Microenvironmental Defects Research Articles

Modeling Defective Erythropoiesis in Myelodysplastic Syndrome (MDS)

Anemia is a significant clinical component of MDS, which results from defective erythropoiesis. By 30 weeks of age, a murine model of MDS based on genetic defects in Nup98-HOXD13 (NHD13) recapitulates the peripheral cytopenias seen in patients, including macrocytic anemia (Balderman et al. 2016, Blood 127:616). We found that these defects in the peripheral blood were correlated with decreases in marrow erythropoiesis at the earliest erythroid-specific progenitor (BFU-E), as well as a decrease in the expansion of downstream erythroblast precursors. Along with a cell-intrinsic defect, erythropoiesis in MDS patients could also be influenced by bone marrow microenvironmental changes. To investigate potential cell-intrinsic and microenvironmental defects, we analyzed erythropoiesis in chimeric mice transplanted with both UBC-GFP+ wildtype bone marrow and either 1) cells from NHD13, or 2) another MDS model based on overexpression of Evi1, or 3) unlabeled wildtype cells, which served as controls. In the two chimeric MDS models, cytopenias were not observed until after 15 weeks post-transplantation. Yet as early as 6 weeks post-transplant, analysis of peripheral blood demonstrated that GFP+ wild-type cells contributed disproportionately to circulating red blood cell (RBCs) in both MDS models. In the bone marrow of chimeric mice with normal hematocrits, we found significant decreases in both MDS-derived and wildtype BFU-E. The maintenance of RBC numbers was achieved by extensive expansion of wildtype erythroid precursors. Concurrently, MDS-derived erythroid precursors failed to expand, as seen in the non-chimeric NHD13 model. At later time-points when cytopenias become evident in the periphery of the chimeric MDS mice, erythropoiesis was still provided by wildtype cells, but the bone marrow intermediates were no longer present in sufficient numbers to prevent anemia. The source(s) of this cell-extrinsic inhibition of erythropoiesis during MDS progression may include the expanding myeloid cell populations derived from the MDS clone, which we found both in the non-chimeric NHD13 model, as well as in both chimeric MDS models. Taken together, these data suggest that MDS leads to an intrinsic block in erythropoiesis and to a progressive expansion of myelopoiesis in the marrow. These changes initially lead to a compensatory increase in erythropoiesis from wild-type cells to preserve normal RBC output, which ultimately fails with continued disease progression. DisclosuresNo relevant conflicts of interest to declare.

LB1599 Novel microenvironmental defects revealed by large scale transcriptome sequencing of lesional and nonlesional skin of vitiligo patients

LB1599 Novel microenvironmental defects revealed by large scale transcriptome sequencing of lesional and nonlesional skin of vitiligo patients

3132 - Modeling Defective Erythropoiesis in Myelodysplastic Syndrome (MDS)

Anemia is a significant clinical component of MDS, a clonal disorder predisposing to the development of leukemia. A murine model of MDS based on genetic defects in Nup98-HOXD13 (NHD13) recapitulates the peripheral cytopenias seen in patients, including macrocytic anemia (Balderman, 2016). We found that anemia in NHD13 mice was associated with a significant decrease in erythroid-specific progenitors (BFU-E), a lack of erythroblast precursor expansion, and decreased numbers of erythroblastic islands. To investigate whether microenvironmental defects contribute to development of anemia, we analyzed erythropoiesis in chimeric mice transplanted with UBC-GFP+ wild-type marrow and unlabeled cells from either NHD13 or wild-type mice. Non-anemic NHD13 chimeric mice developed macrocytosis. Surprisingly, almost all circulating red cells were GFP+, indicating their derivation from wild-type erythroblasts. In the marrow of these mice, we found significant decreases in both MDS-derived and wild-type BFU-E, as well as a lack of MDS-derived maturing erythroblasts, indicating that wild-type erythroblasts compensate for an intrinsic defect in clonal erythropoiesis. The progressive development of anemia in NHD13 chimeric mice was associated with failure of wild-type erythroid precursor expansion in the marrow, implicating extrinsic, environmental factors as the cause of anemia. We replicated these results in a complementary, EVI1 overexpression chimeric mouse model of MDS/marrow failure. Anemia in NHD13 chimeric mice was also associated with expansion in the marrow of MDS clone-derived monocytes, which expressed higher levels of several inflammatory cytokines, compared with monocytes from age-matched control mice transplanted with wild-type cells. We also found increased erythroblast-monocyte interactions by imaging flow cytometry, consistent with alterations in the erythroblastic island microenvironment. Taken together, these data indicate that the development of anemia in two independent murine models of MDS is due not only to an intrinsic block of MDS clonal erythropoiesis, but also to an extrinsically-mediated failure of residual, predominantly wild-type, erythropoiesis.

