Splenic Hematopoiesis Research Articles

Hyperlipidemia-induced hematopoiesis is repressed by MLKL in endothelial cells of the splenic niche.

Dysregulation of the hematopoietic niche during hyperlipidemia facilitates pathologic leukocyte production, driving atherogenesis. Although definitive hematopoiesis occurs primarily in the bone marrow, during atherosclerosis this also occurs in the spleen. Cells of the bone marrow niche, particularly endothelial cells, have been studied in atherosclerosis, although little is known about how splenic endothelial cells respond to the atherogenic environment. Here we show unique dysregulated pathways in splenic compared to bone marrow endothelial cells during atherosclerosis, including perturbations of lipid metabolism and endocytic trafficking pathways. As part of this response, we identify the mixed lineage kinase domain-like (MLKL) protein as a repressor of splenic, but not bone marrow, myelopoiesis. Silencing MLKL in splenic endothelial cells results in inefficient endosomal trafficking and lipid accumulation, ultimately promoting the production of myeloid cells that participate in plaque development. These studies identify endocytic trafficking by MLKL as a key mechanism of splenic endothelial cell maintenance, splenic hematopoiesis and, subsequently, atherosclerosis.

Deciphering the differential impact of thrombopoietin/MPL signaling on hematopoietic stem/progenitor cell function in bone marrow and spleen

Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help to develop novel therapeutic approaches for hematopoietic disorders.

Persistent hypersplenism following successful hematopoietic stem cell transplantation: unraveling the cellular components of extramedullary residual disease and donor hematopoiesis—a case report

In this study, we report an unprecedented case of graft failure following hematopoietic stem cell transplantation (HSCT) in a patient with a myeloproliferative neoplasm (MPN) who had achieved bone marrow (BM) remission. We outline the intricate cellular components of splenic hematopoiesis, which are derived from both the recipient and the donor, underpinning the persistence of fibrosis. Our investigation characterizes the distinct cellular populations that play a pivotal role in graft exhaustion and introduces an unusual hypothesis regarding the perpetuation of a malignant niche in extramedullary sites within the context of HSCT.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

Hematopoiesis in the spleen after engraftment in unrelated cord blood transplantation evaluated by 18F-FLT PET imaging.

Cord blood is an important donor source for allogeneic hematopoietic stem cell transplantation (allo-HSCT), with its unique composition and quality of hematopoietic cells. The proliferation site and potency of infused hematopoietic stem cells in humans may vary between stem cell sources. We investigated this possibility in a prospective, exploratory study to assess hematopoietic dynamics using the radiopharmaceutical 3'-deoxy-3'-18F-fluorothymidine (18F-FLT), a thymidine analog used in positron emission tomography imaging, before allo-HSCT and on days 50 and 180 after allo-HSCT. We evaluated 11 patients with hematological malignancies who underwent allo-HSCT [five with peripheral blood stem cell transplantation (PBSCT) and six with unrelated cord blood transplantation (UCBT)]. Before allo-HSCT, 18F-FLT uptake did not differ between the two groups. At day 50, 18F-FLT uptake in the spleen was significantly greater in the UCBT group than in the PBSCT group (p = 0.0043), with no difference in whole-body bone marrow. At day 180, the differences in spleen uptake had diminished, and there were no differences between groups in whole-body bone marrow or the spleen, except for the sternum. The persistence of splenic hematopoiesis after engraftment in the UCBT group may reflect the complex systemic homing and proliferation mechanisms of cord blood hematopoietic cells.

