Upregulation Of CD36 Research Articles

CD14+HLA-DRlow/- expression: A novel prognostic factor in chronic lymphocytic leukemia.

Currently, no prognostic factors exist for determining the host immune status of chronic lymphocytic leukemia (CLL) patients. Therefore, the present report analyzed cluster of differentiation 14 (CD14)+ human leukocyte antigen (HLA)-DRlow/− myeloid-derived suppressor cells (MDSC) from 49 CLL patients and demonstrated that these cells were significantly expanded in all CLL patients when compared with monoclonal B cell lymphocytosis patients and healthy volunteers. Furthermore, upregulation of CD14+HLA-DRlow/− MDSCs was correlated with CLL tumor progression and a poor prognosis for CLL patients, and CD14+HLA-DRlow/− MDSCs were significantly correlated with the presence of CD4+ T and CD5+CD19+ cells in CLL patients, which could significantly inhibit the CD4+ T-cell immune response, contributing to CLL cell progression in CLL patients.

Up-Regulation of Prok-2 in Granulocytes Is Present BOTH in MGUS and MM

IntroductionOur previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM.AimProviding a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM.MethodsUsing oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age.Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set).ResultsWe found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories:i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1);ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta).In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation).In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics.ConclusionGranulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM. DisclosuresMusto:Celgene: Honoraria; Janssen: Honoraria.

Re-Educating Myeloma Associated Macrophages with Lenalidomide

Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear.Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry.Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells.Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells. DisclosuresNo relevant conflicts of interest to declare.

Cerium dioxide nanoparticles selectively up-regulate C-C chemokine receptor 2 and CD16 expression on human monocytes

Abstract Cerium dioxide nanoparticles (CeO2 NPs) are known as scavengers of reactive oxygen species for the coexistence of Ce3+/ Ce4+ oxidation states. Cell treatments with CeO2 NPs often lead to controversial pro-inflammatory and anti-inflammatory results. The aim of the study was to investigate the immune events following the administration of ceria nanoparticles to THP-1 monocytes. To address this issue, we performed flow cytometry, chemotaxis and ELISA experiments on THP-1 monocytes treated with different concentrations of CeO2 NPs. CeO2 nanoparticle treatments induced a significant pro-inflammatory C-C chemokine receptor 2 (CCR2) up-regulation within the first 6 hours lasting over-expressed for 24 hours. Differently, CCR5 showed no response at any concentration tested. Enhanced chemotaxis towards the CCR2 specific ligand MCP-1 reinforced the observation demonstrating a functional immune outcome. The pro-inflammatory profile of the treated monocytes was also supported by CD16 up-regulation but no differences in CX3CR1 or other monocyte receptors, like CD11b and CD14, were detectable. Moreover, CeO2 NPs exposure did not promote any release of inflammatory cytokines suggesting a specific and direct effect of the nanoparticles on CCR2 and CD16. Our in vitro results reveal a specific role of CeO2 NPs in the up-regulation of CCR2, which might contribute to increase the pro-inflammatory monocyte/macrophage migration toward the sites of CCL2 expression.

Adipogenic changes of hepatocytes in a high-fat diet-induced fatty liver mice model and non-alcoholic fatty liver disease patients.

Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis associated with liver inflammation. As NAFLD progresses, triglycerides increase within hepatocytes, causing typical vacuoles that resemble adipocytes. However, whether these morphological changes in hepatocytes indicate potential functional changes is unclear. C57BL/6J mice were fed a high-fat diet (HFD) containing 42% fat. Markers for adipocytes in the liver were measured using real-time PCR, Western blot, and double immunofluorescent labeling. Cytokines in cell culture supernatants were quantified with ELISA. To determine the macrophage phenotype, hepatic classical M1 markers and alternative M2 markers were analyzed. After a 24-week feeding period, adipocyte markers aP2 and PPARγ increased at both the mRNA and protein level in the liver of HFD-fed mice. FITC-labeled aP2 and rhodamine-labeled albumin were both stained in the cytoplasm of steatotic hepatocytes as observed under confocal laser scanning microscopy. Cell membrane-bound E-cadherin and albumin expression were reduced in steatotic hepatocytes compared to controls. However, hepatic adiponectin and adiponectin receptor-2 expression decreased with upregulation of hepatic CD36, suggesting impaired adiponectin activity in livers of HFD-fed mice. Moreover, steatotic primary hepatocytes not only released pro-inflammatory cytokines such as TNFα, MCP-1, IL-6, and IL-18, but also could activate macrophages when co-cultured in vitro. In vivo, hepatic expression of M1 genes such as iNOS and TNFα was markedly increased in HFD-fed mice. In contrast, hepatic expression of M2 genes such as Arg1 and CD206 was significantly reduced. Specifically, the ratio of TNFα to CD206 in HFD-fed mice was notably upregulated. Overexpression of adipocyte-specific genes in hepatocytes and their secretory function and epithelial phenotype impairment in NAFLD cause functional changes in steatotic hepatocytes aside from morphological changes. This suggests that adipogenic changes in hepatocytes are involved in pathogenesis of NAFLD.

