Hematopoietic Response Research Articles

Activation Of TLR4-MyD88 Pathway Impairs HSC Function During Acute Inflammation

Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. High mortality rates in sepsis (200,000/yr in the USA) are associated with the host's failure to eradicate pathogens due to the lack of neutrophils, excessive pro-inflammatory cytokines, tissue damage and multiple organ failure. While most studies have focused on the late consequences of sepsis, little is known about the changes occurring in the bone marrow (BM) at early stages of hematopoiesis and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, which closely recapitulate lethal sepsis in burn patients, we have previously reported that HSC undergo a significant expansion in the BM associated with a block of myeloid differentiation. Furthermore, we found that expanded HSC were unable to generate the downstream progenitors (common myeloid progenitors and granulocytes/monocytes progenitors) necessary to produce neutrophils, and had reduced self-renewal. All these effects were TLR4-dependent. TLR4 is activated by bacterial LPS and signal through two major pathways: TRIF-dependent and MyD88-dependent. In this study, we show the different contribution of the TLR4-TRIF and the TLR4-MyD88-dependent pathways to the BM response to Pseudomonas aeruginosa LPS. LPS challenge conducted on TRIF-null and MyD88-null mice demonstrated that TRIF was involved in the expansion of the HSC pool, but did not play a major role in the myelosuppression, whereas MyD88 activation was required for LPS-induced myeloid suppression. Moreover, we found that the impaired engraftment at long-term observed in wild-type LPS-challenged HSC during transplantation was rescued by MyD88 loss of function. Taken together, our results indicate a distinct role of theTLR4-TRIF and -MyD88 pathways in the regulation of the primitive stem/progenitor pool during sepsis and provide insights for a better understanding of the molecular mechanisms leading to neutropenia. Disclosures:No relevant conflicts of interest to declare.

Blood Scent

Blood cell production is tightly regulated by cell-intrinsic mechanisms and environmental factors. The study by Utpal Banerjee and colleagues and colleagues reveals that, in Drosophila, olfactory signals control hematopoietic progenitor maintenance, thus uncovering a physiological link between sensory perception and hematopoietic response to environmental stress.

Short-term sonic-hedgehog gene therapy to mitigate myelosuppression in highly irradiated monkeys: hype or reality?

The protection of hematopoietic stem and progenitor cells and their environment is required for recovery from radiation-induced (RI) myelosuppression. To achieve this goal, we propose a new gene therapy strategy based on local and short-term synthesis and expression of Sonic hedgehog morphogene (Shh) at the niche level. We investigated the hematopoietic response of 8 Gy gamma-irradiated monkeys to a single intra-osseous injection of multipotent mesenchymal stem cells (adipocyte-derived stem cells/ASC) transduced with a Shh pIRES2 plasmid (3+/-0.4 × 10(6) cells/kg on day (D) 2; n=4). Control animals were injected with mock-ASCs (n=4). Two controls died from radiation toxicity on D19 and D196, whereas all Shh-ASC treated monkeys fully recovered. Thrombocytopenia (4.75+/-1.8 days versus 10+/-2.2 days, platelet count <20 × 10(9)/L), neutropenia (14.2 +/-1 days versus 17.7 +/-2.6 days, ANC count<0.5 × 10(9)/L) and anemia (15.5 +/-3.6 days versus 50.7 +/-31 days, Hb less than 10 g/dL) duration were reduced in Shh-ASC animals. Areas under the curve of platelets (P<0.05), ANCs (P=0.06) and RBC/Hb between D0 and D30 were higher in Shh-ASC injected animals. Globally this study suggests that Shh may represent a new factor to counteract RI-myelosuppression.

