Macrophage Progenitor Cells Research Articles

TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9−/− mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9−/− mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Heparin binding proteins on monocyte cell surfaces regulates pre-inflammatory responses in diabetes.

Many diabetic complications, such as renal and cardiovascular disease, share a common association with extensive and chronic inflammation due to infiltration by activated leukocytes that originate from the bone marrow (BM). Our previous study demonstrated that macrophage progenitor cells that divided in hyperglycemia induced intracellular synthesis of hyaluronan and became pro-inflammatory macrophages (Mpi), and that the presence of low concentrations of heparin (~50 nM) prevented the intracellular HA synthesis and promoted the formation of tissue repair macrophages (Mtr). However, the molecular mechanism underlying heparin's role is still unknown. This study showed that heparin can be internalized by dividing monocyte progenitor cells. Further, there are two most abundant heparin binding proteins, alpha-enolase (ENO-1) and cofilin-1, identified on monocyte cell surfaces. In addition to their conventional roles inside of cells, ENO-1 and cofilin-1 can be found on cell surfaces and are also involved in autoimmune diseases. Thus, this study provides new insight into heparin's role in regulating monocyte and macrophage function.

Abstract 1129: Targeting tissue-resident macrophage progenitors restores anti-tumor immunity and suppresses tumor growth

Abstract Macrophages are innate immune cells that accumulate rapidly in tumors, where they promote immune suppression, tumor growth and resistance to checkpoint inhibitor therapy. As these cells play critical roles in tumor progression, they are the focus of efforts to develop novel cancer therapeutics. Strategies to inhibit macrophage accumulation to suppress tumor growth by blocking their recruitment from circulation have been only partially effective. In this project, we examined the origins of tumor associated macrophages using lineage tracing, flow cytometry, and single cell sequencing. We found that large proportions of macrophages in subcutaneous and genetically engineered mouse tumors do not derive from Ly6C+ or Ly6C- or CCR2+ monocytes. Instead, a large population of CCR2-Ly6C-F4/80hi macrophages that are CD206+MerTK+ are profoundly immune suppressive resident macrophages that arise from a transcriptionally related proliferative progenitor subpopulation rather than by trafficking from the circulation. By comparing gene expression profiles of resident and recruited macrophages in subcutaneous, alveolar, central nervous and renal normal and tumor tissues, we identified generic tissue resident macrophage and recruited signatures that reveal that resident macrophages are more immune suppressive than recruited macrophages and are associated with worse outcomes in cancer patients. Tissue resident macrophages express signatures of Myc-driven proliferation, cholesterol metabolism and profound immune suppression while bone marrow derived macrophages express mixed signatures of inflammation and immune suppression. Genetic and pharmacological inhibition of c-Kit/mCsf1R, cMyc and cyclin-dependent kinases suppressed the expansion of these macrophages, thereby limiting their accumulation in tumors, promoting anti-tumor adaptive immunity and inhibiting tumor progression in mouse models of cancer. We identified a novel cKit/Csf1r/Cdk19 inhibitor that suppressed tissue resident macrophage accumulation and potently inhibited tumor growth. These results identify tissue resident macrophage progenitor cells as key targets for the development of therapeutic strategies to stimulate anti-cancer immunity. Citation Format: Hui Chen, Marc Paradise, Ryan Shepard, Guangfang Wang, Mark Onaitis, Judith Varner. Targeting tissue-resident macrophage progenitors restores anti-tumor immunity and suppresses tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1129.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

The features of tumor niche cell composition in invasive breast ductal carcinoma of no special type

Breast cancer is the most common cancer and the leading cause of cancer death in woman of childbearing age. Tumor progression depends on the character of stromal-parenchymal interactions. Tumor microenvironment exerts a key influence on tumor progression. Tumor niche is an important element of the tumor microenvironment. According to existing ideas, tumor niche consists on immune cells and bone marrow progenitor cells. The present study describes the parameters of tumor niche in invasive breast carcinoma of no special type (IC-NST), associated with lymph node metastases. The purpose of the study was to investigate the features of tumor niche cell composition in IC-NST. Material and methods. The study included 128 patients with IC-NST (T1–3N0–3M0), who underwent total mastectomy or partial mastectomy with axillary lymph node dissection. The age of the patients ranged from 29 to 90. Histological examination of surgical specimens was carried out in accordance with standard methods. Suspensions of fresh frozen tumor surgical specimens were prepared for the hematopoetic progenitor cells identification. The antibodies against CD34, CD133, CD90, CD11B, CD45, AND CD202 were applied. Results. The study showed that the total number of hematopoietic stem and progenitor cells and macrophage progenitor cells in an amount exceeding 1.24 cells per 100 tumor cells was associated with the risk of developing lymph node metastases and large tumor size. Conclusion. The results obtained may be useful for understanding the role of tumor niche in tumor growth and lymph node metastasis of IC-NST.

