Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers

Adipose Tissue-Derived Mesenchymal Stem Cells Increase Skin Allograft Survival and Inhibit Th-17 Immune Response

Adipose tissue-derived mesenchymal stem cells (ADSC) exhibit immunosuppressive capabilities both in vitro and in vivo. Their use for therapy in the transplant field is attractive as they could render the use of immunosuppressive drugs unnecessary. The aim of this study was to investigate the effect of ADSC therapy on prolonging skin allograft survival. Animals that were treated with a single injection of donor allogeneic ADSC one day after transplantation showed an increase in donor skin graft survival by approximately one week. This improvement was associated with preserved histological morphology, an expansion of CD4+ regulatory T cells (Treg) in draining lymph nodes, as well as heightened IL-10 expression and down-regulated IL-17 expression. In vitro, ADSC inhibit naïve CD4+ T cell proliferation and constrain Th-1 and Th-17 polarization. In summary, infusion of ADSC one day post-transplantation dramatically increases skin allograft survival by inhibiting the Th-17 pathogenic immune response and enhancing the protective Treg immune response. Finally, these data suggest that ADSC therapy will open new opportunities for promoting drug-free allograft survival in clinical transplantation

Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa

Conditional mutagenesis in the immune system: targeting the expression of the iCre2 recombinase to neutrophils and macrophages

Conditional mutagenesis allows the introduction of tissue specific mutations in the mouse and is of crucial importance in converting genome sequence information into functional data for biomedical research. Mice expressing the Cre recombinase in a spatially controlled manner are essential in creating such conditional knock-outs. A wide variety of Cre mice have been generated, but there is a distinct lack of models expressing the recombinase faithfully and at high levels in cells of the innate immune system. To address this need, three target genes, Itgb2l, Marco and Msr1, were chosen to create novel neutrophil and macrophage specific knock-in models harbouring iCre2, a recombinase engineered for increased expression levels. Two strategies were employed. Initially gene specific bacterial artificial chromosomes in which the iCre2 fragment replaced the endogenous translation start codon were created by Red/ET recombineering. Utilization of these BAC vectors for embryonic stem cell targeting successfully created knock-ins but the identification of homologous recombinants was complicated by the vectors’ large size. As the discovery of mutations impeding iCre2 functionality in the knock-in lines necessitated repeating the vector creation process, novel shorter vectors were designed. These vectors achieved targeting frequencies of around 10% and facilitated the isolation and verification of 9 Itgb2l and Marco specific iCre2 knock-in murine embryonic stem cell lines on the 129 genetic background. To determine tissue specific iCre2 expression before generating mouse models, an in vitro haematopoietic differentiation system, utilising three-dimensional embryoid body formation and selective expansion of progenitors in the presence of IL-3 and MCSF, was adapted. Embryonic stem cells were successfully differentiated into macrophages as assessed by CD11b and F4/80 marker expression. Collectively, this work has established the foundations for obtaining viable myeloid specific Cre producer mouse strains and discusses the potential of their future application in elucidating the role of macrophages and neutrophils in innate immune function

Modulators of the Acute Inflammatory Response: A Dissertation

Acute inflammatory response is caused by the rapid recruitment of leukocytes, mainly neutrophils and monocytes, from blood to the tissue site. Diverse agents, including invading pathogens, injured or dead cells, and other irritants, may stimulate this response. In the ensuing inflammatory response, the recruited leukocytes and their secreted molecules help in eliminating or containing the injurious agents and promoting tissue regeneration. But often this response is imprecise and can lead to bystander tissue damage. Unchecked neutrophil activation is implicated in the pathology of many inflammatory conditions. An in-depth understanding of the pathways regulating this response, therefore, becomes critical in identifying therapeutic targets for these diseases. In this study, we investigate the role of intestinal commensal bacteria in regulating the acute inflammatory response. Furthermore, we examine the mechanism by which Interleukin-1 (IL-1) controls the inflammatory response to sterile agents. Inflammatory responses have been studied in the context of host defense against pathogens. However, we report that the innate immune system needs to be primed by intestinal flora to enable neutrophil recruitment to diverse microbial or sterile inflammatory signals. This priming requires myeloid differentiation primary response gene (88) (MyD88) signaling. In antibiotic-treated mice, which have depleted intestinal flora, we show that neutrophils get released into the blood from the bone marrow, but have a specific defect in migration into the inflammed tissue. This deficiency can be restored by pre-stimulating the mice with a purified MyD88 ligand. Despite having reduced number of infiltrating neutrophils, antibiotic-treated mice make higher levels of pro-inflammatory cytokines in the tissue, after inflammatory challenge. This suggests that antibiotic-treated mice produce some anti-inflammatory molecule(s) that counteract the effect of the pro-inflammatory cytokines. However, this effect is not due to the overproduction of the anti-inflammatory cytokine, Interleukin-10 (IL-10). In summary, our findings highlight the role of commensals in the development of acute inflammatory responses to microbial and sterile particles. The inflammatory response to sterile dead cells has been shown to be critically dependent upon IL-1. However, several key aspects of the IL-1 signaling cascade including the source of IL-1 and the cellular target of IL-1 were unresolved. We find that in most cases, the injured cells are not a major contributor of IL-1 that is required to propagate the inflammatory signal. On the contrary, we demonstrate that both the isoforms of IL-1, IL-1α/IL-1β are generated by bone marrow-derived, tissue-resident responding cells, upon sensing the injury. We also sought to determine the identity of the cellular target of IL-1 signaling. Previous studies have shown that for cell death-induced neutrophil recruitment, interleukin-1 receptor (IL-1R) expression is required on parenchymal cells. To identify this parenchymal cell, we are currently in the process of making the conditional knockout mouse of IL-1R. The latter would facilitate the parenchymal tissue-specific deletion of IL-1R. In summary, this study reports our progress in unraveling key aspects of IL-1 signaling during sterile inflammation. Taken together, we have identified key modulators of the acute inflammatory response and their mechanisms of regulation. These findings would facilitate the development of new therapies for inflammatory diseases triggered by both microbe and sterile agents

