Bone Marrow Transplantation Studies Research Articles

COVID-19 disease in children and adolescents following allogeneic hematopoietic stem cell transplantation: A report from the Turkish pediatric bone marrow transplantation study group.

Data on the risk factors and outcomes for pediatric patients with SARS-CoV-2 infection (COVID-19) following hematopoietic stem cell transplantation (HSCT) are limited. The study aimed to analyze the clinical signs, risk factors, and outcomes for ICU admission and mortality in a large pediatric cohort who underwent allogeneic HSCT prior to COVID-19 infection. In this nationwide study, we retrospectively reviewed the data of 184 pediatric HSCT recipients who had COVID-19 between March 2020 and August 2022. The median time from HSCT to COVID-19 infection was 209.0 days (IQR, 111.7-340.8; range, 0-3845 days). The most common clinical manifestation was fever (58.7%). While most patients (78.8%) had asymptomatic/mild disease, the disease severity was moderate in 9.2% and severe and critical in 4.4% and 7.6%, respectively. The overall mortality was 10.9% (n: 20). Deaths were attributable to COVID-19 in nine (4.9%) patients. Multivariate analysis revealed that lower respiratory tract disease (LRTD) (OR, 23.20, p: .001) and lymphopenia at diagnosis (OR, 5.21, p: .006) were risk factors for ICU admission and that HSCT from a mismatched donor (OR, 54.04, p: .028), multisystem inflammatory syndrome in children (MIS-C) (OR, 31.07, p: .003), and LRTD (OR, 10.11, p: .035) were associated with a higher risk for COVID-19-related mortality. While COVID-19 is mostly asymptomatic or mild in pediatric transplant recipients, it can cause ICU admission in those with LRTD or lymphopenia at diagnosis and may be more fatal in those who are transplanted from a mismatched donor and those who develop MIS-C or LRTD.

Abstract 2962: Chemotherapy induced senescence drives peripheral neuropathy

Abstract Chemotherapy is a mainstay of cancer therapy. Unfortunately, while chemotherapy can profoundly impact disease free survival, it is often accompanied with devastating side effects including hair loss, bone loss, cognitive impairment (referred to as "brain fog"), and peripheral neuropathies. While many of these side effects may subside after the completion of chemotherapy, some persist for a lifetime, substantially compromising the quality of life for survivors. Particularly insidious is chemotherapy-induced peripheral neuropathy (CIPN), which can impair a patient's ability to care for themselves. Shockingly, in over 30% of patients, CIPN persists even after the cessation of chemotherapy, subjecting them to a lifetime of numbness and/or pain. Paclitaxel (PTX), a common treatment for breast cancer, induces CIPN in 61-92% of patients depending on the administered dose. Unfortunately, there are currently no preventative or curative measures for CIPN resulting in side effects that persist for months or even years after the cessation of chemotherapy. Considering chemotherapy can trigger cellular senescence, leading to the release of a diverse array of factors collectively known as the senescence-associated secretory phenotype (SASP) that are capable of negatively influencing neighboring cells. To determine if senescence played a role in CIPN we treated with PTX and found that animals displayed reduced axon innervation in the paws and diminished paw withdrawal responses during the von Frey assay, characteristic symptoms of CIPN. To determine if senescent drove PTX-induced CIPN, we utilized the INKATTAC (INK) mouse, a model in which a p16INK4a promoter drives an inducible suicide transgene to eliminate p16+ senescent cells following treatment with AP20187 (AP). Employing the INK mouse, we successfully prevented CIPN by eliminating PTX-induced senescent cells. Furthermore, through bone marrow (BM) transplant studies using BM from INK mice into wildtype recipients, we failed to prevent CIPN. This result establishes that senescent immune cells, do not contribute to CIPN. In an independent approach, we found that senolytic drugs (kill senescent cells), such as dasatinib and quercetin (DQ) or ABT737 (ABT), effectively prevented and reversed CIPN in mice. Additionally, since the p38MAPK pathway has been shown to regulate SASP mRNA stability, we treated with p38 and MK2 inhibitors and found that inhibition prevented CIPN in mice. To address the mechanism behind CIPN we have carried out single cell RNA sequencing (scRNA-seq) to identify the cell population senescing in response to chemotherapy. These analyses will allow us to understand the mechanisms that drive CIPN and may lead to new treatments for patients suffering from CIPN. Citation Format: Taylor Malachowski, Ganesh Kumar Raut, Xianmin Luo, Satarupa Mullick Bagchi, Sheila Stewart. Chemotherapy induced senescence drives peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2962.

