Bone Marrow Transplantation In Mice Research Articles

Abstract LT03: C/EBPα Acts as an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

Abstract The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα’s separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα’s DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα’s RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations. It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together though, we demonstrate that C/EBPα is also an RNA binding protein with unique functions distinct from its DNA binding activity that is essential for monocyte maturation, differentiation and mobilization. Citation Format: Mahmoud A. Bassal, Yanzhou Zhang, Yanjing V. Liu, Virginia Camacho, Simone Ummarino, Robert S. Welner, Daniel G Tenen. C/EBPα Acts as an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT03.

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Bahcc1 is critical for the aberrant epigenetic program in a mouse model of MLL-ENL–mediated leukemia

AbstractIn leukemogenesis, genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) drives individual context-dependent programs of malignant transformation. In light of the various differentiation stages of HSPCs based on a recently revised definition using CD150/CD48, our analyses showed that a subpopulation of long-term repopulating HSCs was most susceptible to MLL-ENL–mediated transformation. An analysis of the molecular mechanism identified Bromo-adjacent homology domain and coiled-coil containing 1 (Bahcc1), which encodes a reader molecule of trimethylated histone H3 lysine 27 (H3K27me3), as a candidate gene involved in distinct susceptibility to leukemic transformation. Interestingly, Bahcc1 was previously reported to be highly expressed in acute myeloid leukemia (AML) with an unfavorable prognosis, including some cases of MLL-rearranged AML. We found that MLL-ENL upregulated Bahcc1 through binding to its promoter, and that Bahcc1 was involved in MLL-ENL–mediated immortalization at least partly through repression of H3K27me3-marked Cdkn1c. Analyses using bone marrow transplantation in mice showed that depletion of Bahcc1 suppressed the leukemogenic activity of MLL-ENL. In a public database, high BAHCC1 expression was found to be associated with a poor prognosis in pediatric AML, in which BAHCC1 expression was significantly lower in MLL-AF9-AML than in other MLL-fusion-AML. These findings shed light on the distinct immortalization potential of HSPCs and suggest a novel MLL-fusion-Bahcc1 axis, which may lead to development of molecular targeted therapy against MLL-fusion–mediated leukemia.

CD155 is essential for skeletal muscle regeneration by regulating satellite cell proliferation and differentiation.

CD155, a member of the immunoglobulin superfamily, is closely related to cell proliferation, adhesion, and migration. CD155 is overexpressed on the surface of cancer cells to promote cell proliferation and is upregulated in damaged tissues as a stress-induced molecule. The process of skeletal muscle regeneration after injury is complex and involves injurious stimulation and subsequent satellite cell proliferation. However, the role of CD155 in this process remains unelucidated. This study aimed to explore the role of CD155 in injured skeletal muscle regeneration and to clarify its effect on satellite cell proliferation and differentiation. Here, quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence results indicated that CD155 expression in satellite cells increased after skeletal muscle injury. CD155 knockout in mice impaired the regeneration of skeletal muscle. A bone marrow transplantation mouse model was constructed and revealed that CD155 on skeletal muscle tissues, not immune cells, affected muscle regeneration. Invitro, CD155 knockdown in myoblasts inhibited their proliferation and differentiation. The transcriptomic analysis also indicated that CD155 absence can impair the biological proliferation and differentiation process of myoblasts. Our research demonstrates that CD155 directly promotes injured muscle regeneration by regulating satellite cell proliferation and differentiation, which may be a potential therapeutic molecule for skeletal muscle injury.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

BackgroundG-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism.MethodsThe expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl4) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription‑polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.ResultsWe found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl4-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca2+-JNK/NF-κB pathways, while promoted the expression of TGF-β through the Gαq-Ca2+-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo.ConclusionsGPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

Characterization of the long-term effects of lethal total body irradiation followed by bone marrow transplantation on the brain of C57BL/6 mice