Effect of influenza A infection on maturation and function of neonatal monocyte-derived dendritic cells.

Dendritic cells (DC) are essential for the first-line innate defense against influenza infection. The greater susceptibility to severe influenza infection in young infants and neonates may be attributed in part to their defective DC function. We sought to investigate the effect of influenza A virus (IAV) infection on the maturation, apoptosis, and function of monocyte-derived dendritic cells (MoDCs) from umbilical cord blood (UCB) and compared this with responses from adult peripheral blood (APB). Our findings were as follows. First, MoDCs derived from UCB showed deficient CD40, CD80, CD86, and HLA-DR upregulation following IAV infection compared to APB MoDCs. Second, IAV induced a multiplicity of infection (MOI)-dependent increase of apoptosis in UCB MoDCs, similar to that observed with APB. Third, the ability of UCB MoDCs to uptake dextran is decreased following IAV infection. Fourth, deficient TNF-α, but not IL-6, IFN-α response was induced by IAV infection of UCB MoDCs. Fifth, the ability of UCB MoDCs to promote allogeneic CD3 T-cell proliferation is inhibited by IAV infection. Taken together, we demonstrated a differential response of UCB and APB MoDCs following IAV infection, which may contribute in part to the increased susceptibility to severe influenza infection observed in young infants and neonates.

CD36 Upregulation Mediated by Intranasal LV-NRF2 Treatment Mitigates Hypoxia-Induced Progression of Alzheimer's-Like Pathogenesis

There is extensive evidence that oxidative stress induces cellular dysfunction in the brain and plays a critical role in Alzheimer's disease (AD) pathogenesis. Hypoxia increases factors involved in oxidative stress injury and contributes to the onset and progression of AD. Nuclear factor erythroid 2-related factor 2 (NRF2), a major component regulating antioxidant response, is attenuated in the AD brain. Importantly, NRF2 directly regulates the alternative first exons of CD36, an important participant in oxidative and inflammatory processes. To explore the effects of hypoxia-induced deterioration of AD-like pathogenesis and investigate the correlation between hypoxia-induced NRF2 signal alterations and CD36 expression, we examined the NRF2 signaling, CD36, and oxidative stress events in hypoxia-treated APPswe/PSEN1dE9 (APP/PS1) mice brain. We observed that hypoxia treatment increased oxidative stress, exacerbated inflammation, and aggravated learning defects in aged APP/PS1 mice. Microglia from hypoxia-treated mice brain exhibited marked reduction in CD36 expression and inhibition of β-amyloid (Aβ) degradation. Accordingly, hypoxia treatment caused a decrease in transactivation of NRF2 target genes in the aging mouse brain. Intranasal administration with a lentiviral vector encoding human NRF2 increased CD36 expression, ameliorated the weak antioxidant response triggered by hypoxia, diminished Aβ deposition, and improved spatial memory defects. In this study, we demonstrated for the first time that NRF2 intranasal treatment-induced increases of CD36 could enhance Aβ clearance in AD transgenic mouse. These results suggest that targeting NRF2-mediated CD36 expression might provide a beneficial intervention for cognitive impairment and oxidative stress in AD progression.