Id1 Ablation Rescues Clonal Hematopoiesis and Delays Disease Progression in Tet2 Null Mice

Hematopoietic malignancies are thought to emerge through the gradual acquisition of genetic mutations within hematopoietic stem and progenitor cells (HSPCs). Some mutations impart a selective proliferative/growth advantage to HSPCs, which expand over time and contribute to a substantial percentage of mature blood cells. This increased proliferation and expansion is termed clonal hematopoiesis (CH) and is a preleukemic phase that is associated with an increased risk of accumulating additional mutations and developing leukemia. These mutations are also associated with increased inflammatory stress and increased cytokine production (“cytokine storm”) in the hematopoietic microenvironment which can contribute to clonal hematopoiesis. Inhibitor of DNA binding 1 (ID1) protein is induced by hematopoietic growth factors and other mediators of inflammatory stress and promotes HSPC proliferation by regulating E proteins and their targets. HSCs that lack Id1 are protected from chronic proliferative stress and exhaustion during serial BMT, and other chronic physiologic stress including genotoxic and inflammatory stress, and aging. ID1 is frequently overexpressed in hematopoietic malignancies and enforced expression of Id1 in HSPCs promotes a myeloproliferative disease in mice suggesting that ID1 may drive clonal expansion and regulate the growth of some hematologic malignancies. Mutations in the Ten-Eleven-Translocation-2 ( Tet2) (Tet methylcytosine dioxygenase 2) gene are frequently observed in patients with CH, and mice that lack Tet2 show clonal expansion, increased mutational loads, genomic instability that results in myeloid and lymphoid malignancies after a long latency. Here, we show that ablation of Id1 rescues clonal hematopoiesis and delays disease progression in Tet2 -/- mice. We found that Tet2 -/- HSPCs express high levels of Id1. Therefore, we genetically ablated Id1 in Tet2 -/- mice and generated Tet2 -/-;Id1 -/- (dKO) mice and performed competitive bone marrow transplantation studies (BMT) to study the intrinsic role of these genes in hematopoietic development. We found that genetic ablation of Id1 in Tet2 -/- HSPCs reduces HSPC proliferation/expansion and myeloid skewing by flow cytometry. dKO mice showed reduced spleen size and weight indicating that reducing ID1 levels in Tet2 -/- mice rescues extramedullary hematopoiesis. We confirmed that reducing ID1 levels in Tet2 -/- mice reduces clonal expansion of Tet2 -/- BMCs using limited number of donor BMCs in competitive BMT. Furthermore, we performed functional assays including colony forming assays (CFU-C) and single cell (SC) assays to assess the proliferation of these cells and found that loss of Id1 in Tet2 -/- mice reduced 1) the number of CFU-C's and the serial replating ability of Tet2 -/- HSPCs, and 2) progenitor expansion in SC assays. Moreover, dKO recipient mice BMCs showed increased apoptosis and senescence compared to Tet2 -/-BMCs, which could contribute to reduced expansion. Surprisingly, Id1 ablation in Tet2 -/- mice also resulted in reduced number of chromosomal aberrations indicating that reducing ID1 levels in Tet2 -/- mice rescues genomic instability. Furthermore, dKO recipient mice showed delayed onset of disease in a model of chronic proliferative stress. Mechanistically, we found that p16 expression was significantly increased in the dKO recipient mice, which could account for reduced expansion and increased apoptosis and senescence. shRNA mediated knockdown of p16 in dKO lineage negative cells reduced apoptosis and senescence in these cells confirming the role of p16 in these phenotypes. We also found that 53BP1 and Rad51 expression was reduced in the dKO recipient mice, which could lead to improved DNA repair and genomic stability. Thus, Id1 may represent a potential therapeutic target to reduce clonal expansion and delay the onset of leukemia.