Coste-efectividad del tratamiento de la anemia inducida por quimioterapia con agentes estimuladores de la eritropoyesis

ObjectiveThis study evaluates the efficiency of the erythropoiesis-stimulating agents in the management of chemotherapy-induced anaemia (CIA) in Spain. CIA affects 68% of the patients treated with chemotherapy in Europe.MethodA systematic review and a decision tree were developed based on the efficacy of weekly darbepoetin-alfa, darbepoetin-alfa every three weeks, weekly epoetin-alfa and weekly epoetin-beta and the associated resources (drugs, administration, visits and blood tests) during a 12-week treatment. Perspective: Public health system. Costs were expressed in 2012 euros. Sensitivity analysis: efficacy variations, unit costs, iron use, and variations in the rate of patients with dose increases.ResultsAccording to published data, the hematopoietic response rate at week 16 (proportion of patients achieving hemoglobin ≥12 g/dL or a Hb increase of ≥2 g/dL from baseline) is 73% for patients treated with darbepoetin-alfa, and 68% for patients treated with weekly epoetin-alfa and weekly epoetin-beta. Among epoetin-treated patients, 29.63% required a dose increase, and the average amount of micrograms needed for each treatment was 1,865, 1,679, 2,433, and 2,042 (1 μg=200 IU) for weekly darbepoetin-alfa, darbepoetin-alfa every three weeks, weekly epoetin-alfa and weekly epoetin-beta, respectively. Average direct costs were €3,419, €3,478, €3,499, and €4,075 for weekly darbepoetin-alfa, darbepoetin-alfa every three weeks, weekly epoetin-alfa, and weekly epoetin-beta, respectively. The average cost per response was €4,684, €4,764, €5,145, and €5,992, respectively. On average, darbepoetin-alfa was more effective and less costly than weekly epoetin-alfa and weekly epoetin-beta in sensitivity analyses.ConclusionDarbepoetin-alfa showed to be an efficient treatment for the public health system to treat CIA, and was, on average, more effective and less costly than epoetin-alfa and epoetin-beta.

The effects of R683S (G) genetic mutations on the JAK2 activity, structure and stability

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL.

Pathophysiology and management of thrombocytopenia in bone marrow failure: possible clinical applications of TPO receptor agonists in aplastic anemia and myelodysplastic syndromes

Aplastic anemia is a bone marrow failure syndrome that causes pancytopenia and can lead to life-threatening complications. Bone marrow transplantation remains the standard of care for younger patients and those with a good performance status but many patients may not have a suitable donor. Immunosuppressive therapy is able to resolve cytopenias in a majority of patients with aplastic anemia but relapses are not uncommon and some patients remain refractory to this approach. Patients may require frequent blood and platelet transfusion support which is expensive and inconvenient. Life-threatening bleeding complications still occur despite prophylactic platelet transfusion. Thrombopoietin (TPO) mimetics, such as romiplostim and eltrombopag, were developed to treat patients with refractory immune thrombocytopenia but are now being investigated for the treatment of bone marrow failure syndromes. TPO is the main regulator for platelet production and its receptor (c-Mpl) is present on megakaryocytes and hematopoietic stem cells. Trilineage hematopoietic responses were observed in a recent clinical trial using eltrombopag in patients with severe aplastic anemia refractory to immunosuppression suggesting that these agents can provide a new therapeutic option for enhancing blood production. In this review, we discuss these recent results and ongoing investigation of TPO mimetics for aplastic anemia and other bone marrow failure states like myelodysplastic syndromes. Clonal evolution or progression to acute myeloid leukemia remains a concern when using these drugs in bone marrow failure and patients should only be treated in the setting of a clinical trial.

