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The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2., Here, the authors generate a replication-competent VSV based vaccine expressing SARS-CoV-2 spike protein and show protection in the hamster model with one dose. Analysis of the antibody response in mice shows induction of neutralizing antibodies and suggests a desirable Th1-biased response to the vaccine.

INTRODUCTION: Most animal handling procedures are associated with injuries among veterinary staff and laboratory animal researchers. However, much of the currently available animal handling equipment is inadequate, limiting access to the treated animal or making workflow cumbersome. Moreover, restraining animals to perform procedures, such as blood collection or injection, elicits stress in both the animal and the worker. Herein, we present 4 home-built restraint and blood collection devices in extensive use in our institute. METHODS: Animal laboratory workers and experienced veterinarians regularly using the devices (n = 14) were asked to complete a survey ranking the contribution of the devices to worker safety and procedural efficiency. RESULTS: The overwhelming majority of responders (≥75%) associated all 4 devices with substantial improvements in worker safety and procedural efficiency. There were no reports of impaired workflow or safety when using the devices. DISCUSSION: Infection and exposure control may be implemented on various levels, including use of safer procedures, such as injection and blood collection devices. The presented intuitive handling and restraint devices allow the animal worker/researcher to perform various procedures safely and efficiently while eliciting less animal and worker stress. The devices can be easily adjusted to accommodate animal size and disease status. CONCLUSION: The current devices will serve as prototypes for design of devices for larger laboratory animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause for the ongoing COVID-19 pandemic1. The continued spread of SARS-CoV-2 along with the imminent flu season increase the probability of influenza-SARS-CoV-2 dual infection which might result in a severe disease. In this study, we examined the disease outcome of influenza A virus (IAV) and SARS-CoV-2 co-infection in K18-hACE2 mice. Our data indicates that IAV-infected mice are more susceptible to develop severe disease upon co-infection with SARS-CoV-2 two days post influenza infection. This co-infection results in severe morbidity and nearly uniform fatality as compared to the non-fatal influenza disease, or the partial fatality of SARS-CoV-2 alone. Co-infection was associated with elevated influenza viral load in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevented the severe disease and mortality. These data provide an experimental support that flu intervention by prior vaccination may be valuable in reducing the risk of sever Flu - SARS-CoV-2 comorbidity, and highlight the importance of vaccination.

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage (&ldquo, phage&rdquo, ) therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood.

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-ΔG-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-ΔG-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-ΔG-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters’ lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-ΔG-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

The generation of adaptive immunity by vaccination is usually a prolonged process that requires multiple dosing over several months. Hence, vaccines are administered for disease prevention a relatively long time prior to possible infection as opposed to post-exposure prophylaxis, which typically requires rapid intervention such as antibiotic therapy. The emergence of pathogens resistant to common antibiotic treatments has prompted the search for alternative therapeutic strategies. We previously demonstrated that vaccination of mice with the F1 capsular antigen of Yersinia pestis elicits specific and effective yet, unexpectedly, rapid anti-plague immunity. Here, we show by applying genetic and immunological approaches that the F1 antigen is targeted by peritoneal innate-like B1b cells that generate a prompt T-independent (TI) anti-F1 humoral response. The rapid F1-mediated defense response was diminished in Xid (Btkm) mice in which B1 cell numbers and activity are limited. Binding of fluorophore-labeled F1 to peritoneal B1b cells was detected as soon as 6 h post vaccination, emphasizing the high speed of this process. By assessing the ability to achieve rapid immunity with monomerized F1, we show that the natural polymeric structure of F1 is essential for (i) rapid association with peritoneal B1b cells, (ii) early induction of anti-F1 titers and (iii) rapid TI immunity in the mouse model of bubonic plague. These observations shed new light on the potential of novel as well as well-known protective antigens in generating rapid immunity and could be implemented in the rational design of future vaccines., Bubonic plague: Mechanisms of a rapid antibody response Full acquired immunity normally takes weeks to develop, however it’s possible to raise rapid (days) protective antibody responses against the bubonic plague bacterium Yersinia pestis by targeting its F1 capsular antigen. Emanuelle Mamroud and colleagues at the Institute of Biological Research in Israel use genetic and functional studies to understand the mechanism of this rapid anti-F1 antigen vaccine response. Subcutaneous vaccination with polymeric F1 antigen within hours results in selective binding to peritoneal B1b cells. Within seven days this generates an antibody response protective against fully virulent Y. pestis. This protection is spleen- and T-helper cell-independent but requires B1b cells. The form of F1 is also important, with monomerized antigen resulting in significantly inferior protection. These findings highlight the potential of post-exposure prophylaxis and the insights into its mechanism having wider implications for vaccine development.