Neuropeptide Y Attenuates Aging-Induced Bone Marrow Niche Defects and Rejuvenate Aged Hematopoietic Stem/Progenitor Cells

Aging-associated defects in hematopoiesis are linked to declining in hematopoietic stem cell (HSC) self-renewal and multilineage differentiation capacity. The bone marrow (BM) microenvironment/niche has been suggested to influence HSC aging. However, the factors/mechanisms whereby the BM niche mediates hematopoietic stem and progenitor (HSPC) aging are poorly understood. We recently discovered that neuropeptide Y (NPY) plays a crucial role in HSPC trafficking by regulating the BM vascular gateway function (PMCID: PMC5707149). NPY is one of the most abundant neurotransmitters released in the BM by sympathetic nerve fibers. In this study, we found that the aging-induced deficit of NPY levels in the BM plays a critical role in niche defects and HSPC aging by impairing the molecular pathways involved in regulating oxidative stress, senescence, and stemness. The measurement of NPY levels in the BM of young (3-4 months) and aged (18-23 months) mice showed lower NPY levels in aged mice than in young mice (2.2-fold), which was associated with a decrease in mesenchymal stem cells (MSC) (2-fold, p=0.008) and endothelial cells (EC) (1.5-fold, p<0.05) numbers in the BM. In addition, we found that circulating NPY levels decline in the older human population (65-83 years) than in young/ middle age (23-55 years) individuals (1.5-fold, p=0.01). We hypothesized if aging-induced NPY deficit contributes to BM niche defects and HSPC aging, then NPY supplementation may attenuate/revert these processes. To explore this possibility, we administered exogenous NPY in aged mice (100ng/mouse) for 15 consecutive days and evaluated HSPC and niche cells. NPY supplementation in aged mice (18-23 months) substantially reverted aged HSPC phenotype, including NPY decreased the accumulation of HSC-enriched SLAM LSK (Lin-Sca-1+c-Kit+CD48-CD150+) in the BM (1.8-fold, p<0.05) and increased the HPC clonal expansion (1.9-fold, p<0.05) measured by flow cytometry and CFU-C assay, respectively. Competitive BM transplantation studies using donor cells from young, aged, or NPY-treated aged mice into lethally irradiated syngeneic mice showed myeloid biased repopulation from aged mice donor cells than young mice donor cells, and that was attenuated in NPY-treated aged mice donor cells (33%, p=0.003). In addition, NPY substantially reverted aging-related reduction in BM MSC (CD45-Ter119-CD31-Nestin+PDGFR+CD51+) and EC (CD45-Ter119-CD31+VE-cadherin+) numbers, and BM MSC clonal expansion (2-fold, p<0.05) as measured by CFU-F assay. To further validate NPY's role in the aged BM niche and HSPC function, we used NPY gene overexpressing and knockout (KO) mouse models. Compared to wild-type (WT) aged mice, NPY overexpressing aged mice showed a substantial increase in BM niche MSC numbers (1.4-fold, P=0.03) and improved HSPC clonal expansion (2.5-fold, P=0.002). In contrast, 16-18 months old NPY KO mice showed enhanced aging than aged WT mice, reflected by fewer MSC counts and lower HSPC clonal expansion (MSC: 1.6-fold, and CFU-F: 1.5-fold, <0.05). Oxidative stress and senescence are the hallmarks of aging. To examine if NPY attenuates HSPC aging by controlling oxidative and senescence, we measured reactive oxygen species (ROS) and senescence in SLAM-LSK of young, aged, and NPY-treated aged mice using CellROX and anti-p16 antibodies, respectively. The SLAM-LSK from aged mice showed higher expression of ROS (9-fold, p=0.03) and p16 than young mice (1.7-fold). However, ROS and p16 expression were decreased in NPY-treated aged mice (ROS: 2-fold, and p16: 1.3-fold, p<0.05). To identify the molecular pathways involved in NPY-mediated attenuation of HSPC aging, we performed RNA sequencing analysis of SLAM-LSK cells. Aged mice SLAM LSK showed impaired expression of genes involved in pathways regulating antioxidant defense, DNA damage repair and stemness than in young mice SLAM LSK. However, NPY treatment in aged mice substantially restored these pathways in SLAM LSK. In conclusion, our studies suggest that an aging-induced deficit in NPY levels in the BM is a crucial lesion contributing to niche defects and HSPC aging. Thus, NPY supplementation or protection of NPY-producing nerve fibers in the BM can attenuate/restore aging-induced niche damage and rejuvenate aged HSPC.

