Synovial 5-Lipoxygenase-Derived Oxylipins Define a Lympho-Myeloid-Enriched Synovium.

OBJECTIVE: Oxylipins are bioactive lipids derived from polyunsaturated fatty acids (PUFAs) that modulate inflammation and may remain overexpressed in refractory synovitis. In plasma, they could also be biomarkers of synovial pathology. The aim of this study is to determine if synovial oxylipins in inflamed joints correlate with plasma oxylipins and with synovial histologic patterns. METHODS: Patients with established rheumatoid or psoriatic arthritis with active disease despite treatment were recruited, and paired synovial tissue (ST) and plasma were collected. Oxylipins were determined by liquid chromatography with tandem mass spectrometry and were classified into groups according to their PUFA precursor and enzyme. The expression of CD20, CD68, CD3, and CD138 was obtained to describe synovial histology. Cell-specific expression of oxylipin-related genes was identified by examining available synovial single-cell RNA sequencing data. RESULTS: We included a total of 32 ST and 26 paired-plasma samples. A total of 71 oxylipins were identified in ST, but only 24 were identified in plasma. Only levels of 9,10-dihydroxyoctadecenoic acid and tetranor-Prostaglandin FM had a significant positive correlation between plasma and ST. Several oxylipins and oxylipin-related genes were differentially expressed among synovial phenotypes. Specifically, several 5-lipoxygenase (LOX)-derived oxylipins were statistically elevated in the lympho-myeloid phenotype and associated with B cell expression in rheumatoid arthritis samples. CONCLUSION: The lack of correlation between ST and plasma oxylipins suggests that ST lipid profiling better characterizes active pathways in treated joints. Synovial 5-LOX-derived oxylipins were highly expressed in lympho-myeloid-enriched synovium. Combination therapy with 5-LOX inhibitors to improve refractory inflammation may be needed in patients with this histologic group.

CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Protocol for the analysis of hematopoietic lineages in the whole kidney marrow of adult zebrafish.

The whole kidney marrow (WKM) is the site for hematopoiesis in the adult zebrafish. Here, we present a protocol for analyzing hematopoietic lineages in the WKM of adult zebrafish. We describe steps for the isolation of hematopoietic cells from the WKM, the downstream analysis of total marrow cellularity, and analysis of cell populations by flow cytometry. We then detail procedures for May-Grünwald-Giemsa staining for analysis of cellular morphology and phenotyping. For complete details on the use and execution of this protocol, please refer to Mahony et al.1.

Dual inhibition of glycolysis and glutaminolysis for synergistic therapy of rheumatoid arthritis.

BACKGROUND: Synovial fibroblasts in rheumatoid arthritis (RAFLS) exhibit a pathological aberration of glycolysis and glutaminolysis. Henceforth, we aimed to investigate if dual inhibition of these pathways by phytobiological compound c28MS has the potential of synergistic therapy for arthritis by targeting both glucose and glutamine metabolism. METHODS: The presence of HK2 and GLS across various cell types and associated gene expression in human synovial cells and a murine model of arthritis was evaluated by scRNA-seq. The metabolic profiling of RAFLS cells was done using H1-nuclear magnetic resonance spectroscopy under glycolytic and glutaminolytic inhibitory conditions by incubating with 3-bromopyruvate, CB839, or dual inhibitor c28MS. FLS functional analysis was conducted under similar conditions. ELISA was employed for the quantification of IL-6, CCL2, and MMP3. K/BxN sera was administered to mice to induce arthritis for in vivo arthritis experiments. RESULTS: scRNA-seq analysis revealed that many fibroblasts expressed Hk2 along with Gls with several genes including Ptgs2, Hif1a, Timp1, Cxcl5, and Plod2 only associated with double-positive fibroblasts, suggesting that dual inhibition can be an attractive target for fibroblasts. Metabolomic and functional analysis revealed that c28MS decreased the aggressive behavior of RAFLS by targeting both upregulated glycolysis and glutaminolysis. c28MS administered in vivo significantly decreased the severity of arthritis in the K/BxN model. CONCLUSION: Our findings imply that dual inhibition of glycolysis and glutaminolysis could be an effective approach for the treatment of RA. It also suggests that targeting more than one metabolic pathway can be a novel treatment approach in non-cancer diseases.

Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes.

Background: Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. Methods: HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Results: Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Conclusion: Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency.

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies.

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow-stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFß stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics. See related commentary by Derecka and Crispino, p. 263. This article is highlighted in the In This Issue feature, p. 247.