P-226 A role for transcriptional adaptor SSBP2 in hematopoietic stem cell maintenance and stress response

P-226 A role for transcriptional adaptor SSBP2 in hematopoietic stem cell maintenance and stress response

MyD88 Signaling in CD4 T Cells Promotes IFN-γ Production and Hematopoietic Progenitor Cell Expansion in Response to Intracellular Bacterial Infection

Hematopoietic stem and progenitor cell (HSPC) phenotype and function can change in response to infectious challenge. These changes can be mediated by cytokines, IFNs, and pathogen-associated molecules, via TLR, and are thought to promote tailored immune responses for particular pathogens. In this study, we investigated the signals that activate HSPCs during ehrlichiosis, a disease characterized by profound hematopoietic dysfunction in both humans and mice. In a mouse model of ehrlichiosis, we observed that infection-induced proliferation of bone marrow HSPCs was dependent on IFN-γ signaling and was partially dependent on MyD88. However, MyD88 was not required in HSPCs for their expansion during infection, because similar frequencies of MyD88-deficient and wild-type HSPCs proliferated in mixed bone marrow chimeric mice. MyD88-deficient mice exhibited low serum and bone marrow concentration of IFN-γ compared with wild-type mice. We next identified CD4 T cells as the primary cells producing IFN-γ in the bone marrow and demonstrated a nonredundant role for CD4-derived IFN-γ in increased HSPCs. Using mixed bone marrow chimeric mice, we identified a requirement for MyD88 in CD4 T cells for increased T-bet expression, optimal IFN-γ production, and CD4 T cell proliferation. Our data demonstrate an essential role for CD4 T cells in mediating HSPC activation in response to bacterial infection and illustrate a novel role for MyD88 signaling in CD4 T cells in this process. These findings further support the idea that IFN-γ production is essential for HSPC activation and hematopoietic responses to infection.

Disruption of E627 and R683 interaction is responsible for B-cell acute lymphoblastic leukemia caused by JAK2 R683G(S) mutations

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in hematopoietic and immune responses. The acquired JAK2 R683G(S) somatic mutations are detected in 15% of patients with B-cell acute lymphoblastic leukemia (B-ALL) and are presumed to be a biomarker for B-ALL. However, how JAK2 R683G(S) mutations lead to B-ALL is still unclear. Our results indicated that the E627 and R683 interaction played a vital role in JAK2 autoinhibition. Mutations (R683S, R683G and E627A) disrupting this interaction led to JAK2 constitutive activation, while mutations (R683K, E627D) restoring this interaction decreased its activity. Furthermore, spectroscopy experiments implied that disruption of the E627 and R683 interaction abolished JH1/JH2 domain interactions and forced the JH1 domain into the open, active conformation. Mutations abolishing this interaction promoted the proliferation of Ba/F3 cells. The results herein may provide clues to understanding the mechanism of JAK2 R683G(S) mutation-associated B-ALL.

Low dose lenalidomide induction followed by autologous transplantation in untreated patients with myeloma is associated with adequate collection of haematopoietic and dendritic cell precursors and high response rates

Low dose lenalidomide induction followed by autologous transplantation in untreated patients with myeloma is associated with adequate collection of haematopoietic and dendritic cell precursors and high response rates

An exploratory phase I trial of immunochemoradiotherapy in locally advanced and borderline resectable (LA/BR) pancreatic adenocarcinoma (PC).

