Promotes Monocyte Recruitment Research Articles

Hypoxia-induced miR-210-3p expression in lung adenocarcinoma potentiates tumor development by regulating CCL2 mediated monocyte infiltration.

In most cancers, tumor hypoxia downregulates the expression of C-C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is not yet clear. We compared noncancerous and lung-adenocarcinoma human samples for hypoxia-inducible factor 1-alpha (HIF-1A), microRNA-210-3p (mir-210-3p), and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia-induced miR-210-3p in the regulation of CCL2 expression and macrophage polarization. HIF-1Α stabilization increases miR-210-3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR-210-3p directly binds to the 3'untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR-210-3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR-210-3p mimic-transfected HIF-1A silenced cancer cells. In contrast, inhibition of miR-210-3p in HIF-1A-overexpressed cells markedly restored monocyte migration, highlighting a direct link between the miR-210-3p level and tumor monocyte burden. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an antitumor M1 phenotype. Anti-hsa-miR-210-3p-locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR-210-3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Unraveling the role of galectin-3 in cardiac pathology and physiology.

Galectin-3 (Gal-3) is a carbohydrate-binding protein with multiple functions. Gal-3 regulates cell growth, proliferation, and apoptosis by orchestrating cell-cell and cell-matrix interactions. It is implicated in the development and progression of cardiovascular disease, and its expression is increased in patients with heart failure. In atherosclerosis, Gal-3 promotes monocyte recruitment to the arterial wall boosting inflammation and atheroma. In acute myocardial infarction (AMI), the expression of Gal-3 increases in infarcted and remote zones from the beginning of AMI, and plays a critical role in macrophage infiltration, differentiation to M1 phenotype, inflammation and interstitial fibrosis through collagen synthesis. Genetic deficiency of Gal-3 delays wound healing, impairs cardiac remodeling and function after AMI. On the contrary, Gal-3 deficiency shows opposite results with improved remodeling and function in other cardiomyopathies and in hypertension. Pharmacologic inhibition with non-selective inhibitors is also protective in cardiac disease. Finally, we recently showed that Gal-3 participates in normal aging. However, genetic absence of Gal-3 in aged mice exacerbates pathological hypertrophy and increases fibrosis, as opposed to reduced fibrosis shown in cardiac disease. Despite some gaps in understanding its precise mechanisms of action, Gal-3 represents a potential therapeutic target for the treatment of cardiovascular diseases and the management of cardiac aging. In this review, we summarize the current knowledge regarding the role of Gal-3 in the pathophysiology of heart failure, atherosclerosis, hypertension, myocarditis, and ischemic heart disease. Furthermore, we describe the physiological role of Gal-3 in cardiac aging.

Association of Circulating Neutrophils with Relative Volume of Lipid-Rich Necrotic Core of Coronary Plaques in Stable Patients: A Substudy of SMARTool European Project

Coronary atherosclerosis is a chronic non-resolving inflammatory process wherein the interaction of innate immune cells and platelets plays a major role. Circulating neutrophils, in particular, adhere to the activated endothelium and migrate into the vascular wall, promoting monocyte recruitment and influencing plaque phenotype and stability at all stages of its evolution. We aimed to evaluate, by flow cytometry, if blood neutrophil number and phenotype-including their phenotypic relationships with platelets, monocytes and lymphocytes-have an association with lipid-rich necrotic core volume (LRNCV), a generic index of coronary plaque vulnerability, in a group of stable patients with chronic coronary syndrome (CCS). In 55 patients, (68.53 ± 1.07 years of age, mean ± SEM; 71% male), the total LRNCV in each subject was assessed by a quantitative analysis of all coronary plaques detected by computed tomography coronary angiography (CTCA) and was normalized to the total plaque volume. The expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, CXCR4 and CD41a cell surface markers was quantified by flow cytometry. Adhesion molecules, cytokines and chemokines, as well as MMP9 plasma levels, were measured by ELISA. On a per-patient basis, LRNCV values were positively associated, by a multiple regression analysis, with the neutrophil count (n°/µL) (p = 0.02), neutrophil/lymphocyte ratio (p = 0.007), neutrophil/platelet ratio (p = 0.01), neutrophil RFI CD11b expression (p = 0.02) and neutrophil-platelet adhesion index (p = 0.01). Significantly positive multiple regression associations of LRNCV values with phenotypic ratios between neutrophil RFI CD11b expression and several lymphocyte and monocyte surface markers were also observed. In the bivariate correlation analysis, a significantly positive association was found between RFI values of neutrophil-CD41a+ complexes and neutrophil RFI CD11b expression (p < 0.0001). These preliminary findings suggest that a sustained increase in circulating neutrophils, together with the up-regulation of the integrin/activation membrane neutrophil marker CD11b may contribute, through the progressive intra-plaque accumulation of necrotic/apoptotic cells exceeding the efferocytosis/anti-inflammatory capacity of infiltrating macrophages and lymphocytes, to the relative enlargement of the lipid-rich necrotic core volume of coronary plaques in stable CAD patients, thus increasing their individual risk of acute complication.

