Bone Marrow Cell Transplantation Research Articles

Functional loss of inactive rhomboid-like protein 2 mitigates obesity by suppressing pro-inflammatory macrophage activation-triggered adipose inflammation

ObjectiveChronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation. MethodsMice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet (SCD) or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone marrow cells were isolated for the further detection. ResultsAfter 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2 knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2 overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. We further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in the myeloid cells showed less severe adipose inflammation and insulin resistance than control groups. Conversely, transplantation of bone marrow cells from normal mice to iRhom2 KO mice unleashed severe systemic inflammation and metabolic dysfunction after HFD ingestion. ConclusionWe identified iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues may indirectly restrain inflammation and insulin resistance via blocking crosslinks between macrophages and adipocytes. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction.

Protective role of myelocytic nitric oxide synthases in pulmonary hypertension due to lung diseases and/or hypoxia

Nitric oxide (NO), formed from NO synthases (NOSs), plays a pathogenetic role in pulmonary hypertension (PH). However, the role of NO/NOSs in bone marrow (BM) cells in PH remains to be clarified. We addressed this point in clinical and basic studies. We demonstrated that, in 36 consecutive patients with idiopathic pulmonary fibrosis, pulmonary artery systolic pressure is inversely correlated with NOx levels in the bronchoalveolar lavage fluid, suggesting reduced pulmonary NO production in group III PH. We then revealed that transplantation of BM cells from mice lacking all NOSs aggravates hypoxia-induced PH in wild-type (WT) mice, and transplantation of BM cells from the WT mice ameliorates hypoxia-induced PH in the NOSs-/- mice, indicating a protective role of myelocytic NOSs in the pathogenesis of PH. Immune and inflammatory mechanisms appeared to be involved in the aggravation of hypoxia-induced PH caused by transplantation of BM cells from the NOSs-/- mice. Our findings provide novel insights into the cellular and molecular basis of group III PH.

Role of hematopoietic aging in cognitive decline

We recently demonstrated that transplantation of young bone marrow cells rescued hippocampal learning and memory in old recipient mice. Our study also implicated CCL11/eotaxin-1 in microglial reactivity during aging, which likely underlies neurotoxicity and synapse loss. CCL11 was reduced in the circulation of old recipients of young bone marrow, which likely contributed to their improved cognitive function. Thus, targeting CCL11 or its receptor may be an effective strategy for the treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

T-lymphopoiesis is Severely Compromised in Ubiquitin-Green Fluorescent Protein Transgenic Mice

Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.

Hemodynamic and clinical effectiveness of the bone marrow cell transplantation in treatment of critical ischemia of the lower extremities

Hemodynamic and clinical effectiveness of the bone marrow cell transplantation in treatment of critical ischemia of the lower extremities

Role of SOCS2 in the Regulation of Immune Response and Development of the Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Experimental Autoimmune Encephalomyelitis (EAE) is the most widely used animal model for the study of MS. The Suppressor of Cytokine Signaling (SOCS) 2 protein plays a critical role in regulating the immune responses. The role of SOCS2 during EAE has not been explored. EAE was induced in WT and SOCS2−/− mice using myelin oligodendrocyte glycoprotein (MOG35-55) peptide. Brain and spinal cord were examined during the peak (day 14) and recovery phase (day 28) of the disease. SOCS2 was upregulated in the brain of WT mice at the peak and recovery phase of EAE. The development of the acute phase was slower in onset in SOCS2−/− mice and was associated with reduced number of Th1 (CD3+CD4+IFN-γ+) cells in the spinal cord and brain. However, while in WT mice, maximal clinical EAE score was followed by a progressive recovery; the SOCS2−/− mice were unable to recover from locomotor impairment that occurred during the acute phase. There was a prolonged inflammatory response (increased Th1 and decreased Th2 and T regulatory cells) in the late phase of EAE in the CNS of SOCS2−/− mice. Transplantation of bone marrow cells from SOCS2−/− into irradiated WT mice resulted in higher lethality at the early phase of EAE. Altogether, these results suggest that SOCS2 plays a dual role in the immune response during EAE. It is necessary for damage during the acute phase damage but plays a beneficial role in the recovery stage of the disease.