Abstract 442: D-4F, an Apolipoprotein A-I Mimetic Peptide, Protects Macrophages From Oxidized Low-Density Lipoprotein--Induced Apoptosis by Inhibiting the Endoplasmic Reticulum Stress-C/EBP Homologous Protein Pathway

Objective: D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, exerts a variety of atheroprotective functions similar to apoA-I, the major protein component of high density lipoprotein (HDL), including acting as an antioxidant, mediating cholesterol efflux from foam cells and direct anti-inflammatory effects. Our previous studies have demonstrated that endoplasmic reticulum (ER) stress promotes macrophage-derived foam cell formation by upregulating CD36 expression and mediates oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis. The goal of this study was to investigate the protective effect of D-4F on ox-LDL-induced macrophage cytotoxicity and specifically the ER stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis. Methods and Results: Treatment with D-4F (12.5, 25 and 50 mg/L) attenuated ox-LDL (100 mg/L)-induced cholesterol accumulation in RAW264.7 macrophages and foam cell formation in a dose-dependent manner. Similar to tunicamycin (TM), a classical ER stress inducer, ox-LDL reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of ox-LDL (100 mg/L) and TM (5 mg/L) were remarkably inhibited by D-4F treatment. Interestingly, we found that D-4F also significantly suppressed the ox-LDL- and TM-induced CD36 upregulation and activation of ER stress signaling events, including the phosphorylation of inositol-requiring enzyme 1 (IRE1) and nuclear translocation of activating transcription factor 6 (ATF6). In addition, exposure of RAW264.7 macrophages to ox-LDL or TM resulted in a significant increase in the expression of CHOP, a proapoptotic transcription factor regulated by IRE1 and ATF6 under conditions of ER stress. D-4F blocked these effects in a dose-dependent manner. Moreover, administration of apoE –/– mice with D-4F (1 mg/kg per day) suppressed apoptosis and the upregulation of CD36, phospho-IRE1, GRP78 and CHOP in macrophage-dense atherosclerotic lesions. Conclusion: These data indicate that D-4F can protect macrophages from ox-LDL-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ER stress-CHOP signaling pathway.

Notch modulates human NK cell differentiation through regulation of inhibitory killer immunoglobulin-like receptor (KIR) expression and acquisition of effector function (TUM2P.890)

Abstract The Notch signaling pathway plays a substantial role on human NK cell development, however the role of Notch on KIR upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood derived NK cells or sorted KIR- peripheral blood NK cells were cultured with IL-15 for 7 days in the presence or absence of gamma-secretase inhibitor, known to inhibit Notch signaling. Inhibition of Notch signaling significantly decreased KIR expression. Conversely, co-culturing the same cells with OP9 cells bearing Notch ligands enhanced KIR expression. Overexpression of the active portion of Notch on cord blood derived NK cells after 28 days of culture resulted in a 2-fold increase in KIR expression indicating that Notch signaling plays a direct, cell intrinsic, role in KIR regulation. By knocking down delta-like 1 (DLL1) on NK cells and co-culturing them with OP9 cells expressing Notch ligands we show that DLL1 mediated cis-inhibition controls KIR expression. Notch signaling also enhances CD16 upregulation that precedes KIR expression. More important than receptor expression is function. We found that Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even on post-transplant samples where NK cell function is inherently defective. Given these attributes of Notch signaling, we propose Notch agonists can enhance NK cell maturation and tumor killing in a post-transplant setting.

Endoplasmic Reticulum Stress Promotes Macrophage-derived Foam Cell Formation by Up-regulating Cluster of Differentiation 36 (CD36) Expression