ALKBH5-mediated m6 A demethylation of TIRAP mRNA promotes radiation-induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma.

Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6-methyladenylate methylation (m6 A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation-induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC-educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated. Radiation-induced ALKBH5 expression in HSC mediated m6 A demethylation of toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF-κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up-regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown-combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation-induced liver injury and poor tumour response to radiotherapy. Collectively, irradiation up-regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.

Di-n-butyl phthalate promotes monocyte recruitment via miR-137-3p-SP1-MCP-1 pathway

Since non-covalent bound character and widespread application in numerous products, people are exposed to di-n-butyl phthalate (DBP) at low levels through various ways. Epidemiological studies suggested an association between DBP exposure and atherosclerosis (AS). Still, molecular mechanisms remain unclear. This study aimed to explore the effects of DBP on monocyte recruitment, a key and initial step of AS. EA.hy926 cells were treated with DBP (10−9–10−5 M) or DMSO as control. Chemotaxis assay was applied to investigate THP-1 recruitment. Expression of mRNA /miRNAs and proteins were measured by qRT-PCR and Western blotting, respectively. Levels of monocyte chemotactic protein 1 (MCP-1) in supernatant were detected by ELISA assay. Receptor internalization assay was performed to confirm C-C chemokine receptor type 2 (CCR2) subcellular localization in THP-1 cells and the binding between CCR2 and MCP-1. Dual-luciferase reporter assay was used to analyze the combination between miR-137-3p and specificity protein 1 (SP1), as well as SP1 and MCP-1. Results showed that number of recruited THP-1 cells after EA.hy926 cells treated by DBP was significantly higher than that in the control group due to promoted MCP-1 expression. In addition, expression of MCP-1 was regulated through miR-137-3p-SP1 cascade. Besides, overexpression of miR-137-3p reversed the increased number of recruited THP-1 cells. Our results implied that DBP might promote THP-1 recruitment by targeting miR-137-3p-SP1-MCP-1 in EA.hy926 cells.

CCR2 promotes monocyte recruitment and intestinal inflammation in mice lacking the interleukin-10 receptor

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

The receptor of the colony-stimulating factor-1 (CSF-1R) is a novel prognostic factor and therapeutic target in follicular lymphoma

Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures (n = 19) and in vivo mouse co-xenografts demonstrated that FL-FDC crosstalk favors tumor growth and, via the secretion of CCL2 and CSF-1, promotes monocyte recruitment, differentiation, and polarization towards an M2-like protumoral phenotype. Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy.

Visfatin increases ICAM-1 expression and monocyte adhesion in human osteoarthritis synovial fibroblasts by reducing miR-320a expression.