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1‐like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6‐RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non‐haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4‐family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l‐deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

IRhom2 Ablation Mitigates Obesity by Suppressing Pro-Inflammatory Macrophage Activation-Triggered Adipose Inflammation

Chronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation. Mice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone-marrow cells were isolated for the further detection. After 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2-knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2-overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. Also, we further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in myeloid cells showed less severe adipose inflammation and insulin resistance than that of control groups. Conversely, transplantation of bone-marrow cells from normal mice to iRhom2 KO mice unleashed a severity of systemic inflammation and metabolic dysfunction after HFD ingestion. We identify iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues inhibited inflammation and insulin resistance. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction. Funding Statement: This work was jointly supported by (1) National Natural Science Foundation of China (NSFC Grant No.: 81703527); (2) Chongqing Research Program of Basic Research and Frontier Technology (Grant No.: cstc2017jcyjAX0356, cstc2018jcyjA3686, cstc2018jcyjAX0784, cstc2018jcyjA1472, cstc2018jcyjAX0811, cstc2018jcyjA3533 and KJZD-M201801601); (3) School-level Research Program of Chongqing University of Education (Grant No.: KY201710B and 17GZKP01); (4) Advanced Programs of Post-doctor of Chongqing (Grant No.: 2017LY39); (5) Science and Technology Research Program of Chongqing Education Commission of China (Grant No.: KJQN201901608, KJQN201901615, KJ1601402) and (6) Children's Research Institute of National Center for Schooling Development Programme and Chongqing University of Education (Grant No.: CSDP19FSO1108). Declaration of Interests: Minxuan Xu, Chenxu Ge, Yuting Qin, Deshuai Lou, Qiang Li, Jing Feng, Yekuan Wu , Linfeng Hu, Bochu Wang & Jun Tan declare that they have no conflict of interest. Ethics Approval Statement: All research procedures associating with mice were approved by the Institutional Animal Care and Use Committee in Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health in 1996. The protocols used in this study were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People's Republic of China.

Successful Bone Marrow Transplantation Using an Immunomyelosuppressive Conditioning in Patients with Severe Congenital Neutropenia: The Results of a Single-Institute

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by recurrent bacterial infections from early infancy due to severe chronic neutropenia. Majority of SCN patients have benefitted by the treatment with granulocyte colony-stimulating factor (G-CSF). However, patients on long-term G-CSF therapy have a relative risk of developing myelodysplastic syndrome/acute myeloid leukemia. The only curable treatment for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning regimens have been applied to the treatment for SCN patients prior to malignant transformation. However, the optimal conditioning of HSCT for SCN patients has not been established. In this study, we conducted bone marrow cell transplantations (BMT) in 16 patients with SCN using an immunomyelosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. A total of 17 BMT procedures were performed in 16 patients with SCN from 2008 to 2019. Five of 16 patients had experienced the engraftment failure of initial HSCT and 4 of them were referred to our hospital for re-transplantation. Fifteen of 16 patients had a heterozygous mutation in the ELANE gene. Bone marrow cells (BM) were obtained from 6 HLA-matched related, 3 HLA-matched unrelated, and 8 HLA-mismatched unrelated (7/8 antigens) donors, respectively. Conditioning regimen consisted of fludarabine, cyclophosphamide, melphalan, total body irradiation (3.6 Gy) with or without antithymocyte globulin. Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 24 days in all patients. Two patients developed graft failure on day 40 and day 90, respectively, after the temporal engraftment. However, both patients were rescued by second BMT from different HLA-matched unrelated donors receiving the same conditioning regimen. Four patients who received BMT from HLA-matched related donors developed stable mixed chimerism without neutropenia in peripheral blood for 3 to 10 years. Although one patient who received donor lymphocyte infusion due to mixed chimerism developed grade II acute GVHD and limited chronic GVHD, the others did not develop severe GVHD. All patients are alive for 6 months to 11 years after BMT with no signs of severe infections or transplantation-related morbidity. Similar conditioning regimen has been applied to BMT for 35 patients with chronic granulomatous disease (CGD) in our hospital. In that study 4 male adulthood patients with CGD already fathered each child by their wives through spontaneous pregnancy, implying the successful preservation of patients' fertility. Collectively, our results demonstrate that BMT with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. The excellent results in our cohort suggest that indications for proceeding to HSCT could be extended to patients without malignant transformation.The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

