James Palis

Adult platelets are relatively enriched in immune related molecules compared to neonatal platelets, but neonatal platelets express some growth factors and enzymes at comparatively higher levels. This makes a prediction of platelet-immune cell interaction outcomes in neonates a challenge, as they are likely dependent on the cell type and tissue environment at the time of injury or infection. Our past studies revealed that the transfusion of adult but not neonatal platelets into thrombocytopenic neonatal mice led to an acute increase in monocyte trafficking. We have now found that neonatal, but not adult platelets, induce monocytes to a Myeloid Derived Suppressor Cell (MDSC) phenotype, typified by increased PD-L1, that limits T-cell activation in vitroand in vivo. Monocytes prior incubated with neonatal, but not adult platelets, or platelet releasates, limited T-cell activation in vitro. Using an in vivo asthma-like model we also found that the treatment of mice with monocytes prior incubated with neonatal platelet releasates limited T-cell activation in a asthma-like model. Platelet-driven effects were dependent on neonatal platelets producing more PGE2 that signaled through monocyte EP4. These studies indicate that neonatal platelets have immune limiting roles in the post-natal period by indirectly limiting T-cell responses, perhaps contributing to the adverse outcomes of platelet transfusions to neonates.

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using in vivo models, we uncover divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1, which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

In this issue of Blood Advances, Winward et al1 determined that (1) maturing erythroid progenitors normally undergo a progressive shortening of the cell cy

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using novel in vivo models, we uncover strikingly divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1 , which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1, a Polycomb Repressive Complex 1 member, is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts, its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts, which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy, along with repressive histone marks, are present at known BMI1 target genes, including the INK-ARF locus, consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications, including key regulators of cholesterol homeostasis, along with functional studies, suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses.

Myelodysplastic syndromes (MDS) are age-related diseases characterized by bone marrow (BM) dysfunction and an increased risk for developing acute leukemia. Although there is growing evidence that highlight the crucial role of the BM microenvironment (BMME) in MDS, the specific influence of inflammation on BMME changes and the potential benefits of targeting cytokines therapeutically remain to be elucidated. We previously found that interleukin-1 (IL-1) is a driver of aging phenotypes of the BMME and hematopoietic stem and progenitor cells (HSPCs). In this study, BM samples from patients with MDS demonstrated upregulated levels of IL-1 family cytokines, including IL-18. Using highly purified primary BM-derived mesenchymal stromal cells (MSCs), both IL-1b and IL-18 were found to exert direct effects on MSCs, thus influencing their ability to support HSPCs and erythroid progenitors. This confirms the significant involvement of both these IL-1 family cytokines in regulating the BM niche. Furthermore, targeting IL-1 receptor type 1 mitigated these aging phenotypes in older mice. We subsequently employed an age-appropriate murine model of MDS by transplanting NUP98-HOXD13 transgenic mice (NHD13Tg) cells into aged wild-type mice. Treatment with inhibitors that targeted IL-1 receptor-associated kinase 4 (IRAK4) and NOD-like receptor family pyrin domain containing 3 (NLRP3) reversed the proliferation of dysfunctional MSCs and enhanced their functionality. In addition, IRAK4 inhibition selectively suppressed MDS clonal cells while sparing non-MDS cells in the BM. These findings suggest that targeting IL-1 signaling holds promise for MDS treatment by addressing the underlying myeloid malignancy and restoring the altered BMME via BM-MSCs.

Tubular aggregate myopathy (TAM) is a heritable myopathy primarily characterized by progressive muscle weakness, elevated levels of creatine kinase (CK), hypocalcemia, exercise intolerance, and the presence of tubular aggregates (TAs). Here, we generated a knock-in mouse model based on a human gain-of-function mutation which results in a severe, early-onset form of TAM, by inducing a glycine-to-serine point mutation in the ORAI1 pore ( Orai1 ^G100S/+ or GS mice). By 8 months of age, GS mice exhibited significant muscle weakness, exercise intolerance, elevated CK levels, hypocalcemia, and robust TA presence. Unexpectedly, constitutive Ca ²⁺ entry in mutant mice was observed in muscle only during early development and was abolished in adult skeletal muscle, partly due to reduced ORAI1 expression. Consistent with proteomic results, significant mitochondrial damage and dysfunction was observed in skeletal muscle of GS mice. Thus, GS mice represent a powerful model for investigation of the pathophysiological mechanisms that underlie key TAM symptoms, as well as those compensatory responses that limit the damaging effects of uncontrolled ORAI1-mediated Ca ²⁺ influx.

Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short lived, it has been assumed that extravascular lung Mks are constantly “seeded” from the BM. To investigate lung Mk origins and how origin affects their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered via the oropharyngeal route. Labeled lung Mks were present for up to 4 months, while BM Mks had a lifespan of less than 1 week. In a parabiosis model, lung Mks were partially replaced over 1 month from a circulating source. Unlike tissue-resident macrophages, using MDS1 ^-Cre-ERT2 TdTomato mice, we found that lung Mks arose from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks were derived from a Flt3-independent lineage that did not go through a multipotent progenitor. CFSE labeling to track lung Mk–derived platelets showed that approximately 10% of circulating platelets were derived from lung-resident Mks at steady state, but in sterile thrombocytopenia this was doubled (~20%). Lung-derived platelets were similarly increased in a malaria infection model ( Plasmodium yoelii ) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3 ^– BM source, are long-lived, and contribute more platelets during thrombocytopenia.

We previously characterized lung megakaryocytes (Mks) as largely extravascular cells with an immune modulatory phenotype (Pariser et al., 2021). Because bone marrow (BM) Mks are relatively short lived, it is assumed that extravascular lung Mks are constantly ‘seeded’ from the BM, but there are no experimental data to validate this concept. To investigate lung Mk origin and how their origin may impact lung Mk functions, we developed novel models using CFSE dye delivered oropharyngeal (OP) and biotin labeling to specifically label lung Mks and identify lung Mk derived platelets. Labeled lung Mks were present for up to four months, while BM Mks had a less than 1 week lifespan. In a parabiosis model, lung Mks were only partially replaced by a circulating source over a 1-month time period. Unlike tissue resident lung macrophages, we determined using MDS1 ^-Cre-ERT2 TdTomato mice that lung Mks arise from a hematopoietic stem cell (HSC) source. However, studies with FlkSwitch mTmG mice showed that lung Mks originate from a Flt3-negative cell lineage, that does not go through a multipotent progenitor stage. CFSE labeling of lung cells enabled us to track lung Mk-derived platelets and we found that about 10% of circulating platelets at steady-state are lung resident Mk-derived, but in the context of sterile thrombocytopenia there was a doubling of lung Mk derived platelets (about 20%). Lung-derived platelets were similarly increased in a murine malaria infection model ( Plasmodium yoelii ) typified by chronic thrombocytopenia. Taken together, our studies indicate that lung Mks arise from a Flt3-negative, HSC-dependent pathway and contribute relatively more platelets during thrombocytopenia.