Pathophysiological events that modulate the progression of structural changes in osteoarthritis (OA) include monocyte adhesion and infiltration, and synovial inflammation. In particular, the adhesion protein intercellular adhesion molecule type 1 (ICAM-1) promotes monocyte recruitment into the synovial tissue. Visfatin is an adipocyte hormone that promotes the release of inflammatory cytokines during OA progression. We report that visfatin enhances ICAM-1 expression in human OA synovial fibroblasts (OASFs) and facilitates the adhesion of monocytes with OASFs. AMPK and p38 inhibitors, as well as their respective siRNAs, attenuated the effects of visfatin upon ICAM-1 synthesis and monocyte adhesion. We also describe how miR-320a negatively regulates visfatin-induced promotion of ICAM-1 expression and monocyte adhesion. We detail how visfatin affects ICAM-1 expression and monocyte adhesion with OASFs by inhibiting miR-320a synthesis via the AMPK and p38 signaling pathways.

P0068APABETALONE, A CLINICAL STAGE BET INHIBITOR, REDUCES THE PRO-INFLAMMATORY RESPONSE IN PBMCS FROM FABRY DISEASE PATIENTS ON ERT THERAPY

Abstract Background and Aims Fabry disease (FD) is a rare X-linked recessive genetic disorder caused by mutations in the α-galactosidase A gene. Absence or defects of this lysosomal enzyme lead to globotriaosylceramide (Gb3) accumulation. Gb3 disrupts basic cellular metabolic processes, inducing oxidative stress and promoting inflammation. Systemically, Gb3 accumulation leads to organ damage throughout the body with an increase in renal, cardiac, cerebrovascular and skin complications. Enzyme replacement therapy (ERT) is current standard of care. This therapy may reduce Gb3 deposits but the impact on cardiac and kidney function is unclear based on the limited studies. Further, ERT is less effective in the late phases of FD, partially due to the development of uncontrolled inflammation and fibrosis. Apabetalone is a clinical stage orally administered bromo and extra terminal domain inhibitor (BETi) that has beneficial effects on vascular inflammation and vascular calcification through an epigenetic pathway. Based on its mechanism of action and impact on multiple disease drivers in both preclinical and clinical studies, apabetalone has therapeutic potential to modulate inflammation in FD patients on ERT therapy. Method PBMCs and neutrophils were isolated from fresh blood of FD patients ± ERT therapy using density gradient centrifugation. The baseline immune status of PBMCs was compared in naïve FD patients (without ERT, n=3) and ERT treated patients (n=8) via flow cytometry. PBMCs or neutrophils from ERT patients were treated ex vivo with 5µM or 20µM apabetalone ± LPS stimulation. Expression of key pro-inflammatory mediators was analyzed with real time quantitative PCR. The production of reactive oxygen species (ROS) in neutrophils was assessed by flow cytometry. Results The PBMC inflammatory profile in baseline showed monocytes from ERT patients produced lower amounts of pro-inflammatory mediators (TNFα and IL6) than naïve patients (∼60%, trending p≤0.06 and p≤0.08 respectively). However, surface abundance of CCR2, a chemokine receptor for the MCP1 chemokine that promotes monocyte recruitment to local inflamed tissue during inflammatory process, was ∼2-fold greater on monocytes from ERT subjects than the naïve controls (trending p≤0.06). Gene expression of MCP1 was approximately 5-fold higher in PBMC from ERT patients versus naïve controls (p=0.01). In response to ex vivo LPS stimulation, PBMCs from FD patients on ERT showed a robust induction of pro-inflammatory responses with increases in gene expression of MCP1, IL12B, TNFA and IL6. Ex vivo treatment with apabetalone (5µM) blocked the LPS-induction of MCP1 gene expression (∼90%, p&amp;lt;0.0001), and suppressed the upregulation of IL12B, TNFA and IL6 by 85%, 27% and 17%, respectively (p&amp;lt;0.0001,p=0.04,p=0.004). In response to LPS stimulation, neutrophils induced reactive oxygen species (ROS), an indicator of oxidative damage caused by intracellular Gb3 deposit in FD patients, by ∼6 fold. Apabetalone treatment reduced this induction by 32% and 62% at 5µM and 20 µM, respectively (p=0.01, p=0.0007). Conclusion Monocytes from FD patients on ERT therapy display an enhanced expression of the CCR2-MCP-1 axis as compared to naïve controls, indicating the potential for immune cell tissue infiltration and local inflammation. Apabetalone counters this increase by preventing MCP1 gene transcription. Apabetalone also reduces PBMC mediated pro-inflammatory gene transcription (TNFA, IL12B, IL6) and neutrophil mediated ROS production in response to LPS stimulation. Therefore, apabetalone treatment may reduce pathological inflammation in FD patients and thus complement ERT to optimize patient outcomes, which will be tested further with warranted clinical studies.