The Role of Macrophages in Bone Marrow Injury and Hematopoietic Reconstitution

Introduction Successful homing, engrafment and effective hematopoietic recovery after hematopoietic stem cell transplantation (HSCT) are strictly regulated by various hematopoietic microenvironment cells. Increasing evidence shows that macrophages (MФs), one of the most important component niche cells are crucial for the haematopoietic regulation. MФs depletion can enhance hematopoietic stem cell mobilization. Our previous study showed that MФs ameliorate bone marrow inflammatory injury and promote hematopoiesis in mice after allo-HSCT, but its role in syngeneic HSCT and acute bone marrow injury is still unknown. Our aim is to explore the role of macrophages in acute bone marrow injury and hematopoietic reconstitution after isogenic hematopoietic stem cell transplantation and sublethal dose irradiation in vivo. Methods BALB/c male mice at 8-10 weeks were irradiated with 60 Co 7.5 Gy and 3.0 Gy, respectively, and then isogenic hematopoietic stem cell transplantation model and sublethal-dose bone marrow injury model were constructed. The transplantation model mice were randomly divided into total body irradiation group (TBI group), bone marrow cell transplantation group (BMT group), bone marrow cell transplantation + Clodronate Liposomes injection group (BMT+Clod-Lip group), bone marrow cell transplantation + PBS Liposomes injection group ( BMT + PBS-Lip group), and normal control group (Normal group). The sublethal-dose experimental mice were randomly divided into the total body irradiation group (TBI group), the whole body irradiation + Clodronate Liposomes injection group (TBI+Clod-Lip group), the whole body irradiation + PBS Liposomes injection group (TBI+PBS-Lip group), and normal control group (Normal group). Mice in Clod-Lip group were injected with Clodronate Liposomes for several specific times to deplete macrophages until the specimens were obtained. Mice in PBS-Lip group were injected PBS Liposomes as controls.Then, the living conditions and body weight changes of the mice were observed and the survival rates of mice in different experimental groups were recorded. Peripheral blood and bone marrow in each group were collected at the corresponding detection time, blood routine analyzer was used to detect blood routine changes, HE staining was used to observe bone marrow damage, and flow cytometry was used to analyze changes in macrophages, hematopoietic stem/progenitor cells and their subgroups such as myeloid cells, megakaryocytes, and nucleated red blood cells in bone marrow. Results Depletion of bone marrow macrophages could reduce the survival rate of hematopoietic stem cell transplantation mice. The pathological results of bone marrow showed that bone marrow injury were heaviest on the 7th day in all three transplantation groups, and then gradually alleviated. The recovery of the BMT+Clod-Lip group was inferior to that of the BMT+PBS-Lip group and the BMT group at the corresponding time point. Depletion of macrophages increased the percentage of myeloid cells in the bone marrow and the number of white blood cells in the peripheral blood, reduced the total number of bone marrow cells, the proportion of hematopoietic stem cells and megakaryocytes in the bone marrow, and delayed recovery of red blood cells and platelets in peripheral blood; Depletion of bone marrow macrophages could also reduce survival rate of sublethal dose irradiation mice, delayed the repair of pathological damage of bone marrow, and increase the proportion of progenitor cells, CMP, GMP, myeloid cells and the number of peripheral white blood cells ,increase the proportion of hematopoietic stem cell apoptosis, reduce the total number of bone marrow cells, the proportion of hematopoietic stem cells, MEP, megakaryocytes and nucleated red blood cells in the bone marrow, delayed peripheral blood recovery of red blood cells and platelets. Conclusion In the isogenic hematopoietic stem cell transplantation model and the sublethal dose irradiation mouse model, the removal of mouse bone marrow macrophages could affect the survival rate of transplanted mice, aggravate the pathological damage of bone marrow, increase the number of GMP and white blood cells, and reduce the total number of bone marrow cells, the number of hematopoietic stem cells and MEP cells. Macrophage depletion was not conducive to the recovery of peripheral blood red blood cells and platelets. Disclosures No relevant conflicts of interest to declare.

Hispanic Race Is Associated with Increased Risk of Severe Sepsis in 1913 Hospitalizations of Children with AML and Infection

Hispanic Race Is Associated with Increased Risk of Severe Sepsis in 1913 Hospitalizations of Children with AML and Infection

TGF-β Signaling Contributes to Myelofibrosis and Clonal Dominance of Myeloproliferative Neoplasms

