Human Peripheral Blood Macrophages Research Articles

Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

Abstract 4556: Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma

Abstract RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [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 4556.

Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of β-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1β. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Extracellular vesicles derived from Hepatitis-D Virus infected cells induce a proinflammatory cytokine response in human peripheral blood mononuclear cells and macrophages

Extracellular vesicles derived from Hepatitis-D Virus infected cells induce a proinflammatory cytokine response in human peripheral blood mononuclear cells and macrophages

Liraglutide Attenuates Preestablished Atherosclerosis in Apolipoprotein E-Deficient Mice via Regulation of Immune Cell Phenotypes and Proinflammatory Mediators.

We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 μg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1β secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.

Effects of Gastric Cancer Cell-Derived Exosomes on the Immune Regulation of Mesenchymal Stem Cells by the NF-kB Signaling Pathway.

Mesenchymal stem cells (MSCs) are important components of the tumor microenvironment, which play an important role in tumor development. Exosomes derived from tumor cells can affect the biological characteristics of MSCs. Our study examined the effects of exosomes derived from gastric cancer cells on MSC immunomodulatory functions. Exosomes were extracted from gastric cancer cell line AGS (AGS-Exos) and cultured with MSCs. MSCs were then cocultured with both human peripheral blood mononuclear cells and macrophages [phorbol-12-myristate-13-acetate (PMA)-stimulated THP1 cells]. The activation levels of T cells and macrophages were detected by flow cytometry and real-time quantitative polymerase chain reaction (RT-PCR). Changes in the MSC signaling pathway after AGS-Exos stimulation were studied using RNA Chip, and the molecular mechanisms of functional change in MSCs were studied by inhibiting the signaling pathway. MSCs treated with AGS-Exos could promote macrophage phagocytosis and upregulate the secretion of proinflammatory factor, and promote the activation of CD69 and CD25 on the surface of T cells. RNA Chip results indicated the abnormal activation of the NF-kB signaling pathway in MSCs after AGS-Exos stimulation, and this was verified by the identification of key proteins in the pathway using western blot analysis. After NF-kB signaling pathway inhibition, the effect of MSCs stimulated by AGS-Exos on T cells and macrophages was markedly weakened. Therefore, AGS-Exos affected the immunomodulation function of MSCs through the NF-kB signaling pathway, which enhanced the ability of MSCs to activate immune cells, maintain the inflammatory environment, and support tumor growth.

Human Peripheral Blood Macrophages As a Model for Studying Glucocerebrosidase Dysfunction

Decreased activity of glucocerebrosidase (GCase) as a result of mutations in the GBA gene causes Gaucher’s disease (GD), which belongs to the group of lysosomal storage disorders. The risk of Parkinson’s disease in homo- and heterozygous carriers of GBA mutations is elevated seven- to eightfold. Screening of novel compounds designed to enhance GCase activity requires development of in vitro models based on primary cell cultures obtained from patients carrying GBA mutations. In this work, the efficiency of different methods used to culture peripheral blood macrophages of GD patients and control subjects was compared, and GCase activity and lysosphingolipid concentrations were evaluated using tandem mass spectrometry (HPLC‒MS/MS) in dried cell spots. For the first time, the efficacy of restoring the activity of mutant GCase has been assessed in primary macrophages of GD patients cultured in the presence of pharmacological GCase chaperones isofagomine and ambroxol. Based on these results, a convenient method of in vitro screening of candidate pharmacological agents designed to increase GCase activity can be proposed.

Abstract A169: Macrophage targeted zoledronic acid conjugated biomineral nanoparticles for γδT-cell-based cancer immunotherapy

