Sowing the Seeds of a Fruitful Harvest: Hematopoietic Stem Cell Mobilization

Hematopoietic stem cell transplantation is the only curative option for a number of malignant and non-malignant diseases. As the use of hematopoietic transplant has expanded, so too has the source of stem and progenitor cells. The predominate source of stem and progenitors today, particularly in settings of autologous transplantation, is mobilized peripheral blood. This review will highlight the historical advances which lead to the widespread use of peripheral blood stem cells for transplantation, with a look towards future enhancements to mobilization strategies

Defining the mechanism of prostaglandin E₂-enhanced hematopoietic stem and progenitor cell homing

Indiana University-Purdue University Indianapolis (IUPUI)Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of hematological disorders. However, to be effective, transplanted HSCs must efficiently “home” to supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution, or in some cases, bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation efficiency. We have previously shown that ex vivo treatment of mouse or human HSCs with 16-16 dimethyl PGE2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting in part from upregulation of surface CXCR4 expression. We now show that pulse-treatment of mouse or human HSPCs with dmPGE2 stabilizes HIF1α in HSPCs, and that similar treatment with the hypoxia mimetic DMOG produces analogous effects to dmPGE2 on HSPC CXCR4 expression and homing. This suggests that HIF1α is responsible for PGE2’s enhancing effects on HSPCs. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP), confirms the requirement of HIF1α for dmPGE2-enhanced migration and CXCR4 upregulation. Additionally, we confirm the requirement for HIF1α in dmPGE2-enhanced in vivo homing using a conditional knockout mouse model of HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after PGE2 treatment. Interestingly, we also observe an increase in the small GTPase Rac1 after dmPGE2 treatment, as well as a defect in PGE2-enhanced migration and CXCR4 expression in Rac1 knockout HSPCs. Using state-of-the-art imaging technology we, confirm an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment that likely explains enhanced sensitivity of PGE2-treated HSPCs to SDF-1. Taken together, these results define a precise mechanism through which ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing, and describe a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation

Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of immunologic disorders. For effective transplant, HSCs must traffic from the peripheral blood to supportive bone marrow niches. We previously showed that HSC trafficking can be enhanced by ex vivo treatment of hematopoietic grafts with 16-16 dimethyl prostaglandin E2 (dmPGE2). While exploring regulatory molecules involved in dmPGE2 enhancement, we found that transiently increasing the transcription factor hypoxia-inducible factor 1-α (HIF1α) is required for dmPGE2-enhanced CXCR4 upregulation and enhanced migration and homing of stem and progenitor cells and that pharmacologic manipulation of HIF1α is also capable of enhancing homing and engraftment. We also now identify the specific hypoxia response element required for CXCR4 upregulation. These data define a precise mechanism through which ex vivo pulse treatment with dmPGE2 enhances the function of hematopoietic stem and progenitor cells; these data also define a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation

Resident alveolar macrophageâ derived vesicular SOCS3 dampens allergic airway inflammation

Resident alveolar macrophages (AMs) suppress allergic inflammation in murine asthma models. Previously we reported that resident AMs can blunt inflammatory signaling in alveolar epithelial cells (ECs) by transcellular delivery of suppressor of cytokine signaling 3 (SOCS3) within extracellular vesicles (EVs). Here we examined the role of vesicular SOCS3 secretion as a mechanism by which AMs restrain allergic inflammatory responses in airway ECs. Bronchoalveolar lavage fluid (BALF) levels of SOCS3 were reduced in asthmatics and in allergenâ challenged mice. Ex vivo SOCS3 secretion was reduced in AMs from challenged mice and this defect was mimicked by exposing normal AMs to cytokines associated with allergic inflammation. Both AMâ derived EVs and synthetic SOCS3 liposomes inhibited the activation of STAT3 and STAT6 as well as cytokine gene expression in ECs challenged with ILâ 4/ILâ 13 and house dust mite (HDM) extract. This suppressive effect of EVs was lost when they were obtained from AMs exposed to allergic inflammationâ associated cytokines. Finally, inflammatory cell recruitment and cytokine generation in the lungs of OVAâ challenged mice were attenuated by intrapulmonary pretreatment with SOCS3 liposomes. Overall, AM secretion of SOCS3 within EVs serves as a brake on airway EC responses during allergic inflammation, but is impaired in asthma. Synthetic liposomes encapsulating SOCS3 can rescue this defect and may serve as a framework for novel therapeutic approaches targeting airway inflammation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/1/fsb220322-sup-0001-FigS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/2/fsb220322.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/3/fsb220322_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/4/fsb220322-sup-0005-TableS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/5/fsb220322-sup-0003-FigS3.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/6/fsb220322-sup-0004-FigS4.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154378/7/fsb220322-sup-0002-FigS2.pd