174 Background: The primary aim of this exploratory trial was to determine the feasibility and safety of chemoradiotherapy (CRT) with combinatorial immunotherapy in LA or BR PC. Methods: 10 patients (pts) with untreated, LA (n=6) or BR (n=4) biopsy-proven PC received gemcitabine (GEM) in combination with telomerase peptide vaccine (GV1001) and sargramostim (GM-CSF) both by intradermal injection. Tadalafil was taken once daily throughout the course of therapy for myeloid derived suppressor cell inhibition. The 2nd month of treatment included external beam radiation therapy (50.4 Gy, 28 fractions) and concurrent twice-weekly GEM 50 mg/m2. Disease response was assessed at 12 weeks. Following recovery from either concurrent CRT or resection, 8 weeks of GV1001 + GM-CSF, tadalafil and GEM completed treatment. Peripheral blood was analyzed by quantitative multiparameter whole blood flow cytometry of immune cell subtypes. Samples were also cryo-preserved for future analysis of peripheral blood mononuclear cell (PBMC) populations and ELISPOT analysis of antigen-specific T cell numbers. Tumor resection specimens were sectioned for immunohistological analysis of macrophage and T cell infiltration. Results: 9 of 10 pts completed therapy to assess response. Grade (Gr) 3-4 hematologic adverse events (AE) were observed during CRT. Immune-related AE were infrequent and mild. 3 of 4 pts with BR disease and 1 with initial LA (unresectable) disease achieved R0 resection. No unanticipated surgical complications were observed. Changes in monocytes and granulocytes occurred throughout treatment and may correlate with clinical benefit. At least 50% reduction in serum CA19-9 was observed in 8 pts. Partial response (n=2) or stable disease (n=7) was achieved in 9 pts. Conclusions: Combinatorial immunotherapy concurrent with GEM-based CRT is feasible and appears safe in LA or BR PC. Initial biochemical responses as well as disease control were observed in the majority of pts. Preliminary immune correlative data suggest a relationship between hematopoietic populations and clinical response.

Tie2+ Bone Marrow Endothelial Cells Regulate Hematopoietic Stem Cell Regeneration Following Radiation Injury

Hematopoietic stem cells (HSCs) reside in proximity to bone marrow endothelial cells (BM ECs) and maintenance of the HSC pool is dependent upon EC-mediated c-kit signaling. Here, we used genetic models to determine whether radioprotection of BM ECs could facilitate hematopoietic regeneration following radiation-induced myelosuppression. We developed mice bearing deletion of the proapoptotic proteins, BAK and BAX, in Tie2(+) ECs and HSCs (Tie2Bak/Bax(Fl/-) mice) and compared their hematopoietic recovery following total body irradiation (TBI) with mice which retained Bax in Tie2(+) cells. Mice bearing deletion of Bak and Bax in Tie2(+) cells demonstrated protection of BM HSCs, preserved BM vasculature, and 100% survival following lethal dose TBI. In contrast, mice that retained Bax expression in Tie2(+) cells demonstrated depletion of BM HSCs, disrupted BM vasculature, and 10% survival post-TBI. In a complementary study, VEcadherinBak/Bax(Fl/-) mice, which lack Bak and Bax in VEcadherin(+) ECs, also demonstrated increased recovery of BM stem/progenitor cells following TBI compared to mice which retained Bax in VEcadherin(+) ECs. Importantly, chimeric mice that lacked Bak and Bax in HSCs but retained Bak and Bax in BM ECs displayed significantly decreased HSC content and survival following TBI compared to mice lacking Bak and Bax in both HSCs and BM ECs. These data suggest that the hematopoietic response to ionizing radiation is dependent upon HSC-autonomous responses but is regulated by BM EC-mediated mechanisms. Therefore, BM ECs may be therapeutically targeted as a means to augment hematopoietic reconstitution following myelosuppression.

Abstract P5-20-03: A phase 2, double-blind, randomized, placebo-controlled, dose-finding study of sotatercept for the treatment of patients with chemotherapy-induced anemia and metastatic breast cancer.

