Hematology
Abstract
Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds-/- mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days post fertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently have stunted growth and shows signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf and inhibition of proliferation correlating with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on wildtype fish. Starved wildtype fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation – similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS.
Authors
Usua Oyarbide, Arish N. Shah, Wilmer Amaya-Mejia, Matthew Snyderman, Margaret Kell, Daniela Allende, Eliezer Calo, Jacek Topczewski, Seth Corey
Abstract
Myelodysplastic syndromes (MDS) are clonal malignant hematopoietic disorders in the elderly characterized by ineffective hematopoiesis. This is accompanied by an altered bone microenvironment, which contributes to MDS progression and higher bone fragility. The underlying mechanisms remain largely unexplored. Here, we show that myelodysplastic NUP98‑HOXD13 (NHD13) transgenic mice display an abnormally high number of osteoblasts, yet a higher fraction of nonmineralized bone, indicating delayed bone mineralization. This was accompanied by high fibroblast growth factor-23 (FGF-23) serum levels, a phosphaturic hormone that inhibits bone mineralization and erythropoiesis. While Fgf23 mRNA expression was low in bone, brain, and kidney of NHD13 mice, its expression was increased in erythroid precursors. Coculturing these precursors with WT osteoblasts induced osteoblast marker gene expression, which was inhibited by blocking FGF-23. Finally, antibody-based neutralization of FGF-23 in myelodysplastic NHD13 mice improved bone mineralization and bone microarchitecture, and it ameliorated anemia. Importantly, higher serum levels of FGF‑23 and an elevated amount of nonmineralized bone in patients with MDS validated the findings. C‑terminal FGF‑23 correlated negatively with hemoglobin levels and positively with the amount of nonmineralized bone. Thus, our study identifies FGF-23 as a link between altered bone structure and ineffective erythropoiesis in MDS with the prospects of a targeted therapeutic intervention.
Authors
Heike Weidner, Ulrike Baschant, Franziska Lademann, Maria G. Ledesma Colunga, Ekaterina Balaian, Christine Hofbauer, Barbara M. Misof, Paul Roschger, Stéphane Blouin, William G. Richards, Uwe Platzbecker, Lorenz C. Hofbauer, Martina Rauner
Abstract
Ribosomopathies are congenital disorders caused by mutations in the genes encoding ribosomal and other functionally related proteins. They are characterized by anemia, other hematopoietic and developmental abnormalities, and p53 activation. Ribosome assembly requires coordinated expression of many ribosomal protein (RP) genes; however, the regulation of RP gene expression, especially in hematopoietic stem cells (HSCs), remains poorly understood. MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1 deficiency is driven by p53; however, the mechanisms of p53 activation remained unclear. Here, we describe the transcriptome of Mysm1-deficient mouse HSCs and identify MYSM1 genome-wide DNA binding sites. We establish a direct role for MYSM1 in RP gene expression and show a reduction in protein synthesis in Mysm1–/– HSCs. Loss of p53 in mice fully rescues Mysm1–/– anemia phenotype but not RP gene expression, indicating that RP gene dysregulation is a direct outcome of Mysm1 deficiency and an upstream mediator of Mysm1–/– phenotypes through p53 activation. We characterize a patient with a homozygous nonsense MYSM1 gene variant, and we demonstrate reduced protein synthesis and increased p53 levels in patient hematopoietic cells. Our work provides insights into the specialized mechanisms regulating RP gene expression in HSCs and establishes a common etiology of MYSM1 deficiency and ribosomopathy syndromes.
Authors
Jad I. Belle, HanChen Wang, Amanda Fiore, Jessica C. Petrov, Yun Hsiao Lin, Chu-Han Feng, Thi Tuyet Mai Nguyen, Jacky Tung, Philippe M. Campeau, Uta Behrends, Theresa Brunet, Gloria Sarah Leszinski, Philippe Gros, David Langlais, Anastasia Nijnik
Abstract
Multiple organ failure in sepsis is a progressive failure of several interdependent organ systems. Liver dysfunction occurs early during sepsis and is directly associated with patient death; however, the underlying mechanism of liver dysfunction is unclear. Platelet transfusion benefits patients with sepsis, and inhibition of complement activation protects liver function in septic animals. Herein, we explored the potential link between platelets, complement activation, and liver dysfunction in sepsis. We found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) exacerbated liver dysfunction in early sepsis. Platelet CLEC-2–deficient mice exhibited higher complement activation, more severe complement attack in the liver, and lower plasma levels of complement inhibitors at early time points after E. coli infection. Circulating monocytes expressed the CLEC-2 ligand podoplanin in early sepsis, and podoplanin binding induced release of complement inhibitors from platelets. Injection of complement inhibitors released from platelets reduced complement attack and attenuated liver dysfunction in septic mice. These findings indicate a new function of platelets in the regulation of complement activation during sepsis.
