Inhibition of 15-PGDH Protects Mice from Immune-Mediated Bone Marrow Failure.

Aplastic anemia (AA) is a human immune-mediated bone marrow failure syndrome that is treated by stem cell transplantation for patients who have a matched related donor and by immunosuppressive therapy (IST) for those who do not. Responses to IST are variable, with patients still at risk for prolonged neutropenia, transfusion dependence, immune suppression, and severe opportunistic infections. Therefore, additional therapies are needed to accelerate hematologic recovery in patients receiving front-line IST. We have shown that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH) with the small molecule SW033291 (PGDHi) increases bone marrow (BM) prostaglandin E2 levels, expands hematopoietic stem cell (HSC) numbers, and accelerates hematologic reconstitution following murine BM transplantation. We now report that in a murine model of immune-mediated BM failure, PGDHi therapy mitigated cytopenias, increased BM HSC and progenitor cell numbers, and significantly extended survival compared with vehicle-treated mice. PGDHi protection was not immune-mediated, as serum IFN-γ levels and BM CD8+ T lymphocyte frequencies were not impacted. Moreover, dual administration of PGDHi plus low-dose IST enhanced total white blood cell, neutrophil, and platelet recovery, achieving responses similar to those seen with maximal-dose IST with lower toxicity. Taken together, these data demonstrate that PGDHi can complement IST to accelerate hematologic recovery and reduce morbidity in severe AA.

Type I IFNs drive hematopoietic stem and progenitor cell collapse via impaired proliferation and increased RIPK1-dependent cell death during shock-like ehrlichial infection.

Type I interferons (IFNα/ß) regulate diverse aspects of host defense, but their impact on hematopoietic stem and progenitor cells (HSC/HSPCs) during infection remains unclear. Hematologic impairment can occur in severe infections, thus we sought to investigate the impact of type I IFNs on hematopoiesis in a tick-borne infection with a virulent ehrlichial pathogen that causes shock-like disease. During infection, IFNα/ß induced severe bone marrow (BM) loss, blunted infection-induced emergency myelopoiesis, and reduced phenotypic HSPCs and HSCs. In the absence of type I IFN signaling, BM and splenic hematopoiesis were increased, and HSCs derived from Ifnar1-deficient mice were functionally superior in competitive BM transplants. Type I IFNs impaired hematopoiesis during infection by both limiting HSC/HSPC proliferation and increasing HSPC death. Using mixed BM chimeras we determined that type I IFNs restricted proliferation indirectly, whereas HSPC death occurred via direct IFNαR -mediated signaling. IFNαR-dependent signals resulted in reduced caspase 8 expression and activity, and reduced cleavage of RIPK1 and RIPK3, relative to Ifnar1-deficient mice. RIPK1 antagonism with Necrostatin-1s rescued HSPC and HSC numbers during infection. Early antibiotic treatment is required for mouse survival, however antibiotic-treated survivors had severely reduced HSPCs and HSCs. Combination therapy with antibiotics and Necrostatin-1s improved HSPC and HSC numbers in surviving mice, compared to antibiotic treatment alone. We reveal two mechanisms whereby type I IFNs drive hematopoietic collapse during severe infection: direct sensitization of HSPCs to undergo cell death and enhanced HSC quiescence. Our studies reveal a strategy to ameliorate the type I IFN-dependent loss of HSCs and HSPCs during infection, which may be relevant to other infections wherein type I IFNs cause hematopoietic dysfunction.

Hematopoietic stem cell loss and hematopoietic failure in severe aplastic anemia is driven by macrophages and aberrant podoplanin expression.

Severe aplastic anemia (SAA) results from profound hematopoietic stem cell loss. T cells and interferon gamma (IFNγ) have long been associated with SAA, yet the underlying mechanisms driving hematopoietic stem cell loss remain unknown. Using a mouse model of SAA, we demonstrate that IFNγ-dependent hematopoietic stem cell loss required macrophages. IFNγ was necessary for bone marrow macrophage persistence, despite loss of other myeloid cells and hematopoietic stem cells. Depleting macrophages or abrogating IFNγ signaling specifically in macrophages did not impair T-cell activation or IFNγ production in the bone marrow but rescued hematopoietic stem cells and reduced mortality. Thus, macrophages are not required for induction of IFNγ in SAA and rather act as sensors of IFNγ. Macrophage depletion rescued thrombocytopenia, increased bone marrow megakaryocytes, preserved platelet-primed stem cells, and increased the platelet-repopulating capacity of transplanted hematopoietic stem cells. In addition to the hematopoietic effects, SAA induced loss of non-hematopoietic stromal populations, including podoplanin-positive stromal cells. However, a subset of podoplanin-positive macrophages was increased during disease, and blockade of podoplanin in mice was sufficient to rescue disease. Our data further our understanding of disease pathogenesis, demonstrating a novel role for macrophages as sensors of IFNγ, thus illustrating an important role for the microenvironment in the pathogenesis of SAA.

