietin, iron trafficking pathways, and erythroid progenitor growth. Bone marrow was also obtained from healthy donors from a commercial source (n = 8). During admission, trauma patients had a median of 625 ml operative blood loss and 5 units of red blood cell transfusions, and Hb decreased from 10.5 to 9.3 g/dl. Compared with hip repair, trauma patients had higher median plasma norepinephrine (21.9 vs. 8.9 ng/ml) and hepcidin (56.3 vs. 12.2 ng/ml) concentrations (both P < 0.05). Bone marrow erythropoietin and erythropoietin receptor expression were significantly increased among patients undergoing hip repair (23% and 14% increases, respectively; both P < 0.05), but not in trauma patients (3% and 5% increases, respectively), compared with healthy control subjects. Trauma patients had lower bone marrow transferrin receptor expression than did hip repair patients (57% decrease; P < 0.05). Erythroid progenitor growth was decreased in trauma patients (39.0 colonies per plate; P < 0.05) compared with those with hip repair (57.0 colonies per plate; P < 0.05 compared with healthy control subjects) and healthy control subjects (66.5 colonies per plate). Severe blunt trauma was associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, erythroid progenitor growth suppression, and persistent injury-associated anemia. Clinical trial registered with www.clinicaltrials.gov (NCT 02577731).

Activation of the vitamin D receptor transcription factor stimulates the growth of definitive erythroid progenitors

AbstractThe pathways that regulate the growth of erythroid progenitors are incompletely understood. In a computational analysis of gene expression changes during erythroid ontogeny, the vitamin D receptor (Vdr) nuclear hormone receptor transcription factor gene was identified in fetal and adult stages, but not at the embryonic stage of development. Vdr was expressed in definitive erythroid (EryD) progenitors and was downregulated during their maturation. Activation of Vdr signaling by the vitamin D3 agonist calcitriol increased the outgrowth of EryD colonies from fetal liver and adult bone marrow, maintained progenitor potential, and delayed erythroid maturation, as revealed by clonogenic assays, suspension culture, cell surface phenotype, and gene expression analyses. The early (cKit+CD71lo/neg), but not the late (cKit+CD71hi), EryD progenitor subset of LinnegcKit+ cells was responsive to calcitriol. Culture of cKit+CD71lo/neg progenitors in the presence of both vitamin D3 and glucocorticoid receptor ligands resulted in an increase in proliferation that was at least additive compared with either ligand alone. Lentivirus shRNA-mediated knockdown of Vdr expression abrogated the stimulation of early erythroid progenitor growth by calcitriol. These findings suggest that Vdr has a cell-intrinsic function in early erythroid progenitors. Targeting of downstream components of the Vdr signaling pathway may lead to new approaches for the expansion of erythroid progenitors ex vivo.

Persistent injury-associated anemia in aged rats

BackgroundHypercatecholaminemia and bone marrow dysfunction have been implicated in the pathophysiology of persistent-injury associated anemia. The elderly may be vulnerable to this phenomenon due to high basal and peak catecholamine levels, impaired erythroid progenitor growth, and baseline anemia. We hypothesized that aged F344-BN rats subjected to severe trauma and chronic stress would have persistent injury-associated anemia. MethodsMale F344-BN rats age 25months were randomly allocated to: naïve (n=8), lung contusion (LC, n=9), LC followed by daily chronic restraint stress (LC/CS, n=9), LC followed immediately by hemorrhagic shock (LCHS, n=8), and LCHS followed by daily CS (LCHS/CS, n=8). Urine norepinephrine was measured on days one and seven. Locomotor testing was performed on day five. Bone marrow cellularity, hematopoietic progenitor growth, and peripheral blood hemoglobin levels were assessed at sacrifice on day seven. Data are presented as mean±standard deviation, *p<0.05 vs. naïve. ResultsNorepinephrine levels (ng/mL) were significantly elevated one day after LCHS (420±239* vs. naïve: 97±71) and LCHS/CS (375±185*), and remained significantly elevated on day seven for LCHS/CS (359±99*), but not LCHS (212±130). On locomotor testing, groups subjected to CS traveled shorter distances at lower velocities and spent less time in the center of the cage. Colony forming units-erythroid (colonies/plate), representing late erythroid progenitors, were significantly decreased after LC/CS (40±1* vs. naïve: 47±4), LCHS (40±1*), and LCHS/CS (38±3*). LCHS/CS animals had significantly lower hemoglobin (g/dL) than naïve animals (13.3±1.3* vs. naïve: 15.2±0.9). ConclusionsPersistent injury-associated anemia occurs in aged rats. Further research is needed to determine whether the pathophysiology of this phenomenon differs from that of younger rats, and to translate these findings to elderly trauma patients.