Purpose Total body irradiation (TBI) followed by bone marrow transplantation (BMT) is used in pre-clinical research to generate mouse chimeras that allow to study the function of a protein specifically on immune cells. Adverse consequences of irradiation on the juvenile body and brain are well described and include general fatigue, neuroinflammation, neurodegeneration and cognitive impairment. Yet, the long-term consequences of TBI/BMT performed on healthy adult mice have been poorly investigated. Material and Methods We developed a robust protocol to achieve near complete bone marrow replacement in mice using 2x550cGy TBI and evaluated the impact of the procedure on their general health, mood disturbances, memory, brain atrophy, neurogenesis, neuroinflammation and blood-brain barrier (BBB) permeability 2 and/or 16 months post-BMT. Results We found a persistent decrease in weight along with long-term impact on locomotion after TBI and BMT. Although the TBI/BMT procedure did not lead to anxiety- or depressive-like behavior 2- or 16-months post-BMT, long-term spatial memory of the irradiated mice was impaired. We also observed radiation-induced impaired neurogenesis and cortical microglia activation 2 months post-BMT. Moreover, higher levels of hippocampal IgG in aged BMT mice suggest an enhanced age-related increase in BBB permeability that could potentially contribute to the observed memory deficit. Conclusions Overall health of the mice did not seem to be majorly impacted by TBI followed by BMT during adulthood. Yet, TBI-induced alterations in the brain and behavior could lead to erroneous conclusions on the function of a protein on immune cells when comparing mouse chimeras with different genetic backgrounds that might display altered susceptibility to radiation-induced damage. Ultimately, the BMT model we here present could also be used to study the related long-term consequences of TBI and BMT seen in patients.

C/EBPα Acts As an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

Heterogeneous Intercellular Communication of Hematopoietic Stem Cells in the Mouse Bone Marrow

Introduction.Hematopoietic stem cells (HSCs) are responsible for maintaining the homeostasis of the blood and immune systems. This process must rapidly adapt to bleeding, infection, injury, and disease. HSCs reside in specialized microenvironments in the bone marrow, called “niche”. Several bone marrow cell types have been proposed as the key components of the niche. It is well established that HSCs rely on molecular signals from the niche cells to survive, migrate, proliferate, and differentiate. Recent studies suggest that HSCs differentiate heterogeneously. Additionally, the bone marrow is a heterogeneous environment consisting of more than thirty cell types. This study will test the hypothesis that individual HSCs are engaged in heterogeneous intercellular signaling in the bone marrow. Materials and Methods.We utilized CellChat (Jin et al., 2021), a database for ligand-receptor pairs and associated pathways, to predict cell-cell signaling at the single cell level. We used single cell RNA sequencing data from mouse hematopoietic stem and progenitor cells, blood and immune cells, and non-hematopoietic cells from the bone marrow to determine putative cell-cell interactions. The predicted signaling interactions were then adjusted based on the cell-cell spatial interaction revealed by PhenoCycler measurement (Akoya Biosciences). Mouse bone marrow transplantation experiments were performed to test the functional significance of the identified signaling pathway interactions. Results.We found that individual HSCs send and receive different levels of molecular signals across various signaling pathways. In addition to the expected interactions with the known HSC niche cells, we also found new interactions between HSCs and their progeny cells including myeloid progenitors and immune cells. These cell-cell signaling interactions are consistent with the spatial interaction frequencies of the corresponding cell types as revealed by our imaging data from the PhenoCycler analysis. Further, we found that some signaling pathways are positively or negatively correlated across individual HSCs, indicating complex intercellular signaling networks. The most significant negative correlation was found between HSCs sending MHC-I signals and sending PARs signals (Figure). Moreover, this MHC-I/PARs signaling correlation network also involves signaling from the HSC niche, including those that are known to play roles in HSC homing such as ESAM and VCAM. Therefore, we tested the engraftment efficiency of HSCs that receive PARs and those that do not using the HSC mouse transplantation model. We found that PAR1 + HSCs engraft much less efficiently. Finally, we present the HSC Interaction Data Explorer (HIDE), a database that comprehensively depicts the interactions between HSCs and other bone marrow cells including the involved signaling pathways, ligands and receptors of the pathways, and the pathway correlations. Conclusion.Our study uncovered novel interactions of HSCs with myeloid progenitor cells and immune cells, which were validated by spatial interaction data. These interactions indicate feedback signaling in regulating blood cell regeneration. Our findings further revealed signaling pathway correlation networks across individual HSCs, and we showed their functional significance using transplantation experiments. Finally, we present HIDE as a resource that informs the interactions between HSCs and various bone marrow cells, the relevant signaling pathways, and the pathway correlations. Figure Legend. HSC signaling correlation networks. Each node shows a signaling pathway and direction (e.g., HSCs sending MHC-I). The edges represent the spearman correlation score.