TGF-β expression is elevated in most cases of myeloproliferative neoplasms (MPNs). However, the contribution of TGF-β to disease pathogenesis is not well understood. Prior studies have shown that TGF-β regulates hematopoietic stem cell (HSC) quiescence. There also is published evidence that increased TGF-β may contribute to myelofibrosis. However, this is controversial, as recent studies have implicated other inflammatory cytokines in the development of myelofibrosis. Here, we test two specific hypotheses. First, we hypothesize that increased TGF-β signaling in mesenchymal stromal cells (MSCs) is required for the development of myelofibrosis. Second, we hypothesize that Jak2 mutated HSCs are resistant to the growth suppressive effect of TGF-β, resulting in a competitive advantage that contributes to their clonal expansion in MPN and clonal hematopoiesis. To test the first hypothesis, we abrogated TGF-β signaling in mesenchymal stem/progenitor cells by deleting Tgfbr2 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow. Osx-Cre was induced at birth (by removal of doxycycline), since we recently reported that the post-natal loss of TGF-β signaling in mesenchymal stromal cells has no discernible effect on basal hematopoiesis or the stem cell niche. We transplanted bone marrow cells from UBC-CreERT2; Jak2V617F mice or c-kit+ cells transduced with MPLW515L retrovirus into irradiated wildtype or Osx-Cre; Tgfbr2f/fmice. For the Jak2V617F model, mice were treated with tamoxifen 6 weeks post transplantation to induce mutant Jak2 expression. Of note, elevated Tgfb1 was present in both MPN models. MPLW515L induced a rapidly fatal MPN with reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype was observed in Osx-Cre; Tgfbr2f/f recipients. However, myelofibrosis, as measured by reticulin staining and Collagen III (Col3a1) mRNA expression, was reduced, but not completely abrogated in Osx-Cre; Tgfbr2f/fmice. Likewise, in the Jak2V617F MPN model, the hematopoietic phenotype was similar in wildtype and Osx-Cre; Tgfbr2f/fmice. Although overt myelofibrosis was not observed in this MPN model, increased RNA and protein expression of Collagen III were detected in the bone marrow. Although still above baseline, Col3a1 expression was significantly reduced in Osx-Cre; Tgfbr2f/frecipient mice. To examine the role of canonical TGF-β signaling in the induction of myelofibrosis, we cultured bone marrow derived MSCs from wildtype and Osx-Cre; Smad4f/f mice, in which canonical TGF-β signaling is abrogated. Treatment of wildtype MSC cultures with TGF-β1 induced the expression of fibrosis associated genes, including Col1a1 and Acta2, and down-regulated expression of key niche factors, including Cxcl12, Scf, and Bglap. Surprisingly, TGF-β1-induced expression of Col1a1 was intact in Smad4-deleted MSC cultures. Conversely, treatment of wildtype MSC cultures with the JNK inhibitor SP600125 abrogated TGF-β1-induced expression of Col1a1. Collectively, these data suggest that TGF-β signaling in MSCs contributes to the development of myelofibrosis in MPN through activation of the non-canonical JNK pathway. To test the second hypothesis, we set up a competitive transplantation assays using wildtype and Jak2V617F hematopoietic cells in which Tgfbr2 had been deleted to abrogate TGF-β signaling in HSCs. Specifically, we transplanted UBC-CreERT2; Jak2V617F; Tgfbr2f/f and UBC-CreERT2; Tgfbr2f/f bone marrow cells at 1:5 ratio into lethally irradiated wild type recipients. For control, we transplanted a 1:5 ratio of UBC-CreERT2; Jak2V617F to wild type cells. Mice were treated with tamoxifen to activate Cre expression 6 weeks post transplantation. Complete blood counts and donor chimerism were measured 6, 10, 14 and 18 weeks after tamoxifen. As expected, in the control group, donor chimerism with Jak2 mutated cells increased over time in myeloid lineages. In contrast, in the Tgfbr2-deleted group, no expansion of Jak2 mutated cells was observed. These data support our hypothesis that Jak2 mutated HSCs are resistant to the growth suppressive effect of TGF-β, providing a selective advantage that contributes to their clonal expansion in MPN and possibly clonal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Combined Heterozygous Loss of Tet2 and Dnmt3a Along with Expression of Flt3ITD/WT results in Acute Myeloid Leukemia Which Responds to a Combination of FLT3 Inhibitor, APE1 Inhibitor and Decitabine