Restoration of the Bone Marrow Microenvironment Improves Hematopoietic Function in a Murine Model of Myelodysplastic Syndrome

While data suggest that, in the myelodysplastic syndromes (MDS), the bone marrow microenvironment (BMME) is abnormal, the lack of examination of the BMME in a robust in vivo model has limited progress in the understanding of reciprocal MDS-BMME interactions. If microenvironmental defects contribute to disease progression, targeting the BM niche may offer an alternative approach for therapeutic benefit. We sought to define the MDS BMME in a well-established transgenic murine model that recapitulates hallmark features of human MDS. In this model, hematopoietic tissue-specific expression of the NUP98-HOXD13 (NHD13) fusion gene is driven by Vav regulatory elements, resulting in peripheral cytopenias by 16 weeks of age and mortality from transformation to leukemia at a median time of 11 months of age. Mice were analyzed at 15-36 weeks of age, when the MDS phenotype is prominent in the absence of leukemia. Flow cytometric quantification of BM stroma in 23-week old NHD13 mice showed a 6.5-fold increase in frequency of CD51+/Sca1- osteoblastic cells (OBC) compared to WT (p<0.05). CD51+/Sca1+ multipotent stromal cells (MSC) and CD31+/Sca1+ endothelial cells were also significantly increased in NHD13 compared to WT mice. This was not due to loss of hematopoietic cells in the marrow of NHD13 mice. While an expansion of functional MSCs and osteoblastic cells could result in skeletal changes, micro CT imaging of the femora and tibiae of 20-week old NHD13 mice revealed no differences in skeletal parameters compared to WT mice. These data suggest that the expanded osteolineage cells in NHD13 mice are not functional bone-forming cells. While stromal populations were not altered in bone-associated cells of 23-week old NHD13 compared to WT mice, 36-week old NHD13 mice also showed increased bone-associated OBCs, MSCs, and endothelial cells. Therefore, there are significant time-dependent shifts in critical stromal populations in this in vivo model of MDS, which may contribute to an abnormal BMME. To determine if the MDS BMME contributes to hematopoietic failure, NHD13 BM (CD45.2) was transplanted with WT competitor BM (CD45.1) in a 1:1 ratio into lethally irradiated NHD13 or WT (CD45.2) recipients, thus exposing the same MDS hematopoietic cells to either MDS or WT microenvironments. Using this transplantation paradigm, we previously reported improvement of hematopoiesis when NHD13 BM is exposed to a WT BMME. Surprisingly, CD45.1+ WT competitor-derived cells exhibited myeloid skewing when transplanted into NHD13 recipients compared to WT recipients, suggesting that interaction of WT BM with an MDS BMME can induce myeloid skewing, a feature of the NHD13 model. NHD13 BM was next transplanted non-competitively into lethally irradiated NHD13 and WT mice. At 10 weeks post-transplant, WT recipients had a 2.5-fold increase in peripheral leukocytes (p<0.05), significant improvement of anemia, and significant mitigation of BM long term-HSC loss compared to NHD13 recipients, suggesting that WT BMME components can rescue hematopoietic function in MDS. Together, our studies strongly suggest that a murine model recapitulates MDS microenvironmental abnormalities, and that exposure of MDS hematopoietic cells to a non-malignant microenvironment is sufficient to improve hematopoietic function. Thus, improvement of the BM microenvironment represents a novel therapeutic strategy to ameliorate hematopoietic function in MDS. DisclosuresBecker:Millenium: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties.

Concise review: diabetes, the bone marrow niche, and impaired vascular regeneration.