Abstract Background: Sotatercept is a recombinant activin receptor IIA (ActRIIA) ligand trap and is comprised of the extracellular domain of ActRIIA linked to the Fc domain of human IgG1 (ActRIIA-IgG1). Results of preclinical and early clinical studies provide evidence that sotatercept increases the concentration of hemoglobin (Hb) in blood and may constitute a novel treatment for chemotherapy-induced anemia (CIA), a condition that results in significant morbidity. Methods: This study evaluated the safety of, and hematopoietic response to, sotatercept in patients with CIA and metastatic breast cancer (MBC). Response was defined as increase in Hb ≥1 g/dL for ≥28 consecutive days during treatment or ≤2 months following the last dose, in the absence of red blood cell (RBC) transfusion or treatment with an erythropoiesis stimulating agent (ESA). If RBC transfusion or treatment with an ESA was required, no Hb measurements within 28 days of the RBC transfusion or administration of ESA were used to determine the Hb response. Subjects were randomized to subcutaneous injection of sotatercept (0.1, 0.3, or 0.5 mg/kg) or placebo and were treated on day 1 of each of four 28-day cycles. Results: Thirty (30) subjects were enrolled and treated (5 placebo, 25 sotatercept). Increases in mean Hb in the 0.3 and 0.5 mg/kg groups were greater than in 0.1 mg/kg and placebo groups. Increases peaked approximately 15 days following treatment and declined over the remaining interval between treatments. Among 23 subjects with confirmed CIA and administered ≥1 dose and followed for ≥57 days (per-protocol analysis), 5/18 (28%) administered sotatercept responded (0/5 [0%] at 0.1 mg/kg; 3/9 [33%] at 0.3 mg/kg; 2/4 [50%] at 0.5 mg/kg) vs 1/5 [20%] administered placebo. Among 13 nonresponders administered sotatercept, 5 (39%) experienced ≥1 dose interruption/reduction, as per protocol, due to elevated Hb. The incidence of adverse events (AEs) was consistent with that reported in patients with MBC. No dose-limiting or dose-related toxicity was observed. Conclusions: Sotatercept demonstrated dose-dependent hematopoietic activity in subjects with CIA and MBC. The safety findings were generally consistent with the known safety profile of sotatercept. These data suggest that a greater dose of sotatercept or a dose interval &amp;lt;28 days might result in a more sustained, and greater rate of, hematopoietic response. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-03.

Chlorella vulgaris treatment ameliorates the suppressive effects of single and repeated stressors on hematopoiesis

The reports regarding the mutual influence between the central nervous system and the immune system constitute a vast and somewhat controversial body of literature. Stress is known to disturb homeostasis, impairing immunological functions. In this study, we investigated the hematopoietic response of Chlorella vulgaris (CV)-treated mice exposed to single (SST) and repeated stress (RST). We observed a reduction in the numbers of hematopoietic progenitors (HP) in the bone marrow and long-term bone marrow cultures (LTBMC) using flow cytometry and a coinciding decrease in the number of granulocyte-macrophage colonies (CFU-GM) after treatment with both stressors, but SST caused a more profound suppression. We observed a proportional increase in the colony-stimulating activity (CSA) of the serum of animals subjected to SST or RST. In the bone marrow, SST and RST induced a decrease in both mature myeloid and lymphoid populations but did not affect pluripotent hematopoietic progenitors (Lin(-)Sca-1(+)c-kit(+), LSK), and again, a more profound suppression was observed after SST. We further quantified the levels of interleukin-1α (IL-1α) and interleukin-6 (IL-6) and the number of myeloid cells in LTBMC. Both SST and RST reduced the levels of these cytokines to similar degrees. The myeloid population was also reduced in LTBMC, and SST induced a more intense suppression. Importantly, CV treatment prevented the changes produced by SST and RST in all of the parameters evaluated. Together, our results suggest that CV treatment is an effective tool for the prophylaxis of myelosuppression caused by single or repeated stressors.

Microbial Impact on Hematologic Homeostasis

AbstractAbstract SCI-40During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation. Disclosures:No relevant conflicts of interest to declare.

Correction of Anemia in the Frail Elderly (CAFÉ): Results of a Randomized, Double-Blind, Placebo-Controlled Study with Darbepoetin Alfa in Elderly Patients with Chronic Unexplained Anemia