Activation of the inflammasome drives peritoneal deterioration in a mouse model of peritoneal fibrosis.

During peritoneal dialysis (PD), the peritoneum is exposed to a bioincompatible dialysate, deteriorating the tissue and limiting the long-term effectiveness of PD. Peritoneal fibrosis is triggered by chronic inflammation induced by a variety of stimuli, including peritonitis. Exposure to PD fluid alters peritoneal macrophages phenotype. Inflammasome activation triggers chronic inflammation. First, it was determined whether inflammasome activation causes peritoneal deterioration. In the in vivo experiments, the increased expression of the inflammasome components, caspase-1 activity, and concomitant overproduction of IL-1β and IL-18 were observed in a mouse model of peritoneal fibrosis. ASC-positive and F4/80-positive cells colocalized in the subperitoneal mesothelial cell layer. These macrophages expressed high CD44 levels indicating that the CD44-positive macrophages contribute to developing peritoneal deterioration. Furthermore, intravital imaging of the peritoneal microvasculature demonstrated that the circulating CD44-positive leukocytes may contribute to peritoneal fibrosis. Bone marrow transplantation in ASC-deficient mice suppressed inflammasome activation, thereby attenuating peritoneal fibrosis in a high glucose-based PD solution-injected mouse model. Our results suggest inflammasome activation in CD44-positive macrophages may be involved in developing peritoneal fibrosis. The inflammasome-derived pro-inflammatory cytokines might therefore serve as new biomarkers for developing encapsulating peritoneal sclerosis.

Impaired thymic iNKT cell differentiation at early precursor stage in murine haploidentical bone marrow transplantation with GvHD.

Early recovery of donor-derived invariant natural killer T (iNKT) cells are associated with reduced risk of graft-versus-host disease (GvHD) and overall survival. Patients with severe GvHD, however, had much slower iNKT cell reconstitution relative to conventional T cells. To characterize the delay of iNKT cell reconstitution and explore its possible causes, we used a haploidentical bone marrow transplantation (haplo-BMT) mouse model with GvHD. We found the delayed recovery of thymic and peripheral iNKT cell numbers with markedly decreased thymic NKT1 subset in GvHD mice. The defective generation of thymic iNKT precursors with egress capability contributed to the reduced peripheral iNKT cells in GvHD mice. We further identified intermediate NK1.1- NKT1 precursor subpopulations under steady-state conditions and found that the differentiation of these subpopulations was impaired in the thymi of GvHD mice. Detailed characterization of iNKT precursors and thymic microenvironment showed a close association of elevated TCR/co-stimulatory signaling provided by double positive thymocytes and macrophages with defective down-regulation of proliferation, metabolism, and NKT2 signature in iNKT precursor cells. Correspondingly, NKT2 but not NKT1 differentiation was favored in GvHD mice. These data underline the important roles of TCR and co-stimulatory signaling in the differentiation of thymic iNKT subsets under transplantation conditions.

Effects of different allo-Treg/allo-NK ratios on graft-versus-host disease in transplanted mice