Heterozygous mutations in FLT3ITD, TET2 and DNMT3A are associated with human neoplasms. Whole exome sequencing of patients demonstrate co-occurrence of several of these mutations in myeloid malignancies; however it is unclear if and how these mutations cooperate and how does this cooperation manifest in disease. We assessed the consequence of co-existence of TET2, DNMT3A and FLT3ITD/WT mutations on AML development and overall survival. Mice were bred to obtain eight distinct genotypes WT, Tet2+/-, Flt3ITD/WT, Dnmt3aFlox/- MxCre, double het Tet2+/-; Dnmt3aFlox/- MxCre (TD), double het Tet2+/-; Flt3ITD/WT (TF), double het Flt3ITD/WT: Dnmt3aFlox/- MxCre (FD) and triple het Tet2+/-; Flt3ITD/WT; Dnmt3aFlox/+ MxCre (TFD) mice. These mice were treated with poly:IC and monitored for survival. Of the 8 genotypes examined, only TFD mice succumbed by 150 days. Peripheral blood (PB) counts, BM cellularity and splenomegaly were significantly higher in TFD mice compared to FD and TF mice. The absolute number of LSK cells in the BM was highest in TFD mice, although TF and FD mice also showed an increase relative to controls. A similar increase in the frequency of GMPs was noted in both double and triple heterozygous mice relative to controls as well as single heterozygous groups. An increase in Gr-1/Mac-1 cells was observed in TF and FD mice as well as in TFD mice relative to controls. To assess if the observed AML phenotype in TFD mice is transplantable, we transplanted BM cells from the 8 genotypes into lethally irradiated hosts. Recipients with TFD BM succumbed to rapid AML development and died within 45 days of transplantation. In contrast, none of the other recipients died during the entire monitoring period. A significant increase in PB neutrophil and monocyte counts with a significant reduction in red blood cells, and platelets counts was noted in TFD mice compared to other groups. Furthermore, a significant increase in BM cellularity, Lin- Kit+ progenitors, frequency and absolute number of LSK cells was also noted in mice with TF and FD BM but most significantly in recipients that received TFD BM. Given that the disease manifestation was qualitatively similar between FD, TF and TFD mice, although the severity greatest in TFD mice, we asked if these differences were due to quantitative or qualitative differences in gene expression between the various groups. RNA-seq analysis revealed distinct differences between WT and FD, TF and TFD BM cells. We observed 2328, 2168 and 1787 up-regulated genes and 1861, 1770 and 1430 downregulated genes in FD, TF and TFD vs. WT, respectively. The variations among FD, TF and TFD groups was smaller compared to the differences between WT and FD, TF and TFD groups, in which TF and TFD were more similar to each other compared to DF. Most of the cytokines regulating the differentiation of myeloid cells were downregulated in FD, TF and TFD, suggesting that all the mutant groups lost their differentiation ability. Of note, cytokines specific to stem cells and those involved in the differentiation of GMPs were all upregulated. Given the RNA-seq profile, we assessed the impact of using a combination of drugs that target Flt3ITD, inflammation as well as methylation status of cells in TFD mice. A cohort of mice was treated with a combination of AC220 (10 mg/kg, orally; Flt3 inhibitor), E3330 (20 mg/kg, i.p.; APE1 inhibitor; regulates the redox function of multiple transcription factors including NFkB and Stat3) and 2-dexoy-aza-cytidine (0.5 mg/kg, i.p). A significant correction in all PB, spleen and BM parameters was observed including in the frequency of Lin- Kit+ and LSK cells, HSCs, HPC1 and peripheral Gr1/Mac1 double positive cells in TFD mice. Collectively, these results demonstrate that a combination of three drugs targeting three different aspects of disease manifestation in TFD mice can significantly impact the leukemia progression both at the level of very primitive stem and progenitor cells as well as more mature myeloid and lymphoid cells. Disclosures No relevant conflicts of interest to declare.

Fli-1 Transcriptionally Integrates Microenvironmental Cues Sensing By Self-Renewing Hematopoietic Stem and Progenitor Cells