Single-Lineage Bone Marrow Failure Driven By 2 Novel PI3KCD Mutations

Five genetic mutation on PI3KCD are known so far to cause the Activated-Phosphoinositide-3-kinaseδ-Syndrome (APDS) a Primary Immune-Deficiency characterized by lymphopenia/hypogammaglobulinemia, respiratory infections, bronchiectasias, gut/pulmonary infiltrates, splenomegaly, autoimmunity and predisposition to malignancies. We report 2 patients carrying 2 novel mutation of PI3KCD gene presenting with a singlelineage bone marrow failure.Case1: A9-months-old girl with splenomegaly,steroid-responsive autoimmune haemolytic anemia and immune thrombocytopenia,severe persistant neutropenia due to marrow myeloid precursors aplasiaresistant to multiple-agents immunosuppression,recurrent life-threatening infections was referred to our Unit. No myeloid precursors at any stage of maturation were found in the bone marrow aspiration. Growth of both homologous and heterologous myeloid progenitor cells was hampered by patient's marrow plasma.Most common causes of congenital neutropenias were excluded. Sirolimus partially and transiently normalized neutrophil count but after yet another life-threatening sepsis neutropenia relapsed. NGS analysis showed the presence of a novel mutation of PI3KCD gene (H273Y) affecting the enzyme's catalytic domain. She then received analpha-beta-CD19-depleted haplo-identical SCT from her mother (infused18.6x106/kgCD34+,2x104/kgAlpha/beta-T-cells) conditioned withATG-Fludarabine-Melphalan.Neutrophils and platelets recoveredat d+26and+10 respectively.Mixed-chimerismpersistedduring post-transplant andneutropenia relapsed on day +177 when full recipient chimerismwas documented witnessing for rejection.She then underwent anotherhaplo-SCTfrom the mother conditioned with Fludarabine-Melphalan-Campath(infused3x106/kgCD34+, 1.5x105/kgAlfa/beta-T-cells).Neutrophil and platelets recovered at d+18 and +13, respectively.Neither toxicity nor GvHD occurred after both transplants.Case 2:A 1 year-old girl presented with Pure Red Cell Aplasia (PRCA) associated with AHIA. No erythroid precursors were seen in the marrow and progenitor colony studies showed that the patient's plasma had an inhibitory effect on the growth of both homologous and control erythroid progenitors. At presentation she also had worsening seizures due to into acute disseminated encephalomyelitis that promptly recovered after few doses of intra-venous immunoglobulins. PRCA/AIHA subsided to combination therapy with Rituximab and Sirolimus and after 2 years of complete remission new episodes of seizures appeared requiring a change of the anti-comitial treatment which by interfering with Sirolimus serum levels caused a relapse of anemiathat was successfully rescued by the addition of monthly IVIG administration. Based on the low Ig levels at the onset of the disease, the response to both Sirolimus (suggesting a mTOR pathway involvement) and IVIG, , we performed Sanger PCR on PI3KCD gene and detected the S277M mutation. To confirm the pathogenetic role of this mutation, we studied the expression of AKT protein by Western blot analysis that showed an increased phosphorilation compared to a same age-healthy control.This report indicates that thesenew PI3KCD mutations may generate a novel APDS clinical phenotype, consisting of severe single-lineage marrow aplasia,and therefore widen the spectrum of diseases underlying single lineage marrow failure in children. Sirolimus and SCT represent valid options for the treatments of the disease. DisclosuresNo relevant conflicts of interest to declare.