To investigate the effects of allo-Treg cells, allo-NK cells, and their mixtures in different proportions on Graft-versus-host disease (GVHD) in bone marrow transplant mouse model. In this study, C57BL/6 mice were used as donors, and 6 Gy dose of 60Co γ was used as the receptor of BALB/c mice. The recipient mice were divided into NC (normal saline), CON (bone marrow cells), NK (bone marrow cells + NK cells), Treg (bone marrow cells + Treg cells), NK+ Treg (1:1) (bone marrow cells +1:1 ratio of Treg cells, NK cells), and NK+ Treg (6:1) (bone marrow cells +1:6 ratio of Treg cells, NK cells), according to the different injection mode through the tail vein. The differences of white blood cell (WBC), platelet (PLT), clinical manifestations, and GVHD score of target organs (liver, lung, small intestine) in each group after transplantation were observed, and the differences of chimerism rate and survival rate in each group at 28 days after transplantation were compared. The interaction between Treg cells and NK cells in different proportions (1:1, 1:2, 1:6, 1:12) was investigated in vitro in mouse erythroleukemia (MEL) cells of mouse erythroleukemia. The results showed that at the 28th day of transplantation, the clinical manifestations and GVHD scores of target organs of mice in NK+ Treg (1:1) group and NK+ Treg (6:1) group were significantly lower than other groups (P < 0.05); the WBC and PLT counts were significantly higher than other groups (P < 0.05), and the survival time was significantly longer than other groups (P < 0.05); the clinical manifestations and GVHD scores of each target organ in NK+ Treg (1:1) group were significantly lower than those in NK+ Treg (6:1) group (P < 0.05); the chimerism rate of each group was >90% on day 28 after transplantation. In vitro experiments showed that the inhibition of Treg cells on NK cell killing activity was dose-dependent, and the proportion of 1:6 and 1:12, killing activity of NK cell was significantly lower than that of groups 1:1 and 1:2 (P < 0.05), which showed that allo-NK and allo-Treg alone had a significant effect on the improvement of GVHD after transplantation, and Treg cells inhibited the killing activity of NK cells by direct contact and showed a dose-dependent effect.

System analysis of Huang-Lian-Jie-Du-Tang and their key active ingredients for overcoming CML resistance by suppression of leukemia stem cells

BackgroundBCR-ABL1-based resistance to imatinib, mainly resulting from BCR-ABL1 mutations, is largely solved after second- and third-generation tyrosine kinase inhibitors (TKIs) are discovered. Nonetheless, imatinib resistance without BCR-ABL1 mutations, including intrinsic resistance induced by stem cells within chronic myeloid leukemia (CML), remains the major clinical challenge for many patients. PurposeTo study the key active ingredients and corresponding target proteins in Huang-Lian-Jie-Du-Tang (HLJDT) against BCR-ABL1-independent CML resistance to therapeutics, and then explore its mechanism of against CML drug resistance. MethodsCytotoxicity of HLJDT and its active ingredients in BCR-ABL1-independent imatinib resistance cells was analyzed through MTT assay. The cloning ability was measured through soft agar assay. Monitoring therapeutic effect on Xenografted mice CML model by in vivo imaging technology and mice survival time. Predicting the potential target protein binding sites by the technology of photocrosslinking sensor chip, molecular space simulation docking, and use Surface Plasmon Resonance (SPR) technology . Flow cytometry to detect the ratio of stem progenitor cells (CD34+). Constructing bone marrow transplantation mice CML leukemia model, detect the effects on leukemia stem cells LSK (Lin-\\ Sca-1+ \\C-kit+) self-renewal. ResultsTreatment with HLJDT, berberine and baicalein inhibited cell viability and colony formation of BCR-ABL1-independent imatinib-resistant cells in vitro while prolonging survival in mouse with CML xenografts and transplatation CML-like mouse models in vivo. JAK2 and MCL1were identified as targets of berberine and baicalein. JAK2 and MCL1 are involved in multi-leukemia stem cell-related pathways. Moreover, the ratio of CD34+ cells in resistant CML cells is higher than in treatment-sensitive CML cells. Treatment with BBR or baicalein partially suppressed CML leukemic stem cells (LSCs) self-renewal in vitro and in vivo. ConclusionFrom the above, we concluded that HLJDT and its key active ingredients (BBR and baicalein) allowed to overcome imatinib resistance with BCR-ABL1 independent by eradication of LSCs by targeting the JAK2 and MCL1 protein levels. Our results lay the foundation for applying HLJDT in patients with TKI-resistant CML.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