IL-3 orchestrates ulcerative colitis pathogenesis by controlling the development and the recruitment of splenic reservoir neutrophils

Inflammatory bowel diseases (IBDs) are a global health issue with an increasing incidence. Although the pathogenesis of IBDs has been investigated intensively, the etiology of IBDs remains enigmatic. Here, we report that interleukin-3 (Il-3)-deficient mice are more susceptible and exhibit increased intestinal inflammation during the early stage of experimental colitis. IL-3 is locally expressed in the colon by cells harboring a mesenchymal stem cell phenotype and protects by promoting the early recruitment of splenic neutrophils with high microbicidal capability into the colon. Mechanistically, IL-3-dependent neutrophil recruitment involves CCL5+ PD-1high LAG-3high Tcells, STAT5, and CCL20 and is sustained by extramedullary splenic hematopoiesis. During acute colitis, Il-3-/- show, however, increased resistance to the disease as well as reduced intestinal inflammation. Altogether, this study deepens our understanding of IBD pathogenesis, identifies IL-3 as an orchestrator of intestinal inflammation, and reveals the spleen as an emergency reservoir for neutrophils during colonic inflammation.

Abstract 102: Hyperlipidemia-induced Hematopoiesis is Repressed by MLKL in Endothelial Cells of the Splenic Niche

The mixed lineage kinase domain-like protein (MLKL) is well-known for its role in the execution of necroptotic cell death. We showed that antisense oligonucleotide knockdown of MLKL ( Mlkl KD) in Apoe -knockout mice reduces plaque necrotic core, as predicted, but not total plaque area. Additionally, Mlkl KD expanded splenic hematopoietic stem and progenitor cells (HSPCs) and mature myeloid cells known to drive atherogenesis. Thus, we hypothesized that MLKL limits splenic hematopoiesis and thereby immune cell contributions to the growing atheroma. To test whether hematopoietic or non-hematopoietic MLKL expression was responsible for HSPC expansion, we transplanted Ldlr -knockout mice with Mlkl -knockout bone marrow and found no changes in hematopoiesis, suggesting that MLKL functions in other cells in the splenic niche. Flow cytometry and immunofluorescence staining during a time course of atherosclerosis revealed a decrease of splenic endothelial cells that was correlated with a reduction of MLKL endogenously or by Mlkl KD, suggesting that MLKL maintains splenic endothelial cells. Both in vivo and in vitro we observed increased lipid accumulation and concomitant defects in cell cycle in Mlkl KD splenic endothelial cells. Similar to our previous work in macrophages, Mlkl KD led to an accumulation of CHMP4B in cultured splenic endothelial cells, suggesting aberrant endocytic trafficking, which has also been linked to dysfunctional proliferation. Furthermore, there was greater production of immune cells derived from transplanted CD45.1 HSPCs in Mlkl KD recipient spleens. Co-culture with Mlkl KD splenic endothelial cells increased HSPC expression of pStat5, a marker of activation, which was coupled with an increase in myeloid colony formation, as compared to co-culture with control splenic endothelial cells. Remarkably no changes in HSPC regulation were found upon Mlkl KD in bone marrow endothelial cells. In conclusion, we demonstrate a novel role for MLKL in regulating the balance of HSPCs, specifically through preservation of splenic endothelial cells that repress hematopoiesis, highlighting the importance of spleen lipid metabolism and its impact on inflammation that potentiates atherogenesis.

Unique Presentations of Burkholderia gladioli Infections in Several Strains of Immunocompromised Mice.