Early sensing of bacterial infection and the immediate recruitment of neutrophils to the lung is a major and decisive stage of the innate immune response to pulmonary bacterial infections. This chapter details the preparation of lung tissue suspensions from mice infected intra-nasally (I.N.) with the plague bacterium Yersinia pestis to study in vivo neutrophil responses to the infection. The samples were used for the quantification of neutrophil levels and for the characterization of the pro-inflammatory response required for neutrophil recruitment to the lung. The specific requirements for performing the procedures under Biosafety Level 3 containment and the proper handling and sterilization of the samples are discussed.

We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis., Author summary We have previously shown that the NlpD lipoprotein, which is involved in the regulation of cell morphogenesis, is essential for virulence of the plague bacteria, Yersinia pestis. To uncover the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis as well as phenotypic and virulence evaluation analyses of the nlpD and related mutants. The study reveals a new role for the Y. pestis NlpD lipoprotein in iron assimilation and Tat system activity.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing contagious disease. In the plague mouse model, a single immunization with the EV76 live attenuated Y. pestis strain rapidly induced the expression of hemopexin and haptoglobin in the lung and serum, both of which are important in iron sequestration. Immunization against a concomitant lethal Y. pestis respiratory challenge was correlated with temporary inhibition of disease progression. Combining EV76-immunization and second-line antibiotic treatment, which are individually insufficient, led to a synergistic protective effect that represents a proof of concept for efficient combinational therapy in cases of infection with antibiotic-resistant strains.

Large numbers of rodents are often used in the study of disease progression and in the evaluation of its potential treatments. To avoid subjective observation and to minimize home cage interference, we developed a computerized home cage monitoring system (HCMS100) based on a standard cage rack adapted with a single laser beam and a detector mounted on each cage, enabling to monitor mice movements based on laser beam interruptions. This retrofit system provided continuous and uninterrupted monitoring of spontaneous movement of a group of mice in a home cage. Validity was evaluated using disease state induced by LPS modelling bacterial infection and by influenza virus. RESULTS: Spontaneous activity of different number of mice (2-8) per cage showed the expected circadian rhythm with increased activity during the night, and its extent dependent on the number of mice in the cage. Females and males show similar circadian rhythm. Intranasal LPS administration and pulmonary infection with live influenza virus resulted in major reduction of mice activity along disease progression. Increase in activity over time was a good indicator of the recovery process from both LPS exposure and the flu infection. CONCLUSIONS: HCMS100 was shown to be a reliable, inexpensive, easy to use system that requires no changes in the common housing of various experimental animals (mice, hamsters, rats etc.). With minimal intervention, HCMS100 provides a continuous record of group activity with clear pattern of circadian rhythm, allowing long term recording of home cage activity even in restricted access environments.

Early sensing of bacterial infection and the immediate recruitment of neutrophils to the lung is a major and decisive stage of the innate immune response to pulmonary bacterial infections. This chapter details the preparation of lung tissue suspensions from mice infected intra-nasally (I.N.) with the plague bacterium Yersinia pestis to study in vivo neutrophil responses to the infection. The samples were used for the quantification of neutrophil levels and for the characterization of the pro-inflammatory response required for neutrophil recruitment to the lung. The specific requirements for performing the procedures under Biosafety Level 3 containment and the proper handling and sterilization of the samples are discussed.

Pneumonic plague is an infectious disease characterized by rapid and fulminant development of acute pneumonia and septicemia that results in death within days of exposure. The causative agent of pneumonic plague, Yersinia pestis (Y. pestis), is a Tier-1 bio-threat agent. Parenteral antibiotic treatment is effective when given within a narrow therapeutic window after symptom onset. However, the non-specific "flu-like" symptoms often lead to delayed diagnosis and therapy. In this study, we evaluated inhalational gentamicin therapy in an infected mouse model as a means to improve antibiotic treatment efficacy. Inhalation is an attractive route for treating lung infections. The advantages include directly dosing the main infection site, the relative accessibility for administration and the lack of extensive enzymatic drug degradation machinery. In this study, we show that inhalational gentamicin treatment administered 24 h post-infection, prior to the appearance of symptoms, protected against lethal intranasal challenge with the fully virulent Y. pestis Kimberley53 strain (Kim53). Similarly, a high survival rate was demonstrated in mice treated by inhalation with another aminoglycoside, tobramycin, for which an FDA-approved inhaled formulation is clinically available for cystic fibrosis patients. Inhalational treatment with gentamicin 48 h post-infection (to symptomatic mice) was also successful against a Y. pestis challenge dose of 10 i.n.LD50. Whole-body imaging using IVIS technology demonstrated that adding inhalational gentamicin to parenteral therapy accelerated the clearance of Y. pestis from the lungs of infected animals. This may reduce disease severity and the risk of secondary infections. In conclusion, our data suggest that inhalational therapy with aerosolized gentamicin may be an effective prophylactic treatment against pneumonic plague. We also demonstrate the benefit of combining this treatment with a conventional parenteral treatment against this rapidly progressing infectious disease. We suggest the inhalational administration route as a clinically relevant treatment modality against pneumonic plague and other respiratory bacterial pathogens.