Oxidized low-density lipoprotein (ox-LDL) up-regulates CD36, a scavenger receptor responsible for macrophage uptake of ox-LDL without limitation. However, the precise underlying mechanism is not completely understood. Our previous study has demonstrated that ox-LDL induces endoplasmic reticulum (ER) stress in macrophages. The goal of this study was to explore the exact relationship between ER stress and macrophage-derived foam cell formation and whether ER stress would be involved in ox-LDL-induced CD36 up-regulation. Our results showed that ox-LDL-induced lipid accumulation in macrophages was promoted synergistically by ER stress inducer tunicamycin (TM), while attenuated by ER stress inhibitor 4-phenylbutyric acid (PBA). Ox-LDL caused CD36 up-regulation with concomitant activation of ER stress as assessed by phosphorylation of inositol-requiring kinase/endonuclease-1 (IRE-1) and protein kinase-like ER kinase (PERK), up-regulation of X-box-binding protein 1 (XBP1) and glucose-regulated protein 78 (GRP 78), and nuclear translocation of activating transcription factor 6 (ATF6). TM not only up-regulated CD36 alone but also synergized with ox-LDL to increase CD36 expression. Alleviation of ER stress with PBA and siRNA against ATF6, IRE1, and GRP78 mitigated ox-LDL-induced CD36 protein up-regulation. Moreover, administration of apoE(-/-) mice with PBA suppressed the up-regulation of CD36, phospho-IRE1, and GRP78 in macrophage-dense atherosclerotic lesions and in peritoneal macrophages. Additionally, CD36 silencing attenuated ox-LDL-induced nuclear translocation of ATF6, phosphorylation of IRE1 and up-regulation of XBP1 and GRP78. These data indicate that CD36-mediated ox-LDL uptake in macrophages triggers ER stress response, which, in turn, plays a critical role in CD36 up-regulation, enhancing the foam cell formation by uptaking more ox-LDL.

CD137 Accurately Identifies and Enriches for Naturally Occurring Tumor-Reactive T Cells in Tumor

Upregulation of CD137 (4-1BB) on recently activated CD8(+) T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137(pos) TILs were sorted and evaluated for antitumor activity in vitro and in vivo. Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzyme-digested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137(pos) TILs, but not PD-1(pos) or PD-1(neg) CD137(neg) cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8(+) TILs upregulated CD137 expression after incubation with HLA-matched, MART-expressing cancer cells and antigen-specific effector function was restricted to the CD137(pos) subset in vitro. CD137(pos) TILs also mediated superior antitumor effects in vivo, compared with CD137(neg) TILs. Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials.

Krüppel-like factor 2 promotes liver steatosis through upregulation of CD36

The Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, apoptosis, development, and responses to external stress. In the present study, we aim to investigate the roles of KLF2 in hepatic steatosis. Our results showed that mRNA and protein levels of KLF2 were significantly elevated in livers from obese mice. Adenoviruses-mediated overexpression of KLF2 induced accumulation of triglycerides in C57BL/6 mice, whereas KLF2 silencing ameliorates hepatosteatosis in ob/ob mice. At the molecular level, our data established CD36 as a novel transcriptional target of KLF2. KLF2 upregulated CD36 expression through a consensus binding site on its proximal promoter region. Additionally, the steatotic effect of KLF2 was dramatically inhibited in CD36-null mice. Therefore, our study reveals a novel link between KLF2-induced hepatic triglyceride accumulation and the expression of CD36.

Combined effect of sesamin and soybean phospholipid on hepatic fatty acid metabolism in rats.

We studied the combined effect of sesamin (1:1 mixture of sesamin and episesamine) and soybean phospholipid on lipid metabolism in rats. Male rats were fed diets supplemented with 0 or 2 g/kg sesamin, and containing 0 or 50 g/kg soybean phospholipid, for 19 days. Sesamin and soybean phospholipid decreased serum triacylglycerol concentrations and the combination of these compounds further decreased the parameter in an additive fashion. Soybean phospholipid but not sesamin reduced the hepatic concentration of triacylglycerol. The combination failed to cause a strong decrease in hepatic triacylglycerol concentration, presumably due to the up-regulation of Cd36 by sesamin. Combination of sesamin and soybean phospholipid decreased the activity and mRNA levels of hepatic lipogenic enzymes in an additive fashion. Sesamin strongly increased the parameters of hepatic fatty acid oxidation enzymes. Soybean phospholipid increased hepatic activity of 3-hydroxyacyl-CoA dehydrogenase although it failed to affect the activity of other enzymes involved in fatty acid oxidation. Sesamin strongly increased hepatic concentration of carnitine. Sesamin and soybean phospholipid combination further increased this parameter, accompanying a parallel increase in mRNA expression of carnitine transporter. These changes can account for the strong decrease in serum triacylglycerol in rats fed a diet containing both sesamin and soybean phospholipid.

Resveratrol counteracts inflammation in human M1 and M2 macrophages upon challenge with 7-oxo-cholesterol: potential therapeutic implications in atherosclerosis.