Four strains of experimentally naïve mice (NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ [NSG], NOD. Cg- Rag1 tm1Mom Il2rg tm1Wjl /SzJ [NRG], B6.129S(Cg)-Stat1 tm1Dlv/J [STAT1 -/-], and B6.129S7- Ifngr1 tm1Agt/J[IFNγR -/-] housed in a barrier facility developed unusual and seemingly unrelated clinical signs. Young NSG/NRG mice (n = 49, mean age = 4 ± 0.4 mo) exhibited nonspecific clinical signs of moderate-to-severe lethargy, hunched posture, decreased body condition, and pallor. In contrast to the NSG/NRG mice, the STAT1-/- and IFNγ R-/- mice (n = 5) developed large subcutaneous abscesses on the head and neck. These mice were euthanized, and samples were collected for culture. NSG/NRG mice had moderate-markedly enlarged livers (20 of 49, 40%) and spleens (17 of 49, 35%). The livers contained multiple, variably-sized, tan regions throughout all lobes. Histology revealed necrotizing hepatitis (13 of 17, 77%), splenic and hepatic extramedullary hematopoiesis (17 of 17, 100%), glomerular histiocytosis (6 of 17, 35%), and metritis (6 of 11, 55%) with perivascular inflammation, suggesting hematogenous spread Differentials for these lesions included mouse hepatitis virus, ectromelia virus, Pseudomonas aeruginosa, Salmonella spp., and Clostridium piliforme. Burkholderia gladioli was cultured from liver lesions and subcutaneous abscesses and confirmed with 16S ribosomal RNA sequencing. After completing systematic testing of the environment, failure of the water autoclave cycle was suspected as the cause of the outbreak. To address the situation, individually ventilated racks were sanitized and new breeders were purchased; these actions dramatically reduced B. gladioli infections. The current literature contains few reports of B. gladioli infections in immunocompromised mice, and its typical presentation is torticollis and rolling. B. gladioli infection is a potential differential for subcutaneous abscesses, hepatitis, and splenomegaly in immunocompromised mice. Careful monitoring of sterilization techniques is essential to prevent such infections in a barrier facility.

Activated CMPs Act in Concert with the Spleen and Accelerate Hematopoietic Inflation after Transplantation

Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After transplantation, injected HSCs rapidly expand to reconstitute the entire blood system. This early post-transplant "hematopoietic inflation" is a crucial phase in sustaining life. However, the time course of hematopoietic inflation and the spatial transition within the whole body have remained a mystery. The discovery of colonies forming in the spleen early after HSC transplantation in the 1960s has been a strong advocate of the existence of HSCs, but biological role of transient nature of spleen colonies has not been understood. In this study, we elucidated the spatiotemporal dynamics of hematopoietic inflation in the same individual in a murine HSC transplantation model. Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize hematopoietic inflation in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and each progenitor cell has a different hematopoietic inflation pattern. We found that activated common myeloid progenitors (aCMPs) use the spleen as a site for hematopoietic inflation, producing much more cells early after transplantation than normal CMPs. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. In the allogeneic transplantation setting, universal exCMPs could sustain life for a long time by a serial transplantation. Human exCMPs could be easily expanded as well, and the clinical use of human exCMPs is highly anticipated as a supportive therapy after HSCT. Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that aCMPs and the spleen cooperated to contribute to hematopoietic inflation post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1418: EXPANDED COMMON MYELOID PROGENITORS ACT IN CONCERT WITH THE SPLEEN AND SUPPORT EARLY HEMATOPOIETIC RECOVERY

Background: Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After intravenous injection, HSCs are thought to home directly toward the bone marrow niche, and their homing mechanisms and interactions with the microenvironment have been intensively studied. However, the time course of the transplanted HSCs to reconstitute the entire blood system and the spatial transition within the whole body have remained a mystery. Aims: We aim to elucidate the spatiotemporal dynamics of hematopoiesis in the same individual in a murine HSC transplantation model. Methods: Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize HSC migration in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Results: Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and we found that common myeloid progenitors (CMPs) use the spleen as a site for hematopoiesis, producing many cells early after transplantation. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. Image:Summary/Conclusion: Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that CMPs and the spleen cooperated to contribute to early hematopoiesis post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery.