Authors
Zhanli Xie, Bojing Shao, Christopher Hoover, Michael McDaniel, Jianhua Song, Miao Jiang, Zhenni Ma, Fei Yang, Jingjing Han, Xia Bai, Changgeng Ruan, Lijun Xia
Abstract
Acute gastrointestinal Graft-versus-Host-Disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem-cell transplantation (alloSCT). It is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFNγ, IL-17A or GM-CSF, and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between T-helper states during priming in mesenteric lymph nodes (mLN) and effector function in the GI tract remain undefined at genome-scale. We applied scRNA-seq and computational modelling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T-cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we inferred an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 prior to gut migration and failed to express cytokines therein. Nevertheless, they exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq suggested divergence of allo-reactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogenous priming in vivo. These findings, the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.
Authors
Jessica A. Engel, Hyun Jae Lee, Cameron G. Williams, Rachel D. Kuns, Stuart Olver, Lianne I.M. Lansink, Megan S.F. Soon, Stacey B. Andersen, Joseph E. Powell, Valentine Svensson, Sarah A. Teichmann, Geoffrey R. Hill, Antiopi Varelias, Motoko Koyama, Ashraful Haque
Abstract
NOD-like receptor 12 (NLRP12) is a member of the nucleotide-binding domain and leucine-rich repeat containing receptor inflammasome family that plays a central role in innate immunity. We previously showed that DNA damage upregulated NLRP12 in hematopoietic stem cells (HSCs) of mice deficient in the DNA repair gene Fanca. However, the role of NLRP12 in HSC maintenance is not known. Here, we show that persistent DNA damage–induced NLRP12 improves HSC function in both mouse and human models of DNA repair deficiency and aging. Specifically, treatment of Fanca–/– mice with the DNA cross-linker mitomycin C or ionizing radiation induces NLRP12 upregulation in phenotypic HSCs. NLRP12 expression is specifically induced by persistent DNA damage. Functionally, knockdown of NLRP12 exacerbates the repopulation defect of Fanca–/– HSCs. Persistent DNA damage–induced NLRP12 was also observed in the HSCs from aged mice, and depletion of NLRP12 in these aged HSCs compromised their self-renewal and hematopoietic recovery. Consistently, overexpression of NLRP12 substantially improved the long-term repopulating function of Fanca–/– and aged HSCs. Finally, persistent DNA damage–induced NLRP12 maintains the function of HSCs from patients with FA or aged donors. These results reveal a potentially novel role of NLRP12 in HSC maintenance and suggest that NLRP12 targeting has therapeutic potential in DNA repair disorders and aging.
Authors
Qiqi Lin, Limei Wu, Zhilin Ma, Fabliha Ahmed Chowdhury,1, Habibul Hasan Mazumder, Wei Du
Abstract
Insufficient O2 supply is frequently associated with fetal growth restriction (FGR), a leading cause of perinatal mortality and morbidity. Although the erythrocyte is the most abundant and only cell type to deliver O2 in our body, its function and regulatory mechanism in FGR remain unknown. Here, we report that genetic ablation of mouse erythrocyte equilibrative nucleoside transporter 1 (eENT1) in dams, but not placentas or fetuses, results in FGR. Unbiased high-throughput metabolic profiling coupled with in vitro and in vivo flux analyses with isotopically labeled tracers led us to discover that maternal eENT1–dependent adenosine uptake is critical in activating AMPK by controlling the AMP/ATP ratio and its downstream target, bisphosphoglycerate mutase (BPGM); in turn, BPGM mediates 2,3-BPG production, which enhances O2 delivery to maintain placental oxygenation. Mechanistically and functionally, we revealed that genetic ablation of maternal eENT1 increases placental HIF-1α; preferentially reduces placental large neutral aa transporter 1 (LAT1) expression, activity, and aa supply; and induces FGR. Translationally, we revealed that elevated HIF-1α directly reduces LAT1 gene expression in cultured human trophoblasts. We demonstrate the importance and molecular insight of maternal eENT1 in fetal growth and open up potentially new diagnostic and therapeutic possibilities for FGR.