AbstractAbstract 5153 Objectives:To evaluate the effect of darbepoetin alfa treatment on hemoglobin (Hb) levels in frail, community dwelling elderly patients with chronic unexplained anemia and its correlation with the presence of comorbidities, serum baseline erythropoietin concentrations and renal function. Design:A 24 weeks, prospective, randomized (1:1), double-blind, placebo-controlled clinical trial. Participants:Eighty community-dwelling, consenting pre-frail or frail (Hopkins Frailty Index score 1 to 3) patients 70 years or older with chronic anemia (Hb <11. 5 g/dL). Intervention:Subcutaneous darbepoetin alfa or placebo weekly for 24 weeks with a 22 week open-label extension. Initial dose 60 μg every other week (bi-weekly). Measurements:Hemoglobin, Erythropoietin serum levels, estimated glomerular filtration rate, Charlson Comorbidity Index and Age Adjusted Comorbidity Index scores. Results:80 subjects were enrolled, and complete data was analyzed. Of those enrolled, most were White (86. 25%) and male (58. 75%) and had a low comorbidity score, and a mean age ± standard deviation 81. 34 ± 6. 34. Mean baseline Hb was 10. 93 ± 0. 57 g/dL (9. 1–11. 9). Erythropoietin baseline levels (mU/mL) for placebo arm were 21. 4 ± 15 and 17. 6 ± 12. 4 for treatment arm. Estimated Glomerular filtration rates at baseline, mL/min per 1. 73m2 were 58. 7 ± 22. 4 and 50. 5 ± 17. 7 for placebo and treatment arms, respectively. During the observation period, there was a significantly greater hematopoietic response (mean 1. 13 ± 0. 59 g/dL) in the participants treated with darbepoetin alfa than in those receiving placebo (0. 3 ± 0. 18 g/dL). All participants (100%) responded to darbepoetin alfa treatment by week 8, attaining the target Hb level of 12 g/dL and maintaining those levels throughout the observation period. The average bi-weekly dose of darbepoetin alfa required to achieve the hemoglobin goal of 12 g/dL by week 8 was 63. 1 ± 3. 5 μg. After the eight week, 77% required less than 40. 46 ± 5. 4/week, and 23 patients (58. 9%) were maintained on weekly average dosages of less than 35 μg/week. The mean bi-weekly dose of darbepoetin alfa during the 24-week observation period was 74. 7 ± 12. 4 μg subcutaneously. The calculated average weekly dose of darbepoetin alfa needed to increase the hemoglobin levels by 1 g/dL was 40. 63μg/Hb/wk. Among the participants receiving placebo, only 3 out of 41 (7. 31%) patients had levels of Hb 12 g/dL or greater at end of the study (week 24) (P 0. 00006). This response was maintained and more significant among patients with lower age-adjusted comorbidity scores (<9) irrespective of gender, renal function and baseline erythropoietin levels. No significant differences were found between treatment and placebo on high comorbidity scores (≥9). Darbepoetin alfa was well tolerated and no major adverse events were reported in this group of patients with Hb target level of 12 g/dL. Conclusion:In this trial involving predominantly older white community dwelling patients with anemia, a direct relationship existed between increases in Hb during darbepoetin alfa therapy, this finding being more evident in the groups with lower age-adjusted comorbidity scores. Relatively lower dosages of darbepoetin alfa were required to attain a durable Hb response, with a good safety profile. Disclosures:Off Label Use: To evaluate the effect of darbepoetin alfa treatment on hemoglobin (Hb) levels in frail, community dwelling elderly patients with chronic unexplained anemia and its correlation with the presence of comorbidities, serum baseline erythropoietin concentrations and renal function.

TGFβ Restores Hematopoietic Homeostasis After Chemotherapy.