Osteoblastic expansion induced by parathyroid hormone receptor signaling in murine osteocytes is not sufficient to increase hematopoietic stem cells.

Microenvironmental expansion of hematopoietic stem cells (HSCs) is induced by treatment with parathyroid hormone (PTH) or activation of the PTH receptor (PTH1R) in osteoblastic cells; however, the osteoblastic subset mediating this action of PTH is unknown. Osteocytes are terminally differentiated osteoblasts embedded in mineralized bone matrix but are connected with the BM. Activation of PTH1R in osteocytes increases osteoblastic number and bone mass. To establish whether osteocyte-mediated PTH1R signaling expands HSCs, we studied mice expressing a constitutively active PTH1R in osteocytes (TG mice). Osteoblasts, osteoclasts, and trabecular bone were increased in TG mice without changes in BM phenotypic HSCs or HSC function. TG mice had progressively increased trabecular bone but decreased HSC function. In severely affected TG mice, phenotypic HSCs were decreased in the BM but increased in the spleen. TG osteocytes had no increase in signals associated with microenvironmental HSC support, and the spindle-shaped osteoblastic cells that increased with PTH treatment were not present in TG bones. These findings demonstrate that activation of PTH1R signaling in osteocytes does not expand BM HSCs, which are instead decreased in TG mice. Therefore, osteocytes do not mediate the HSC expansion induced by PTH1R signaling. Further, osteoblastic expansion is not sufficient to increase HSCs.

Concise review: Current concepts in bone marrow microenvironmental regulation of hematopoietic stem and progenitor cells.

Hematopoietic stem cell (HSC) behavior is governed in large part by interactions of the blood system with the bone microenvironment. Increasing evidence demonstrates the profound role the local HSC microenvironment or niche plays in normal stem cell function, in therapeutic activation and in the setting of malignancy. A number of cellular and molecular components of the microenvironment have been identified thus far, several of which are likely to provide exciting therapeutic targets in the near future. Clinically effective strategies for niche manipulation, however, require careful study of the interaction of these niche components. Some of the key findings defining these regulatory interactions are explored in this concise review, with special emphasis on potential translational applications.

Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis.

Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. Although sex differences have been implicated in differential hematopoietic function in males versus females, the mediators responsible for these effects require further study. Here, we characterized hematopoiesis at a steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared with male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery, we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shape normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.

15-PGDH regulates hematopoietic and gastrointestinal fitness during aging.

Emerging evidence implicates the eicosanoid molecule prostaglandin E2 (PGE2) in conferring a regenerative phenotype to multiple organ systems following tissue injury. As aging is in part characterized by loss of tissue stem cells' regenerative capacity, we tested the hypothesis that the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) contributes to the diminished organ fitness of aged mice. Here we demonstrate that genetic loss of 15-PGDH (Hpgd) confers a protective effect on aging of murine hematopoietic and gastrointestinal (GI) tissues. Aged mice lacking 15-PGDH display increased hematopoietic output as assessed by peripheral blood cell counts, bone marrow and splenic stem cell compartments, and accelerated post-transplantation recovery compared to their WT counterparts. Loss of Hpgd expression also resulted in enhanced GI fitness and reduced local inflammation in response to colitis. Together these results suggest that 15-PGDH negatively regulates aged tissue regeneration, and that 15-PGDH inhibition may be a viable therapeutic strategy to ameliorate age-associated loss of organ fitness.

Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

CCR5 maintains macrophages in the bone marrow and drives hematopoietic failure in a mouse model of severe aplastic anemia.

Severe aplastic anemia (SAA) is an acquired, T cell-driven bone marrow (BM) failure disease characterized by elevated interferon gamma (IFNγ), loss of hematopoietic stem cells (HSCs), and altered BM microenvironment, including dysfunctional macrophages (MΦs). T lymphocytes are therapeutic targets for treating SAA, however, the underlying mechanisms driving SAA development and how innate immune cells contribute to disease remain poorly understood. In a murine model of SAA, increased beta-chemokines correlated with disease and were partially dependent on IFNγ. IFNγ was required for increased expression of the chemokine receptor CCR5 on MΦs. CCR5 antagonism in murine SAA improved survival, correlating with increased platelets and significantly increased platelet-biased CD41hi HSCs. T cells are key drivers of disease, however, T cell-specific CCR5 expression and T cell-derived CCL5 were not necessary for disease. CCR5 antagonism reduced BM MΦs and diminished their expression of Tnf and Ccl5, correlating with reduced frequencies of IFNγ-secreting BM T cells. Mechanistically, CCR5 was intrinsically required for maintaining BM MΦs during SAA. Ccr5 expression was significantly increased in MΦs from aged mice and humans, relative to young counterparts. Our data identify CCR5 signaling as a key axis promoting the development of IFNγ-dependent BM failure, particularly relevant in aging where Ccr5 expression is elevated.

IL-18R-mediated HSC quiescence and MLKL-dependent cell death limit hematopoiesis during infection-induced shock.