Prompt and effective elicitation of protective immunity is highly relevant for cases of rapidly deteriorating fatal diseases, such as plague, which is caused by Yersinia pestis. Here, we assessed the potential of a live vaccine to induce rapid protection against this infection. We demonstrated that the Y. pestis EV76 live vaccine protected mice against an immediate lethal challenge, limiting the multiplication of the virulent pathogen and its dissemination into circulation. Ex vivo analysis of Y. pestis growth in serum derived from EV76-immunized mice revealed that an antibacterial activity was produced rapidly. This activity was mediated by the host heme- and iron-binding proteins hemopexin and transferrin, and it occurred in strong correlation with the kinetics of hemopexin induction in vivo. We suggest a new concept in which a live vaccine is capable of rapidly inducing iron nutritional immunity, thus limiting the propagation of pathogens. This concept could be exploited to design novel therapeutic interventions.

The role of corticosterone (Cort), the immune system's major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment. © 2013 Macmillan Publishers Limited All rights reserved.

BACKGROUND Plague is initiated by Yersinia pestis, a highly virulent bacterial pathogen. In late stages of the infection, bacteria proliferate extensively in the internal organs despite the massive infiltration of neutrophils. The ineffective inflammatory response associated with tissue damage may contribute to the low efficacy of antiplague therapies during late stages of the infection. In the present study, we address the possibility of improving therapeutic efficacy by combining corticosteroid administration with antibody therapy in the mouse model of bubonic plague. METHODS Mice were subcutaneously infected with a fully virulent Y. pestis strain and treated at progressive stages of the disease with anti-Y. pestis antibodies alone or in combination with the corticosteroid methylprednisolone. RESULTS The addition of methylprednisolone to antibody therapy correlated with improved mouse survival, a significant decrease in the amount of neutrophils and matrix metalloproteinase 9 in the tissues, and the mitigation of tissue damage. Interestingly, the combined treatment led to a decrease in the bacterial loads in infected organs. CONCLUSIONS Corticosteroids induce an unexpectedly effective antibacterial response apart from their antiinflammatory properties, thereby improving treatment efficacy.

Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1+/c-Kit+/Lin− cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell–rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1+/c-Kit+/Lin− cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma . The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.

The signal-recognition particle (SRP) mediates the translocation of membrane and secretory proteins across the endoplasmic reticulum upon interaction with the SRP receptor. In trypanosomes, the main RNA molecule is the spliced-leader (SL) RNA, which donates the SL sequence to all messenger RNA through trans-splicing. Here, we show that RNA interference silencing of the SRP receptor (SRalpha) in Trypanosoma brucei caused the accumulation of SRP on ribosomes and triggered silencing of SL RNA (SLS). SLS was elicited due to the failure of the SL RNA-specific transcription factor tSNAP42 to bind to its promoter. SL RNA reduction, in turn, eliminated mRNA processing and resulted in a significant reduction of all mRNA tested. SLS was also induced under pH stress and might function as a master regulator in trypanosomes. SLS is reminiscent of, but distinct from, the unfolded protein response and can potentially act as a new target for parasite eradication.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1+/c-Kit+/Lin− (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P1, or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit+/Lin− cells expressing surface S1P1 in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin+ mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P1 axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal infection (i.n.) of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 hours post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.

The hallmark of hematopoietic stem and progenitor cells (HSPCs) is their motility, which is essential for their function, such as recruitment upon demand. Stromal Derived Factor-1 (SDF-1, CXCL12) and its major receptor CXCR4 play major roles in stem cell motility and development. In vitro migration assays, implicating either gradients or cell surface-bound forms of SDF-1, are easy to perform and provide vital information regarding directional and random stem cell motility, which correlate with their repopulation potential in clinical and experimental transplantations. In vivo stem cell homing to the bone marrow, their retention, engraftment, and egress to the circulation, all involve SDF-1/CXCR4 interactions. Finally, other stem cell features such as stem cell survival and proliferation, are also dependent on the SDF-1/CXCR4 axis.