Causes of Mortality and Pathological Findings in European Hedgehogs (Erinaceus europaeus) Admitted to a Wildlife Care Centre in Southwestern France from 2019 to 2020

The European hedgehog (Erinaceus europaeus) is a nocturnal, solitary and non-territorial mammal endemic across Europe and central Asia. Due to population decline in recent decades in Europe, this species was declared as protected and registered as 'Least Concern' on the International Union for Conservation of Nature Red List of Threatened Species. Previous studies pointed to the possible contribution of human activities to hedgehog mortality but few reports provide a complete overview of the pathological processes associated with mortality of the European hedgehog. The aim of this study was to identify the causes of mortality and pathological findings in 35 wild adult European hedgehogs that died spontaneously or were euthanized at the Wildlife Care Centre of the Ecole Nationale Vétérinaire de Toulouse between 2019 and 2020. The main causes of mortality were trauma (41%) including collision (9%), predation (9%), trapping (6%) and trauma of unknown origin (17%). Infectious diseases associated with systemic and local bacterial infections accounted for 34% of the cases and included cutaneous wounds (23%), mandibular abscesses and botryomycosis (23%), exudative bronchopneumonia (9%), suppurative rhinitis (7%) and otitis (3%). One hedgehog (3%) had a large subcutaneous tumour consistent with a malignant peripheral nerve sheath tumour. Parasitism was associated with mortality in 11% of cases with severe debilitation, massive infestation and active larval migration. Incidental findings included splenic and hepatic extramedullary haematopoiesis (100% and 69%, respectively), pulmonary crenosomiasis (91%), gastrointestinal capillariasis (61%), renal lipofuscinosis (59%), chronic renal infarction and interstitial nephritis (50%). These data emphasize the need for further study of hedgehog mortality and of the genetic, behavioural and environmental factors that may contribute to population decline.

A Murine Model with JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm

IntroductionAlthough murine models have provided unequivocal evidence that the JAK2V617F mutation is able to cause myeloproliferative neoplasms (MPNs), there is significant heterogeneity in disease phenotypes between different murine models and none has been able to recapitulate both the myeloproliferative phenotype and cardiovascular pathology seen in patients with MPNs. In addition, these murine models were mostly followed for less than 3-9 months and how aging affects MPN disease progression has not been studied. Endothelial cells (ECs) are an essential component of the hematopoietic niche, and they have been shown to express the JAK2V617F mutation in patients with MPNs. In this study, we investigated how MPN progresses in the JAK2V617F-bearing vascular niche during aging.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells (including HSPCs) and vascular ECs (Tie2FF1).ResultsThe Tie2FF1 mice developed essential thrombocythemia to primary myelofibrosisdisease transformation with extramedullary splenic hematopoiesis during 18-month follow up. No evidence of leukemia transformation was observed in the Tie2FF1 mice. (Figure 1)Hematopoietic colony formation assays, flow cytometry analysis, and in vitro culture experiments revealed that there was a loss of both HSPC number and HSPC function in the marrow of old Tie2FF1 mice during aging, mimicking the advanced phases of myelofibrosis. In contrast, the spleen of old Tie2FF1 mice was able to maintain the expansion of JAK2V617F mutant hematopoiesis during aging and MPN disease progression. (Figure 2)These differences between marrow and spleen hematopoiesis in the old Tie2FF1 mice prompted us to investigate how aging affects the JAK2V617F mutant hematopoiesis differently in the marrow and spleen. We found that, although the JAK2V617F mutant HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) from old Tie2FF1 mice were more proliferative than wild-type HSCs in both the marrow and spleen, mutant marrow HSCs were more apoptotic and senescent than wild-type HSCs in the marrow while mutant spleen HSCs were relatively protected in the spleen. Examination of the hematopoietic vascular niche revealed that marrow ECs (CD45 -CD31 +) were significantly decreased in old Tie2FF1 mice compared to age-matched control mice; in contrast, spleen ECs were significantly expanded and less senescent in old Tie2FF1 mice compared to control mice. Therefore, the different vascular niche function of the marrow and spleen could contribute to the decreased marrow hematopoiesis and expanded splenic hematopoiesis we have observed in the Tie2FF1 mice during aging. (Figure 3)Previously, we reported that the Tie2FF1 mice developed spontaneous heart failure with thrombosis, vasculopathy, and cardiomyopathy at 20wk of age. Here, we followed the cardiovascular function of Tie2FF1 mice during aging. At 18mo of age, the Tie2FF1 mice continued to demonstrate a phenotype of dilated cardiomyopathy with a moderate but significant decrease in left ventricular ejection fraction and an increase in left ventricular volume and mass compared to age-matched control mice. Histology examination revealed spontaneous thrombosis in the right ventricle, pulmonary arteries, both main (epicardial) coronary arteries and scattered coronary arterioles (microvessels) in the old Tie2FF1 mice, while age-matched Tie2-cre control mice had no evidence of spontaneous thrombosis in their heart or lungs. Despite these cardiovascular dysfunctions, there was no difference in body weight nor was there any increased incidence of sudden death between the old Tie2FF1 mice and control mice. These findings suggested that there was a persistent but compensated cardiomyopathy and heart failure in the Tie2FF1 mice during aging. (Figure 4)ConclusionCompared to other MPN murine models reported so far, the Tie2FF1 mice is the first MPN murine model that faithfully recapitulated almost all the key features of the human MPN diseases. Considering the presence of the JAK2V617F mutation in microvascular ECs isolated from patients with MPNs and the recapitulation of all the key features of human MPN diseasesby the Tie2FF1 mice, the roles of endothelial dysfunction in the hematologic and cardiovascular pathogenesis of MPN shall be further investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Murine Model With JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm.