Macrophage-restricted overexpression of glutaredoxin 1 protects against atherosclerosis by preventing nutrient stress-induced macrophage dysfunction and reprogramming

Background and aimsDeficiency in the thiol transferase glutaredoxin 1 (Grx1) in aging mice promotes, in a sexually dimorphic manner, dysregulation of macrophages and atherogenesis. However, the underlying mechanisms are not known. Here we tested the hypothesis that macrophage-restricted overexpression of Grx1 protects atherosclerosis-prone mice against macrophage reprogramming and dysfunction induced by a high-calorie diet (HCD) and thereby reduces the severity of atherosclerosis. MethodsWe generated lentiviral vectors carrying cluster of differentiation 68 (CD68) promoter-driven enhanced green fluorescent protein (EGFP) or Grx1 constructs and conducted bone marrow (BM) transplantation studies to overexpress Grx1 in a macrophage-specific manner in male and female atherosclerosis-prone LDLR−/− mice, and fed these mice a HCD to induce atherogenesis. Atherosclerotic lesion size was determined in both the aortic root and the aorta. We isolated BM-derived macrophages (BMDM) to assess protein S-glutathionylation levels and loss of mitogen-activated protein kinase phosphatase 1 (MKP-1) activity as measures of HCD-induced thiol oxidative stress. We also conducted gene profiling on these BMDM to determine the impact of Grx1 activity on HCD-induced macrophage reprogramming. ResultsOverexpression of Grx1 protected macrophages against HCD-induced protein S-glutathionylation, reduced monocyte chemotaxis in vivo, limited macrophage recruitment into atherosclerotic lesions, and was sufficient to reduce the severity of atherogenesis in both male and female mice. Gene profiling revealed major sex differences in the transcriptional reprogramming of macrophages induced by HCD feeding, but Grx1 overexpression only partially reversed HCD-induced transcriptional reprogramming of macrophages. ConclusionsMacrophage Grx1 plays a major role in protecting mice atherosclerosis mainly by maintaining the thiol redox state of the macrophage proteome and preventing macrophage dysfunction.

Effect of omega-3 fatty acid diet on prostate cancer progression and cholesterol efflux in tumor-associated macrophages-dependence on GPR120.

Preclinical and clinical translational research supports the role of an ω-3 fatty acid diet for prostate cancer prevention and treatment. The anti-prostate cancer effects of an ω-3 diet require a functional host g-protein coupled receptor 120 (GPR120) but the underlying effects on the tumor microenvironment and host immune system are yet to be elucidated. Friend leukemia virus B (FVB) mice received bone marrow from green fluorescent protein (GFP) labeled GPR120 wild-type (WT) or knockout (KO) mice followed by implanting Myc-driven mouse prostate cancer (MycCap) allografts and feeding an ω-3 or ω-6 diet. Tumor associated immune cells were characterized by flow cytometry, and CD206+ tumor infiltrating M2-like macrophages were isolated for gene expression studies. MycCap prostate cancer cell conditioned medium (CM) was used to stimulate murine macrophage cells (RAW264.7) and bone marrow-derived (BMD) macrophages to study the effects of docosahexanoic acid (DHA, fish-derived ω-3 fatty acid) on M2 macrophage function and cholesterol metabolism. The bone marrow transplantation study showed that an ω-3 as compared to an ω-6 diet inhibited MycCaP allograft tumor growth only in mice receiving GPR120 WT but not GPR120 KO bone marrow. In the ω-3 group, GPR120 WT BMD M2-like macrophages infiltrating the tumor were significantly reduced in number and gene expression of cholesterol transporters Abca1, Abca6, and Abcg1. RAW264.7 murine macrophages and BMDMs exposed to MycCaP cell CM had increased gene expression of cholesterol transporters, depleted cholesterol levels, and were converted to the M2 phenotype. These effects were inhibited by DHA through the GPR120 receptor. Host bone marrow cells with functional GPR120 are essential for the anticancer effects of dietary ω-3 fatty acids, and a key target of the ω-3 diet are the M2-like CD206+ macrophages. Our preclinical findings provide rationale for clinical trials evaluating ω-3 fatty acids as a potential therapy for prostate cancer through inhibition of GPR120 functional M2-like macrophages.

U-Statistics for left truncated and right censored data

The analysis of left truncated and right censored data is very common in survival and reliability analysis. In lifetime studies patients are often subject to left truncation in addition to right censoring. For example, in bone marrow transplant studies based on International Bone Marrow Transplant Registry (IBMTR), the patients who die while waiting for the transplants will not be reported to the IBMTR. In this paper, we develop novel U-statistics under left truncation and right censoring. We prove the n -consistency of the proposed U-statistics. We derive the asymptotic distribution of the U-statistics using the counting process technique. As an application of the U-statistics, we develop a simple non-parametric test for testing the independence between time to failure and cause of failure in competing risks when the observations are subject to left truncation and right censoring. The finite sample performance of the proposed test is evaluated through a Monte Carlo simulation study. Finally, we illustrate our test procedure using the lifetime data of transformers.