Enzyme-Cleaved Bone Marrow Transplantation Improves the Engraftment of Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC) therapy is a promising approach to curing bone diseases and disorders. In treating genetic bone disorders, MSC therapy is local or systemic transplantation of isolated and in vitro proliferated MSC rather than bone marrow transplantation. Recent evidence showed that bone marrow MSC engraftment to bone regeneration has been controversial in animal and human studies. Here, our modified bone marrow transplantation (BMT) method solved this problem. Like routine BMT, our modified method involves three steps: (i) isolation of bone marrow cells from the donor, (ii) whole-body lethal irradiation to the recipient, and (iii) injection of isolated bone marrow cells into irradiated recipient mice via the tail vein. The significant modification is imported at the bone marrow isolation step. While the bone marrow cells are flushed out from the bone marrow with the medium in routine BMT, we applied the enzymes' (collagenase type 4 and dispase) integrated medium to wash out the bone marrow cells. Then, cells were incubated in enzyme integrated solution at 37°C for 10minutes. This modification designated BMT as collagenase-integrated BMT (c-BMT). Notably, successful engraftment of bone marrow MSC to the new bone formation, such as osteoblasts and chondrocytes, occurs in c-BMT mice, whereas routine BMT mice do not recruit bone marrow MSC. Indeed, flow cytometry data showed that c-BMT includes a higher proportion of LepR+, CD51+, or RUNX2+ non-hematopoietic cells than BMT. These findings suggested that c-BMT is a time-efficient and more reliable technique that ensures the disaggregation and collection of bone marrow stem cells and engraftment of bone marrow MSC to the recipient. Hence, we proposed that c-BMT might be a promising approach to curing genetic bone disorders. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

E3 ligase RNF99 negatively regulates TLR-mediated inflammatory immune response via K48-linked ubiquitination of TAB2.

Innate immunity is the first line to defend against pathogenic microorganisms, and Toll-like receptor (TLR)-mediated inflammatory responses are an essential component of innate immunity. However, the regulatory mechanisms of TLRs in innate immunity remain unperfected. We found that the expression of E3 ligase Ring finger protein 99 (RNF99) decreased significantly in peripheral blood monocytes from patients infected with Gram negative bacteria (G-) and macrophages stimulated by TLRs ligands, indicating the role of RNF99. We also demonstrated for the first time, the protective role of RNF99 against LPS-induced septic shock and dextran sodium sulfate (DSS)-induced colitis using RNF99 knockout mice (RNF99-/-) and bone marrow-transplanted mice. In vitro experiments revealed that RNF99 deficiency significantly promoted TLR-mediated inflammatory cytokine expression and activated the NF-κB and MAPK pathways in macrophages. Mechanistically, in both macrophages and HEK293 cell line with TLR4 stably transfection, RNF99 interacted with and degraded TAK1-binding protein (TAB) 2, a regulatory protein of the kinase TAK1, via the lysine (K)48-linked ubiquitin-proteasomal pathway on lysine 611 of TAB2, which further regulated the TLR-mediated inflammatory response. Overall, these findings indicated the physiological significance of RNF99 in macrophages in regulating TLR-mediated inflammatory reactions. It provided new insight into TLRs signal transduction, and offered a novel approach for preventing bacterial infections, endotoxin shock, and other inflammatory ills.

Peptidylarginine deiminase 4 deficiency in bone marrow cells prevents plaque progression without decreasing atherogenic inflammation in apolipoprotein E-knockout mice.

Despite multiple studies in the past, the role of peptidylarginine deiminase 4 (PAD4) in atherosclerosis is currently insufficiently understood. In this regard, PAD4 deletion or inhibition of enzymatic activity was previously reported to ameliorate disease progression and inflammation. Besides, strong influence of neutrophil extracellular traps (NETs) on atherosclerosis burden has been proposed. Here, we studied the role of PAD4 for atherogenesis and plaque progression in a mouse model of atherosclerosis. Lethally irradiated ApoE -/- mice were reconstituted with ApoE -/-/Pad4 -/- bone marrow cells and fed a high-fat diet (HFD) for 4 and 10 weeks, respectively. PAD4 deficiency did not prevent the development of atherosclerotic lesions after 4 weeks of HFD. However, after 10 weeks of HFD, mice with bone marrow cells-restricted PAD4 deficiency displayed significantly reduced lesion size, impaired lipid incorporation, decreased necrotic core area and less collagen when compared to ApoE -/- bone marrow-transplanted mice as demonstrated by histological staining. Moreover, flow cytometric analysis and quantitative real-time PCR revealed different macrophage subsets in atherosclerotic lesions and higher inflammatory response in these mice, as reflected by increased content of M1-like macrophages and upregulated aortic expression of the pro-inflammatory genes CCL2 and iNOS. Notably, diminished oxLDL uptake by in vitro-polarized M1-like macrophages was evidenced when compared to M2-like cells. These results suggest that pharmacological inhibition of PAD4 may impede lipid accumulation and lesion progression despite no beneficial effects on vascular inflammation.