Macrophages consist of two main subsets: the proinflammatory M1 subset and the anti-inflammatory M2 one. 7-oxo-cholesterol, the most abundant cholesterol autoxidation product within atherosclerotic plaque, is able to skew the M1/M2 balance towards a proinflammatory profile. In the present study, we explored the ability of the polyphenolic compound resveratrol to counteract the 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages. Resveratrol-pretreated human monocyte-derived M1 and M2 macrophages were challenged with 7-oxo-cholesterol and analyzed for phenotype and endocytic ability by flow cytometry, for metalloproteinase- (MMP-) 2 and MMP-9 by gelatin zymography, and for cytokine, chemokine, and growth factor secretome by a multiplex immunoassay. We also investigated the NF-κB signaling pathway. In the M1 subset, resveratrol prevented the downregulation of CD16 and the upregulation of MMP-2 in response to 7-oxo-cholesterol, whereas in M2 macrophages it prevented the upregulation of CD14, MMP-2, and MMP-9 and the downregulation of endocytosis. Resveratrol prevented the upregulation of several proinflammatory and proangiogenic molecules in both subsets. We identified modulation of NF-κB as a potential mechanism implicated in 7-oxo-cholesterol and resveratrol effects. Our results strengthen previous findings on the immunomodulatory ability of resveratrol and highlight its role as potential therapeutic or preventive compound, to counteract the proatherogenic oxysterol signaling within atherosclerotic plaque.

Epinephrine enhances the response of macrophages under LPS stimulation.

Trauma associated with infection may directly trigger a neuroendocrine reaction in vivo while the hormone epinephrine is known to mediate immune responses to inflammation after injury. However, the role of epinephrine during the earliest stage of trauma still remains unclear. We therefore explored the role of epinephrine on activated macrophages under LPS stimulation in vitro as well as the mechanisms underlying its effect. Dose- and time-dependent effects of epinephrine on macrophage immune function were assessed after LPS activation. We also employed CD14 siRNA interference to investigate whether CD14 played a role in the mechanism underlying the effect of epinephrine on LPS-induced macrophage responses. Our results showed that epinephrine pretreatment (10 ng/mL) significantly promoted immune responses from LPS stimulated macrophages, including phagocytic rate, phagocytic index, TNFα/IL-1β/IL-10 secretion, and CD14 expression (P < 0.05). Moreover, TNFα/IL-1β/IL-10 levels attained their peak value 1 hour after incubation with 10 ng/mL epinephrine (P < 0.05), and CD14 siRNA transfection dramatically decreased phagocytosis and cytokine secretion by LPS-activated macrophages (P < 0.05). We therefore conclude that 10 ng/mL epinephrine enhances immune responses from macrophages under LPS stimulation and that the underlying mechanism may relate to CD14 upregulation on the surface of macrophages.

The influence of Ouabain on human dendritic cells maturation.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Rosiglitazone-induced CD36 up-regulation resolves inflammation by PPARγ and 5-LO-dependent pathways

PPARγ-achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5-LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ-induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5-LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5(-/-) mice exposed to distal pMCAO. BMMs were obtained from Lys-M Cre(+) PPARγ(f/f) and Lys-M Cre(-) PPARγ(f/f) mice. Stereological counting of double-immunofluorescence-labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5-LO gene, which is induced in neurons by PPARγ activation and at least by one of its products--LXA4--which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5-LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.

