Immune-mediated Bone Marrow Failure Research Articles

Tetramethylpyrazine promotes bone marrow repair in a C57 mouse model of X-rayinduced immune-mediated bone marrow failure

To observe the therapeutic effect of tetramethylpyrazine on immune-mediated bone marrow failure (BMF) induced by different doses of X-ray exposure in C57 mice. C57BL6 mice were randomized into 4 groups, including a blank control group and 3 X-ray exposure groups with X-ray exposure at low (5.0 Gy), moderate (5.75 Gy), and high (6.5 Gy) doses. After total body irradiation with 0.98 Gy/min X-ray. The mice as recipient received injections of 4×106 lymphocytes from DBA/2 mice via the tail vein within 4 h. The survival rate of the recipient mice, peripheral blood cell counts, bone marrow nucleated cell count, and bone marrow pathology were examined at 14 days after the exposure. In the subsequent experiment, C57 mice were exposed to 5.0 Gy X-ray and treated with intraperitoneal injection of tetramethylpyrazine at the low (5 mg/mL), moderate (10 mg/mL), or high (20 mg/mL) doses (12 mice in each group) for 14 consecutive days, and the changes in BMF were observed. X-ray exposure, especially at the high dose, resulted in significantly lowered survival rate in the mouse models of BMF at 14 days. As the X-ray dose increased, the mice showed significantly reduced peripheral blood counts of red blood cells, white blood cells, platelets and lowered bone marrow nucleated cell counts with obvious bone marrow congestion and reduction of nucleated cells (P < 0.05 or 0.001). In the mice exposed to 5.0 Gy X-ray, tetramethylpyrazine at the high dose most obviously increased bone marrow nucleated cells (P < 0.01) and red blood cells (P < 0.001), and even at the low dose, tetramethylpyrazine significantly increased the counts of white blood cells (P < 0.05) and platelets (P < 0.01) following the exposure. Tetramethylpyrazine dose-dependently alleviated bone marrow hyperemia, increased bone marrow nucleated cell counts, and lowered Fas protein expression in the bone marrow. X-ray irradiation at 5.0 Gy is suitable for establish mouse models of immune-mediated BMF. Tetramethylpyrazine promotes bone marrow repair by regulating Fas cell apoptosis signals, which further expands the traditional Chinese medicine theory of "removing blood stasis to create new."

Research progress of abnormal immunologic mechanism in acquired aplastic anemia

Aplastic anemia (AA) is considered as an immune-mediated bone marrow failure syndrome, characterized by peripheral pancytopenia and destruction of hematopoietic stem/progenitor cells. Acquired aplastic anemia (aAA) is more common in clinic. Abnormal immunity is one of the major factors mediating the pathogenesis of aAA. This review discusses the current understanding of the immunobiology in aAA, so as to clarify the immune mechanism of this disease, and to provide a theoretical basis for the clinical treatment of aAA. Key words: Anemia, aplastic; Immunity; Immunosuppressive agent; Children; Acquired aplastic anemia

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ

AbstractThe proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ–mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.

Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

AbstractInterferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. [Display omitted] DisclosuresNakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Sirolimus Augments Hematopoietic Stem and Progenitor Cell Recovery from Hematopoietic Stress