Nociceptive Nerves Facilitate Hematopoietic Stem/Progenitor Cell Homing to Bone Marrow By Tuning Microcirculation

The homing process of hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) is critical for engraftment after BM transplantation (BMT). Nociceptive nerves in BM reportedly regulate the mobilization of HSPCs from BM by directly supplying calcitonin gene-related peptide (CGRP) to HSPCs. However, it remains unclear whether nociceptive nerves control HSPC dynamics other than egress from BM, e.g., homing to BM. Here, we show that nociceptive nerves facilitate HSPCs homing to BM by controlling microcirculation. To assess the effects of CGRP on the BM microenvironment, we immunostained BM sections of mice and found that BM arteries, but not sinusoids, expressed CGRP receptors. We next employed intravital imaging system that visualized calvarial BM vessels and combined it with the transcalvarial administration (TCA), a method that enables manipulation of local BM microcirculation. TCA of CGRP receptor antagonist constricted BM arteries and decreased sinusoidal flux and shear stress, that impeded transendothelial migration (TEM) of transplanted HSPCs into BM, suggesting that CGRP released from nociceptive nerves supports local BM microcirculation and facilitates TEM of transplanted HSPCs into BM. In addition, systemic administration of CGRP receptor antagonist and pharmacological and genetic ablation of nociceptive nerves significantly decreased the homing efficiency of transplanted HSPCs to BM. Moreover, CGRP administration restored homing efficiency in nociceptive denervated-mice, and improved homing efficiency of wild-type mice. We next examined whether the improved homing efficiency of HSPCs with CGRP administration was mediated via arteries or via hematopoietic cells. CGRP is sensed by a heterodimeric CGRP receptor composed of calcitonin receptor-like receptor (CALCRL) and receptor activity modifying protein 1 (RAMP1), and hematopoietic cells reportedly express both CALCRL and RAMP1. We prepared Ramp1-/- mice to examine the homing efficiency of Ramp1-deficient hematopoietic cells. We found that the homing efficiency of hematopoietic cells from Ramp1-/- mice was not impaired upon transplantation into wild-type recipients. These results suggest that CGRP acts on the BM microenvironments, not on hematopoietic cells, to improve the homing efficiency. To determine the relationship between homing control by nociceptive nerves and survival after transplantation, we analyzed survival after BMT in mice in which nociceptive nerves were denervated. We found that pharmacological and genetic denervation of nociceptive nerves in recipient mice significantly reduced survival after BMT. Finally, to determine whether the intake of spicy food that activates nociceptive nerves enhances the efficiency of BMT, mice were fed with food containing capsaicin and subjected to homing experiments. HSPC homing efficiency to BM of mice fed with capsaicin diet was significantly improved compared to that of mice with normal diet. This study reveals a novel function of nociceptive nerves in regulating BM microcirculation and HSPC homing, and suggests that nociceptive nervous system could be a new therapeutic target for improving BMT efficiency.