Abstract 9623: Stimulatory Effects of Cardiotrophin-1 on Atherosclerosis

Cardiotrophin-1 (CT-1), a cytokine belonging to the interleukin-6 superfamily, was recently shown to be expressed in the intima of early atherosclerotic lesions in the human carotid artery. CT-1 stimulates pro-atherogenic molecule expression in human vascular endothelial cells (EC) and monocyte migration. However, it has not been reported whether CT-1 accelerates atherosclerosis. This study was performed to examine the stimulatory effects of CT-1 on human macrophage foam cell formation, human EC proliferation, and the migration and proliferation of human vascular smooth muscle cells (VSMCs) in vitro, and on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice in vivo. CT-1 was expressed at high levels in ECs and macrophages in both humans and ApoE-/- mice. CT-1 significantly enhanced oxidized low-density lipoprotein-induced foam cell formation associated with increased levels of CD36 and acyl-CoA:cholesterol acyltransferase-1 (ACAT1) expression in human monocyte-derived macrophages. CT-1 also significantly stimulated the migration and proliferation of human aortic VSMCs, but not EC proliferation. Four-week-infusion of CT-1 into ApoE-/- mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration and VSMC content in the aortic wall. Activation of inflammasome, such as apoptosis-associated speck-like protein containing a caspase recruitment domain, nuclear factor-κB, and cyclooxygenase-2, was observed in exudate peritoneal macrophages from ApoE-/- mice infused with CT-1. Infusion of anti-CT-1 neutralizing antibody alone into ApoE-/- mice significantly suppressed monocyte/macrophage infiltration in atherosclerotic lesions. These results indicate that CT-1 accelerates the development of atherosclerotic lesions by stimulating the inflammasome and foam cell formation associated with CD36 and ACAT1 up-regulation in macrophages and VSMC migration and proliferation, contributing to the occurrence of atherosclerotic vascular diseases. Blockade of cardiotrophin-1 is expected to emerge as a new line of therapy against atherosclerosis and its related diseases.

Macrophages Engulf Tumor B Cells Coated With Rituximab More Effectively Under The Hypoxic Condition

Growing evidence has been accumulated indicating that the tumor microenvironment plays a pivotal role in the pathogenesis of various tumors. Macrophages are one of the most important players in such a microenvironment, and those residing in tumor tissues are called “Tumor-Associated Macrophages (TAM)”. The TAM content predicts survival in follicular lymphoma patients following treatment, and a high TAM count was associated with a poor clinical course after conventional chemotherapy before the rituximab era. However, immunochemotherapy with rituximab is able to circumvent the unfavorable outcome associated with a high TAM count. This finding thus favors the hypothesis that TAM may act synergistically with anti-CD20–targeted therapy. Hypoxia is a key feature of the microenvironment of cancer cells, and actively participates in tumor progression. In this study, we investigated the impact of hypoxia on the interaction between rituximab and TAM. Macrophages were prepared by culturing isolated monocytes from adult volunteers at the Hokkaido Blood Bank in the presence of M-CSF for seven days in a humidified incubator containing 20% O2, 5% CO2 and 75% N2 (normoxic macrophages) or 1% O2, 5% CO2 and 94% N2(hypoxic macrophages). We used CD20-positive B cells from patients with chronic lymphocytic leukemia (CLL B cells) as target cells. When CLL B cells were opsonized with rituximab, the cell-binding assay showed that the hypoxic macrophages bound more effectively than normoxic macrophages (29.2±0.6% and 18.5±0.9%, respectively, p&lt;0.01). We then performed phagocytosis assays using standard cytospin techniques and microscopic evaluation of stained phagocytic cells. Again, the percentage of phagocytosis of CLL B cells in the presence of rituximab was significantly higher under hypoxic conditions compared to those cultured under normoxic conditions (22.5±1.3% and 13.9±0.9%, respectively, p&lt;0.01). We next investigated the expression of Fc receptors on macrophages cultured in the different oxygen conditions. A flow cytometric analysis revealed that the expression levels of CD16, but not CD32 or CD64, were enhanced on hypoxic macrophages compared to normoxic macrophages, and this upregulation of CD16 was considered to contribute to the increased binding and phagocytosis of macrophages under hypoxic conditions. The phenotype and functional activities of macrophages can be modulated by a number of cytokines and microbial products. Among these cytokines, IL-10 was reported to be the best inducer of macrophages with high phagocytic activity. When IL-10 was added in the culture 24 hours before the phagocytosis assay, the phagocyte rate of hypoxic, but not normoxic, macrophages was significantly enhanced. Intriguingly, the expression levels of CD16 on hypoxic macrophages were upregulated by IL-10 treatment, and this upregulation was not seen in the normoxic macrophages. Taken together, these findings indicate that the hypoxic microenvironment facilitated the rituximab-mediated binding and phagocytic activity of TAM through the upregulation of CD16 expression. IL-10 could therefore be a possible effector modulator that enhances the activity of rituximab in the hypoxic microenvironment. Disclosures: No relevant conflicts of interest to declare.