The hallmark of hematopoietic stem and progenitor cells (HSPCs) is their motility, which is essential for their function, such as recruitment upon demand. Stromal Derived Factor-1 (SDF-1, CXCL12) and its major receptor CXCR4 play major roles in stem cell motility and development. In vitro migration assays, implicating either gradients or cell surface-bound forms of SDF-1, are easy to perform and provide vital information regarding directional and random stem cell motility, which correlate with their repopulation potential in clinical and experimental transplantations. In vivo stem cell homing to the bone marrow, their retention, engraftment, and egress to the circulation, all involve SDF-1/CXCR4 interactions. Finally, other stem cell features such as stem cell survival and proliferation, are also dependent on the SDF-1/CXCR4 axis.

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1–MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF–mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti–MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP–deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF–induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.

Abstract 553 Egress of hematopoietic stem and progenitor cells (HSPC) from the bone marrow (BM) reservoir to the circulation in steady state conditions is a key requirement for normal hematopoiesis. It is dramatically enhanced by G-CSF-induced mobilization, which is widely used for clinical HSPC transplantation. An interplay between cytokines, chemokines (mainly SDF-1 (CXCL12) and its major receptor CXCR4), adhesion molecules, matrix metalloproteinases and neurotransmitters, tightly regulate HSPC egress and mobilization. Recent observations indicate an essential role for sphingolipids, and particularly sphingosine-1-phosphate (S1P) and its major receptor S1P1 in leukocyte trafficking in vivo. Furthermore, several pharmacological agents that target S1P and S1P1 attenuate development of autoimmune and cardiovascular diseases as well as cancer. Based on these findings, we hypothesized that HSPC motility, both in steady state and in stress-induced conditions, is regulated by S1P/S1P1 signaling. We found that cells expressing S1P1 receptor are mainly located near sinusoids in the murine BM, suggesting involvement of S1P/S1P1 axis in HSPC steady state egress. To identify the role of S1P1 in HSPC homeostatic release, we injected mice with the inhibitor FTY720 and discovered a significant decrease in primitive Sca-1+/c-Kit+/Lineage- (SKL) cell numbers in the peripheral blood along with their accumulation in the BM, 24 hr post a single i.p injection. To examine the S1P/S1P1 axis involvement in stress induced mobilization, we tested S1P levels following G-CSF administration. S1P concentrations were decreased in BM supernatants and increased in the peripheral blood, suggesting the formation of a gradient towards the blood, with a potential HSPC mobilization capacity. Accordingly, a 5-fold decreased transcription level of sphingosine kinase 1 (Sphk1, S1P producing enzyme) and a milder increased transcription level of sphingosine phosphatase 1 (SPP1, S1P degrading enzyme) were observed in the BM of G-CSF treated mice. These changes in both S1P modulating enzymes expression levels were mediated by mTOR signaling, independent of the PI3K pathway. Another effect of G-CSF mobilization was enhancing the percentage of BM HSPC expressing surface S1P1 receptor, which was abolished upon inhibition of mTOR by Rapamycin. These findings imply that the reduction in S1P BM levels enabled increased S1P1 receptor expression and HSPC recruitment to the blood. Co-injections of FTY720 with G-CSF revealed decreased numbers of primitive SKL and immature colony-forming cells in the blood, indicating reduced HSPC mobilization. Accordingly, administration of G-CSF to Sphk1 KO mice, which have low S1P plasma concentrations, led to decreased mobilization of primitive SKL cells and progenitors to the blood. We also investigated the cross talk between S1P/S1P1 and SDF-1/CXCR4 axes. Disruption of the S1P/S1P1 axis during G-CSF administration (by co-injections of FTY720 or by using Sphk1 KO mice) reduced HSPC mobilization however, BM mononuclear cells obtained from these mice exhibited enhanced migration to a gradient of SDF-1 in vitro. These results imply that SDF-1/CXCR4 activation is not sufficient for HSPC mobilization. Previously, we have shown that CXCR4 neutralizing antibodies co-administrated on days 4 and 5 of G-CSF treatment, significantly but not completely inhibited HSPC mobilization (Petit et al., Nat Immunol, 2002). Interestingly, such treatment in Sphk1 KO mice completely inhibited mobilization and increased primitive SKL cells in the BM. These results suggest that S1P/S1P1 axis has an important role in parallel to SDF-1/CXCR4 axis during stress-induced mobilization since inactivation of both pathways resulted in total abrogation of HSPC recruitment to the blood. Finally, we show that S1P can inhibit SDF-1 transcription in murine BM stromal cells via activation of the p38/Akt/mTOR signaling pathway. Since SDF-1 reduction in the BM is essential for HSPC mobilization, S1P-induced inhibition of its transcription allows the progenitor cells to detach and migrate. Taken together, our findings reveal involvement of S1P and its major receptor S1P1 in HSPC egress and stress-induced mobilization. These findings may help broaden our understanding regarding the mechanisms behind HSPC motility and thus improve clinical mobilization protocols and drug development based on targeting the S1P/S1P1 axis. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