MP06-15 MYOFIBROBLASTS CYTOKINES SECRETED BY THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA PROMOTE MONOCYTE RECRUITMENT AND INDUCE POLARIZATION OF THP1 TO M2-TYPE MACROPHAGES

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-15 MYOFIBROBLASTS CYTOKINES SECRETED BY THE PROGRESSION OF BENIGN PROSTATIC HYPERPLASIA PROMOTE MONOCYTE RECRUITMENT AND INDUCE POLARIZATION OF THP1 TO M2-TYPE MACROPHAGES Muqiu Zhang* Muqiu Zhang*Muqiu Zhang* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Introduction and Objective: Our previous studies, stroma–epithelium ratios and collagen deposition were prevalent in rapidly-progressed BPH patients (age ≤50 years old) . Meanwhile, we also detected that increased M2 macrophage infiltration in rapidly-progressed BPH patients, M2 macrophage induces myofibroblast phenotype during the progression of benign prostatic hyperplasia. The present study mainly investigates if myofibroblasts cytokines secreted by the progression of benign prostatic hyperplasia may promote monocyte recruitment and induce polarization of THP1 to M2-type macrophages. METHODS: Methods: We separated the human primary prostate fibroblast (PrPF) cells from the early progressed BPH (PrPF-early), age-matched prostate (PrPF-control), and elderly BPH tissues (PrPF-old). In vitro, we uesd flow cytometry to detect CD163 and CD206 (M2-type macrophages’ markers ) expression of THP-1 cells after co-culture 4days with three groups of human primary prostate fibroblast, respectively. Quantitative PCR was used to identify cytokines secreted by three groups of human primary prostate fibroblast. RESULTS: Results: THP-1 was recruited by human primary prostate fibroblast of rapidly-progressed BPH than in the other two groups. Significant increase in the percentage of M2 macrophages after co-culture with PrPF-early. Cytokines MCP-1 and IL-6 were increased in primary prostate fibroblast of rapidly-progressed BPH. Primary prostate fibroblast of rapidly-progressed BPH could recruit THP-1 and induce polarization of THP1 to M2-type macrophages. CONCLUSIONS: Conclusion: The early-progressed BPH fibroblasts recruit THP-1 and induce polarization of THP1 to M2-type macrophages. Source of Funding: no © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e56-e57 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Muqiu Zhang* More articles by this author Expand All Advertisement PDF downloadLoading ...

Targeted Inhibition of CD47-Sirp Alpha Requires Fc-Fc Gamma Receptor Interactions to Maximize Phagocytosis in T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Monoclonal antibodies (mAbs) that inhibit engagement of the “don't eat me” signal CD47 with SIRPα on myeloid cells may induce phagocytosis and have exhibited promise in multiple cancers. We dissected the role of CD47 blockade in opsonization and phagocytosis-induction through Fc-Fc gamma receptor (FcγR) interactions. CD47 was expressed at higher levels on 22 TCL cell lines by flow cytometry compared to quiescent T cells from 10 healthy donors (mean fluorescent intensity, 176.9 versus 69.85; Welch t-test p=0.002). We determined tumor cell-specific protein expression of CD47 by calculation of H scores in a tissue microarray (TMA) of 68 primary TCLs. The common subtypes (PTCL-NOS (n=16), AITL (n=19), and ALCL (n=12)) each had heterogeneous CD47 expression that was not associated with progression-free or overall survival. We utilized B6H12, an anti-CD47 mAb to assess apoptosis, antibody-dependent cell mediated-cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) of TCL cells (HuT-78, HH, Myla, SU-PM2, KIJK, MAC-2A, SMZ1 and K-299) and no increase was observed except for ADCC by murine FcγRIII-expressing engineered Jurkat cells. Upon addition of B6H12 or SRF231, a fully human IgG4 mAb against CD47 that is currently in a Phase I clinical trial (NCT03512340), phagocytosis of CFSE+ TCL cells with human monocyte-derived macrophages (hMDMs) and murine bone-marrow derived macrophages (mBMDMs) was increased. In contrast, induction of phagocytosis of resting T cells from healthy donors was tested using B6H12 and no increase was observed (22.7 and 19.5%). A flow cytometry-based receptor occupancy assay demonstrated a linear correlation between receptor binding and phagocytosis induction in Jurkat T-cell lymphoblastic leukemia cells. hMDMs failed to induce phagocytosis of TCL cells with F(ab')2 portion of B6H12 suggesting complete dependence on Fc receptors. In contrast, mBMDM-mediated phagocytosis of TCL cells with the F(ab')2 portion of B6H12 (mean, 21.5%) was increased compared to negative control but reduced compared to full length (mean, 33.4%), suggesting both Fc-dependent and Fc-independent mechanisms. mBMDMs from FcγR knockout mice demonstrated a statistically significant decrease in phagocytosis (compared to wild-type mBMDMs) of Jurkat, HuT-78, MAC2A and K-299 cells with B6H12, confirming a role for Fc-FcγR engagement. Phagocytosis of tumor cells during CD47 blockade may depend on the ability of SLAMF7 to interact with the integrin Mac-1 (Chen J, Nature 2017). Phagocytosis assays of TCL cells with mBMDMs from Mac-1 knockout mice with full length and F(ab')2 B6H12 demonstrated a reduction in phagocytosis using Mac-1 knockout macrophages (compared to wild-type) only with full-length B6H12. Thus, the Mac-1 contribution to phagocytosis induced by B6H12 in this context is Fc-dependent. We tested SRF231 in vivo using patient-derived xenograft (PDX) models of systemic hepatosplenic (HSTL), transformed cutaneous, and NKTCL. After 10 days, SRF231-treated mice had remarkable reductions in tumor burden compared to untreated mice, across multiple compartments. SRF231 treatment resulted in prolonged survival despite a short course of therapy (Figure 1). Bone marrow from xenografted mice, collected at day 11 post-treatment with SRF231, had significantly increased cytokines known to promote monocyte recruitment. Ex-vivo phagocytosis assays using mBMDMs and hMDMs, enhanced engulfment of HSTL, T-PLL and anaplastic large cell-lymphoma PDX harvested cells upon exposure to SRF231. Finally, we observed that in our HSTL PDX model, depletion of macrophages with clodronate completely abrogated the antitumor efficacy of SRF231 while depletion of neutrophils with anti-Ly6G Ab had no effect, highlighting macrophages as the essential effectors of phagocytosis in this context. In summary, monoclonal antibodies that disrupt CD47-SIRPα have striking in vitro and in vivo preclinical efficacy but require interactions with human FcγR on macrophages to maximize efficacy. [Display omitted] DisclosuresPeluso:Surface Oncology: Employment, Equity Ownership. O'Connor:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Weinstock:Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy.

Smooth muscle glucose metabolism promotes monocyte recruitment and atherosclerosis in a mouse model of metabolic syndrome.

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor-deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.

C-Reactive Protein in Atherothrombosis and Angiogenesis.