Abstract P2061: The Long Non-coding RNA Schlafenlnc As A Regulator Of Cardiac Resident Macrophage Function

Introduction: Cardiac resident macrophages (crMΦs) constitute up to 5% of cells in the murine heart and were shown to play key roles in cardiac homeostasis and disease. Long non-coding RNAs (lncRNAs) are regulatory molecules that impact characteristics such as cell identity, proliferation or migration. However, the function of lncRNAs in crMΦ remains enigmatic. Objective: We sought to identify crMΦ-specific lncRNAs and analyze their function in vitro and in vivo to understand their role during health and cardiac disease. Methods and Results: Using RNASeq (>100 million reads/sample) of purified murine crMΦs and single cell Seq of total murine myocardium in health and disease, we could identify the lncRNA Schlafenlnc as a highly enriched and abundant lncRNA in crMΦs. Employing the CRISPR-Cas system we successfully deleted the full Schlafenlnc locus in a macrophage progenitor cell line. Next, we performed RNASeq of Schlafenlnc -/- macrophages and could observe 2,660 significantly deregulated genes that were enriched in genes associated with chemotaxis and migration. In line with these findings, Schlafenlnc -/- macrophages displayed decreased chemotaxis as well as adhesion using cell-based assays. Furthermore, using RNA-pulldown experiments followed by mass spectrometry analysis and we could identify 27 interaction partners of Schlafenlnc , which are involved in processes such as mRNA processing, transcriptional regulation and alternative splicing. Finally, we are currently using cardiac functional measurements, macrophage stainings as well as single cell Seq during health and cardiac disease to analyze the function of Schlafenlnc in vivo. Conclusion: In this study, we could identify the crMΦ-specific lncRNA Schlafenlnc as a critical regulator of macrophage migratory functions. Therapeutic targeting of the evolutionary conserved lncRNA Schlafenlnc might therefore be beneficial in the treatment of inflammatory cardiac diseases.

Fish telocytes and their relation to rodlet cells in ruby-red-fin shark (rainbow shark) Epalzeorhynchos frenatum (Teleostei: Cyprinidae)

Telocytes comprise the major constituents of the supportive interstitial framework within the various organs. They form a 3D network between different types of stromal and non-stromal cells, which makes them distinctively vital. We have previously explored the origin of the peculiar rodlet cells, especially on their differential stages in aquatic species. The current study aimed at highlighting the relation of telocytes with different rodlet stages. Samples of fish, olfactory organs, and gills were processed for semi thin sections, transmission electron microscopy, and immunohistochemistry. It was evident in the study that telocytes formed a 3D interstitial network, entrapping stem cells and differentiating rodlet cells, to establish direct contact with stem cells. Differentiated stem cells and rodlet progenitor cells, practically in the granular and transitional stages, also formed ultrastructure junctional modifications, by which nanostructures are formed to establish cell contact with telocytes. Telocytes in turn also connected with macrophage progenitor cells. Telocytes (TCs) expressed CD34, CD117, VEGF, and MMP-9. In conclusion, telocytes established direct contact with the stem and rodlet cells in various differential stages. Telocytes may vitally influence stem/progenitor cell differentiation, regulate rodlet cell function, and express MPP-9 that may regulate immune cells functions especially, including movement and migration ability.

Discussion: Docosahexaenoic Acid Improves Diabetic Wound Healing in a Rat Model by Restoring Impaired Plasticity of Macrophage Progenitor Cells.

Discussion: Docosahexaenoic Acid Improves Diabetic Wound Healing in a Rat Model by Restoring Impaired Plasticity of Macrophage Progenitor Cells.

Docosahexaenoic Acid Improves Diabetic Wound Healing in a Rat Model by Restoring Impaired Plasticity of Macrophage Progenitor Cells.