The myeloproliferative neoplasms (MPNs) are characterized by an expansion of the neoplastic hematopoietic stem/progenitor cells (HSPC) and an increased risk of cardiovascular complications. The acquired kinase mutation JAK2V617F is present in hematopoietic cells in a majority of patients with MPNs. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can also be detected in patients with MPNs. In this study, we show that a murine model with both JAK2V617F-bearing hematopoietic cells and JAK2V617F-bearing vascular ECs recapitulated all the key features of the human MPN disease, which include disease transformation from essential thrombocythemia to myelofibrosis, extramedullary splenic hematopoiesis, and spontaneous cardiovascular complications. We also found that, during aging and MPN disease progression, there was a loss of both HSPC number and HSPC function in the marrow while the neoplastic hematopoiesis was relatively maintained in the spleen, mimicking the advanced phases of human MPN disease. Different vascular niche of the marrow and spleen could contribute to the different JAK2V617F mutant stem cell functions we have observed in this JAK2V617F-positive murine model. These results indicate that the spleen is functionally important for the JAK2V617F mutant neoplastic hematopoiesis during aging and MPN disease progression. Compared to other MPN murine models reported so far, our studies demonstrate that JAK2V617F-bearing vascular ECs play an important role in both the hematologic and cardiovascular abnormalities of MPN.

Bone marrow and splenic metabolic activity by 18F-FDG PET/CT are associated with noncalcified coronary burden and lipid-rich necrotic core in psoriasis