Sirolimus is an approved drug to prevent solid organ transplant rejection and to treat lymphangioleiomyomatosis. Sirolimus also has been employed in a broad range of clinical circumstances, including induction of tolerance in nonmyeloablative HLA-mismatched bone marrow (BM) transplantation and treatment of graft-versus-host disease following allogeneic stem cell transplantation. Sirolimus suppresses mammalian target of rapamycin (mTOR), a serine-threonine protein kinase that responds to multiple signals to regulate cellular metabolism, survival, and growth. We recently reported the application of sirolimus on murine models of immune-mediated BM failure (Feng X, Haematologica, 2017;102:1691-1703) in which sirolimus alleviated pancytopenia and attenuated BM destruction by depleting active cytotoxic T cells and expanding regulatory T cells. Controls for these experiments suggested that sirolimus might also act directly to preserve hematopoietic stem and progenitor cells (HSPCs) in non-immune BM damage and hematopoietic cell injury. In the current study, we systematically tested the effects of sirolimus on HSPC regeneration following irradiation and exposure to cytotoxic drugs. B6 mice subjected to 5 Gys total body irradiation (TBI) showed large declines in HSPC numbers, which were abrogated by sirolimus at 2 mg/Kg/day by intraperitoneal (i.p.) injection for 13 days: we observed significant increases (2-4-fold) in the proportions and absolute numbers of HSPCs relative to TBI controls when animals were evaluated at 14 days (Fig. 1A). Sirolimus did not alter peripheral blood cell counts or BM cellularity. Functionally, BM cells from sirolimus-treated TBI mice contained 2-3-fold more colonies than did marrow of TBI controls. In a competitive repopulation stem cell assay, recipients of sirolimus-treated TBI donor BM cells had enhanced donor engraftment, as measured in recipient animals' peripheral blood and BM. There was also an increased recovery of donor HSPCs in recipient BM, relative to recipients of TBI-only donor BM cells, evaluated at 7 months after donor cell infusion. In other experiments, sirolimus at the same dose following busulfan (10 mg/kg i.p. three times weekly for 10 injections; Fig. 1B) or 5-fluorouracil (150 mg/Kg on days 0, 15, and 29 respectively; Fig. 1C) induced similar improved HSPC regeneration. Sirolimus-mediated HSPC regeneration was associated with c-Kitup-regulation in BM cells. In a PCR-based transcriptome assay of hematopoiesis genes performed on sorted Lin-CD150+ BM cells, sirolimus-treated TBI mice showed a 90% increase in c-Kit mRNA relative to TBI controls. Expression of other stem cell and transcription factor genes, including Cd34, Gata2,Angpt1, and Etv6, was also increased, while expression of T cell differentiation and Notch signaling genes, such as Cd2, Cd4, Cd8a, Tnfsf11, Jag1a, and Il-1a, was decreased in sirolimus-treated TBI mice relative to TBI controls. In parallel experiments, sirolimus had no effect on HSPCs in steady-state B6 mice that were not subjected to hematopoietic injury. Sirolimus stimulated HSPC expansion in mice carrying the Wv mutation in the c-Kit locus (Fig. 1D). To test the potential utility of sirolimus in humans, we pre-irradiated human BM CD34+ cells with 5 Gys to induce injury, then transplanted the radiated cells into immunodeficient NSG mice, followed by sirolimus treatment (2 mg/Kg/day i.p. for 4 injections). Sirolimus significantly increased engraftment of human CD45+ cells in recipient blood and BM at 5 weeks relative to the mice without sirolimus treatment (Fig. 1E). In summary, sirolimus augments c-Kit expression and HSPC recovery in response to hematopoietic stress. This novel functional property detected in murine models and xenotransplantation of human cells suggests potential clinical applicability of sirolimus to ameliorate hematopoietic cell injury and accelerate HSPC regeneration after BM hematopoietic injury. [Display omitted] DisclosuresYoung:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding.

Interleukin-18 plays a dispensable role in murine and likely also human bone marrow failure

Interleukin-18 (IL-18), also known as interferon-gamma (IFN-γ)-inducing factor, is involved in Th1 responses and regulation of immunity. Accumulating evidence implicates IL-18 in autoimmune diseases, but little is known of its role in acquired aplastic anemia (AA), the immune-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). IL-18 protein levels were significantly elevated in sera of severe AA (SAA) patients, including both responders and nonresponders assayed before treatment, and decreased after treatment. IL-18 receptor (IL-18R) was expressed on HSPCs. Co-culture of human BM CD34+ cells from healthy donors with IL-18 upregulated genes in the helper T-cell and Notch signaling pathways and downregulated genes in the cell cycle regulation, telomerase, and IL-6 signaling pathways. Plasma IL-18 levels were also elevated in murine models of immune-mediated BM failure. However, deletion of IL-18 in donor lymph node cells or deletions of either IL-18 or IL-18R in recipients did not attenuate elevations of circulating IFN-γ, tumor necrosis factor-alpha, or IL-6, nor did they alleviate BM failure. In summary, our findings suggest that, although increased circulating IL-18 is a feature of SAA, it may reflect an aberrant immune response but be dispensable to the pathogenesis of AA.