C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

Papillary renal cell carcinoma-derived chemerin, IL-8, and CXCL16 promote monocyte recruitment and differentiation into foam-cell macrophages

Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma. The only curative treatment available for pRCC is radical surgery. If the disease becomes widespread, neither chemo- nor radiotherapy will have therapeutic effect, hence further research on pRCC is of utmost importance. Histologically, pRCC is characterized by a papillary growth pattern with focal aggregation of macrophages of the foam cell phenotype. In other forms of cancer, a clear role for tumor-associated macrophages during cancer growth and progression has been shown. Although the presence of foamy macrophages is a histological hallmark of pRCC tumors, little is known regarding their role in pRCC biology. In order to study the interaction between pRCC tumor and myeloid cells, we established primary cultures from pRCC tissue. We show that human pRCC cells secrete the chemokines IL-8, CXCL16, and chemerin, and that these factors attract primary human monocytes in vitro. RNAseq data from The Cancer Genome Atlas confirmed a high expression of these factors in pRCC tissue. Conditioned medium from pRCC cultures induced a shift in human monocytes toward the M2 macrophage phenotype. In extended cultures, these macrophages became enlarged and loaded with lipids, adopting the foam cell morphology found in pRCC tissue. These results show for the first time that pRCC primary tumor cells secrete factors that attract and differentiate monocytes into anti-inflammatory tumor-associated macrophages with foam cell histology.

Corticosterone mobilizes monocytes into circulation and induces endothelial adhesion molecule expression to cause monocyte recruitment to the brain during stress

Repeated social defeat stress causes release of bone marrow-derived monocytes into circulation and increases endothelial cell adhesion molecule expression to promote monocyte recruitment to the brain. Sympathetic activation during stress has been previously associated with increased circulating monocytes in the blood. However, stress also activates the hypothalamic-pituitary-adrenal axis to secrete corticosterone (cort). To understand the role of cort in immune dysregulation during stress, adrenalectomized (ADX) mice and non-ADX mice were exposed to six days of repeated social defeat stress. ADX mice produced the same levels of bone marrow monocytes as non-ADX mice but released fewer cells into circulation during stress. We show here for the first time that the chemokine CXCL12, involved in retention of bone marrow cells, was reduced during stress - an effect prevented by ADX. Stress-induced increases in monocytes and intercellular adhesion molecule 1 (ICAM1) expression in the brain were also prevented by ADX. Notably, ADX independently altered key regulatory genes (CX3CR1, nr4a1) in microglia. ADX prevented morphological remodeling of microglia during stress but did not prevent expression of proinflammatory cytokines. Overall, we show here that corticosterone promotes mobilization of monocytes in the periphery, and their recruitment to the brain via endothelial cell adhesion molecule expression. Furthermore, physiological corticosterone is necessary for microglia homeostasis, and corticosterone depletion and over-production can both lead to microglial dysregulation in the brain.

IL-33 Drives Monocyte Recruitment to Lung Interstitium through Chemokine Upregulation.

Tissue infiltration by circulating monocytes is a critical step in the initiation and augmentation of type 2 inflammatory responses in the lungs. Our studies demonstrate that IL-33−/− mice have a defect in monocyte extravasation from the vasculature to the lung interstitium during induction of type 2 inflammatory responses. This result suggests that monocyte migration to the lungs is IL-33 dependent, and we found that administration of exogenous recombinant IL-33 is sufficient to restore monocyte localization to the lung interstitium. Further investigation of the effect of early administration of recombinant IL-33 on the lungs identified upregulation of multiple chemokines including the monocyte chemoattractants CCL2, CCL7, and CCL22. Importantly, blockade of G-protein coupled receptor–dependent signaling, and thereby chemokine receptor activity, inhibited IL-33–driven monocyte recruitment. CCR2 deficiency prevented recruitment of monocytes to the lung extravascular space during allergic sensitization, and resulted in reduced eosinophilia after allergen challenge. Thus, IL-33 plays a critical role in the initiation of type 2 inflammatory responses by inducing upregulation of chemokines that promote monocyte recruitment to the lung interstitium.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6-/- mice contain less inflammatory (CCR2hiCX3CR1lo) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2hiCX3CR1lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2hiCX3CR1lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis.