P38 mitogen-activated protein kinase inhibition enhances in vitro erythropoiesis of Fanconi anemia, complementation group A–deficient bone marrow cells

Bone marrow failure in Fanconi anemia (FA) has been linked in part to overproduction of inflammatory cytokines, to which FA stem and progenitor cells are hypersensitive. In cell lines and murine models p38 mitogen-activated protein kinase (MAPK)-dependent tumor necrosis factor α (TNF-α) overexpression can be induced by the Toll-like receptors (TLRs) 4 and 7/8 ligands Lipopolysaccharide (LPS) and R848. Ex vivo exposure of FA stem cells to TNF-α suppresses their replication and selects preleukemic clones. Here we show that inhibition of p38 MAPK also reduces TLR4 and 7/8-mediated TNF-α production in primary human FA complementation group A (FANCA)-deficient monocytes from nine patients and demonstrate that, while p38 MAPK inhibition also enhances clonal growth of FANCA-deficient erythroid progenitors, the effect was mediated indirectly by the influence of the inhibitor on auxiliary cells, not erythroid colony-forming units themselves. Taken together, these results support the view that inhibition of the p38 MAPK pathway in monocytes may improve hematopoiesis in FANCA patients.

Azacitidine Treatment in High Risk Myelodysplastic Patients in Complete Haematological Remission Reverts Mesenchymal Stem Cells to a Normal Phenotype

Introduction. Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective bone marrow hematopoiesis, peripheral blood cytopenias, and a substantial risk for progression to acute myeloid leukemia. Mesenchymal stem cells (MSCs) isolated from bone marrow of patients affected by myelodysplastic syndromes (MDS) play a critical role in myelodysplastic microenvironment showing altered structural epigenetic and functional features.Methods. In this work we evaluated the effect of azacitidine treatment on MSC-MDS. In particular, we analyzed MSC-MDS from 24 high-risk patients at diagnosis and after azacitidine treatment, studying their morphology, proliferative potential, cell cycle activity and their capacity to support haematopoiesis.Results. MDS-MSCs at diagnosis appeared larger and flattened, achieved confluence at a significantly lower rate than donors and displayed reduced proliferative capacity. In particular 40% of samples were unable to expand.This reduced proliferative capacity of MSC-MDS at diagnosis suggested changes in the cell cycle activity. Therefore we studied the gene expression profiles of 37 regulatory genes, observing CDKN2B up-regulation in MDS-MSCs (8 times higher than donors).Notably, after azacitidine treatment MDS-MSCs of patients who reached complete haematological remission (MDS-MSCs-CR) reverted to the typical BM-MSC morphology and recovered a proliferative potential similar to normal BM-MSC achieving confluence at a significantly higher rate.Molecular analysis on MDS-MSC-CR revealed a significant reduction in the expression level of CDKN2B showing correlation between cell cycle progression and expression level of this gene.Moreover, to study the long-term hematopoietic maintaining ability, MDS-MSCs at diagnosis were cultured with CD133+ cells, and they showed a decreased ability to support the growth of myeloid and erythroid progenitors. Conversely, MSC-MDS-CR showed an increased capacity to support haematopoiesis similar to healthy donors.Conclusion. We showed that MDS-MSCs at diagnosis were structurally and functionally altered while MSC-MDS-CR after azacitidine revert to a normal phenotype. It has been supposed that healthy MSCs adopt MDS-MSCs like molecular features when exposed to haematopoietic MDS cells.Our results may confirm these data suggesting that myelodysplastic cells can alter bone marrow microenvironment interacting with MSC and affecting their normal role and functionality. DisclosuresNo relevant conflicts of interest to declare.