Diabetes mellitus is a global health problem that results in multiorgan complications leading to high morbidity and mortality. Until recently, the effects of diabetes and hyperglycemia on the bone marrow microenvironment-a site where multiple organ systems converge and communicate-have been underappreciated. However, several new studies in mice, rats, and humans reveal that diabetes leads to multiple bone marrow microenvironmental defects, such as small vessel disease (microangiopathy), nerve terminal pauperization (neuropathy), and impaired stem cell mobilization (mobilopathy). The discovery that diabetes involves bone marrow-derived progenitors implicated in maintaining cardiovascular homeostasis has been proposed as a bridging mechanism between micro- and macroangiopathy in distant organs. Herein, we review the physiological and molecular bone marrow abnormalities associated with diabetes and discuss how bone marrow dysfunction represents a potential root for the development of the multiorgan failure characteristic of advanced diabetes. The notion of diabetes as a bone marrow and stem cell disease opens new avenues for therapeutic interventions ultimately aimed at improving the outcome of diabetic patients.

CrbpI regulates mammary retinoic acid homeostasis and the mammary microenvironment

Cellular retinol-binding protein, type I (CrbpI), encoded by retinol-binding protein, type 1 (Rbp1), is a chaperone of vitamin A (retinol) that is epigenetically silenced in ~25% of human breast cancers. CrbpI delivers vitamin A to enzymes for metabolism into an active metabolite, all-trans retinoic acid (atRA), where atRA is essential to cell proliferation, apoptosis, differentiation, and migration. Here, we show the effect of CrbpI loss on mammary atRA homeostasis using the Rbp1(-/-) mouse model. Rbp1(-/-) mouse mammary tissue has disrupted retinoid homeostasis that results in 40% depleted endogenous atRA. CrbpI loss and atRA depletion precede defects in atRA biosynthesis enzyme expression. Compensation by CrbpIII as a retinoid chaperone does not functionally replace CrbpI. Mammary subcellular fractions isolated from Rbp1(-/-) mice have altered retinol dehydrogenase/reductase (Rdh) enzyme activity that results in 24-42% less atRA production. Rbp1(-/-) mammary tissue has epithelial hyperplasia, stromal hypercellularity, increased collagen, and increased oxidative stress characteristic of atRA deficiency and early tissue dysfunction that precedes tumor formation. Consistent with the findings from the Rbp1(-/-) mouse, tumorigenic epithelial cells lacking CrbpI expression produce 51% less atRA. Together, these data show that CrbpI loss disrupts atRA homeostasis in mammary tissue, resulting in microenvironmental defects similar to those observed at the early stages of tumorigenesis.

Dynamics of Human Prothymocytes and Xenogeneic Thymopoiesis in Hematopoietic Stem Cell-Engrafted Nonobese Diabetic-SCID/IL-2rγnull Mice

To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors invitro and their recovery in transplanted recipients invivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Getting back at nature: understanding thymic development and overcoming its atrophy

T cell development is a complex and tightly regulated process involving reciprocal interactions between the thymic stroma and differentiating thymocytes. Normal thymic function is critical for immunity and microenvironmental defects predispose to dysregulation in the T cell compartment. Thymic structure and function are also severely damaged by chemotherapy and pre-transplant conditioning. Furthermore, poor immune competence with ageing is closely linked to thymic atrophy. Overcoming such thymic defects would have immediate application in many diseases, especially the recovery of cancer patients from cytotoxic treatment. Reversing the thymus ageing process via inhibition of atrophic factors such as sex steroids or administration of thymopoietic growth factors is one possible approach. Moreover, it is becoming clear a common thymic epithelial progenitor exists, raising the possibility for de novo thymus generation using emerging stem cell and tissue engineering technologies. Achievement of this goal will open up many avenues for the application of thymus-based immune rejuvenation and manipulation.