Severe infection can dramatically alter blood production, but the mechanisms driving hematopoietic stem and progenitor cell (HSC/HSPC) loss have not been clearly defined. Using Ixodes ovatus Ehrlichia (IOE), a tick-borne pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, type I and II interferons (IFNs) promoted loss of HSPCs via increased cell death and enforced quiescence. IFN-αß were required for increased interleukin 18 (IL-18) expression during infection, correlating with ST-HSC loss. IL-18 deficiency prevented BM aplasia and increased HSC/HSPCs. IL-18R signaling was intrinsically required for ST-HSC quiescence, but not for HSPC cell death. To elucidate cell death mechanisms, MLKL- or gasdermin D-deficient mice were infected; whereas Mlkl-/- mice exhibited protected HSC/HSPCs, no such protection was observed in Gsdmd-/- mice during infection. MLKL deficiency intrinsically protected HSCs during infection and improved hematopoietic output upon recovery. These studies define MLKL and IL-18R signaling in HSC loss and suppressed hematopoietic function in shock-like infection.

Polymer Microparticles Prolong Delivery of the 15-PGDH Inhibitor SW033291.

As the prevalence of age-related fibrotic diseases continues to increase, novel antifibrotic therapies are emerging to address clinical needs. However, many novel therapeutics for managing chronic fibrosis are small-molecule drugs that require frequent dosing to attain effective concentrations. Although bolus parenteral administrations have become standard clinical practice, an extended delivery platform would achieve steady-state concentrations over a longer time period with fewer administrations. This study lays the foundation for the development of a sustained release platform for the delivery of (+)SW033291, a potent, small-molecule inhibitor of the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) enzyme, which has previously demonstrated efficacy in a murine model of pulmonary fibrosis. Herein, we leverage fine-tuned cyclodextrin microparticles-specifically, ß-CD microparticles (ß-CD MPs)-to extend the delivery of the 15-PGDH inhibitor, (+)SW033291, to over one week.

Therapeutic targeting of 15-PGDH in murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by interstitial remodeling and pulmonary dysfunction. The etiology of IPF is not completely understood but involves pathologic inflammation and subsequent failure to resolve fibrosis in response to epithelial injury. Treatments for IPF are limited to anti-inflammatory and immunomodulatory agents, which are only partially effective. Prostaglandin E2 (PGE2) disrupts TGFß signaling and suppresses myofibroblast differentiation, however practical strategies to raise tissue PGE2 during IPF have been limited. We previously described the discovery of a small molecule, (+)SW033291, that binds with high affinity to the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and increases PGE2 levels. Here we evaluated pulmonary 15-PGDH expression and activity and tested whether pharmacologic 15-PGDH inhibition (PGDHi) is protective in a mouse model of bleomycin-induced pulmonary fibrosis (PF). Long-term PGDHi was well-tolerated, reduced the severity of pulmonary fibrotic lesions and extracellular matrix remodeling, and improved pulmonary function in bleomycin-treated mice. Moreover, PGDHi attenuated both acute inflammation and weight loss, and decreased mortality. Endothelial cells and macrophages are likely targets as these cell types highly expressed 15-PGDH. In conclusion, PGDHi ameliorates inflammatory pathology and fibrosis in murine PF, and may have clinical utility to treat human disease.

Hematopoietic Stem Cell Regulation by Type I and II Interferons in the Pathogenesis of Acquired Aplastic Anemia.

Aplastic anemia (AA) occurs when the bone marrow fails to support production of all three lineages of blood cells, which are necessary for tissue oxygenation, infection control, and hemostasis. The etiology of acquired AA is elusive in the vast majority of cases but involves exhaustion of hematopoietic stem cells (HSC), which are usually present in the bone marrow in a dormant state, and are responsible for lifelong production of all cells within the hematopoietic system. This destruction is immune mediated and the role of interferons remains incompletely characterized. Interferon gamma (IFNγ) has been associated with AA and type I IFNs (alpha and beta) are well documented to cause bone marrow aplasia during viral infection. In models of infection and inflammation, IFNγ activates HSCs to differentiate and impairs their ability to self-renew, ultimately leading to HSC exhaustion. Recent evidence demonstrating that IFNγ also impacts the HSC microenvironment or niche, raises new questions regarding how IFNγ impairs HSC function in AA. Immune activation can also elicit type I interferons, which may exert effects both distinct from and overlapping with IFNγ on HSCs. IFNα/ß increase HSC proliferation in models of sterile inflammation induced by polyinosinic:polycytidylic acid and lead to BM aplasia during viral infection. Moreover, patients being treated with IFNα exhibit cytopenias, in part due to BM suppression. Herein, we review the current understanding of how interferons contribute to the pathogenesis of acquired AA, and we explore additional potential mechanisms by which interferons directly and indirectly impair HSCs. A comprehensive understanding of how interferons impact hematopoiesis is necessary in order to identify novel therapeutic approaches for treating AA patients.