AbstractAbstract 2344 Background:Hematopoietic stem cells (HSCs) are required for lifelong blood cell production and, to prevent exhaustion, the majority of HSCs are deeply quiescent during steady-state hematopoiesis. Paracrine factors produced by specialized bone marrow niche cells maintain HSC quiescence. Yet, evolution demands a rapid hematopoietic response to stressors such as infection, bleeding or toxin exposure. These triggers set off a remarkable adaptation in hematopoiesis that sacrifices HSPC quiescence, and the protection it affords, to meet an urgent need for new blood cell production. A great deal is known about how the HSPCs are mobilized during these periods of stress but mechanisms driving the return to steady-state hematopoiesis after stress have not been identified. Method/Results:Because TGFβ is one of few negative regulators of hematopoiesis, we measured the levels of active TGFβ and assessed the effects of its downstream signaling during hematopoietic recovery after chemotherapy. During late hematopoietic regeneration, as hematopoietic homeostasis is restored, the levels of active TGFβ spike in whole bone marrow and downstream signaling (as reported by Smad2 phosphorylation) increases in hematopoietic stem and progenitor cells (HSPCs). To assess how TGFβ signaling alters hematopoietic regeneration, we administered myelosuppressive chemotherapy to mice and then, during recovery, treated cohorts with either an antibody that specifically neutralizes active TGFβ (1D11), an isotype control antibody (13C4) or nothing at all. We found that TGFβ blockade accelerated blood count and bone marrow regeneration, and promoted HSCP self-renewal/proliferation by delaying the return of HSPCs to quiescence (G0). In contrast, TGFβ blockade during homeostasis elevates neither blood counts nor bone marrow cellularity and fails to induce HSPCs to emerge from quiescence. Conclusions:The de facto paradigm is that homeostasis is passively reestablished as stress mediators normalize. Our data strongly suggest that this paradigm is incorrect. Rather than being a passive process, it appears that steady-state hematopoiesis is actively reimposed. TGFβ pathway activation marks regenerating HSPCs returning to quiescence and this context-depending signaling helps reestablish homeostasis during recovery from chemotherapy. Together, our data demonstrate that myelosuppression does not drive hematopoiesis using only a cytokine-fueled gas pedal but also taps an active braking mechanism once sufficient recovery has been attained. More directly, our data strongly suggests that TGFb pathway inhibitors could be used to promote multi-lineage hematopoietic reconstitution. This finding significantly extends prior work in the field by opening the door to new, potentially superior, ways to modulate the hematopoietic adaptation to stress. Disclosures:No relevant conflicts of interest to declare.

Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

AbstractAbstract 641Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as ’emergency myelopoiesis’. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4−/−→WT mice) and WT hematopoiesis on a TLR4−/− nonhematopoietic background (WT→TLR4−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date.We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88−/−→Myd88−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, Tek-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b+Gr-1low immature myeloid precursors accompanied by a decrease of BM CD11b+Gr-1high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis. Disclosures:No relevant conflicts of interest to declare.

Stromal SPARC contributes to the detrimental fibrotic changes associated with myeloproliferation whereas its deficiency favors myeloid cell expansion

AbstractIn myeloid malignancies, the neoplastic clone outgrows normal hematopoietic cells toward BM failure. This event is also sustained by detrimental stromal changes, such as BM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We show that the matricellular protein SPARC contributes to the BM stromal response to myeloproliferation. The degree of SPARC expression in BM stromal elements, including CD146+ mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of BM failure characterizing the prototypical myeloproliferative neoplasm primary myelofibrosis. Using Sparc−/− mice and BM chimeras, we demonstrate that SPARC contributes to the development of significant stromal fibrosis in a model of thrombopoietin-induced myelofibrosis. We found that SPARC deficiency in the radioresistant BM stroma compartment impairs myelofibrosis but, at the same time, associates with an enhanced reactive myeloproliferative response to thrombopoietin. The link betwen SPARC stromal deficiency and enhanced myeloid cell expansion under a myeloproliferative spur is also supported by the myeloproliferative phenotype resulting from the transplantation of defective Apcmin mutant hematopoietic cells into Sparc−/− but not WT recipient BM stroma. Our results highlight a complex influence of SPARC over the stromal and hematopoietic BM response in myeloproliferative conditions.

An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response

Hematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control hematopoietic injury response is largely unknown. We report an invivo gain-of-function screen and the identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150 null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150-forced expression. Our data highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of invivo functional screens for studying microRNAs in normal tissue physiology.