Chronic inflammation associated with delayed diabetic wound healing is induced by disturbed polarization of macrophages derived mainly from predisposed progenitor cells in bone marrow. Docosahexaenoic acid plays a critical role in regulating the function of macrophage progenitor cells. The authors evaluated whether docosahexaenoic acid accelerates diabetic wound healing in rats. Streptozotocin-induced diabetic rats divided into control and docosahexaenoic acid-treated groups (n = 10) were subjected to paired dorsal skin wounds. Docosahexaenoic acid (100 mg/kg per day) was orally supplemented 2 weeks before wounding until termination. The wound healing process was recorded 0, 7, and 14 days after wounding. At day 7, blood perfusion was measured by laser Doppler perfusion imaging; angiogenesis was compared using immunofluorescent CD31 and α-smooth muscle actin staining; macrophage polarization was detected using immunofluorescence for CD68, CD206, and inducible nitric oxide synthase. Hematoxylin and eosin staining was used to examine wound healing at day 14. Activation status of macrophages derived from bone marrow cells in normal, diabetic, and docosahexaenoic acid-treated diabetic rats was determined in vitro using Western blotting and enzyme-linked immunosorbent assay. Docosahexaenoic acid significantly accelerated wound healing 7 and 14 days (p < 0.01) after wounding. Increased vessel densities (1.96-fold; p < 0.001) and blood perfusion (2.56-fold; p < 0.001) were observed in docosahexaenoic acid-treated wounds. Immunofluorescence revealed more CD206 and fewer inducible nitric oxide synthase-positive macrophages (p < 0.001) in treated wounds. Furthermore, macrophages derived from diabetic rats expressed higher levels of inducible nitric oxide synthase and tumor necrosis factor-α and lower arginase-1 and interleukin-10 (p < 0.05). Docosahexaenoic acid accelerates diabetic wound healing at least in part by restoring impaired plasticity of macrophage progenitor cells.

Niche Transfer of Small Non-Coding RNAs Regulates Hematopoietic Response to Stress

The bone marrow (BM) niche, is an extrinsic regulator of hematopoietic stem and progenitor cells (HSPCs) (Morrison and Scadden 2014). The niche can drive disease and loss of HSPC function (Raaijmakers et al., 2010; Kode et al., 2014; Dong et al., 2016). Of particular interest, deletion in specific niche cells of the small RNA processing enzymes, Dicer1 and Angiogenin (Ang1) resulted in hematopoietic transformation (Raaijmakers et al., 2010) and loss of HSC quiescence (Goncalves et al., 2016), respectively. We tested mechanisms by which small non-coding RNAs (sncRNA) from niche cells might contribute to HSPC regulation. The transfer of cytoplasmic contents from specific mesenchymal subsets to wild type (WT) transplanted hematopoietic cells was evaluated by GFP. Osteoblastic cell reporter mice (Ocn-GFP, Col1-GFP) transferred 40-fold more GFP to HSPC than mesenchymal stem cells (Nes-GFP) or osteoprogenitors (Osx-GFP). GFP cannot transfer through gap junctions, however 100nM extracellular vesicles (EVs) labeled with the endocytic marker TSG-101 and GFP were found in Ocn-GFP bone marrow plasma. Granulocyte macrophage progenitor (GMPs) cells were the major hematopoietic recipients of osteoblast derived EVs (Fig. 1A). Small RNA analysis of mouse BM EVs demonstrated &gt;85% were tRNA (Fig. 1B) while Northern blot (NB) revealed both tRNAs and stress induced RNAs (tiRNAs) (Fig. 1C). tRNAs were evident in EVs and in EV generating and recipient cells, while tiRNAs were highly enriched in EVs compared to cells (Fig. 1C). Extracellular vesicle recipient and non-recipient GMPs (GMP[GFP+] and GMP[GFP-]) respectively demonstrated a 2-fold enrichment in reads mapping to tRNAs in GMP[GFP+]. Principle component analysis demonstrated that the two populations are distinct in their tRNA content. In addition, twelve specific tRNAs were present at a significantly higher levels in GMP[GFP+]. Gene expression analysis of GMP[GFP+]and GMP[GFP-] revealed an enrichment of protein translation and cell cycle related functions in GMP[GFP+] which were validated using an in vivo protein translation assay and cell cycle analyses. Synthetic oligos corresponding to the sequence of top ten tiRNAs that are enriched in GMP[GFP+] were transfected into primary GMPs. 5'-Pro-CGG-1 and 5'-Cys-GCA-27 increased the rate of protein translation and cellular proliferation in transfected GMPs (Fig. 1D-E). The physiologic relevance of EV transfer was assessed using two stress states, systemic Candida albicans infection and low dose gamma irradiation. The frequency of GMP[GFP+] increased in both states in concert with increased cell proliferation. Therefore, EV transfer of sncRNAs from niche to hematopoietic progenitors in vivo is a rapid means of conveying information between cells. It alters progenitor protein translation and proliferation in a manner responsive to stress. The transfer of 5'tiRNA EV from specific niche cells to specific hematopoietic cells is an additional mechanism for niche regulation of blood cell production. Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder. Scadden:Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Abstract Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed &gt;95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P&lt;0.01, n&gt;4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