Dectin-1 Acts as a Non-Classical Receptor of Ang II to Induce Cardiac Remodeling.

Cardiac remodeling in heart failure involves macrophage-mediated immune responses. Recent studies have shown that a PRR (pattern recognition receptor) called dectin-1, expressed on macrophages, mediates proinflammatory responses. Whether dectin-1 plays a role in pathological cardiac remodeling is unknown. Here, we identified a potential role of dectin-1 in this disease. To model aberrant cardiac remodeling, we utilized mouse models of chronic Ang II (angiotensin II) infusion. In this model, we assessed the potential role of dectin-1 through using D1KO (dectin-1 knockout) mice and bone marrow transplantation chimeric mice. We then used cellular and molecular assays to discover the underlying mechanisms of dectin-1 function. We found that macrophage dectin-1 is elevated in mouse heart tissues following chronic Ang II administration. D1KO mice were significantly protected against Ang II-induced cardiac dysfunction, hypertrophy, fibrosis, inflammatory responses, and macrophage infiltration. Further bone marrow transplantation studies showed that dectin-1 deficiency in bone marrow-derived cells prevented Ang II-induced cardiac inflammation and dysfunction. Through detailed molecular studies, we show that Ang II binds directly to dectin-1, causing dectin-1 homodimerization and activating the downstream Syk (spleen tyrosine kinase)/NF-κB (nuclear factor kappa B) signaling pathway to induce expression of inflammatory and chemoattractant factors. Mutagenesis studies identified R184 in the C-type lectin domain to interact with Ang II. Blocking dectin-1 in macrophages suppresses Ang II-induced inflammatory mediators and subsequent intercellular cross talk with cardiomyocytes and fibroblasts. Our study has discovered dectin-1 as a new nonclassical receptor of Ang II and a key player in cardiac remolding and dysfunction. These studies suggest that dectin-1 may be a new target for treating hypertension-related heart failure.

Hematopoietic Stem Cell Transplantation in People With Active Secondary Progressive Multiple Sclerosis.

Uncontrolled evidence suggests that autologous hematopoietic stem cell transplantation (AHSCT) can be effective in people with active secondary progressive multiple sclerosis (SPMS). In this study, we compared the effect of AHSCT with that of other anti-inflammatory disease-modifying therapies (DMTs) on long-term disability worsening in active SPMS. We collected data from the Italian Bone Marrow Transplantation Study Group and the Italian Multiple Sclerosis Register. Patients were considered eligible if treatment had been started after the diagnosis of SPMS. Disability worsening was assessed by the cumulative proportion of patients with a 6-month confirmed disability progression (CDP) according to the Expanded Disability Status Scale (EDSS) score. Key secondary endpoints were the EDSS time trend after treatment start and the prevalence of disability improvement over time. Time to first CDP was assessed by means of proportional hazard Cox regression models. A linear mixed model with a time × treatment group interaction was used to assess the longitudinal EDSS time trends. Prevalence of improvement was estimated using a modified Kaplan-Meier estimator and compared between groups by bootstrapping the area under the curve. Seventy-nine AHSCT-treated patients and 1975 patients treated with other DMTs (beta interferons, azathioprine, glatiramer-acetate, mitoxantrone, fingolimod, natalizumab, methotrexate, teriflunomide, cyclophosphamide, dimethyl fumarate, and alemtuzumab) were matched to reduce treatment selection bias using propensity score and overlap weighting approaches. Time to first CDP was significantly longer in transplanted patients (hazard ratio [HR] = 0.50; 95% CI = 0.31-0.81; p = 0.005), with 61.7% of transplanted patients free from CPD at 5 years. Accordingly, EDSS time trend over 10 years was higher in patients treated with other DMTs than in AHSCT-treated patients (+0.157 EDSS points per year compared with -0.013 EDSS points per year; interaction p < 0.001). Patients who underwent AHSCT were more likely to experience a sustained disability improvement: 34.7% of patients maintained an improvement (a lower EDSS than baseline) 3 years after transplant vs 4.6% of patients treated by other DMTs (p < 0.001). The use of AHSCT in people with active SPMS is associated with a slowing of disability progression and a higher likelihood of disability improvement compared with standard immunotherapy. This study provides Class III evidence that autologous hematopoietic stem cell transplants prolonged the time to CDP compared with other DMTs.