Abstract 840 Hematopoietic stem and progenitor cell (HSPC) regulation involves the integration of signals emanating from the nervous system and various bone marrow (BM) cells. Stress signals are produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis via secretion of corticosterone (Cort in mice is the equivalent to cortisol in humans). The major stress hormone, Cort, is secreted in a circadian manner and is known to cause bone loss in supraphysiologic levels. However, whether Cort plays a role in regulation of HSPC and their interactions with the BM microenvironment is currently unknown. Moreover, potentially undesired effects of clinical glucocorticoid administration on HSPC function have not been investigated. We hypothesized that Cort acts in a circadian and dose dependent manner to orchestrate and integrate HSPC function and bone turnover, which are essential for host defense and homeostasis. To determine the roles of circadian Cort oscillations and effects of aberrant Cort regulation on HSPC biology, we utilized CRFR1-/- (R1KO) mice, which lack corticotropin releasing factor receptor 1, a known HPA axis receptor. These mice lack circadian Cort rhythms and have only residual plasma Cort levels. We found that R1KO mice have higher levels of hematopoietic progenitors and the more primitive SKL cells in their BM and blood compared to WT mice. Cort levels measured in the BM of R1KO mice were continuously low, contrasted to the normal and circadially oscillating levels detected in WT mice. As previously established, interactions of the chemokine SDF-1 (CXCL12), the pivotal regulator of stem cell migration and quiescence, with its major receptor CXCR4, are perturbed by various immune-mediated stress signals. Interestingly, the levels of SDF-1 were constantly high in the BM of R1KO mice with no typical circadian peaks. Furthermore, SKL cell release to the blood, expected to follow BM SDF-1-circadian oscillations, as previously shown by Frenette's group, was also constantly increased. Concomitantly, CXCR4 expression in R1KO SKL cells was reduced and the cells demonstrated higher proliferation rates with superior long-term repopulation capacity over WT transplanted cells. Rescue treatment of R1KO mice attained by adding Cort to their drinking water restored their BM SKL and SDF-1 levels to the basal values of WT mice. Increasing Cort levels continuously in the drinking water of WT mice diminished their BM phenotype, including long-term engraftment potential. In vitro cultures of normal BM cells yielded higher colony formation capacity when stimulated with low Cort levels (10-8M). Conversely, high Cort levels (10-5M) inhibited colony formation. Next, we examined microenvironmental components with HSPC support potential. R1KO mice had more primitive stromal cells (CD45-/CD11b-/CD29+/Sca-1+) in their BM. Expression of osteocalcin and osterix, genes characterizing mature osteoblasts, was reduced. Osteoclast maturation was also attenuated, as indicated by increased Gr-1-/CD11b-/c-Kit+ monocyte precursors and reduced transcription of the genes RANK and cathepsin K, both associated with mature osteoclasts and bone resorption. R1KO mice had fewer active osteoclasts in their bones and lower bone turnover rates as indicated by calcein/alizarin bone labeling. In addition, μCT analyses demonstrated a dramatic reduction in femoral trabecules and bone volume. Looking for mediating mechanisms, we found that R1KO BM mononuclear cells highly express Notch1 and its target gene Hes1, known to be involved in regulation of stem cell self renewal. Both genes reverted back to normal expression levels after adding Cort to the drinking water. The signaling Notch1 intracellular domain (NICD), a product of Notch1 cleavage by γ-secratase, was also upregulated in R1KO BM hematopoietic and stromal precursors. Finally, inhibition of Notch1 activation by adding the γ-secratase inhibitor DAPT to BM cultures prevented the stimulating effect of low Cort on colony formation, suggesting that this effect is mediated by Notch1 signaling. Taken together, our study suggests that abolishment of circadian Cort production coupled with continuous low Cort levels increases HSPC proliferation directly and indirectly via Notch1 signaling, while impairing bone maintenance and structure. We propose Cort oscillations and levels to be critical for integrating balanced bone turnover and HSPC function. Disclosures: No relevant conflicts of interest to declare.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor–induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell–derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1+/c-Kit+/Lin− cells and interfered with their enhanced chemotactic migration to stromal cell–derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met–mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.