The role of transforming growth factor (TGF)-β in the infarcted myocardium.

The adult mammalian heart has negligible regenerative capacity. Following myocardial infarction, sudden necrosis of cardiomyocytes triggers an intense inflammatory reaction that clears the wound from dead cells and matrix debris, while activating a reparative program. A growing body of evidence suggests that members of the transforming growth factor (TGF)-β family critically regulate the inflammatory and reparative response following infarction. Although all three TGF-β isoforms (TGF-β1, -β2 and -β3) are markedly upregulated in the infarcted myocardium, information on isoform-specific actions is limited. Experimental studies have suggested that TGF-β exerts a wide range of actions on cardiomyocytes, fibroblasts, immune cells, and vascular cells. The findings are often conflicting, reflecting the context-dependence of TGF-β-mediated effects; conclusions are often based exclusively on in vitro studies and on associative evidence. TGF-β has been reported to modulate cardiomyocyte survival responses, promote monocyte recruitment, inhibit macrophage pro-inflammatory gene expression, suppress adhesion molecule synthesis by endothelial cells, promote myofibroblast conversion and extracellular matrix synthesis, and mediate both angiogenic and angiostatic effects. This review manuscript discusses our understanding of the cell biological effects of TGF-β in myocardial infarction. We discuss the relative significance of downstream TGF-β-mediated Smad-dependent and -independent pathways, and the risks and challenges of therapeutic TGF-β targeting. Considering the high significance of TGF-β-mediated actions in vivo, study of cell-specific effects and dissection of downstream signaling pathways are needed in order to design safe and effective therapeutic approaches.

Harnessing the Tumor Microenvironment for the Treatment of Double Hit Lymphoma

Double Hit Lymphomas (DHL) constitute 6-9% of all newly-diagnosed Diffuse Large B-cell Lymphomas (DLBCL) and are defined by MYC translocation in concert with BCL2 (80%) or BCL6 (20%) rearrangement. DHL is characterized by poor upfront response to standard chemoimmunotherapies. The development of more effective treatment for DHL has been limited by the lack of models that accurately recapitulate the biology of this disease. To faithfully model bona fide human DHL, we generated two patient-derived xenografts (PDXs) in NSG mice from patients with DHL who relapsed after or were refractory to R-CHOP. Both PDXs had low/negative expression of surface CD20, in line with previous reports of DHL, and high surface expression of CD52. Importantly, these features recapitulated the original tumor phenotypes. We reasoned that immunologic approaches that target DHLs may help overcome their resistance to cytotoxic chemotherapy. Given the surface expression of CD52 on both PDXs, we treated xenografted mice with the anti-CD52 antibody Alemtuzumab (2x5mg/kg) when >2% tumor was detected in peripheral blood, a time corresponding to marked splenomegaly and ~30% bone marrow involvement. In both models, Alemtuzumab markedly reduced tumor involvement in the peripheral blood and spleen; however, the bone marrow was largely refractory to Alemtuzumab treatment. This is in line with previous results (Pallasch et al. Cell 2014) who also observed bone marrow-specific resistance to Alemtuzumab using a humanized transgenic model of DHL. In the latter model, Alemtuzumab resistance was successfully overcome by high-dose cyclophosphamide (CTX, 100mg/kg), which induced a tumor secretory response that induced macrophage-mediated phagocytosis of tumor cells. Thus, we treated both PDX models with vehicle, CTX 100mg/kg, Alemtuzumab or CTX+Alemtuzumab. While CTX monotherapy was able to partially reduce bone marrow tumor burden, in both PDXs, a single dose of CTX+Alemtuzumab was highly synergistic and eradicated nearly all bone marrow disease (Figure 1), which resulted in significantly extend murine survival compared to either CTX or Alemtuzumab alone alone (p<0.01). In contrast, high-dose Doxorubicin (DOX, 5mg/kg) had minimal effect on bone marrow DHL and DOX+Alemtuzumab was not synergistic. Strikingly, CD11b+F4/80+CD11c- macrophages comprised 20-40% of bone marrow cellularity 8 days after CTX treatment of xengorafted mice. DHL cells isolated from treated mice were next examined for changes in surface protein expression. At 16 hours after treatment, bone marrow DHL cells in mice receiving CTX (but not those receiving DOX or vehicle) had significantly reduced levels of surface CD47, the “don’t eat me” signal that suppresses macrophage phagocytosis. By 48 hours after treatment, bone marrow DHL cells in mice receiving CTX (but not those receiving DOX or vehicle) had significantly increased expression of the pro-phagocytic proteins CD31 and Calreticulin. Next, we performed cytokine array analysis on bone marrow from mice engrafted with DHL PDXs at 16 hours after treatment. CTX-treated (but not vehicle- or DOX-treated) bone marrow had significantly increased levels of human IL-16 and CCL4, cytokines known to promote monocyte recruitment, as well as VEGF, PDGF and TNFα. Ex-vivo phagocytosis assays using murine bone marrow-derived macrophages confirmed that recombinant human VEGF and TNFα each enhanced macrophage phagocytosis of Alemtuzumab coated Raji lymphoma cells. Findings from RNA-seq studies on tumor cells and macrophages from the spleen and bone marrow as well as therapy induced changes in these respective niches will be further discussed at the meeting. In summary, we have demonstrated that high-dose CTX induces a phenotypic and secretory response from human DHL cells in vivo that promotes phagocytosis of Alemtuzumab-opsinized cells. As a result, we are initiating a clinical trial of this combination in patients with relapsed/refractory DHL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cardiac fibroblast cytokine profiles induced by proinflammatory or profibrotic stimuli promote monocyte recruitment and modulate macrophage M1/M2 balance in vitro