During adulthood and embryogenesis, fate decisions of hematopoietic stem and progenitor cells (HSPCs), such as specification, self-renewal, and differentiation are tightly regulated by their neighboring niche cells. Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPCs. Yet, currently the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown. Friend Leukemia integration-1 (Fli-1), is an ETS transcription factor expressed by vascular beds and hematopoietic lineages. Fli-1 belongs to the "heptad factors" which are hypothesized to specify and sustain a hematopoietic cell fate. While Fli-1 overexpression is linked to leukemia, the functional role Fli-1 plays in HSPC specification and maintenance remains undefined. We show that inducible deletion of Fli-1 using a Rosa-CreERT2 transgenic adult mice (Fli-1ROSAΔ), results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1ROSAΔ bone marrow (BM) cells into WT recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion, resulted with the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre- or post-transplant), we observed defective ability of Fli-1ROSAΔ HSPCs to lodge, engraft, and to sustain hematopoiesis post repopulation. Fli-1 deficient HSPCs exhibited reduced quiescent cell cycling status, a hallmark of stemness, and displayed enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognized cell-autonomous HSPC functions. To determine whether Fli-1 modulates HSPC specification, Fli-1 was conditionally deleted using a developmental VE-cadherin (CDH5)-Cre transgenic model (Fli-1CDH5Δ). This resulted with premature mortality of Fli-1CDH5Δ embryos, accompanied with a hemorrhagic phenotype. Reduced numbers of hematopoietic cells were still detected in the AGM of e10.5 Fli-1CDH5Δ embryos. Conditional Fli-1 deletion using a developmental hematopoietic Vav-1 Cre transgenic model (Fli-1Vav-1Δ) resulted again with premature mortality. Reduced presence of embryonic Fli-1Vav-1Δ liver HSPCs was observed at e12.5. We also applied two in vitro co-culture systems, to study Fli-1 in endothelial to hematopoietic transition (EHT). First, isolated hemogenic endothelial cells (HEC) from WT and Fli-1ROSAΔ embryos were co-cultured with AGM-derived vascular niche. HECs isolated from Fli-1ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, we have applied an endothelial to hematopoietic reprogramming system in which isolated lung ECs are virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells. Both WT and Fli-1ROSAΔ ECs were able to acquire a hemogenic like state resulting with a final capacity to convert into hematopoietic cells. Again, Fli-1ROSAΔ cells displayed lesser numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Indeed, reduced expansion capacity was observed both for mature CD45+ and for HSPC derived from Fli-1CDH5Δ AGM region. Adult Fli-1ROSAΔ HSPCs exhibited the same niche-dependent expansion defect. Induction of Fli-1 deletion in vitro in adult HSPCs revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1ROSAΔ HSPCs in the presence of a vascular niche. Hence, Fli-1 is essential for HSPC expansion rather than hematopoietic specification. Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1ROSAΔ HSPCs, revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Unexpectedly, H3K27Ac analysis, a marker for transcriptional priming, revealed increased global acetylation of Fli-1ROSAΔ HSPCs' chromatin. Therefor, Fli-1 may not only perform as transcription activator, but foremostly as a genomic suppressor via modulation of histone acetylation status. Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal, may promote an improved expansion protocol of human HSPC pre-transplantation, and provide additional insights for microenvironmental sensing by Fli-1-dependent leukemic cells. Disclosures No relevant conflicts of interest to declare.

Role of B7H3/IL-33 Signaling in Pulmonary Fibrosis-induced Profibrogenic Alterations in Bone Marrow.

Rationale: The impact of lung insult on the bone marrow (BM) and subsequent disease is unknown.Objectives: To study alterations in the BM in response to lung injury/fibrosis and examine their impact on subsequent lung insult.Methods: BM cells from control or bleomycin-treated donor mice were transplanted into naive mice, which were subsequently evaluated for bleomycin-induced pulmonary fibrosis. In addition, the effect of prior bleomycin treatment on subsequent fibrosis was examined in wild-type and B7H3-knockout mice. Samples from patients with idiopathic pulmonary fibrosis were analyzed for potential clinical relevance of the findings.Measurements and Main Results: Recipient mice transplanted with BM from bleomycin-pretreated donors showed significant exacerbation of subsequent fibrosis with increased B7H3+ cell numbers and a T-helper cell type 2-skewed phenotype. Pretreatment with a minimally fibrogenic/nonfibrogenic dose of bleomycin also caused exacerbation, but not in B7H3-deficient mice. Exacerbation was not observed if the mice received naive BM cell transplant after the initial bleomycin pretreatment. Soluble B7H3 stimulated BM Ly6Chi monocytic cell expansion in vitro and caused similar expansion in the lung in vivo. Notably, soluble B7H3 was elevated in plasma of patients with idiopathic pulmonary fibrosis and in BAL fluid in those with acute exacerbation. Finally, ST2 deficiency diminished the bleomycin-induced B7H3 and IL-13 upregulation, suggesting a role for type 2 innate lymphoid cells.Conclusions: Pulmonary fibrosis caused significant alterations in BM with expansion and activation of monocytic cells, which enhanced fibrosis when transplanted to naive recipients with potential mediation by a novel role for B7H3 in the pathophysiology of pulmonary fibrosis in both mice and humans.