CXCR2 expression on granulocyte and macrophage progenitors under tumor conditions contributes to mo-MDSC generation via SAP18/ERK/STAT3

Myeloid-derived suppressor cells (MDSCs) comprise a critical component of the tumor environment and CXCR2 reportedly plays a key role in the pathophysiology of various inflammatory diseases. Here, CXCR2 expression on granulocyte and macrophage progenitor cells (GMPs) was found to participate in myeloid cell differentiation within the tumor environment. In CXCR2-deficient tumor-bearing mice, GMPs exhibited fewer macrophage and dendritic cell progenitor cells than wild-type tumor-bearing mice, thereby decreasing monocytic MDSCs (mo-MDSCs) expansion. CXCR2 deficiency increased SAP18 expression in tumor-bearing mice, which reduced STAT3 phosphorylation through restraining ERK1/2 activation. Our findings reveal a critical role for CXCR2 in regulating hematopoietic progenitor cell differentiation under tumor conditions, and SAP18 is a key negative regulator in this process. Thus, inhibiting CXCR2 expression may alter the tumor microenvironment and attenuate tumor progression.

Deciphering liver environmental signaling pathways for Kupffer cell identity

Abstract Functional specialization of tissue resident macrophages occurs through environmental signals controlling activity and/or expression of transcription factors. Kupffer cells are resident macrophages in the hepatic sinusoids and have critical roles in the innate immune response and iron metabolism. Here, we characterize transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. Nr1h3 encoding LXRα is rapidly and highly induced in repopulating liver macrophages, suggesting its induction plays a crucial role in Kupffer cell differentiation. Restricted deletion of Nr1h3 in Kupffer cells reveal that it is required for shaping the Kupffer cell-specific enhancer landscape. Further, we obtain evidence that combinatorial interactions of DLL4 and TGF-β/BMP produced by sinusoidal endothelial cells and endogenous LXR ligands are required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of RBPJ through Notch signaling plays a key role in activating poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogram the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Using molecules which mimic these liver environment signals, we show that it is possible to induce Kupffer cell-specific genes in mouse bone marrow progenitor cells and human monocytes in vitro. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Knockout rat models mimicking human atherosclerosis created by Cpf1-mediated gene targeting

The rat is a time-honored traditional experimental model animal, but its use is limited due to the difficulty of genetic modification. Although engineered endonucleases enable us to manipulate the rat genome, it is not known whether the newly identified endonuclease Cpf1 system is applicable to rats. Here we report the first application of CRISPR-Cpf1 in rats and investigate whether Apoe knockout rat can be used as an atherosclerosis model. We generated Apoe- and/or Ldlr-deficient rats via CRISPR-Cpf1 system, characterized by high efficiency, successful germline transmission, multiple gene targeting capacity, and minimal off-target effect. The resulting Apoe knockout rats displayed hyperlipidemia and aortic lesions. In partially ligated carotid arteries of rats and mice fed with high-fat diet, in contrast to Apoe knockout mice showing atherosclerotic lesions, Apoe knockout rats showed only adventitial immune infiltrates comprising T lymphocytes and mainly macrophages with no plaque. In addition, adventitial macrophage progenitor cells (AMPCs) were more abundant in Apoe knockout rats than in mice. Our data suggest that the Cpf1 system can target single or multiple genes efficiently and specifically in rats with genetic heritability and that Apoe knockout rats may help understand initial-stage atherosclerosis.