Alveolar macrophage- derived extracellular vesicles inhibit endosomal fusion of influenza virus

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM- EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.SynopsisExtracellular vesicles are emerging as homeostatic vectors, but poorly understood in influenza infection. Here, alveolar macrophage- derived extracellular vesicles inhibit influenza- endosome fusion in a strain- specific, and pH- dependent manner.Following initial infection of epithelial cells, the influenza virus traffics within host cell endosomes which undergo progressive acidification.Prior to gaining entry into the nucleus for its replication, influenza virus must fuse with endosome membranes- an event initiated at a strain- specific pH.Alveolar macrophages secrete extracellular vesicles which, when internalized by epithelial cells, lead to accelerated acidification of endosomes.Infection of epithelial cells by influenza strains which preferentially fuse with endosome membranes at high pH is inhibited by extracellular vesicles. Infection by influenza strains which fuse at low pH is unaffected by extracellular vesicles.Extracellular vesicles secreted from alveolar macrophages can promote acidification of endosomes in influenza virus- infected epithelial cells to inhibit viral replication.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/5/embj2020105057-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/4/embj2020105057_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/3/embj2020105057.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/2/embj2020105057-sup-0001-Appendix.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/1/embj2020105057.reviewer_comments.pd

Differential Stem and Progenitor Cell Trafficking by Prostaglandin E2

SUMMARY To maintain lifelong production of blood cells, hematopoietic stem cells (HSC) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSC (LT-HSC) reside in several, perhaps overlapping, niches that produce regulatory molecules/signals necessary for homeostasis and increased output following stress/injury 1–5. Despite significant advances in specific cellular or molecular mechanisms governing HSC/niche interactions, little is understood about regulatory function within the intact mammalian hematopoietic niche. Recently, we and others described a positive regulatory role for Prostaglandin E2 (PGE2) on HSC function ex vivo 6,7. While exploring the role of endogenous PGE2 we unexpectedly observed hematopoietic egress after nonsteroidal anti-inflammatory drug (NSAID) treatment. Surprisingly, this was independent of the SDF-1/CXCR4 axis. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin (OPN). Hematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in higher species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced EP4 receptor signaling. These results not only uncover unique regulatory roles for EP4 signaling in HSC retention in the niche but also define a rapidly translatable strategy to therapeutically enhance transplantation

Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a

Signed, sealed, delivered: microenvironmental modulation of extracellular vesicle-dependent immunoregulation in the lung

Unconventional secretion and subsequent uptake of molecular cargo via extracellular vesicles (EVs) is an important mechanism by which cells can exert paracrine effects. While this phenomenon has been widely characterized in the context of their ability to promote inflammation, less is known about the ability of EVs to transfer immunosuppressive cargo. Maintenance of normal physiology in the lung requires suppression of potentially damaging inflammatory responses to the myriad of insults to which it is continually exposed. Recently, our laboratory has reported the ability of alveolar macrophages (AMs) to secrete suppressors of cytokine signaling (SOCS) proteins within microvesicles (MVs) and exosomes (Exos). Uptake of these EVs by alveolar epithelial cells (AECs) resulted in inhibition of pro-inflammatory STAT activation in response to cytokines. Moreover, AM packaging of SOCS within EVs could be rapidly tuned in response to exogenous or AEC-derived substances. In this article we will highlight gaps in knowledge regarding microenvironmental modulation of cargo packaging and utilization as well as EV secretion and uptake. Advances in these areas are critical for improving understanding of intercellular communication in the immune system and for therapeutic application of artificial vesicles aimed at treatment of diseases characterized by dysregulated inflammation

Survivin Modulates Genes with Divergent Molecular Functions and Regulates Proliferation of Hematopoietic Stem Cells through Evi-1

The inhibitor of apoptosis protein Survivin regulates hematopoiesis, although its mechanisms of regulation of hematopoietic stem cells (HSCs) remain largely unknown. While investigating conditional Survivin deletion in mice, we found that Survivin was highly expressed in phenotypically defined HSCs and Survivin deletion in mice resulted in significantly reduced total marrow HSC and progenitor cells (HPC). Transcriptional analysis of Survivin−/− HSCs revealed altered expression of multiple genes not previously linked to Survivin activity. In particular, Survivin deletion significantly reduced expression of the Evi-1 transcription factor indispensable for HSC function, and the downstream Evi-1 target genes Gata2, Pbx1 and Sall2. The loss of HSCs following Survivin deletion and impaired long-term HSC repopulating function could be partially rescued by ectopic Evi-1 expression in Survivin −/− HSCs. These data demonstrate that Survivin partially regulates HSC function by modulating the Evi-1transcription factor and its downstream targets and identify new genetic pathways in HSCs regulated by Survivin