Cyclosporin A inhibits adipogenic differentiation and regulates immunomodulatory functions of murine mesenchymal stem cells

Aplastic anemia (AA) is generally considered as an immune-mediated bone marrow failure syndrome. Several studies show that bone marrow mesenchymal stem cells (BM-MSCs), as key cellular components of the bone marrow microenvironment, are also involved in the pathogenic mechanism of AA. Cyclosporin A (CsA) is a classic immunosuppressive drug for AA, and it specifically inhibits mammalian T cells by preventing activation of transcription factors involved in cytokine gene expression. However, little is known about the effect of CsA on the BM-MSCs. In this study, murine BM-MSCs were stimulated in the presence of CsA. Further, we found that CsA could inhibit murine BM-MSC proliferation and promote BM-MSC apoptosis, what's more CsA could inhibit adipogenic differentiation. Our study also showed that CsA could inhibit interleukin-6 expression in BM-MSCs, while promoting programmed death-ligand 2 expression. In conclusion, our results proposed that CsA may exert an effect on regulating the bone marrow environment by influencing BM-MSCs, which have a beneficial effect on treating AA.

Apoptosis of Platelets Inhibited By Herba Sarcandrae Extract through the Mitochondria Pathway.

The purpose of the present study is to decode the underlying mechanism of Herba Sarcandrae that indicated antipurpuric effect and to unveil one of its primary components, flavonoids, which play an important role. An immune mediated bone marrow failure (BMF) model in mouse was established by infusion thymus suspension cells after radiation in vivo. Platelets isolated in vitro were prepared from normal mice and BMF mice, respectively. The expressions of PS, P-selectin, PAC-1, Bax, Bad, Bid, and caspase-9 were examined by flow cytometry, and alteration of morphology of platelets under different conditions was observed. Our results indicated that the number of platelets was increased by addition of total flavonoids, and some of apoptotic markers such as Bax, Bad, Bid, and Caspase-9 were downregulated. In addition, the phosphatidylserine (PS) exposure on platelets was inhibited by total flavonoids, and the expressions of PAC-1 and P-selectin were decreased. In conclusion, it is suggested that the total flavonoids of Herba Sarcandrae may inhibit the excessive platelet apoptosis through mitochondrial pathway. In addition, activation of platelets may be also involved in mediating apoptosis of platelets.

Tumor Necrosis Factor-α in Target Cells but Not in Effector Cells Plays Critical Roles in Development of Murine Immune-Mediated Bone Marrow Failure