Abstract BS1-1: High resolution intravital imaging of the mechanism of how breast cancer initiates and sustains systemic dissemination

Abstract Multiphoton microscopy of live animals (IVI) has revolutionized our understanding of cancer metastasis. IVI demonstrates that TMEM, the intravasation doorway, composed of a three cell complex: Mena-Hi tumor cell, endothelial cell and Tie2-Hi/VEGF-Hi macrophage, is the only site in breast tumors where tumor cell intravasation occurs1. During tumor progression, macrophage progenitor cells undergo unidirectional transition from migratory to TMEM-associated macrophages2. During and after this macrophage programming, tumor cells migrate with macrophages and chemotax with high persistence to blood vessels under the control of HGF gradients3 arriving at TMEM1. The TMEM structure itself, as well as the signaling pathways unique to tumor cells interacting with TMEM, have been validated as prognostic markers for predicting metastasis in breast cancer patients4-6. These were the first markers of metastasis in clinical use derived from multiphoton intravital imaging. TMEM are found in both primary and metastatic tumor sites of breast cancer7 and in primary and metastatic sites of pancreatic ductal and neuro-endocrine tumors. Clinical trials of TMEM inhibitors, targeting TMEM in both primary and secondary sites8,9, are now underway in breast cancer patients (clinical trials study number NCT02824575). As tumor cells interact with TMEM, MenaINV. expression in the tumor cells occurs in response to macrophage-induced NOTCH signaling10, which drives invadopod assembly/ function, and metastatic seeding11,12. MenaINV expression is necessary for invadopod-mediated transendothelial migration during intravasation at TMEM13 and assures the efficient dissemination of seeding competent and non-dividing tumor cells which, after a delay, contribute to metastatic recurrence at multiple systemic sites. These findings support TMEM as a critical therapeutic target for inhibiting breast cancer metastasis. 1.Harney, A.S. et al. (2015) PMC4560669 2.Arwert, E.N. et al. (2018) PMC5946803 3.Leung, E. et al. (2017) PMC5426963 4.Karagiannis, G.S. et al (2016) PMC4893654 5.Rohan, T.E. et al. (2014) PMC4133559 6.Sparano, J.A. et al. (2017)PMC5678158 7.Entenberg, D. et al. (2018)PMC5755704 8.Harney, A.S. et al. (2017)PMC5669998 9.Karagiannis, G.S. et al. (2017) PMC5592784 10.Pignatelli, J. et al. (2016) PMC5129016 11.Eddy, R.J. et al (2017) PMC5524604 12.Linde, N. et al. (2018) PMC5750231 13.Pignatelli, J. et al.(2014) PMC4266931 Citation Format: Condeelis J. High resolution intravital imaging of the mechanism of how breast cancer initiates and sustains systemic dissemination [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr BS1-1.