Abstract 15362: Checkpoint Kinase 1-Associated Senp2 S344 Phosphorylation Under Laminar Flow Attenuates Endothelial-Mesenchymal Transition and Atherogenesis

Aims: To investigate the role of Checkpoint Kinase 1 (CHK1)-associated SENP2 S344 phosphorylation in laminar flow (LF)-regulated Endothelial-Mesenchymal Transition (EndMT) inhibition and the underlying mechanisms. Methods: We generated deSUMOylation enzyme sentrin-specific isopeptidase 2 (SENP2) S343A (equivalent to human SENP2 S344) knock-in (KI) and endothelial cell (EC)-specific SENP2 knock-out (SENP2 EKO) mice to detect EC activation, EndMT, and atherogenesis in vitro and in vivo . We also performed next-generation RNA sequencing (RNA-seq) analysis in ECs derived from these mice. Results: LF, but not disturbed flow (DF), triggered SENP2 S344 phosphorylation through CHK1. The decreased ERK5- and p53-SUMOylation by LF was abolished in ECs derived from SENP2 S343A KI mice. Both EC apoptosis and VCAM-1 expression were upregulated in SENP2 S344A KI mice. The increased atherogenesis in SENP2 S344A KI was regulated by vascular, but not hematopoietic, cells based on bone marrow transplantation (BMT) study. CHK1 expression was decreased by ionizing radiation (IR), leading to an enhanced ERK5- and p53-SUMOylation as well as EC inflammation. RNA-seq analysis revealed the involvement of SENP2 S344 phosphorylation in changes of fibrotic phenotypes and EndMT-related genes. Further, SENP2 S344A point mutation prevented LF-regulated TGFbR1 SUMOylation inhibition in vitro, as well as hypercholesterolemia-induced EndMT in vivo. Discussions: These data revealed the unique role of CHK1-associated SENP2 S344 phosphorylation in not only LF-regulated EndMT inhibition but also IR-associated EndMT activation and the subsequent atherogenesis. Maintaining CHK1-associated SENP2 S344 phosphorylation can be an ideal approach to mitigate EndMT, which is important in tumor radio-resistance as well as IR-associated cardiovascular disease.

Enrollment Lessons from a Biological Assignment Study of Marrow Transplantation versus Standard Care for Adolescents and Young Adults with Sickle Cell Disease: Considerations for Future Gene and Cellular Therapy Trials

We previously conducted a single-arm feasibility study (STRIDE1) of myeloablative bone marrow transplantation (BMT) in adolescents and young adults with sickle cell disease (SCD). The trial identified donors before entry, enrolled well, and found no unexpected regimen-related toxicity. Although many single-arm studies have been published, there are no controlled trials of either BMT or gene therapy in SCD. Therefore, we designed a comparative trial by biological assignment (available donor versus no donor). This multicenter National Institutes of Health-funded study (Blood and Marrow Transplant Clinical Trials Network 1503; STRIDE2) enrolled patients between 2016 and 2021 at 35 sites. Lagging recruitment led to study closure, and here we report the impediments to accrual. The BMT regimen and entry criteria were from STRIDE1, and 2-year survival was the primary endpoint. To minimize selection bias from prior HLA typing, STRIDE2 excluded individuals with previously identified donors. Accrual was stopped at 69% of target (138 enrolled; assigned 28 with donor, 96 with no donor). Barriers to enrollment included lower than expected frequency of HLA-matched related and unrelated donors; loss of enrollees owing to previously identified donors; conventional care arm dissuading some seeking BMT; challenging short-term endpoints in SCD, including incomplete documentation of sickle pain episodes; state Medicaid (primary insurers of SCD) denial of BMT coverage for adult SCD despite the study having secured Coverage with Evidence Development from the Center for Medicare & Medicaid Services; slowed accrual in 2019 to 2021 during the Coronavirus disease 2019 pandemic; and restriction of BMT resourcing for nonmalignant diseases by academic medical (cancer) centers. Social obstacles and access to BMT centers also limited entry, as did practitioner and participant concerns over suitability, cost, and toxicity. Planning for future controlled trials of curative therapy in SCD and other nonmalignant diseases likely will meet these enrollment challenges. Lessons from this trial may aid the development of future comparative studies.