Tumor necrosis factor alpha (TNF-α) is a critical cytokine in systemic inflammation and a potent inducer of apoptotic cell death. Type 1 cytokines are involved in the pathogenesis of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. Overproduction of interferon gamma (IFN-γ) and TNF-α from activated T cells or bone marrow cells has been proposed as a mechanism of BM failure. We hypothesized that inhibiting the production of TNF-α or blockade of the TNF-α signaling pathway would attenuate hematopoietic cell destruction in murine models of immune BM failure. Therefore, we tested the role of TNF-α in several mouse models of human AA, using C57BL/6 (B6) mice carrying germline deletions of TNF-α (TNF-α-/-) or its receptors including TNF-α receptor superfamily member 1a (TNF-rsf1a-/-), TNF-rsf1b-/-, and TNF-rsf1a-/-1b-/-as donors or recipients, respectively.Lymph node (LN) cells from both TNF-α-/- and TNF-α receptor knock out mice induced fatal BM failure with efficacy similar to LN cells from wild-type B6 donors when infused into CByB6F1 recipients that had received 5 Gy of total body irradiation (TBI), indicating that expression of TNF-α or TNF-α receptors on effector cells did not affect the development of BM failure.However, TNF-α-/- mice as recipients unexpectedly showed resistance to immune-mediated BM failure. Infusion of 5 million LN cells from MHC-mismatched FVB donors to 6.5 Gy TBI pre-conditioned TNF-α-/- recipients did not induce AA: the animals had normal or increased levels of WBC, RBC, platelets, and total BM cells relative to TBI-only TNF-α-/- mice; control wild-type B6 recipient mice developed severe pancytopenia and BM failure after LN infusion at 10 days (Fig. 1A&B). This result suggests that TNF-α production in the recipients had a role in immune attack by effector cells. When residual BM cells were cultured in vitro in cytokine-supplemented methylcellulose media, cells from wild-type BM failure mice, but not from TNF-α-/- recipient mice, showed a significant reduction in colony forming units relative to their TBI controls. By flow cytometry, Fas+ cells and Annexin V-binding cells were increased in BM of wild-type marrow failure mice relative to wild-type TBI controls, but no increase in Fas+ cells and Annexin V-binding cells was observed in TNF-α-/- mice that had received LN infusion. In parallel experiments, injection of FVB LN cells into sub-lethally-irradiated TNF-rsf1a-/-, TNF-rsf1b-/-, or TNF-rsf1a-/-1b-/- mice resulted in BM failure with declines in WBC and platelets as observed in wild-type B6 mice after FVB LN infusion. Plasma levels of both IFN-γ and TNF-α were markedly increased in all the wild-type B6 and TNF-α receptor knock out mice that developed BM failure, but not in TNF-α-/- recipient mice. These data suggest that depletion of TNF-α receptors could not attenuate immune-mediated BM destruction and that TNF-α did not exert its destructive effect on hematopooetic targets through the TNF-α/TNF-rsf1a and 1b pathways.We hypothesized that TNF-α might modulate T cell IFN-γ production to affect immune-mediated BM destruction. TNF-α is mainly produced by activated macrophages, while IFN-γ is mainly secreted by activated T cells. Blockade of either IFN-γ in effector cells or IFN-γ receptor in target cells ameliorates the development of BM failure (Chen J, Blood.2015;126:2621). To test this hypothesis, we first depleted macrophages from CByB6F1 recipient mice with liposomal clodronate before BM failure induction with TBI and B6 LN cell injection. Indeed, removal of macrophages attenuated BM destruction relative to BM failure mice pre-injected with PBS-loaded liposomes without macrophage depletion (Figure 1C). When we injected recombinant TNF-α daily i.v. for 1 week into TNF-α-/- recipients following TBI and FVB LN cell infusion, TNF-α-injected mice had decreased BM cellularity relative to mice that received TBI and LN infusion without TNF-α (Figure 1D). Remarkably, TNF-α injection increased intracellular IFN-γ expression in BM T cells as determined by flow cytometry, indicating that TNF-α was capable of stimulating T cells to secrete IFN-γ.In summary, our study reveals a critical role of TNF-α in target cells but not in effector cells, in the pathogenesis of immune-mediated BM failure by cell-cell “crosstalk” modulation of IFN-γ secretion. [Display omitted] DisclosuresYoung:Novartis/GSK to institute: Research Funding.

Self-Destructive Behavior among Full-Donor Blood and Marrow Grafts and the Association with Long-Term Graft Function

Self-Destructive Behavior among Full-Donor Blood and Marrow Grafts and the Association with Long-Term Graft Function

A composite mouse model of aplastic anemia complicated with iron overload.

Iron overload is commonly encountered during the course of aplastic anemia (AA), but no composite animal model has been developed yet, which hinders drug research. In the present study, the optimal dosage and duration of intraperitoneal iron dextran injection for the development of an iron overload model in mice were explored. A composite model of AA was successfully established on the principle of immune-mediated bone marrow failure. Liver volume, peripheral hemogram, bone marrow pathology, serum iron, serum ferritin, pathological iron deposition in multiple organs (liver, bone marrow, spleen), liver hepcidin, and bone morphogenetic protein 6 (BMP6), SMAD family member 4 (SMAD4) and transferrin receptor 2 (TfR2) mRNA expression levels were compared among the normal control, AA, iron overload and composite model groups to validate the composite model, and explore the pathogenesis and features of iron overload in this model. The results indicated marked increases in iron deposits, with significantly increased liver/body weight ratios as well as serum iron and ferritin in the iron overload and composite model groups as compared with the normal control and AA groups (P<0.05). There were marked abnormalities in iron regulation gene expression between the AA and composite model groups, as seen by the significant decrease of hepcidin expression in the liver (P<0.01) that paralleled the changes in BMP6, SMAD4, and TfR2. In summary, a composite mouse model with iron overload and AA was successfully established, and AA was indicated to possibly have a critical role in abnormal iron metabolism, which promoted the development of iron deposits.