CXCR4 Has a CXCL12-Independent Essential Role in MLL-AF9 Driven Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a clonal hematological disorder associated with poor prognosis, and there is a strong need to develop new therapeutic strategies. AML is propagated by a small population of leukemic cells in the bone marrow with self-renewal capacity, a population termed leukemia stem cells (LSC). To identify in vivo dependencies of LSCs, we conducted a CRISPR/Cas9 drop-out screen targeting cell surface receptors on murine LSCs. To this end, we generated a pooled lentiviral CRISPR library targeting 96 cell surface receptors that are upregulated on murine MLL-AF9 LSC compared to normal granulocyte and macrophage progenitor (GMP) cells based on global gene expression data. The CRISPR library was transduced into Cas9-expressing murine c-Kit+MLL-AF9 AML cells and subsequently injected into sublethally irradiated (600 cGy) C57BL/6 mice. Two weeks later when the mice had developed leukemia, the bone marrow was harvested, and sgRNAs sequenced. A high reproducibility in sgRNA representation was observed between five biological replicates, demonstrating that the screen was robust.The screen ranked CXCR4 as the most critical cell surface receptor on AML stem cells with all five sgRNAs targeting Cxcr4 depleting more than 4-fold in vivo. Cxcr4 has previously been shown to be critical in several types of cancers including hematological malignancies. Moreover, CXCR4 has been shown to be important for homing of normal and leukemia stem cells in the bone marrow. To validate the role of CXCR4 in a microenvironment and cell intrinsic manner in AML, we disrupted Cxcr4 in c-Kit+MLL-AF9 AML cells using lentiviral vectors expressing sgRNAs targeting Cxcr4 and GFP as a marker gene. Whereas Cxcr4 disruption did not affect the growth and survival of the AML cells in in vitro cultures with standard cytokines, a 15-fold depletion (p = 0.006) of Cxcr4 sgRNA-expressing AML cells was observed 12 days post-transplantation in vivo. We next studied whether the loss of CXCR4 on the surface of leukemic cells would translate into a survival difference in vivo. Notably, the mice receiving sorted leukemia cells expressing Cxcr4 sgRNAs did not develop AML, demonstrating that CXCR4 is essential for LSCs in vivo. To investigate if this effect was due to a homing defect, we transplanted Cxcr4 sgRNA-expressing cells into sublethally irradiated mice and measured the number of AML cells in the bone marrow after 24 hours. A 2.5-fold reduction (p = 0.03) of leukemia cells expressing Cxcr4 sgRNA compared to the control sgRNA was observed in the bone marrow, suggesting that the depletion of leukemic cells in vivo was partially due to a homing defect.CXCL12, the most studied ligand of CXCR4, is a homeostatic chemokine widely expressed by several cell types in the bone marrow and exists both in a membrane and soluble form. Although the CXCR4-CXCL12 interaction is critical for the retention of normal hematopoietic stem and progenitor cells in the bone marrow, the role of CXCL12 in AML is less characterised. To study the role of different CXCL12-expressing bone marrow cell populations in AML development, we first transplanted c-Kit+MLL-AF9 leukemia cells into Cxcl12f/f-Tek-Cre+ mice, which are devoid of Cxcl12 expression in endothelial cells, and in Cxcl12f/f-Prx1-Cre+ mice, which lack expression of Cxcl12 in mesenchymal stem cells. No reduction in leukemia levels in the blood and bone marrow was observed with either of these mouse strains as recipients, indicating that Cxcl12 expression by endothelial cells and MSC is not critical for AML development. Next, we repeated the experiment using Cxcl12f/f-Ubc-Cre+ mice, which lack Cxcl12 expression in all cells. Intriguingly, there was no reduction in leukemia levels in these mice. Instead, a significant 2-fold increase in leukemia levels in the peripheral blood (p=0.011) and a 1.5-fold increase in leukemia levels in the bone marrow (p=0.043) were observed 12 days after transplantation. These findings suggest that the CXCR4-CXCL12 interaction is dispensable for AML development and progression.Taken together, we here established an in vivo pooled CRISPR/Cas9 drop-out screen using murine LSCs and identified Cxcr4 as an essential regulator for LSCs. Further validations identified that CXCR4 has a previously uncharacterized CXCL12-independent essential role for MLL-AF9 AML cells. Ongoing studies are aimed at identifying the ligand that is essential for CXCR4 in AML. DisclosuresNo relevant conflicts of interest to declare.

The Depletion of TGF-β Co-Receptor CD109 Induces Erythroid Differentiation of TF-1 Cells: A Model of Preferential Commitment of PIGA-Mutated Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. [Display omitted] DisclosuresNakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Abstract 5006: Kit-dependent tissue resident macrophage progenitors drive cancer progression

Abstract Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that tissue-resident as well as bone marrow-derived macrophages play critical roles in promoting tumor growth. Tissue-resident macrophages were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. Tissue-resident macrophages and bone marrow-derived macrophages rapidly accumulate in tumors where they play independent roles in promoting tumor growth. Tissue-resident macrophages are CD11b+Gr1-F4/80hiCX3CR1hiCCR2-Ki67+ cells that accumulate in tumors independently of trafficking receptors. In contrast, bone marrow-derived CD11b+Gr1+F4/80loCX3CR1loCCR2+ macrophages accumulate in tumors in an integrin α4β1/αLβ2 and CCR2 or CXCR4-dependent manner. Gene expression studies show that tumor-associated tissue-resident macrophages are highly proliferative, immune-suppressive myeloid cells that are less proangiogenic than bone marrow-derived macrophages. Our studies show that tumor cells induce the expansion of tissue-resident macrophage progenitor cells by secreting stem cell factor and mCSF. Here we identify a Kit/KitL-dependent tissue-resident macrophage progenitor that is abundant in tumors but not in normal tissues. Notably, tumor growth and colony-forming activity are significantly inhibited in mice treated with SCF and Kit inhibitors. Tumors adoptively transferred with tissue-resident macrophage progenitor cells exhibited a significant growth advantage over control tumors. Furthermore, Kit inhibitors synergize with other immune therapy regimens to suppress tumor growth. Our studies show that tissue-resident macrophage progenitors promote aggressive tumor growth that can be targeted by Kit/SCF inhibition. Citation Format: Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Judith A. Varner. Kit-dependent tissue resident macrophage progenitors drive cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5006.