Total Marrow Irradiation Reduces Organ Damage in a Bone Marrow Transplant Model of Sickle Cell Disease

Sickle cell disease (SCD) is associated with significant organ pathology. Currently, the only curative option is bone marrow transplantation (BMT) after myeloablative conditioning. However, significant organ damage from myeloablative total body irradiation (TBI) remains one of the major side effects of BMT. To reduce exposure to vital organs, but efficient enough to maintain engraftment, we developed 3D image guided-total marrow irradiation (TMI) for targeted delivery to the bone marrow (BM) and spleen of humanized BERK sickle mice while sparing the vital organs. We hypothesized that TMI will prevent organ damage compared to that induced by TBI during BMT. Sickle mice were treated with: [i] TBI (4:4), 4 Gy to the BM & 4 Gy to the whole body; [ii] TMI (4:0), 4 Gy to BM, 0 Gy to the body, and [iii] TMI (8:2), 8 Gy to BM & 2 Gy to the whole body. After 24h of irradiation, SS mice were transplanted with 5 million donor BM cells from HbAA BERK mice expressing normal human hemoglobin A (n=3/group). Thirty days after BMT mice were euthanized, and organs were necropsied for histopathology. Formalin-fixed paraffin-embedded sections of the lung, liver, kidney, spleen, and bone marrow (femur & tibia) were stained with H&E and Prussian blue (iron deposits). Morphologic findings of vascular congestion, inflammatory infiltrate, and iron deposits were scored from '0' for no lesion up to '6' for severe lesions occupying 90-100% of the field (Manci et al., Blood 2006). Tubular brush border thickness was assessed on periodic acid Schiff's (PAS)-hematoxylin-stained kidney sections using a 0-4 grading scale: no changes ='0'; lesion area <25%=1; 25%-50% = 2; >50% = 3 and 100% = 4 (Kasztan et al. J Am Soc Nephrol. 2017). We observed significantly increased vascular congestion in the liver (p=0.003), and spleen (p=0.013), and inflammatory infiltrates in the lungs (p=0.031) of TBI-treated mice compared to TMI (8:2). Total body irradiation has been shown to induce inflammation with granulocyte infiltration into tissues in association with endothelial damage, vascular dilatation, and congestion. Moreover, increase iron content in BM and serum and disrupted iron metabolism is another known sequala of total body irradiation, which contributes to hemosiderin formation in tissues. We found significant increase in iron deposits in TBI (4:4) vs TMI (8:2) in liver (p=0.001), spleen (p=0.014), and kidney (p=0.043). TBI (4:4) led to more renal tubular lesions (p=0.003) marked by a significant loss in the tubular brush border on PAS-stained kidney sections compared to TMI (8:2). Thus, TMI shows lesser damaging effect on vital organs compared to TBI, while TMI (8:2) showed donor chimerism similar to TBI on days 7 and 14 but higher than TMI (4:0) treated mice. However, the engraftment failed (less than 10% chimerism) in all groups by day 30. To improve engraftment, we transplanted 10- and 25-million donor HbAA BERK BM cells into TMI (4:0) and TMI (8:2) treated sickle mice. TMI (8:2) showed sustained donor chimerism but TMI (4:0) did not. Histopathological analysis 90 days post-BMT showed that TMI (8:2) treatment (n=3) results in significantly lesser organ pathology Vs untreated sickle mice (n=2), as evinced by reduction in: vascular congestion (lung, p=0.014; liver, p=0.014; spleen p= 0.014), inflammatory infiltrate (lung, p= 0.031; liver p= 0.005), liver infarctions (p=0.003) and liver and kidney iron deposits (p=0.041 & 0.021, respectively). Untreated mice showed a significant loss in the tubular brush border compared to TMI (8:2) treatment (p=0.008). BM in TMI (8:2) (cellularity~70%) showed improvement in cellular differentiation and topography in comparison to the untreated (cellularity ~90% ) that were packed with minimal fat spaces, thin trabeculae, and marked erythroid hyperplasia. These data suggest recovery from sickle cell phenotype with TMI-8:2 without significant organ pathology. Additionally, toluidine blue stained skin sections showed that the number (~45 Vs ~15) and % degranulating mast cells (~85 % Vs 50 %) were significantly lower in TMI (8:2) Vs untreated sickle mice (p<0.05), suggesting that TMI (8:2) treatment and successful engraftment of healthy donor cells may reduce inflammation and pain phenotype. We show that dose escalated TMI (8:2) is well tolerated with reduced organ pathology. Therefore, TMI has the potential to improve BMT-related outcomes without an adverse effect on organ damage.