Immune-Mediated Hematopoietic Failure after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT) and often requires stem cell infusion from the same donor when the patient fails to respond to conventional therapies. We screened the peripheral blood (PB) of 14 patients who developed donor-type LGF at 2 to 132 months after allo-SCT for the presence of the markers for immune-mediated bone marrow (BM) failure. Increased glycosylphosphatidyl inositol-anchored protein-deficient (GPI-AP-) leukocytes, which accounted for .009% to 0.147% of the total granulocytes, were detected in 5 patients (severe aplastic anemia, n = 2; follicular lymphoma, n = 1; acute lymphoblastic leukemia, n = 1; myelodysplastic syndromes; n = 1) and 4.7% to 81.2% HLA-allele–lacking leukocytes (HLA-LLs) were detected in 2 patients (acute myelogenous leukemia, n = 1; and myelodysplastic syndromes, n = 1). Three of the 5 patients with increased GPI-AP- leukocytes were treated with antithymocyte globulin (ATG), and 2 patients achieved transfusion independence. These results suggest that immune mechanisms that are similar to acquired aplastic anemia underlie condition of approximately one-half of the patients with donor-type LGF, and that in patients with increased GPI-AP- cells, donor-derived hematopoiesis may be restored by ATG therapy alone without donor stem cell infusion.

Rapamycin is highly effective in murine models of immune-mediated bone marrow failure.

Acquired aplastic anemia, the prototypical bone marrow failure disease, is characterized by pancytopenia and marrow hypoplasia. Most aplastic anemia patients respond to immunosuppressive therapy, usually with anti-thymocyte globulin and cyclosporine, but some relapse on cyclosporine withdrawal or require long-term administration of cyclosporine to maintain blood counts. In this study, we tested efficacy of rapamycin as a new or alternative treatment in mouse models of immune-mediated bone marrow failure. Rapamycin ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of cyclosporine. Rapamycin effectively reduced Th1 inflammatory cytokines interferon-γ and tumor necrosis factor-α, increased the Th2 cytokine interleukin-10, stimulated expansion of functional regulatory T cells, eliminated effector CD8+ T cells (notably T cells specific to target cells bearing minor histocompatibility antigen H60), and preserved hematopoietic stem and progenitor cells. Rapamycin, but not cyclosporine, reduced the proportion of memory and effector T cells and maintained a pool of naïve T cells. Cyclosporine increased cytoplasmic nuclear factor of activated T-cells-1 following T-cell receptor stimulation, whereas rapamycin suppressed phosphorylation of two key signaling molecules in the mammalian target of rapamycin pathway, S6 kinase and protein kinase B. In summary, rapamycin was an effective therapy in mouse models of immune-mediated bone marrow failure, acting through different mechanisms to cyclosporine. Its specific expansion of regulatory T cells and elimination of clonogenic CD8+ effectors support its potential clinical utility in the treatment of aplastic anemia.

The herbal decoction modified Danggui Buxue Tang attenuates immune-mediated bone marrow failure by regulating the differentiation of T lymphocytes in an immune-induced aplastic anemia mouse model.