Genetic disruption of the Gipr in Apoe−/− mice promotes atherosclerosis

ObjectiveThe gut hormone glucose-dependent insulinotropic polypeptide (GIP) stimulates beta cell function and improves glycemia through its incretin actions. GIP also regulates endothelial function and suppresses adipose tissue inflammation through control of macrophage activity. Activation of the GIP receptor (GIPR) attenuates experimental atherosclerosis and inflammation in mice, however whether loss of GIPR signaling impacts the development of atherosclerosis is uncertain. MethodsAtherosclerosis and related metabolic phenotypes were studied in Apoe−/−:Gipr−/− mice and in Gipr+/+ and Gipr−/− mice treated with an adeno-associated virus expressing PCSK9 (AAV-PCSK9). Bone marrow transplantation (BMT) studies were carried out using donor marrow from Apoe−/−:Gipr−/−and Apoe−/−:Gipr+/+mice transplanted into Apoe−/−:Gipr−/− recipient mice. Experimental endpoints included the extent of aortic atherosclerosis and inflammation, body weight, glucose tolerance, and circulating lipid levels, the proportions and subsets of circulating leukocytes, and tissue gene expression profiles informing lipid and glucose metabolism, and inflammation. ResultsBody weight was lower, circulating myeloid cells were reduced, and glucose tolerance was not different, however, aortic atherosclerosis was increased in Apoe−/−:Gipr−/− mice and trended higher in Gipr−/− mice with atherosclerosis induced by AAV-PCSK9. Levels of mRNA transcripts for genes contributing to inflammation were increased in the aortae of Apoe−/−:Gipr−/− mice and expression of a subset of inflammation-related hepatic genes were increased in Gipr−/− mice treated with AAV-PCSK9. BMT experiments did not reveal marked atherosclerosis, failing to implicate bone marrow derived GIPR + cells in the control of atherosclerosis or aortic inflammation. ConclusionsLoss of the Gipr in mice results in increased aortic atherosclerosis and enhanced inflammation in aorta and liver, despite reduced weight gain and preserved glucose homeostasis. These findings extend concepts of GIPR in the suppression of inflammation-related pathophysiology beyond its classical incretin role in the control of metabolism.

N-acetylcysteine attenuates atherosclerosis progression in aging LDL receptor deficient mice with preserved M2 macrophages and increased CD146

Background and aimsInflammation and reactive oxygen species (ROS) are important to the pathogenesis of atherosclerosis. The effect of antioxidants on atherosclerosis is inconsistent, and sometimes controversial. We aimed to test the hypothesis that attenuation of atherosclerosis by N-acetylcysteine (NAC) depends on NAC treatment timing and duration. MethodsMale LDL receptor deficient (LDLR−/−) mice were fed a normal diet (ND) and divided into controls (on ND for 24 months), models 1–2 (at age of 9 months, starting NAC treatment for 3 or 6 months), and model 3 (at age of 18 months, starting NAC treatment for 6 months). To determine if hyperlipidemia compromises NAC treatment outcome, mice were fed a high fat diet (HFD) starting at age of 6 weeks and treated with NAC starting at 9 months of age for 6 months. ResultsNAC treatment for 6 months, not for 3 months, significantly attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions, in aging LDLR−/− mice on ND. NAC had no effect on atherosclerotic lesions in mice on HFD. NAC treatment significantly decreased aortic ROS production, and the levels of inflammatory cytokines in serum and aorta of aging LDLR−/− mice with increased CD146 level. Bone marrow transplantation study with GFP-positive bone marrow cells showed that NAC treatment preserved M2 population and M2 polarization in the aorta of LDLR−/− mice. ConclusionsEarly and adequate NAC treatment could effectively attenuate inflammation and atherosclerosis progression with preserved M2 population and increased CD146 level in aging LDLR−/− mice without extreme hyperlipidemia.