Abstract Background/Introduction Psoriasis is an immune-mediated inflammatory skin condition with an increased risk of myocardial infarction (MI). Elevated bone marrow (BM) and splenic hematopoiesis occurs after MI. In stable patients without chronic inflammation, higher splenic hematopoiesis predicts major adverse cardiovascular events (MACE). Nevertheless, studies in humans investigating these relationships in states of chronic inflammation on coronary artery disease features associated with MACE are limited. Purpose To investigate the relationships between bone marrow and splenic metabolic activity by [18]-fluorodeoxyglucose (FDG) PET/CT and subclinical cardiovascular disease in psoriasis. Methods Healthy participants (N=30) and psoriasis participants (N=210) were age and sex matched. All participants underwent 18FDG PET/CT and CT angiography (Toshiba 320 slice). Coronary artery plaque characteristics were assessed using QAngio CT (Medis, The Netherlands) and lipid rich necrotic core (LRNC) was assessed using vascuCAP (Elucid Bioimaging, Boston, MA). For tissue metabolic activities target-to-background ratio (TBR) was calculated as the ratio of arterial and venous standardized uptake values (SUV). Results The psoriasis cohort was middle aged 49.2 (±SD 11.9) years and predominantly male (64%). Those with psoriasis vs. healthy participants had higher BM (1.58 (IQR 1.35–1.89) vs. 1.23 (IQR 1.14–1.35); p<0.001) and splenic (1.40 (IQR 1.21–1.66) vs.1.17 (IQR 1.11–1.26); p<0.001) metabolic activity. After adjustment for cardiovascular risk factors bone marrow metabolic activity was associated with total burden, non-calcified burden (NCB) and LRNC (β=0.36, β=0.39, β=0.26; all p<0.001) respectively. Similar findings were observed for splenic activity (β=0.33, β=0.36, β=0.36; all p<0.001). In ROC analysis, when comparing area under the curve, BM activity better incrementally predicted non-calcified burden and lipid rich necrotic core compared to splenic activity (Figure). Conclusions BM and splenic metabolic activity are increased in psoriasis. Both are associated with coronary artery disease but there was a slightly stronger association with BM activity compared to splenic activity, These findings warrant further study to understand immune mechanisms underlying these observations. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Heart, Lung and Blood Institute Intramural Research Program in Bethesda, Maryland Figure 1. Median values of NCB and LRNC were used to convert these continuous variables into dichotomous variables such that values ≤ median were designated as 0 and values >median were designated 1. Bone marrow model compared to base model and splenic model added incremental value in predicting NCB (p<0.0001) and LRNC (p=0.0003). Base model: Framingham risk score, lipid treatment, biologic therapy, homeostasis model assessment as an index of insulin resistance (HOMA-IR), low density lipoprotein (LDL).

Abstract MP09: MLKL (Mixed Lineage Kinase Domain-like Protein) Is A Novel Regulator Of The Splenic Microenvironment That Restricts Hematopoiesis During Atherosclerosis

During atherosclerosis, macrophages within the aorta undergo necroptosis, a mode of pro-inflammatory cell death that is triggered by the phosphorylation of RIPK1 & RIPK3 and leads to the activation of the mixed lineage kinase domain-like protein (MLKL) and cell lysis. Our previous work demonstrated that while inhibition of MLKL decreased necroptosis and necrotic core in the plaque, unlike RIPK1 & RIPK3 this inhibition did not lead to overall decrease in plaque area, suggesting that MLKL may in fact participate in other processes that drive atherosclerotic plaque development. Because circulating leukocytes are recruited and contribute to the growing atheroma, and these leukocytes are derived from hematopoietic stem and progenitor cells (HSPCs) found in both the bone marrow (BM) and the spleen, we investigated whether these hematopoietic reservoirs became dysregulated upon loss of MLKL during atherogenesis. To induce atherogenesis, Apoe -knockout mice ( Apoe -/-) were fed a high fat-high cholesterol diet for 16 weeks while receiving weekly subcutaneous administration of antisense oligonucleotides (ASOs) to knockdown MLKL expression. In the Apoe -/- mice receiving MLKL ASO we observed a 3.1-fold increase in splenomegaly compared to the control ASO, specifically with an expansion of the splenic red pulp, the area of splenic hematopoiesis. Furthermore, flow cytometry revealed a significant increase in myeloid HSPCs and mature myeloid populations in the spleen, but not the BM, after MLKL knockdown. However, no changes were observed in these hematopoietic measures after transplantation of Ldlr -knockout mice with MLKL-knockout BM, indicating that MLKL restricts hematopoiesis through its regulation of non-hematopoietic cells of the splenic niche. Indeed, upon MLKL knockdown in the Apoe -/- mice we observed a >50% reduction in splenic endothelial cells, which are required to promote HSPC quiescence and repress hematopoiesis. These data therefore demonstrate a novel role for MLKL on preserving splenic endothelial cells to limit immune cell development during atherosclerosis, and more generally, highlight the importance of the dysregulation of the splenic microenvironment to contributing to the inflammatory processes that drive atherosclerosis.