Virus-Specific T Lymphocytes Maintain Their In Vivo Phenotype and Functional Competence After Automated Processing To Generate a Cellular Product For Immunotherapy

IntroductionAdoptive transfer of virus-specific T cells is an encouraging strategy to manage severe and fatal infections in immunocompromised patients. To generalize this approach, a cGMP- compliant enrichment process of both CD4+ and CD8+ viral-specific T cells is necessary. Here, we used a newly established automated manufacturing process for rapid and efficient ex vivo selection of multi-virus-specific CD4+ and CD8+ T cells. We show how the isolated virus-specific T cells retain their original effector/memory status and their effector functions. MethodLeukapheresis from healthy donors were used as starting material. Multi-virus or cytomegalovirus pp65 peptide-specific T cell products were generated in a novel closed cell-processing device with a fully automated manufacturing procedure. During this process white blood cells were stimulated with either a combination of peptide pools covering cytomegalovirus pp65, Epstein-Barr-Virus EBNA-1, BZLF1, and LMP2, and adenovirus hexon protein (n=6) or with a single pp65 peptide for four hours (n=4). Subsequently, virus-specific CD4+ and CD8+ T cells were magnetically enriched using the IFN-g secretion assay technology. In parallel, the reversible MHC/peptide multimer technology, which is restricted to CD8+ peptide-specific T cell enrichment, was used for comparison in a manual magnetic selection procedure for pp65 peptide-specific CD8+ T cells (n=4). All virus-specific T cell products were rested in vitro in the presence of T-cell-depleted PBMCs without addition of cytokines or antigens for up to 4 days. Expression of CD45RA, CCR7, CD28, CD69, CD137 as well as IFN-g production with and without cognate antigen(s)-restimulation were analyzed by flow cytometry before and up to 4 days after the selection process. ResultsManufacturing of multi-virus and pp65 peptide-specific T cells using the IFN-g secretion assay technology requires a short period of antigen stimulation and IFN-g expression, therefore, up to 96% of T cells produced IFN-g in the enriched fraction. However, after a few days resting phase in culture, IFN-g production decreased drastically. In addition, we detected an upregulation of CD69 and CD137 in the IFN-g enriched T cell products directly and 24 hours after the selection process, respectively. The transient nature of activation could again be confirmed, as both, CD69 and CD137 were downregulated during the resting phase. Results were compared to pp65-peptide specific CD8+ T cell products generated by the MHC/peptide multimer technology, which does not require an antigen incubation step. Activation was also seen for these enriched T cells, even when the MHC/peptide complexes were released, while unprocessed and cultured PBMC did not show IFN-g secretion or activation marker expression; indicating that cell processing and not the culture conditions triggered the activation.To test the functionality of the generated T cell products, we re-incubated three days resting cells with the corresponding antigens. In all samples, independent of the technology used for selection, induction of IFN-g expression in up to 100% of T cells was observed. Thus, T cells in all the products were able to maintain their in vivo imprinted physiological role, i.e. IFN-g production after antigen contact.Furthermore we examined if cell processing influences the effector/memory status of virus-specific T cells. Because the MHC/peptide multimer technology is restricted to the selection of single peptide-specific CD8+ T cells only, we monitored CD45RA, CD28 and CCR7 expression on pp65-peptide specific CD8+ T cells either identified by IFN-g secretion or by MHC/peptide multimer staining before and directly after the enrichment. The frequency of CD45RA+ and CD28+ cell populations varied between the donors and CCR7 was not detected at all, but importantly the enrichment process did not induce phenotypic changes. This result demonstrates the phenotype is stable during the manufacturing process. ConclusionA newly developed automated manufacturing process for direct ex vivo enrichment of multi-virus-specific CD4+ and CD8+ T cell populations via the IFN-g secretion assay technology provides a product for immunotherapy, where the original phenotypic and functional characteristics of the cells are conserved. Hence this cellular product is expected to fight efficiently against viral infections upon adoptive transfer. Disclosures:Richter:Miltenyi Biotec GmbH: Employment. Preussner:Miltenyi Biotec: Employment. Traska:Miltenyi Biotec: Employment. Peters:Miltenyi Biotec: Employment. Oysal:Miltenyi Biotec: Employment. Ruhnke:Miltenyi Biotec: Employment. Brauns:Miltenyi Biotec: Employment. Kramer:Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:Miltenyi Biotec: Employment.