Angelicae Sinensis, Radix Astragali and Rhizoma Coptidis are all herbs of modified Danggui Buxue Tang (DGBX) and are extensively applied herbs in traditional Chinese medicine for the treatment of anemia and inflammation. In this study, immune-induced AA mice were used as an animal model, and the immunosuppressive agent, Ciclosporin A (CsA), was used as a positive control. Multiple pro-inflammatory cytokines were examined by bead-based multiplex flow cytometry. The T-cell subsets were assessed using a fluorescence-activated cell sorter (FACS). Western blot analysis was used to estimate the protein expression levels of specific transcription factors for T helper cells (Th1, Th2 and Th17) and key molecules of the Janus-activated kinase (Jak)/signal transducer and activator of transcription (Stat3) signaling pathway. DGBX treatment could significantly increase the production of whole blood cells in peripheral blood (PB); inhibit the expansion of Th1 and Th17 cells; increase the differentiation of Th2 and Tregs cells; regulate the expression levels of T-bet, GATA-3, RORγ and proinflammatory cytokines; and decrease the expression levels of key molecules in the Jak/Stat signaling pathway. These results indicate that DGBX can regulate the differentiation of T lymphocytes, resulting in immunosuppressive and hematogenic functions on AA mice. DGBX might be a good candidate for inclusion in a randomized study for AA with more data on the possible side effects and doses used in humans. Ultimately, it may be used for applications of traditional medicine against AA in modern complementary and alternative immunosuppressive therapeutics.

Research progress of Blimp-1 regulating T lymphocytes in pathogenesis of aplastic anemia

Aplastic anemia (AA) is considered as an immune-mediated bone marrow failure, but the specific pathogenesis is still under-researched. B lymphocyte-induced maturation protein (Blimp)-1 is a transcriptional repressor encoded by positive regulatory domain (PRDM)1 gene and is required to trigger the maturation of B lymphocytes into antibody secreting cells. Mounting evidence suggests that Blimp-1 plays a common role in the terminal differentiation of multiple T lymphocyte subsets, and functions significantly in the pathogenesis of many immune disorders. Here, we summarized the function of T lymphocytes in AA and the recent advances on the relationships of Blimp-1 and T lymphocytes, and we discuss the role of Blimp-1 regulating T lymphocytes in pathogenesis of AA. Key words: Blimp-1; T-lymphocytes; Anemia, aplastic; B lymphocyte-induced maturation protein-1

Recent advances in understanding clonal haematopoiesis in aplastic anaemia.

Acquired aplastic anaemia (AA) is an immune-mediated bone marrow failure disorder inextricably linked to clonal haematopoiesis. The majority of AA patients have somatic mutations and/or structural chromosomal abnormalities detected as early as at diagnosis. In contrast to other conditions linked to clonal haematopoiesis, the clonal signature of AA reflects its immune pathophysiology. The most common alterations are clonal expansions of cells lacking glycophosphotidylinositol-anchored proteins, loss of human leucocyte antigen alleles, and mutations in BCOR/BCORL1, ASXL1 and DNMT3A. Here, we present the current knowledge of clonal haematopoiesis in AA as it relates to aging, inherited bone marrow failure, and the grey-zone overlap of AA and myelodysplastic syndrome (MDS). We conclude by discussing the significance of clonal haematopoiesis both for improved diagnosis of AA, as well as for a more precise, personalized approach to prognostication of outcomes and therapy choices.

Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia.

Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a−/− mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a−/− mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a−/− lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.

Relatively Low Sensitivity of CD109(-) Hematopoietic Stem/Progenitor Cells (HSPCs) to TGF-β: A Possible Mechanism Responsible for the Preferential Commitment of Piga Mutant HSPCs in Immune-Mediated Bone Marrow Failure

[Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ.[Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells.[Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2).[Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. [Display omitted] [Display omitted] DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Update on the diagnosis and management of paroxysmal nocturnal hemoglobinuria.

Once suspected, the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is straightforward when flow cytometric analysis of the peripheral blood reveals a population of glycosyl phosphatidylinositol anchor protein-deficient cells. But PNH is clinically heterogeneous, with some patients having a disease process characterized by florid intravascular, complement-mediated hemolysis, whereas in others, bone marrow failure dominates the clinical picture with modest or even no evidence of hemolysis observed. The clinical heterogeneity is due to the close, though incompletely understood, relationship between PNH and immune-mediated bone marrow failure, and that PNH is an acquired, nonmalignant clonal disease of the hematopoietic stem cells. Bone marrow failure complicates management of PNH because compromised erythropoiesis contributes, to a greater or lesser degree, to the anemia; in addition, the extent to which the mutant stem cell clone expands in an individual patient determines the magnitude of the hemolytic component of the disease. An understanding of the unique pathobiology of PNH in relationship both to complement physiology and immune-mediated bone marrow failure provides the basis for a systematic approach to management.