A Switch from Cell-Associated to Soluble PDGF-B Protects against Atherosclerosis, despite Driving Extramedullary Hematopoiesis.

Platelet-derived growth factor B (PDGF-B) is a mitogenic, migratory and survival factor. Cell-associated PDGF-B recruits stabilizing pericytes towards blood vessels through retention in extracellular matrix. We hypothesized that the genetic ablation of cell-associated PDGF-B by retention motif deletion would reduce the local availability of PDGF-B, resulting in microvascular pericyte loss, microvascular permeability and exacerbated atherosclerosis. Therefore, Ldlr-/-Pdgfbret/ret mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of Pdgfbret/ret mice, microvessel density and intraplaque hemorrhage were unexpectedly unaffected. Plaque macrophage content was reduced, which is likely attributable to increased apoptosis, as judged by increased TUNEL+ cells in Pdgfbret/ret plaques (2.1-fold) and increased Pdgfbret/ret macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, Pdgfbret/ret plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain Pdgfbret/ret collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in Pdgfbret/ret liver and adipose tissue. While dampening plaque inflammation, Pdgfbret/ret paradoxically induced systemic leukocytosis. The increased incorporation of 5-ethynyl-2′-deoxyuridine indicated increased extramedullary hematopoiesis and the increased proliferation of circulating leukocytes. We concluded that Pdgfbret/ret confers vascular and metabolic effects, which appeared to be protective against diet-induced cardiovascular burden. These effects were unrelated to arterial mesenchymal cell content or adventitial microvessel density and leakage. In contrast, the deletion drives splenic hematopoiesis and subsequent leukocytosis in hypercholesterolemia.

Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy

PurposeSelected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis.Methods and materialsf-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors.Results and conclusionsThe survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Development of multiple features of antigen-induced asthma pathology in a new strain of mast cell deficient BALB/c-KitW-sh/W-sh mice

Mast cell-deficient mice are widely used to identify and quantify contributions of mast cells to diverse biological responses in vivo, including allergic inflammation. However, despite the fact that scores of genes have been identified as modifiers of allergic inflammation, most mast cell-deficient models have been available only on a single genetic background. We transferred the KitW-sh allele onto the BALB/c background to generate BALB/c mast cell-deficient mice (BALB/c-KitW-sh/W-sh). BALB/c-KitW-sh/W-sh mice have dramatically reduced mast cell numbers (0–2% of wild type) in all tissues examined, as well as subtle hematologic differences from the corresponding wild type mice, including splenomegaly with evidence of increased splenic hematopoiesis. We examined in BALB/c-KitW-sh/W-sh mice models of allergic inflammation that are substantially diminished in C57BL/6-KitW-sh/W-sh mast cell-deficient mice. In a model of acute allergic inflammation, i.e., IgE-dependent passive cutaneous anaphylaxis, both ear swelling and leukocyte infiltration were largely or entirely absent in BALB/c-KitW-sh/W-sh mice. In contrast, in two different models of allergic airway inflammation, airway hyperresponsiveness, lung inflammation, and airway remodeling developed robustly in mast cell-deficient BALB/c-KitW-sh/W-sh mice. These results support the conclusion that the importance of mast cell contributions in various models of allergic inflammation may be at least partially determined by genetic background.

Abnormal folate metabolism causes age-, sex- and parent-of-origin-specific haematological defects in mice.

Key points Folate (folic acid) deficiency and mutations in folate‐related genes in humans result in megaloblastic anaemia.Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one‐carbon methyl groups for cellular methylation.In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late‐onset and sex‐specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia.Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific.Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one‐carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age‐, sex‐ and parent‐specific manner.