Abstract 117: Transplantation of Bone Marrow Cells from miR-150-KO Mice Improves Immune Function and Attenuates Arterial Stiffness and Hypertension in Senescence-Accelerated Mice

Background and Objective: Aging is associated with increased prevalence of arterial stiffness and hypertension. The senescence-accelerated mouse (SAMP1) demonstrates early and spontaneous aging phenotypes around 6-8 months of age. In contrast, microRNA-150 knockout (miR-150 KO) slows down the aging process and extends lifespan in mice by 20-30%. The object of this study is to investigate whether transplantation of bone marrow cells (BMCs) from miR-150 KO mice attenuates arterial stiffening and hypertension in SAMP1 mice. Method and Results: Pulse wave velocity (PWV) and blood pressure (BP) levels were increased significantly in the SAMP1 mice compared with the age-matched control (AKR/J) mice (10 months), indicating that SAMP1 mice developed arterial stiffening and hypertension. Interestingly, transplantation of bone marrow cells (BMCs) from miR-150-KO mice decreased PWV and BP in SAMP1 mice to nearly control levels within 3 months. BMC transplantation decreased expression of collagen1, TGFβ1, Scleraxis (collagen gene transcription factor), MMP-2 and MMP-9 but increased expression of elastin in aortas of SAMP1 mice, indicating improved arterial remodeling. BMC transplantation from miR-150-KO mice partially rescued the downregulation of B cells lymphocytes and increased the serum level of IgG and IgM, abolished infiltration of T cells and macrophages, and decreased inflammatory cytokine expression in aortas in SAMP1 mice. In the cell culture study, IgG deficiency increased inflammatory factor cytokine expression in endothelial cells, which can be abolished by addition of IgG protein. Conclusion: BMC transplantation from miR-150-KO mice reversed arterial stiffness and hypertension in SAMP1 mice likely via modulation of the immune function. This study demonstrates, for the first time, that BMC transplantation may be an effective therapeutic strategy for aging-related arterial stiffness and hypertension.

Abstract 301: Bone Marrow Niche-Mediates Cardiac Repair is Associated With Adiponectin in Retnla Deficiency

Background: Resistin like α (Retnla) is known to be upregulated in anti-inflammatory macrophages and to modulate lipid profile in atherosclerosis. However, the role of Retnla is unknown in the cardiac pathology. Methods: Myocardial infarction (MI) was induced in wild type, Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiomyocytes, cardiac fibroblasts, macrophages and bone marrow cells were treated with Retnla protein for mechanistic studies. For an adoptive transfer, bone marrow cells were isolated from KO mice to transplant to the infarcted heart. Results: TUNEL staining showed the number of apoptotic cardiomyocytes was higher in the Retnla KO mice than in the TG mice 1 day after MI. Cardiac function and angiogenesis were significantly better in the Retnla KO group, while were worsen in the TG group. Retnla is an anti-inflammatory marker in macrophages, but the expressions of inflammation-related genes were not different between both groups. Interestingly, however, the numbers of macrophages isolated from bone marrow, peritoneum, and cardiac tissue of Retnla KO mice were higher than in the wild type mice. Moreover, phospho-histone H3-positive proliferating cells in the bone marrow were much more abundant in the Retnla KO mice than in the wild type mice. Adiponectin was highly expressed in bone marrow of KO mice, and we transplanted bone marrow cells from KO mice to recipient wild type mice. Two weeks after MI, recipient group transplanted with bone marrow cells from KO mice showed better angiogenesis, reserved cardiac function, and more anti-inflammatory macrophages. In Retnla-treated cardiomyocytes, bax, phopho-p38, and p21 were notably upregulated, while bcl2 and adiponectin were significantly reduced. To examine whether Retnla acted negatively on adiponectin, we administrated a synthetic adiponectin receptor agonist AdipoRon to Retnla-treated cardiomyocytes. AdipoRon blunted the effect of Retnla on cardiomyocytes. These data showed better cardiac recovery was associated with bone marrow-derived adiponectin in the Retnla KO group. Conclusion: Our study suggested that substantial cardiac recovery was strongly associated with adiponectin derived from highly proliferating bone marrow cells in Retnla KO mice.

Daily bone marrow cell transplantations for the management of fast neurodegenerative processes.

