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Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which-upon activation by the inflammasomes-translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice.

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Cell-lineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER + perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. β-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration. Therefore, quiescent Cxcl12-creER + BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration. Bone marrow stromal cells (BMSCs) lining sinusoidal blood vessels are mesenchymal cells whose function is critical for the skeleton. Here the authors show that quiescent CXCL12-expressing BMSCs can convert into a skeletal stem cell-like state, and differentiate into cortical bone osteoblasts only in response to injury.

Imaging Mass Cytometry (IMC) is an emerging multiplexed imaging technology for analyzing complex microenvironments using more than 40 molecularly-specific channels. However, this modality has unique data processing requirements, particularly for patient tissue specimens where signal-to-noise ratios for markers can be low, despite optimization, and pixel intensity artifacts can deteriorate image quality and downstream analysis. Here we demonstrate an automated content-aware pipeline, IMC-Denoise, to restore IMC images deploying a differential intensity map-based restoration (DIMR) algorithm for removing hot pixels and a self-supervised deep learning algorithm for shot noise image filtering (DeepSNiF). IMC-Denoise outperforms existing methods for adaptive hot pixel and background noise removal, with significant image quality improvement in modeled data and datasets from multiple pathologies. This includes in technically challenging human bone marrow; we achieve noise level reduction of 87% for a 5.6-fold higher contrast-to-noise ratio, and more accurate background noise removal with approximately 2 × improved F1 score. Our approach enhances manual gating and automated phenotyping with cell-scale downstream analyses. Verified by manual annotations, spatial and density analysis for targeted cell groups reveal subtle but significant differences of cell populations in diseased bone marrow. We anticipate that IMC-Denoise will provide similar benefits across mass cytometric applications to more deeply characterize complex tissue microenvironments. Multiplexed imaging technologies can reveal the complex cellular and molecular profiles of tissue. Here, the authors develop and implement a denoising pipeline to significantly enhance imaging mass cytometry quality and improve single-cell analyses.

Targeted deletion of the chemokine Cxcl12 in different bone marrow stromal cell populations shows that distinct niches exist in the bone marrow for haematopoietic stem cells and lineage-committed progenitors. Multiple stem cell niches in bone marrow The chemokine CXCL12 has an important role in maintaining haematopoietic stem cell (HSC) function. Two complementary papers in the issue of Nature study the effects of conditional deletion of Cxcl12 from candidate niche cells in the bone marrow and arrive at similar conclusions despite using different cre and Cxcl12 alleles. Lei Ding and Sean Morrison map CXCL12 expression in different (putative) niche components of the adult mouse bone marrow, showing that deletion of Cxcl12 from endothelial cells, but not Lepr – cre -targeted perivascular stromal cells, contributes to HSC maintenance. These niches do not overlap, indicating compartmentalization in the bone marrow microenvironment. Daniel Link and colleagues deleted Cxcl12 from candidate niche stromal cell populations and found that expression of CXCL12 from osterix-expressing stromal cells supports B-lymphoid progenitors and retains haematopoietic progenitor cells in the bone marrow, whereas its expression from stromal cells in the perivascular region supports HSCs. These insights into the complexity of the HSC niche are of relevance to work on the development of haematopoietic disease. Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts 1 , 2 and perivascular stromal cells including endothelial cells 3 , CXCL12-abundant reticular cells 4 , 5 , leptin-receptor-positive stromal cells 6 , and nestin–green fluorescent protein (GFP)-positive mesenchymal progenitors 7 have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C–X–C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations 7 , 8 , 9 , 10 , 11 . Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of C xcl12 from nestin-negative mesenchymal progenitors using Prx1–cre ( Prx1 also known as Prrx1 ) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

In this study, investigators compared genome sequencing with cytogenetic analysis in 263 patients with acute myeloid leukemia or myelodysplastic syndromes. Prospective sequencing detected new genetic information that was not revealed by cytogenetic analysis in nearly 25% of the patients, which altered the risk category for most of these patients.

Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR+ chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2(-/-) and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2(-/-) neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR+ chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF-induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

Whole-genome sequencing of samples from seven subjects with secondary acute myeloid leukemia identified somatic mutations. These data, together with genotype analysis of the antecedent myelodysplastic syndromes (MDS), revealed the clonal evolution of MDS and secondary AML. The myelodysplastic syndromes, a heterogeneous group of diseases characterized by ineffective hematopoiesis, are the most common cause of acquired bone marrow failure in adults. 1 Secondary acute myeloid leukemia (AML) develops in approximately one third of persons with myelodysplastic syndromes. 2 Clinical discrimination between the myelodysplastic syndromes and secondary AML currently rests predominantly on cytomorphologic analysis, since patients with myelodysplastic syndromes have dysplastic hematopoiesis and a myeloblast count of less than 20%, whereas those with a myeloblast count of 20% or more have AML. Although considerable overlap exists between the spectrum of cytogenetic and molecular lesions seen in the two disorders, there . . .

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre-targeted MSCs prevented the development of myelofibrosis in both MPLW515L and Jak2V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPLW515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPLW515L was intact in Osx-Cre Smad4fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPLW515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

Hematopoietic clones harboring specific mutations may expand over time. However, it remains unclear how different cellular stressors influence this expansion. Here we characterize clonal hematopoiesis after two different cellular stressors: cytotoxic therapy and hematopoietic transplantation. Cytotoxic therapy results in the expansion of clones carrying mutations in DNA damage response genes, including TP53 and PPM1D . Analyses of sorted populations show that these clones are typically multilineage and myeloid-biased. Following autologous transplantation, most clones persist with stable chimerism. However, DNMT3A mutant clones often expand, while PPM1D mutant clones often decrease in size. To assess the leukemic potential of these expanded clones, we genotyped 134 t-AML/t-MDS samples. Mutations in non- TP53 DNA damage response genes are infrequent in t-AML/t-MDS despite several being commonly identified after cytotoxic therapy. These data suggest that different hematopoietic stressors promote the expansion of distinct long-lived clones, carrying specific mutations, whose leukemic potential depends partially on the mutations they harbor. Cellular stressors can impact clonal hematopoiesis. Here, the authors explore the impact of cytotoxic therapy and hematopoietic transplantation on clonal expansion, suggesting different stressors can promote expansion of distinct long-lived clones.