Abstract GS113: Inflammasome Primed Neutrophils Regulate Myocardial Infarction Induced Myelopoiesis

Background: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in-part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β, the effector molecule in granulopoiesis was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the LAD artery, and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, utilizing multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the non-infarcted parabionts. Prior priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Pharmacological and/ or genetic strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (e.g., IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release

Abstract 450: Monocyte-to-endothelial Cell Crosstalk By 27-hydroxycholesterol Promotes Monocyte Vascular Recruitment And Thereby Advances Atherogenesis

How hypercholesterolemia invokes the vascular inflammation that is critical to atherogenesis is poorly understood. Sterol 27-hydroxylase (cyp27a1) converts cholesterol to 27-hydroxycholesterol (27HC), and we previously showed that 27HC is an estrogen receptor alpha (ERα) ligand. Here we determined how 27HC produced by monocytes impacts atherogenesis. In the absence of changes in circulating lipids, monocyte cyp27a1 deletion in apolipoprotein E -/- mice (apoE -/- ) caused a 35% decline in atherosclerotic lesion severity; this was related to decreased macrophage accumulation due to a reduction in lesion monocyte recruitment, which likely occurred as a result of decreases in endothelial cell VCAM-1 and ICAM-1. Experiments in cultured endothelial cells revealed that 27HC liganding of ERα activates NF-κB by causing the disassociation of the cytoplasmic scaffolding protein septin 11 from ERα and resulting septin 11 activation of Jnk kinase signaling. Endothelial septin 11 deletion in apoE -/- mice phenocopied the impact of monocyte cyp27a1 loss, yielding a 37% decrease in lesion severity that was similarly explained by declines in endothelial cell VCAM-1 and ICAM-1, lesion monocyte recruitment and macrophage accumulation. In bone marrow transplant studies the reintroduction of monocyte cyp27a1 caused a 71% increase in lesions which was fully dependent on endothelial cell ERα and septin 11. Furthermore, in apoE -/- mice pharmacologic inhibition of cyp27a1 decreased lesion severity by 41% without altering circulating lipids, causing declines in endothelial cell VCAM-1 and ICAM-1, monocyte recruitment and macrophage accumulation. There was no additivity with monocyte cyp27a1 deletion and cyp27a1 inhibition, indicating that the treatment-related atheroprotection occurs via monocyte cyp27a1 inhibition. These cumulative findings reveal that through a unique pro-inflammatory mechanism involving endothelial cell ERα and septin 11, monocyte-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes monocyte vascular recruitment to advance atherogenesis. Interventions attenuating the crosstalk may complement LDL-directed strategies in the battle against atherosclerosis.

Abstract 383: MLKL (Mixed Lineage Kinase Domain-like Protein) In The Splenic Microenvironment Restrains Hematopoietic Contributions To The Atherosclerotic Plaque

The mixed lineage kinase domain-like protein (MLKL) was first discovered in 2012 as the executioner of necroptosis. In line with this, we showed that MLKL knockdown ( Mlkl KD) by administration of antisense oligonucleotides to atherosclerotic-prone Apoe -knockout ( Apoe -/-) mice did indeed decrease necroptosis and necrotic core in the atherosclerotic plaque. However, there were no changes in overall plaque area, suggesting additional roles of MLKL during atherosclerosis. During atherogenesis, the spleen allows for the expansion of hematopoietic stem and progenitor cells (HSPCs) and mature myeloid cells that drive atherogenesis. Accordingly, we observed splenomegaly with an expansion of splenic HSPCs and mature myeloid cells upon Mlkl KD. However, bone marrow transplantation studies revealed that MLKL did not impact hematopoiesis through its expression in hematopoietic cells. Within hematopoietic reservoirs such as the spleen, HSPCs are regulated by their interaction with microenvironmental cell types, including endothelial cells. We thus hypothesized that MLKL preserves the microenvironment to repress hematopoiesis in the spleen during atherosclerosis. Flow cytometry and immunofluorescence staining during a time course of atherosclerosis revealed a progressive decrease of splenic endothelial cells that was correlated with a reduction of MLKL endogenously or by Mlkl KD, suggesting that splenic MLKL modulates the endothelial cells known to regulate hematopoiesis. RNA sequencing of these splenic endothelial cells revealed two major pathways that were perturbed upon Mlkl KD, including cell cycle and factors related to leukocyte differentiation. Adoptive transfer of CD45.1 HSPCs to CD45.2 Apoe -/- mice during Mlkl KD revealed an expansion of mature immune cells in the spleen as compared to control, together demonstrating the influence of MLKL on the splenic microenvironment and subsequently hematopoiesis. In conclusion, we here demonstrate a novel role for MLKL in regulating the balance of hematopoiesis, specifically through preservation of splenic endothelial cells that promote HSPC quiescence, and more generally highlight the contributions of the spleen and its impact on inflammatory pathways that potentiate atherogenesis.

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1β release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow–derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis.

Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β (interleukin-1β), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1β through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release.

Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension.

[Figure: see text].

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms

IntroductionA potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are...