Hapln1b, a central organizer of the ECM, modulates kit signaling to control developmental hematopoiesis in zebrafish.

During early vertebrate development, hematopoietic stem and progenitor cells (HSPCs) are produced in hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand to the fetal liver and the caudal hematopoietic tissue, in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta must be degraded to enable HSPCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSPC specification has never been addressed. In this study, hapln1b, a key component of the ECM, was specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSPCs in the early embryo, showing that hapln1b is necessary, at the correct level, to specify HSPCs in the hemogenic endothelium. Furthermore, the expression of hapln1b was necessary to maintain the integrity of the ECM through its link domain. By combining functional analyses and computer modeling, we showed that kitlgb interacts with the ECM to specify HSPCs. The findings show that the ECM is an integral component of the microenvironment and mediates the cytokine signaling that is necessary for HSPC specification.

A connexin/ifi30 pathway bridges HSCs with their niche to dampen oxidative stress.

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.

Essential role for Gata2 in modulating lineage output from hematopoietic stem cells in zebrafish.

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.

Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis.

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.

How HSCs Colonize and Expand in the Fetal Niche of the Vertebrate Embryo: An Evolutionary Perspective.

Rare hematopoietic stem cells (HSCs) can self-renew, establish the entire blood system and represent the basis of regenerative medicine applied to hematological disorders. Clinical use of HSCs is however limited by their inefficient expansion ex vivo, creating a need to further understand HSC expansion in vivo. After embryonic HSCs are born from the hemogenic endothelium, they migrate to the embryonic/fetal niche, where the future adult HSC pool is established by considerable expansion. This takes place at different anatomical sites and is controlled by numerous signals. HSCs then migrate to their adult niche, where they are maintained throughout adulthood. Exactly how HSC expansion is controlled during embryogenesis remains to be characterized and is an important step to improve the therapeutic use of HSCs. We will review the current knowledge of HSC expansion in the different fetal niches across several model organisms and highlight possible clinical applications.

bif1, a new BMP signaling inhibitor, regulates embryonic hematopoiesis in the zebrafish.

Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function, si:ch73-299h12.2, which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 (bif1). We identified a bif1 ortholog in Sinocyclocheilus rhinocerous, another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function: bif1 codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis.

Embryonic Microglia Derive from Primitive Macrophages and Are Replaced by cmyb-Dependent Definitive Microglia in Zebrafish.

Microglia, the tissue-resident macrophages of the CNS, represent major targets for therapeutic intervention in a wide variety of neurological disorders. Efficient reprogramming protocols to generate microglia-like cells in vitro using patient-derived induced pluripotent stem cells will, however, require a precise understanding of the cellular and molecular events that instruct microglial cell fates. This remains a challenge since the developmental origin of microglia during embryogenesis is controversial. Here, using genetic tracing in zebrafish, we uncover primitive macrophages as the unique source of embryonic microglia. We also demonstrate that this initial population is transient, with primitive microglia later replaced by definitive microglia that persist throughout adulthood. The adult wave originates from cmyb-dependent hematopoietic stem cells. Collectively, our work challenges the prevailing model establishing erythro-myeloid progenitors as the sole and direct microglial precursor and provides further support for the existence of multiple waves of microglia, which originate from distinct hematopoietic precursors.

Oncostatin M and Kit-Ligand Control Hematopoietic Stem Cell Fate during Zebrafish Embryogenesis.

Understanding the molecular pathways controlling hematopoietic stem cell specification and expansion is a necessary milestone to perform regenerative medicine. Here, we used the zebrafish model to study the role of the ckit signaling pathway in this process. We show the importance of kitb/kitlgb signaling in the specification and expansion of hematopoietic stem cells (HSCs), in the hemogenic endothelium and caudal hematopoietic tissue (CHT), respectively. Moreover, we identified the zebrafish ortholog of Oncostatin M (osm) in the zebrafish genome. We show that the osm/osmr pathway acts upstream of kitb during specification of the hemogenic endothelium, while both pathways act synergistically to expand HSCs in the CHT. Moreover, we found that osm, in addition to its role in promoting HSC proliferation, inhibits HSC commitment to the lymphoid fate. Altogether, our data identified two cytokines, kitlgb and osm, secreted by the vascular niche, that control HSCs during early embryonic development.

tfec controls the hematopoietic stem cell vascular niche during zebrafish embryogenesis.

In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non-cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes.