Human M-CSF Research Articles

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

A29 HIGH DIMENSIONAL SINGLE CELL ANALYSIS OF IL-4 TREATED MACROPHAGES REVEALS A HETEROGENIC PHENOTYPE AND THE ABSENCE OF CD206+ SUBSETS IN CROHN'S DISEASE PATIENTS

Abstract Background Individuals with IBD have a reduced quality of life and while treatment has improved, a cure remains elusive. Interleukin-4 differentiated macrophages (M(IL4)s) from healthy donors and IBD patients in remission promote epithelial wound repair and reduce the severity of colitis in a mouse model: findings that support the autologous transfer of M(IL4)s to treat IBD. However, issues remain if this novel therapy is to advance to clinical evaluation: a much greater understanding of the possible heterogeneity within the human M(IL4) population, the biology of any sub-groups therein and the biology of any M(IL4) subtypes is needed. Purpose To determine if cryopreservation, sex, and disease status in Crohn's disease (CD) impact the phenotype and function of M(IL4) Method Blood mononuclear cells were collected from healthy donors, and individuals with active CD or in remission (n=6/group), and differentiated to macrophages with GMP-grade recombinant human M-CSF (10 ng/ml, 7 days). Macrophages (2.5 x105) were activated with IL-4 (10ng/ml, 48h). Fresh and cryopreserved M(0) and M(IL4) were stained using a panel of 18 metal-conjugated antibodies. Data were acquired on a Helios CyTOFII mass cytometer. FCS files were imported into Cytobank for manual gating (viable CD45+CD3-CD19- singlet cell events) and exported for meta-clustering in R with the CATALYST package using the FlowSOM function with 10 metaclusters. Result(s) FlowSOM analysis on M(IL4) from healthy donors indicated clear differences between M(0) and M(IL4), with 8 metaclusters >1.5% of M(IL4)s: the most abundant metaclusters express high and intermediate CD206, PDL1, CD11b, CD33, CD64, HLA-DR and low CD14 levels. There were no significant differences in these M(IL4)s metaclusters between males and females, or fresh vs. cryopreserved cells using this characterization strategy. M(IL4) from patients with active CD, were markedly different with the absence of three high-intermediate CD206+PDL1+ metaclusters and a general lack of CD206+ metaclusters compared to healthy controls. Surprisingly, and despite M(IL4)s from CD patients in remission showing increased CD206 mRNA, CYTOF analysis revealed that the macrophages were more similar to those from individuals with active disease than those from healthy controls Conclusion(s) Here human macrophages activated with IL4 in vitro are revealed as a heterogeneous population, and subsets characterized by increased high CD206 expression are generally absent in active Crohn’s disease. Intriguing while prior experiments with M(IL4) from CD patients in remission reduced the severity of colitis in rag1-/- mice, the characterization of these cells via 18 protein markers indicates they are more like M(IL4)s from patients with active disease compared to healthy controls. While presenting autologous M(IL4) transfer as a novel approach for IBD, a much fuller understanding of the phenotype and function of M(IL4)s (and sub-populations therein) is required before this would become a therapeutic option Please acknowledge all funding agencies by checking the applicable boxes below CAG Disclosure of Interest None Declared

Humanized Patient-derived Xenograft Models of Disseminated Ovarian Cancer Recapitulate Key Aspects of the Tumor Immune Environment within the Peritoneal Cavity.

huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

A humanized mouse model to study NK cell biology during HIV infection.

NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.

NLRP3-Independent Inflammasome Activation By Proteasome Inhibitors

Introduction Proteasome inhibitors (PIs) such as Bortezomib (BTZ) have revolutionized multiple myeloma (MM) treatment. However, their efficacy is limited by the emergence of drug resistance and treatment-limiting side effects, including peripheral neuropathy and lymphopenia. Treatment resistance in MM has been demonstrated to be promoted by an NLRP3 inflammasome-mediated innate immune response to MM-associated proteins. Thus, we set out to investigate the impact of PI treatment on innate immune cytokine production. Materials and Methods The PIs BTZ, marizomib, epoxomicin and onx-0914 were applied to a variety of inflammasome-competent human immortalized and primary cells, including the cell line THP-1 (monocytic), primary human peripheral blood mononuclear cells (PBMC), M-CSF or ascites-derived macrophages, keratinocytes, and epithelial cells. Inflammasome activation was characterized via Interleukin-1b (IL-1β) release in cellular supernatants (ELISA, all cell types) and ASC-speck formation using an ASC-GFP reporter cell line (THP-1). To characterize the contribution of Nod-like receptor pyrin domain-containing protein 3 (NLRP3) and caspase-1 (CASP-1) to inflammasome formation, NLRP3 and CASP-1-deficient THP1 cells were generated using CRISPR/Cas9-mediated genome engineering, and NLRP3 and caspase-1 were pharmacologically inhibited using MCC-950 (Invitrogen) and VX-765 (Invivogen). Results We report PI-mediated activation of the inflammasome in all cells tested. Treatment with PI induced secretion of IL-1b by THP1 cells and PBMC with an EC50 that was similar to the IC50 in BTZ-sensitive, but not BTZ-resistant, KMS-11 MM cells. We observed hallmarks of canonical inflammasome activation such as the formation of ASC specks in PI-exposed cells. Moreover, pharmacological inhibition of caspase-1 with VX-765 or genetic caspase-1 deficiency abrogated IL-1b. A variety of inflammatory stimuli, including soluble proteins secreted by MM cells, induce inflammasome formation via activation of NLRP3, the only inflammasome receptor currently targetable by a small molecule and investigated in clinical trials. Surprisingly, neither genetic ablation of NLRP3 in THP-1 cells nor MCC-950 treatment of PBMC inhibited IL-1b signaling following BTZ exposure. Conclusion Here, we identify PIs as activators of the inflammasome. In contrast to previously identified myeloma-intrinsic mechanisms leading to inflammasome assembly, PI-mediated IL-1b secretion was independent of NLRP3. Further investigation will include the identification of the receptor mediating BTZ-driven inflammasome assembly in order to identify mechanisms of action that may be amenable to therapeutic intervention.

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

Combined Blockade of CD47-Sirpa Interaction By 5F9 (Magrolimab) and Azacitidine/Venetoclax Therapy Facilitates Macrophage-Mediated Anti-Leukemia Efficacy in AML Pre-Clinical Models

Combined Blockade of CD47-Sirpa Interaction By 5F9 (Magrolimab) and Azacitidine/Venetoclax Therapy Facilitates Macrophage-Mediated Anti-Leukemia Efficacy in AML Pre-Clinical Models

An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundPoor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.AimsTo compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.MethodsThis prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.ResultsAlterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.Summary/ConclusionIn summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2020: Identification of abnormal CSF1 transcripts in tenosynovial giant cell tumors and dose-dependent increase in plasma CSF1 levels in response to pexidartinib treatment

Abstract Background: Tenosynovial giant cell tumors (TGCTs) are characterized by rearrangements of the colony-stimulating factor 1 (CSF1) gene. Dysregulated CSF1 may attract CSF1 receptor (CSF1R)-bearing mononuclear cells that form the bulk of the tumor. CSF1R inhibitors including pexidartinib have been developed and have proven to be effective therapies. Methods: Formalin-fixed paraffin-embedded (FFPE) TGCT specimens from the phase 1 first-in-human study of pexidartinib (NCT01004861) were analyzed using a custom sequencing assay to detect CSF1 genetic alterations. The ArcherDX RNA panel (ArcherDX, Inc., Boulder, CO) consisted of 33 gene-specific primer sets specifically designed to target CSF1, including at least 1 primer set to target each exon/exon boundary as well as primer sets to tile the 3′-untranslated region (3′UTR). Plasma samples from 5 phase 1 trials of pexidartinib across multiple tumor types (NCT02777710, NCT01217229, NCT01525602, NCT01790503, NCT02452424) were assayed for CSF1 protein by solid-phase ELISA (Quantikine® Human MCSF, R&D Systems, Inc., Minneapolis, MN) at baseline and following pexidartinib (200-1200 mg). Results: FFPE TGCT specimens (N=25) were successfully isolated, prepared into libraries, and sequenced; 8 showed evidence of gene rearrangements at the junction of CSF1 exons 5/6, and 15 showed alterations of the CSF1 3′UTR (n=23). All 25 TGCT libraries had higher CSF1 expression, with 24 exceeding 2-fold of the RNA control library. The plasma samples from 132 patients were analyzed for CSF1 protein. Pexidartinib treatment (200-1200 mg) led to a dose-dependent increase in plasma CSF1 by day 8, 15, or 29. Significant (>4-fold) CSF1 elevation was observed at 600 mg or higher doses. Discussion: All 25 analyzed TGCT study patients had tumor tissue with elevated CSF1 ligand transcripts, and 23 had CSF1 genomic alterations identified, thus confirming the TGCT etiology for which pexidartinib therapy was recently approved. The initial discovery of gross chromosomal aberrations involving the CSF1 locus used break-apart FISH probes (West et al. Proc Natl Acad Sci USA. 2006;103:690-695). Details of the CSF1 gene aberrations were obtained here using a sequencing assay suitable for FFPE specimens having partially degraded RNA. Abnormal CSF1 transcripts identified in our study are similar to those published by others (Ho et al. Genes Chromosomes Cancer. 2019[Epub]; Tsuda et al. Int J Cancer. 2019;145:3276-3284) after completion of our work, including both predicted fusion proteins and loss of 3′UTR microRNA negative regulatory sites. Increased plasma CSF1 is a useful pharmacodynamics marker of CSF1R inhibition. One potential mechanism for CSF1 plasma elevations could be the inhibition of liver macrophages, known to express CSF1R and thought to play an essential role in the normal clearance of CSF1 from circulation. Citation Format: Paul Severson, Brian L. West, William D. Tap, Zev A. Wainberg, Sandra Tong-Starksen, Henry H. Hsu, Chao Zhang. Identification of abnormal CSF1 transcripts in tenosynovial giant cell tumors and dose-dependent increase in plasma CSF1 levels in response to pexidartinib treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2020.

Abstract PD8-08: Development of humanized immune DCIS models using patient peripheral blood derived hematopoietic stem cells (CD34+)

Abstract Background: Ductal carcinoma in situ (DCIS) is the most common form of non-invasive breast cancer. To accurately study the natural progression of DCIS lesions in mice, we devised the mouse-intraductal (MIND) animal model, which involves intraductal injection of human DCIS epithelial cells into the mammary ducts of immunocompromised mice. To improve the translational application of the MIND model, we aimed to mimic the natural microenvironment of human DCIS with patient-derived immune cells and assess the role of engrafted immune cells on human DCIS progression. In order to achieve successful engraftment of the entire immune system in mice, we utilized MISTRG mice. These mice were developed by Rongvaux et al., on an immunodeficient (Rag2-/-IL2rγ-/-) background. The genes encoding human M-CSF (M), human IL-3 (I), SIRP1α (S), human thrombopoietin (TPO)(TR), and GM-CSF (G) were knocked into their respective mouse loci. As such, MISTRG mice are highly permissive for human hematopoiesis, supporting the development and function of lymphocytes, monocytes, and natural killer (NK) cells. In contrast, previous studies have used the humanized CD34+ NOD-SCID IL2rγ-/- mice (CD34+NSG), which are unable to support myeloid cell differentiation due to lack of expression of human-specific cytokines. Moreover, prior xenograft studies in the CD34+NSG mice have not used immune cells derived from the same patient as the tumor. Results: Human CD34+ cells derived from patients' peripheral blood were expanded ex vivo ˜100-fold using a novel formulation of culture medium. Transplantation of ex vivo expanded CD34+ cells via tail vein injection of MISTRG mice resulted in the successful engraftment of human immune cells as early as 4 weeks following injection. Successful engraftment was confirmed by flow cytometry using human specific antibodies that recognize human leukocytes (anti-CD45), T cells (anti-CD3), B cells (anti-CD20), and myeloid cells (anti-CD33) in spleen, bone marrow, and peripheral blood of MISTRG mice. Once engraftment was confirmed, DCIS epithelial cells from the same DCIS patients or DCIS cell lines were injected intraductally. Recruitment of patient-derived immune cells to the DCIS lesions was confirmed by immunofluorescence using human-specific antibodies that recognize neutrophils (anti-myeloperoxidase), macrophages (anti-CD68), M2-polarized macrophages (anti-c-MAF), natural killer cells (anti-CD56), dendritic cells (anti-CD21), T cells (anti-CD3) and B cells (anti-CD20). Conclusion: This model represents the first to enable the study of mechanisms of DCIS progression in a manner that fully represents the heterogeneity of human disease, including the influence of the patients' own immune cells on DCIS progression. Citation Format: Behbod F, Harper H, Hansford H, Limback D, Hong Y, Elsarraj H, Ricci LR, Fan F, Tawfik O, May L, Cusick T, Inciardi M, Redick M, Gatewood J, Winblad O, Fields TA, Fabian C, Godwin AK, Fields PE, Meierotto R, Perry J. Development of humanized immune DCIS models using patient peripheral blood derived hematopoietic stem cells (CD34+) [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 PD8-08.

Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

339: Maternal and fetal umbilical cord levels of chemokines in normal and PreE pregnancies - A prospective trial

Preeclampsia (PreE) is the de novo development of hypertension and proteinuria after 20 weeks of gestation affecting 3-8% of pregnancies, and has a link to alterations of feto-placental stress that pass to the offspring causing detrimental effects. We aimed at comparing the clinical outcomes and chemokines from the maternal and cord blood between patients with and without PreE. We recruited 20 normal pregnant (NP) and PreE patients in an IRB approved prospective cohort at our institution. We evaluated maternal age, body mass index (BMI), blood pressure (BP), and gestational age (GA) at delivery along with measured serum levels of six chemokines in maternal-fetal paired samples using ELISA test. The markers are; Human CCL4/MIP-1beta, Human CXCL10/IP-10, Eotaxin/CCL11, RANTES (CCL5), Human MCP1, Human M-CSF. Maternal: The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were higher in PreE (SBP 162 ± 17; DBP 97 ± 12) compared to NP (SBP 121 ± 3; DBP 75 ± 9; p <0.05). GA at delivery in weeks was lower in PreE (35.1 ± 3.8) compared to NP (39.2 ± 0.3; p <0.05), as well as birth weight in PrE (2556 ± 977) compared to NP (3385 ± 328; p <0.011) indicating a link to intrauterine growth restriction (IUGR). We did not find a difference in maternal BMI between the two groups. Biomarkers: For all six chemokines CCL4, CXCL10, CCL11, CCL5, MCP1, and M-CSF, maternal and fetal cord serum chemokines levels differ between subjects with and without PreE (p < 0.001). There is also a difference between the maternal and fetal levels for the six chemokines (<0.01). The fetal levels were compared to associated maternal levels for each of the six chemokines using t test and Mann-Whitney U test. Women with PreE had higher BP, preterm deliveries, and fetal IUGR. However, in this cohort the women were of similar age, size, and parity prior to the onset of pregnancy. The six chemokines in the samples from maternal and fetal circulation are greater in the PreE group, and each of the six chemokines samples from the fetuses had values greater than maternal samples which could implicate placental cells as the source. PreE alters the intrauterine environment and activates the detrimental signaling that is transferred to the fetus resulting in the aforementioned complications.

Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies.

Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n=11) and RA-derived (n=12) EXOs profoundly inhibited osteoclast differentiation (70%, p<0.01; 65%, p<0.01, respectively). In contrast, PsA-derived (n=10) EXOs had a stimulatory effect (75%, p<0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n=5) and RA (n=5)-derived EXOs inhibited (p<0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n=7)-derived EXOs were unable to mediate this effect. Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

Forced expression of human macrophage colony-stimulating factor in CD34+ cells promotes monocyte differentiation in vitro and in vivo but blunts osteoclastogenesis in vitro.

Here, we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in cord blood (CB) CD34+ cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo. Human M-CSF was overexpressed in cord blood CD34+ cells using a lentiviral vector. We show that LV-hM-CSF-transduced CB CD34+ cells expand 3.6- and 8.5-fold more with one or two exposures to the hM-CSF-expressing vector, respectively, when compared to control cells. Likewise, LV-hM-CSF-transduced CB CD34+ cells show significantly higher levels of monocytes. In addition, these cells produced high levels of hM-CSF. Furthermore, they are able to differentiate into functional bone-resorbing osteoclasts in vitro. However, osteoclast differentiation and bone resorption were blunted compared to control CD34+ cells receiving exogenous hM-CSF. NSG mice engrafted with LV-hM-CSF-transduced CB CD34+ cells have physiological levels of hM-CSF production that result in an increase in the percentage of human monocytes in peripheral blood and bone marrow as well as in the spleen, lung and liver. In summary, ectopic production of human M-CSF in CD34+ cells promotes cellular expansion and monocyte differentiation in vitro and in vivo and allows for the formation of functional osteoclasts, albeit at reduced levels, without an exogenous source of M-CSF, in vitro.

Inv(16) AML Engrafts Human Cytokine Knock-in Mice

Favorable-risk acute myeloid leukemia (AML) constitutes up to 40% of newly diagnosed AML cases. However, only 66% of younger and 33% of older patients are alive 3 years post diagnosis. Studying the biology of these entities in vivo has been inherently difficult: in fact, faithful xeno-engraftment of human AML in immunodeficient mice has been limited to higher-risk AML.To address this limitation we hypothesized that a human myelopoiesis-supportive environment may permit xeno-engraftment of favorable-risk AML: we transplanted primary human AML with isolated NPM1 mutation and AML with inv(16) into knock-in (KI) mice with human versions of genes encoding M-CSF, IL-3, GM-CSF and Thrombopoietin. Newborn MISTRG mice (Rongvaux et al. Nat Biotechnol. 2014) were sub-lethally irradiated, intra-hepatically injected with AML blasts and analyzed 16-24 weeks post transplantation.Flow cytometric analysis of bone marrow (BM) and blood of cytokine KI versus NSG mice revealed profound differences: Average engraftment was 36% in the BM of cytokine KI mice compared to only 10% in NSG mice across all patient samples transplanted. In line with engraftment levels, engraftment efficacy was higher in cytokine KI mice. Importantly, average engraftment in cytokine KI mice approached BM and peripheral blast count observed in human AML patients, while NSG engraftment levels were significantly lower. AML engraftment in cytokine KI mice was found to be independent of expression of the SIRPα transgene in mice expressing all four cytokines. NMP1mut AML showed a trend towards higher BM engraftment in MI(S)TRG versus NSG mice. Expression of the NPM1mutallele was more than 3-fold higher in MI(S)TRG mice compared to NSG mice. Performing targeted next generation sequencing in human and engrafted murine BM, we found comparable allelic frequency ranging from 38% to 51% for NPM1 mutation in a patient’s leukemia and engrafted cytokine KI mice. Of note, even the composition of NPM1 mutation subclones was constant between patient and cytokine KI recipients.In contrast to NPM1mut, a striking difference in quantitative inv(16) AML engraftment was observed between cytokine KI and NSG mice, both by aggregate and split-sample analysis. Serial transplantation further validated engraftment of an inv(16) leukemia-initiating cell population in cytokine KI mice. To dissect the contribution of respective single human KI cytokines to inv(16) AML propagation, we analyzed engraftment in Rag2-/-gc-/- mice carrying, in addition to a human SIRPα transgene, further KI genes for human thrombopoietin (STRG), M-CSF and Thrombopoietin (MSTRG), and M-CSF, IL-3, GM-CSF and Thrombopoietin (MI(S)TRG). STRG mice, as NSG mice, did not support engraftment. In contrast, addition of M-CSF in MSTRG mice led to robust engraftment of inv(16) AML, while addition of IL-3 and GM-CSF in MI(S)TRG mice resulted in only marginal, if any, improvement over MSTRG mice. Of note, knock-in of human M-CSF has previously been shown to be sufficiently cross-reactive to rescue the murine M-CSF knock-out phenotype (Rathinam et al. Blood 2011). We thus reasoned that the engraftment advantage observed in MSTRG mice was likely due to human M-CSF effects on blasts and not due to reduction in mouse macrophages. Together, this establishes a central role for M-CSF in maintenance of inv(16) AML.When profiling cytokine receptor expression for TPO, M-CSF, IL-3 and GM-CSF on inv(16) AML blasts in comparison to NPM1mut AML and an aggregate of non-favorable-risk AML cases, using microarray data from an international cohort study (Wouters et al. Blood 2009), inv(16) AML showed significantly higher expression of each of the 4 receptors compared to NPM1mut AML, and significantly higher expression of M-CSFR and GM-CSFR compared to non-favorable-risk AML; the overall highest difference in expression was observed for M-CSFR. Moreover, gene set enrichment analysis demonstrated enrichment of M-CSF-inducible genes in inv(16) AML compared to NPM1mutAML and non-favorable-risk AML cases, confirming increased signaling through the M-CSF pathway.In sum, we here describe the first faithful and highly efficacious xenotransplantation model of favorable-risk, and specifically, inv(16) AML. We further demonstrate a strong dependency of this subtype on M-CSF, a finding that might provide a basis for therapy optimization studies informed by an improved understanding of favorable-risk AML biology. DisclosuresNo relevant conflicts of interest to declare.

Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation.

Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1β, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals. Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF. Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinraprior to stimulation by MSU crystals. Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation. We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1β is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect. Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.

Inv(16) and NPM1mut AMLs engraft human cytokine knock-in mice

AbstractFavorable-risk human acute myeloid leukemia (AML) engrafts poorly in currently used immunodeficient mice, possibly because of insufficient environmental support of these leukemic entities. To address this limitation, we here transplanted primary human AML with isolated nucleophosmin (NPM1) mutation and AML with inv(16) in mice in which human versions of genes encoding cytokines important for myelopoiesis (macrophage colony-stimulating factor [M-CSF], interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) were knocked into their respective mouse loci. NPM1mut AML engrafted with higher efficacy in cytokine knock-in (KI) mice and showed a trend toward higher bone marrow engraftment levels in comparison with NSG mice. inv(16) AML engrafted with high efficacy and was serially transplantable in cytokine KI mice but, in contrast, exhibited virtually no engraftment in NSG mice. Selected use of cytokine KI mice revealed that human M-CSF was required for inv(16) AML engraftment. Subsequent transcriptome profiling in an independent AML patient study cohort demonstrated high expression of M-CSF receptor and enrichment of M-CSF inducible genes in inv(16) AML cases. This study thus provides a first xenotransplantation mouse model for and informs on the disease biology of inv(16) AML.

Abstract 730: The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages

Abstract Tumor-associated macrophages (TAMs) have important roles in the angiogenesis, tumor immunosuppression and tumor infiltration of various cancers. Previously, we reported that a large number of infiltrated tumor promoting CD204+ TAMs within esophageal squamous cell carcinomas (ESCCs) contributed to the significant association of clinicopathological malignancy and poor disease free survival. However, the relevant malignant progression mechanisms have not been studied explicitly yet. To elucidate roles of TAMs in ESCCs, we established an in vitro macrophage model using human peripheral blood acquired by healthy volunteer donors. Peripheral blood monocytes were treated with 25 ng/mL recombinant human M-CSF for 6 days to induce macrophage-like differentiation (macrophage-like cells), then exposed to the conditioned media of TE series ESCC cell lines (TECM) for 2 days to induce TAM-like cells. From the cDNA microarray data between macrophages-like cells treated with and without TECM, we interested in upregulated genes. Among highly expressed genes in TAM-like cells, we focused on neural cell adhesion molecule (NCAM) which was a possibility to act on the regulation between tumor and its surrounding stroma. Gene knockdown of NCAM by siRNA in TAM-like cells revealed significant suppression of cell survival and migration via decrease of PI3K/Akt signaling and downregulation of fibroblast growth factor receptor 1 (FGFR1) expression. Interestingly, fibroblast growth factor-2 (FGF-2) derived from ESCCs promotes FGF2/FGFR1 autocrine loop, and activates FGFR1 signaling in TAM-like cells. Furthermore, we analyzed 70 ESCC tissue samples by immunohistochemistry whether the expression levels of FGF-2 were associated with clinicopathological background factors of ESCC patients. Relationships of the expression levels of FGF-2 in human ESCC stromal cells including macrophages had a correlation with the depth of invasion, blood vessel invasion, TNM classification UICC cancer staging and high number of infiltrating CD163 positive and CD204 positive macrophages as a marker for the M2 macrophage phenotype. These findings may indicate novel roles of FGF-2-NCAM/FGFR1 interplay in ESCCs and its microenvironment including TAMs. Citation Format: NOBUHISA TAKASE, Yuichiro Koma, Noriaki Arai, Himiko Kodaira, Masayoshi Hosono, Yumi Ichihara, Mari Nishio, Manabu Shigeoka, Hiroshi Yokozaki. The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 730.

Conversion of human M-CSF macrophages into foam cells reduces their proinflammatory responses to classical M1-polarizing activation