A novel role for the marrow microenvironment in initiating and sustaining hematopoietic disease

Background: The marrow microenvironment is composed of a complex network of cells and extra cellular matrix that cooperate to regulate normal hematopoiesis. There is growing evidence that microenvironmental defects can contribute to the pathogenesis of hematological malignancies. Objective/methods: We review the role of the microenvironment in inducing and sustaining hematological malignancies. Results/conclusions: Two basic mechanisms could explain the role of microenvironmental defects in the evolution of hematopoietic neoplasms. There is significant data to support the first mechanism, in which the malignant hematopoietic clone induces reversible functional changes in the microenvironment that result in improved growth conditions for the malignant cells. More recent studies from mouse models have indicated that a second mechanism involving primary microenvironmental defects can also result in malignancy.

Alcohol Induces Proliferation of Multipotential Mesenchymal Stromal Cells and Their Differentiation into Adipocytes

During aging, bone marrow (BM) fat increases and hematopoietic stem cells (HSCs) decrease, converting the marrow from red to yellow. Multipotential Mesenchymal Stromal Cells (MMSCs) give rise to various cell types comprising the BM microenvironment, that regulate HSCs differently. MMSC differentiation into osteoblasts, provides a favorable microenvironment where as into adipocytes is less supportive for HSCs. Therefore accumulation of adipocytes in BM may contribute to osteoporosis and hematopoietic deficiencies of aging. In the present study, changes in various stem cell populations with aging in human subjects were studied, BM‐MMSCs were enumerated as either CD45lo Stro‐1+ cells or Fibroblast Colony Forming Cells (FCFC). Stro‐1+ cells declined slightly with age but FCFC showed no significant changes. However, when subjects were segregated into drinkers of alcohol versus non‐drinkers, the former had fewer FCFCs than the latter. The potential basis of this observation was studied by exposing a cloned mouse MMSC cell line, OMA‐AD to various concentrations of ethanol. Lower concentrations (5–50 mM) increased proliferation as compared to controls and also induced adipocytic differentiation. Higher concentrations of ethanol (&gt;300 mM) were progressively toxic to MMSC. Potentially, the loss of FCFC and by implication MMSC, in alcohol drinkers might occur by promotion of differentiation to adipocytes leading to inhibition of osteoblast production. This, in turn, may have implications for impaired fracture repair, increased osteoporosis and hematopoietic microenvironmental defects in the elderly who consume alcohol.

Thymic microenvironment and NZB mice: the abnormal thymic microenvironment of New Zealand mice correlates with immunopathology.

There are distinct microenvironmental abnormalities of thymic architecture in several murine models of SLE defined using immunohistochemistry and a panel of mAb dissected at thymic epithelial markers. To address the issue of the relationship between the thymic microenvironment and autoimmunity, we studied backcross (NZB x NZW) F1 x NZW mice in which 50% of offspring develop nephritis associated with proteinuria and anti-DNA antibodies. We reasoned that if thymic abnormalities are associated with development of disease, the correlation of abnormalities with lupus-like disease in individual backcross mice will form the foundation for identification of the mechanisms involved. In parallel, we directed a genetic linkage analysis, using markers previously shown to be linked to nephritis and IgG autoantibody production, to determine if such loci were similarly associated with microenvironmental changes. Our data demonstrate that all (NZB x NZW) F1 x NZW backcross mice with disease have microenvironmental defects. Although the microenvironmental defects are not sufficient for development of autoimmune disease, the severity of thymic abnormalities correlates with titers of IgG autoantibodies to DNA and with proteinuria. Consistent with past studies of (NZB x NZW) F1 x NZW mice, genetic markers on proximal chromosome 17 (near MHC) and distal chromosome 4 showed trends for linkage with nephritis. Although the markers chosen only covered about 10-15% of the genome, the results demonstrated trends for linkage with thymic medullary abnormalities for loci on distal chromosome 4 and distal chromosome 1. We believe it will be important to define the biochemical nature of the molecules recognized by these mAbs to understand the relationships between thymic architecture and immunopathology.

Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

Mouse hematopoietic stem cells and the interaction ofc‐kirreceptor and steel factor

Hematopoietic stem cells (HSCs) are distinguished from other hematopoietic progenitors in bone marrow by their unique ability to undergo multilineage differentiation and self-renewal. Two mouse mutations, dominant spotting (W) and steel (Sl), have pleiotropic effects on hematopoiesis, gametogenesis, and melanoblast development. These two mutations have been shown to be intrinsic (W) and microenvironmental (Sl) defects. Recently, molecular studies revealed that the W and Sl loci encode the c-kit receptor and steel factor (SLF), respectively. The c-kit receptor is expressed on HSCs and hematopoietic progenitors, while SLF is produced by stromal cells. SLF acts on hematopoietic progenitors synergistically with other growth factors. Here we review the effect of these mutations on mouse hematopoiesis, and show that SLF acts on HSCs and other myeloerythroid progenitors, but that it, in our hands, does not play a critical role in HSC generation or self-renewal. Rather, SLF is the most potent co-mitogen (with IL-1, IL-3, IL-6, G-CSF, GM-CSF, or M-CSF) found that acts on these cells, but the effect of such treatments is the rather specific and massive expansion of myeloerythropoiesis, not lymphopoiesis, and perhaps at the expense of HSC self-renewal.

Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of New Zealand black mice.

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate resulting in a deficiency of B lineage precursors in adult (greater than 15 wk old) animals. To study the characteristics of B lineage cells in young (4 wk) and old (6 mo) NZB mice, bone marrow from these animals was used to initiate long term lymphoid bone marrow cultures (LBMC) that permit the long term maintenance of B cells and their precursors. Age-matched cultures from BALB/c mice and NZB.xid marrow were established in parallel. Primary LBMC were readily established from these strains and showed similar patterns of growth for the 3-mo observation period. No significant differences in numbers of 14.8 positive cells were observed. However, NZB mice at both ages had a higher percentage of membrane IgM (mIgM)-expressing cells. Significant levels of supernatant IgM were found only in cultures of 6-mo NZB and BALB/c mice; levels were highest in NZB culture supernatants and were often more than 500 ng/ml; significant, although much lower, levels of IgG were likewise detected. Lymphoid cells from NZB.xid mice were unable to generate significant levels of IgM in supernatant fluids indicating the effects of the xid gene were displayed in vitro. Autoantibodies were not detected in any of the culture supernatants. Additional evidence for NZB hyperactivity in primary B lymphopoiesis was observed upon initiation of primary myeloid bone marrow cultures (MBMC) from these strains of mice and subsequently transferring them to LBMC conditions. This results in the cessation of myelopoiesis at the initiation of B lymphopoiesis. At the time of converting MBMC to LBMC, cultures of NZB and BALB/c mice morphologically resembled myeloid cultures and had neither B cell colony-forming units nor cells that expressed 14.8 or mIgM. However, following the switch, NZB mice had a 5-fold higher number of B cell colony-forming units. Further, MBMC established from NZB bone marrow cells had a reduced capacity to form colonies in the granulocyte-macrophage colony-forming unit assay. These studies indicate that defects of NZB hemopoietic cells are manifest in vitro and suggest the use of in vitro long term cultures as a valuable technique to further dissect the hematopoietic abnormalities of NZB mice and possible underlying microenvironmental defects.

Influence of islet and bone marrow transplantation on the diabetes and immunodeficiency of BB rats

The results of islet transplantation in spontaneous autoimmune diabetes of BB rats were studied to determine whether this disease process might damage the transplanted islet tissue. Since BB rats are not genetically uniform, syngenetic grafts could not be used; therefore, allograft rejection was prevented by rendering BB rats immunologically tolerant of WF transplantation antigens by neonatal inoculation with bone marrow cells. Despite the resultant tolerant state, which permitted successful engraftment of WF skin allografts, the transplanted islets ameliorated the spontaneous diabetes of BB rats only briefly before they were destroyed by immune insulitis. BB rats from the diabetic stock were found to suffer from abnormalities in T lymphocytes and their subsets as well as defective immune response patterns. When analyzed with monoclonal antibodies specific for rat lymphocyte markers, BB rats of the diabetic stock were found to be lymphocytopenic. There was a reduction in helper T cells and a more severe deficit in the suppressor/cytotoxic subset. BB rats that were inoculated neonatally with bone marrow from normal donors were found to have a strikingly reduced incidence of diabetes. Moreover, the T cell functional, numerical, and microenvironmental defects that were present in noninoculated BB rats were restored in marrow-inoculated BB rats, findings possibly related to the decreased incidence of diabetes.