Systemic influences of mammary cancer on monocytes in mice

SIMPLE SUMMARY: Using a mouse model of breast cancer driven by the mammary epithelial expression of the polyoma middle T oncoprotein in which the tumors progress from benign to malignant metastatic stages, we show that cancer causes an increase in circulating monocytes and a splenomegaly. This increase in monocyte number is due to their increased proliferation in the bone marrow and not turnover rates in the blood. Single cell sequencing also shows that new populations of monocytes do not arise during cancer. Cancer also drives systemic changes in the monocyte transcriptome, with a notable down-regulation of interferon signaling. These systemic influences start in the bone marrow but intensify in the blood. Comparison of cancer prone and cancer resistant mouse inbred strains carrying the same oncogene reveals that the genetic background of the strain causes different monocyte transcriptional changes. Similarly, a comparison of the mouse transcriptome to human breast cancer monocyte profiles indicates limited similarities, to the extent that interferon signaling is enhanced in humans. Systemic responses are different in the same model of cancer on different genetic backgrounds within a species and even greater changes are found across species. These data suggest that at the very least this mouse model will be limited when it comes to exploring the mechanism behind systemic changes in humans. ABSTRACT: There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer

Bone Marrow Angiopoietin/Tie signaling in the control of hematopoietic stem cells

Hematopoietic stem cells (HSC) primarily reside in the bone marrow (BM) and possess the ability of self-renewal and differentiation to any progenitor or mature blood cell through hematopoiesis. Adult HSCs are found in specialized bone marrow niches that are essential for the regulation of quiescence, mobilization and differentiation of HSCs. Multiple studies have attempted to shed light on the complex signaling pathways between stromal and hematopoietic cells of the niche at steady state, inflammation and disease. Endothelial cells (EC) and perivascular stromal niches are known to illicit paracrine signals for the control of HSC maintenance and function. Curative transplantation approaches dwell on the effective activation and mobilization of hematopoietic stem and progenitor cells (HSPC) to the blood circulation. Current pharmaceutical approaches involve the use of mobilizing agent granulocyte-colony stimulating factor (G-CSF). The Angiopoietin/Tie (Ang/Tie) signaling pathway is essential for embryonic blood and lymphatic vessel development and maturation as well as vessel homeostasis in the adult. Few studies attempted to investigate Ang/Tie signaling in the BM stem cell niche. Angiopoietin-1 (Ang1) has been studied in the context of HSC maintenance and quiescence in the BM. It has also been shown that Ang1/Tie2 signaling is important for vascular recovery following BM irradiation. Besides the well-established role of Ang/Tie signaling in EC, Tie2 receptor is also known for its expression in HSPC. These findings led to the hypothesis that Ang/Tie signaling might impact HSPC in the bone marrow niche. The present study investigated Angiopoietin-2 (Ang2) in vivo in the context of HSPC activation, egress and mobilization to the periphery. For this purpose, both genetic approaches as well as pharmaceutical inhibition of Ang2 were employed. Although Ang2 did effect HSPC egress at steady state, Ang2KO mice demonstrated a delayed and reduced HSPC mobilization to the periphery upon G-CSF stimulation. Further dissection of the phenotype revealed that the absence of Ang2 hindered the prompt activation of HSPC rather than the process of mobilization. The bone marrow vasculature and its function seemed unaffected by Ang2 at steady state and upon G-CSF mobilization. Further assessment of Ang2 function on HSPC was carried out in the context of hematopoietic reconstitution upon lethal irradiation. The reduced capability of immune cell reconstitution in Ang2KO mice confirmed the ligand’s importance in replenishing the BM. The next focus of the thesis was elucidating the roles of Tie1 receptor on ECs in the BM in vivo. The investigation of HSPC egress and G-CSF-induced mobilization revealed fewer HSPCs in the periphery. Functional assays on blood vessels revealed that subtle changes in the vasculature are responsible for the reduced ability of HSPC mobilization in Tie1iECKO mice. Finally, since Tie1 receptor is not only expressed in ECs but also in HSPCs, this study involved the investigation of the receptor’s role in progenitor colony formation in vitro and BM reconstitution in vivo. Colony forming unit (CFU) assays revealed that Tie1 deletion on HSPC (Tie1KO) reduced the cells' ability to form differentiated colonies. Serial and competitive transplantations in mice confirmed the reduced ability of Tie1-deleted HSPC to repopulate the myeloid BM compartment of lethally irradiated mice. The present thesis sheds lights on the interactions of blood vessels and HSPCs from an "Ang/Tie-centric" perspective. The experiments have unraveled the contribution of the context-dependent partial agonist Ang2, and the orphan receptor Tie1 in HSPC egress, mobilization and bone marrow reconstitution. These new discoveries are important in elucidating Ang/Tie signaling in the BM and potentially contribute to HSPC mobilization research for the treatment of hematological malignancies