BackgroundIn atherosclerotic lesions, cholesterol-laden macrophage foam cells are formed and exposed to M1- and M2-polarizing factors. However, the effects of the polarizing factors on the proinflammatory and the anti-inflammatory potential of foam cells are not known. ObjectiveTo investigate the effects of M1- and M2-polarizing factors on the expression of pro- and anti-inflammatory genes in cultured human macrophage foam cells. Methods and ResultsHuman monocytes were differentiated into macrophages in the presence of M-CSF, and then converted into cholesterol-loaded foam cells by incubation with acetylated LDL. The generated macrophages and foam cells were polarized into the M1 phenotype by classical activation with LPS and IFN-γ, or into the M2 phenotype by alternative activation with IL-4. When subjected to the M1-polarizing factors, the macrophages responded by typical upregulation of several key proinflammatory genes (TNFA, IL1B, CXCL8, CCL19, and COX2), while the anti-inflammatory genes (MRC1, CCL17, and IL10) displayed variable responses. The foam cells, again, showed a weaker response to the M1-polarizing factors, as indicated by reduced upregulation of the proinflammatory genes, reduced secretion of TNF-α, and a trend towards lower NF-κB activity. When subjected to alternative M2 polarization, both macrophages and foam cells responded by a typical upregulation of the anti-inflammatory genes, which was of equal magnitude in both cell types. ConclusionsConversion of cultured human macrophages into foam cells suppresses their proinflammatory responses to M1-polarizing factors. Thus, in M1-polarizing microenvironments of atherosclerotic lesions, foam cell formation may locally weaken the macrophage-dependent inflammatory component of atherogenesis.

Niche-Dependent Growth of Malignant and Pre-Neoplastic Plasma Cells in Humanized Mice

Multiple myeloma (MM) is characterized by progressive growth of transformed plasma cells (PC) in the bone marrow. In nearly all cases, MM is preceded by clinically asymptomatic precursor states termed as monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic MM (AMM). Almost all prior attempts to study the growth of transformed PCs in vivo have been restricted to patients with clinical MM, and there is an unmet need for in vivo models to understand the biology of precursor states. Major obstacles to xenotransplantation of human cells in immune-deficient mice include murine innate immune rejection, as well as presence of species-restricted non-cross reactive growth factors/cytokines. In order to help overcome these obstacles, we utilized humanized mice in which human versions of 5 genes important for innate immune cell development and hematopoiesis were knocked into their respective murine loci. These mice termed MIS(KI)TRG for human M-CSF, IL-3, GM-CSF, Thrombopoietin and SIRPα knock-in on a Rag2-/-IL-2Rγ-/- background, exhibited superior multi-lineage engraftment of human hematopoietic stem cells including innate immune cells. Interleukin-6 (IL-6) is well established as a critical growth factor for human MM cells and lacks species cross-reactivity. Therefore, we knocked-in human IL-6 to MIS(KI)TRG mice to generate MIS(KI)TRG6 mice that were utilized for these studies.MIS(KI)TRG6 mice were transplanted intra-femoral with bone marrow mononuclear cells isolated from patients with plasma cell disorders (n=27). Growth of tumor cells was monitored by flow cytometry and by ELISA detection of tumor-derived clonal human Ig. We observed successful engraftment of tumor cells following transplantation of purified CD138+ cells as well as CD3-depleted CD138- mononuclear cells or simply CD3-depleted bulk bone marrow mononuclear cells in >80% of experiments. Importantly, the engrafted myeloma cells were primarily restricted to the transplanted bone confirming the niche requirement of these cells. Growth of tumor cells in the contralateral bone was typically observed when samples from patients with more aggressive disease were transplanted. Growth of tumor cells in the spleen was only observed in the setting of patients with circulating phase tumors, such as those with plasma cell leukemia. In contrast to tumor cells, non-malignant cells such as human T, NK or myeloid cells readily migrated to the periphery and were detected in the spleen by flow cytometry. Together these data indicate that the capacity to grow independent of the marrow niche is a late event in the pathogenesis of MM.Importantly, in addition to clinical MM, this model also allowed for the first time, efficient growth of asymptomatic precursor states (MGUS/AMM) in 5 of 5 patients engrafted. Interestingly, tumor cells from MGUS/AMM mediate progressive growth in vivo, indicating that the clonal stability observed in these patients is likely mediated by features extrinsic to the plasma cell clone. Detailed analysis of phenotypic features of engrafted plasma cells by single cell mass cytometry revealed phenotypic similarities between freshly isolated tumor cells and those growing in mice.In summary, our studies demonstrate that humanized MIS(KI)TRG6 mice are an excellent host for in vivo growth of the entire spectrum of human plasma cell tumors, including for the first time, pre-neoplastic states. Our studies provide evidence that clonal stability in MGUS/AMM is likely in part due to growth controls extrinsic to the tumor cells. The capacity to metastasize from bone to bone and eventually to extra-medullary sites is acquired later during evolution of tumors. The ability to faithfully reproduce homing and growth patterns of primary tumor cells will allow detailed evaluation of plasma cell tumors in their natural microenvironment. DisclosuresNo relevant conflicts of interest to declare.