Macrophage polarization plays an essential role in cardiac remodeling after injury, evolving from an initial accumulation of proinflammatory M1 macrophages to a greater balance of anti-inflammatory M2 macrophages. Whether cardiac fibroblasts themselves influence this process remains an intriguing question. In this work, we present evidence for a role of cardiac fibroblasts (CF) as regulators of macrophage recruitment and skewing. Adult rat CF, were treated with lipopolysaccharide (LPS) or TGF-β1, to evaluate ICAM-1 and VCAM-1 expression using Western blot and proinflammatory/profibrotic cytokine secretion using LUMINEX. We performed in vitro migration and adhesion assays of rat spleen monocytes to layers of TGF-β1- or LPS-pretreated CF. Finally, TGF-β1- or LPS-pretreated CF were co-cultured with monocyte, to evaluate their effects on macrophage polarization, using flow cytometry and cytokine secretion.There was a significant increase in monocyte adhesion to LPS- or TGF-β1-stimulated CF, associated with increased CF expression of ICAM-1 and VCAM-1. siRNA silencing of either ICAM-1 or VCAM-1 inhibited monocyte adhesion to LPS-pretreated CF; however, monocyte adhesion to TGF-β1-treated CF was dependent on only VCAM-1 expression. Pretreatment of CF with LPS or TGF-β1 increased monocyte migration to CF, and this effect was completely abolished with an MCP-1 antibody blockade. LPS-treated CF secreted elevated levels of TNF-α and MCP-1, and when co-cultured with monocyte, LPS-treated CF stimulated increased macrophage M1 polarization and secretion of proinflammatory cytokines (TNF-α, IL-12 and MCP-1). On the other hand, CF stimulated with TGF-β1 produced an anti-inflammatory cytokine profile (high IL-10 and IL-5, low TNF-α). When co-cultured with monocytes, the TGF-β1 stimulated fibroblasts skewed monocyte differentiation towards M2 macrophages accompanied by increased IL-10 and decreased IL-12 levels. Taken together, our results show for the first time that CF can recruit monocytes (via MCP-1-mediated chemotaxis and adhesion to ICAM-1/VCAM-1) and induce their differentiation to M1 or M2 macrophages (through the CF cytokine profile induced by proinflammatory or profibrotic stimuli).