Cell therapy has been proven to be a promising treatment for fighting neurodegenerative diseases. As neuronal replacement presents undeniable complications, the neuroprotection of live neurons arises as the most suitable therapeutic approach. Accordingly, the earlier the diagnosis and treatment, the better the prognosis. However, these diseases are commonly diagnosed when symptoms have already progressed towards an irreversible degenerative stage. This problem is especially dramatic when neurodegeneration is aggressive and rapidly progresses. One of the most interesting approaches for neuroprotection is the fusion between healthy bone marrow-derived cells and neurons, as the former can provide the latter with regular/protective genes without harming brain parenchyma. So far, this phenomenon has only been identified in Purkinje cells, whose death is the cause of different diseases like cerebellar ataxias. Here we have employed a model of aggressive cerebellar neurodegeneration, the Purkinje Cell Degeneration mouse, to optimize a cell therapy based on bone marrow-derived cell and cell fusion. Our findings show that the substitution of bone marrow in diseased animals by healthy bone marrow, even prior to the onset of neurodegeneration, is not fast enough to stop neuronal loss in time. Conversely, avoiding bone marrow replacement and ensuring a regular supply of healthy cells through continuous, daily transplants, the neurodegenerative milieu of PCD is enough to attract those transplanted elements. Furthermore, in the most affected cerebellar regions, more than a half of surviving neurons undergo a process of cell fusion. Therefore, this method deserves consideration as a means to impede neuronal cell death.

Innate Lymphoid Cells in the Induction of Obesity

Complex interactions between immune cells are an important component in the induction of obesity. Here, we show that Il2rg-/-Rag2-/- mice lacking all lymphocytes are resistant to diet-induced obesity. Transplantation of bone marrow cells from Rag2-/- mice, which lack only acquired immune cells, into Il2rg-/-Rag2-/- mice abolishes this resistance, indicating a role for innate lymphoid cells (ILCs) in this process. Mice lacking ILC2 or ILC3 cells, but not natural killer cells, are resistant to obesity. Adoptive transfer of naive ILC2s isolated from the small intestine (SI), but not ILC2s from white adipose tissue (WAT), restores the induction of diet-induced obesity in Il2rg-/-Rag2-/- mice. Analysis of transcriptional differences reveals that SI-ILC2s express higher levels of IL-2 than do WAT-ILC2s and that blockade of IL-2 signaling impairs weight gain and reduces the populations of ILC2s and ILC3s in the SI, suggesting a role for the IL-2/ILC2/3 axis in the induction of obesity.

Expression of Vsig4 attenuates macrophage-mediated hepatic inflammation and fibrosis in high fat diet (HFD)-induced mice

The innate immune response contributes to hepatic steatosis and nonalcoholic fatty liver disease (NAFLD). However, the pathogenic mechanism of NAFLD is still poorly understood. The costimulatory molecule V-set and immunoglobulin domain-containing protein-4 (Vsig4), which is exclusively expressed on macrophages, shows significant role in regulating macrophage-mediated inflammation. Here, we attempted to explore if Vsig4 expression was involved in high fat diet (HFD)-induced NAFLD. The results indicated that Vsig4 expression was markedly down-regulated in fatty livers of NAFLD patients and obese mice. Vsig4 knockout accelerated HFD-induced metabolic dysfunction. In addition, the loss of Vsig4 significantly promoted insulin resistance and lipid deposition in liver samples of HFD-challenged mice. Furthermore, HFD-induced inflammation was apparently accelerated in Vsig4 knockout mice by further activating nuclear factor-κB (NF-κB) signaling pathway. Also, Vsig4 deficient mice exhibited greater collagen accumulation in hepatic samples in HFD-challenged mice compared to the WT mice, which was through promoting transforming growth factor-β1 (TGFβ1) signaling. Importantly, we found that lipopolysaccharide (LPS)- or TGFβ1-stimulated inflammation and fibrosis in primary hepatocytes and hepatic stellate cells, respectively, were markedly exacerbated by co-culture with condition medium from bone marrow-derived macrophages (BMDMs) with Vsig4 deficiency. Finally, transplantation of bone marrow cells from control mice to Vsig4-knockout mice restored the severity of steatosis, inflammation and fibrosis after HFD feeding. Therefore, loss of Vsig4 accelerated the severity of lipid deposition, fibrosis and the inflammatory response. Vsig4 could be a therapeutic target for NAFLD treatment.