Authors
Seisuke Sayama, Anren Song, Benjamin C. Brown, Jacob Couturier, Xiaoli Cai, Ping Xu, Changhan Chen, Yangxi Zheng, Takayuki Iriyama, Baha Sibai, Monica Longo, Rodney E. Kellems, Angelo D’Alessandro, Yang Xia
Abstract
Introduction: PD-1 and PD-L1 have been studied interchangeably in the clinic as checkpoints to reinvigorate T cells in diverse tumor types. Data for biologic effects of checkpoint blockade in human premalignancy are limited. Methods: We analyzed the immunologic effects of PD-L1 blockade in a clinical trial of atezolizumab in patients with asymptomatic multiple myeloma (AMM), a precursor to clinical malignancy. Genomic signatures of PD-L1 blockade in purified monocytes and T cells in vivo were also compared to those following PD-1 blockade in lung cancer patients. Effects of PD-L1 blockade on monocyte-derived dendritic cells were analyzed to better understand its effects on myeloid antigen-presenting cells. Results: In contrast to anti-PD-1 therapy, anti-PD-L1 therapy led to a distinct inflammatory signature in CD14+ monocytes and increase in myeloid-derived cytokines (e.g. IL-18) in vivo. Treatment of AMM patients with atezolizumab led to rapid activation and expansion of circulating myeloid cells which persisted in the bone marrow. Blockade of PD-L1 on purified monocyte-derived dendritic cells (DCs) led to rapid inflammasome activation and synergized with CD40L-driven DC maturation, leading to greater antigen-specific T cell expansion. Conclusions: These data show that PD-L1 blockade leads to distinct systemic immunologic effects compared to PD-1 blockade in vivo in humans, particularly manifest as rapid myeloid activation. These findings also suggest an additional role for PD-L1 as a checkpoint for regulating inflammatory phenotype of myeloid cells and antigen-presentation in DCs, which may be harnessed to improve PD-L1-based combination therapies. Trial Registration: NCT02784483 Funding: NCI CA197603, CA238471, CA208328
Authors
Noffar Bar, Federica Costa, Rituparna Das, Alyssa Duffy, Mehmet K. Samur, Samuel S. McCachren, Scott Gettinger, Natalia Neparidze, Terri L. Parker, Jithendra Kini Bailur, Katherine E. Pendleton, Richa Bajpai, Lin Zhang, Mina L. Xu, Tara Anderson, Nicola Giuliani, Ajay K. Nooka, Hearn J. Cho, Aparna Raval, Mala Shanmugam, Kavita M. Dhodapkar, Madhav Dhodapkar
Abstract
The TAFRO clinical subtype of idiopathic multicentric Castleman disease (iMCD-TAFRO) is a rare hematologic illness involving episodic disease flares of thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly (TAFRO) and progressive multiple organ dysfunction. We previously showed that the mTOR signaling pathway is elevated in lymph nodes of iMCD-TAFRO patients and that an mTOR inhibitor is effective in a small cohort of patients. However, the upstream mechanisms, cell types, and mediators involved in disease pathogenesis remain unknown. Here, we developed a targeted approach to identify candidate cellular drivers and mechanisms in iMCD-TAFRO through cellular and transcriptomic studies. Using paired iMCD-TAFRO PBMC samples collected during flare and remission, we identified T cell activation and alterations in NK cell and monocyte subset frequencies during iMCD-TAFRO flare. These changes were associated with increased Type I IFN (IFN-I) response gene signatures across CD8+ T cells, NK cells, and monocytes. Finally, we found that IFN-β stimulation of monocytes and T cells from iMCD-TAFRO patient remission samples induced increased mTOR activation compared with healthy donors, and this was abrogated with either mTORC1 or JAK1/2 inhibition. The data presented here support a potentially novel role for IFN-I signaling as a driver of increased mTOR signaling in iMCD-TAFRO.
Authors
Ruth-Anne Langan Pai, Alberto Sada Japp, Michael Gonzalez, Rozena F. Rasheed, Mariko Okumura, Daniel Arenas, Sheila K. Pierson, Victoria Powers, Awo Akosua Kesewa Layman, Charlly Kao, Hakon Hakonarson, Frits van Rhee, Michael R. Betts, Taku Kambayashi, David C. Fajgenbaum
Abstract
We reported that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in OCLs (MVNP mice) are a Paget’s disease (PD) model, and that osteoclasts (OCLs) from PD patients and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1’s role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice and assessed OCL-IGF1’s effects on bone mass, bone formation rate, ephrinB2/EphB4 expression on OCLs and osteoblasts (OBs) and pagetic bone lesions (PDLs). Forty percent of MVNP mice but no MVNP/Igf1-cKO mice had PDLs. BV/TV was decreased 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO vs. MVNP mice with PDLs and by 45% vs. all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice vs. WT and MVNP mice. MVNP mice had increased ephrinB2 and EphB4 levels in OCLs/OBs vs. WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing ephrinB2/EphB4 expression in vivo, and that OCL-IGF1 may possibly contribute to normal bone remodeling.
Authors
Kazuaki Miyagawa, Yasuhisa Ohata, Jesus Delgado-Calle, Jumpei Teramachi, Hua Zhou, David W. Dempster, Mark A. Subler, Jolene J. Windle, John Chirgwin, G. David Roodman, Noriyoshi Kurihara
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