Abstract Nanoparticle platforms can be engineered to deliver immune-stimulatory molecules to specific immune cell populations, thereby evoke desired response. Here, we present a novel biomineral nanoparticle system for macrophage specific delivery of bisphosphonate molecules to evoke activation of γδ T-cells for cancer immunotherapy. Generally, γδ T-cells therapy is implemented either by ex vivo expansion followed by adoptive transfer or by in vivo activation using intravenous injection of bisphosphonates. One of the major limitations of intravenously injection of bisphosphonates such as zoledronic acid is its unfavorable biodistribution in the skeletal system due to covalent binding with calcium in the bone. In the present work, we have pre-conjugated zoledronic acid in a calcium-containing biomineral nanoparticle (Zol-BM) that has spontaneous specificity towards macrophages in the liver, spleen and lymph nodes. This enabled targeted expression of γδT-cell stimulating ligand, isopentyl pyrophosphate, in macrophages, leading to enhanced activation of γδT-cells. Optimized Zol-BM showed a drug conjugation efficiency of ~95% with particle size ~ 100 nm and zeta potential, -15mV. The compatibility of Zol-BM was tested in primary human peripheral blood mononuclear cells and macrophages cell lines. The biodistribution analysis of Zol-BM in healthy rat models showed accumulation in liver, spleen and lymph nodes which was confirmed by MRI and Prussian-blue analysis. Flow cytometry based γδT-cell proliferation analysis gave ~18% enhancement in γδ T-cell subset compared to untreated control. Thus, we show that by rational design of engineered nanoparticles, specific immuno-stimulatory molecules like zolendronate can be delivered to desired immune cells of interest (macrophages) for activating another sub-population of immune cells such as γδ T-cells, for cancer-immunotherapy applications. Citation Format: Anusha Ashokan, Minu Anoop, Siju Surendran, Aparna Balakrishnan, Ida M. Anna, Anjana Ramkumar, Girish Chundayil Madathil, Vijay Harish, Manzoor Koyakutty. Macrophage targeted zoledronic acid conjugated biomineral nanoparticles for γδT-cell-based cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A169.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. Invitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. Invivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Interactions of Nitroxide-Conjugated and Non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies.

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Exploration of bivalent ligands targeting putative mu opioid receptor and chemokine receptor CCR5 dimerization

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR–CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5–MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5–MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation.

Tomato Aqueous Extract Modulates the Inflammatory Profile of Immune Cells and Endothelial Cells.

Nutrients transiently or chronically modulate functional and biochemical characteristics of cells and tissues both in vivo and in vitro. The influence of tomato aqueous extract (TAE) on the in vitro inflammatory response of activated human peripheral blood leukocytes (PBLs) and macrophages was investigated. Its effect on endothelial dysfunction (ED) was analyzed in human umbilical vein endothelial cells (HUVECs). Murine macrophages (RAW264.7 cells), PBLs and HUVECs were incubated with TAE. They were activated with LPS or TNF-α in order to induce inflammatory processes and ED, respectively. Inflammatory mediators and adhesion molecules were measured by immune assay-based multiplex analysis. Gene expression was quantified by RT-PCR. TAE altered the production of interleukins (IL-1β, IL-6, IL-10, IL-12) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10) in PBLs. TAE reduced ED-associated expression of adhesion molecules (ICAM-1, VCAM-1) in endothelial cell. In macrophages, the production of nitric oxide, PGE2, cytokines and ILs (TNF-α, IL-1β, IL-6, IL-12), which reflects chronic inflammatory processes, was reduced. Adenosine was identified as the main bioactive of TAE. Thus, TAE had cell-specific and context-dependent effects. We infer from these in vitro data, that during acute inflammation TAE enhances cellular alertness and therefore the sensing of disturbed immune homeostasis in the vascular-endothelial compartment. Conversely, it blunts inflammatory mediators in macrophages during chronic inflammation. A novel concept of immune regulation by this extract is proposed.

In Vitro Evaluation of Colloidal Silver on Immune Function: Antilymphoproliferative Activity

Colloidal silver (AgC) is currently used by humans and it can be internalized through inhalation, injection, ingestion, and dermal contact. However, there is limited information about immunological activity; more investigations using colloidal silver are needed. In the present study, the effects of AgC (17.5 ng/mL) on immunological parameters (proliferation and immunophenotyping) using human peripheral blood mononuclear cells (PBMC) and macrophages (phagocytosis) and cytotoxicity on leukemia and lymphoma cancer cell lines (1.75 to 17.5 ng/mL) were investigated. AgC was observed to significantly (p<0.05) decrease interleukin-2 (IL-2) production and proliferation induced by phytohemagglutinin or concanavalin A in PBMC without affecting its cell viability but with cytotoxic effect on cancer cells. IL-2, IL-4, IL-6, IL-10, INF-γ, and IL-17A cytokines production and CD3+, CD3−CD19+, CD3+CD4+, CD3+CD8+, and CD16+CD56+ PBMC phenotypes were not affected by AgC. The present study demonstrates that colloidal silver is harmless and nontoxic to the immune system cells and its ability to interfere with the immune response by decreasing cell proliferation when stimulated with mitogens demonstrated the antilymphoproliferative potential of AgC.

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2014-1658

THP-1 cell line: an in vitro cell model for immune modulation approach.

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. (1)H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or "closed chain" structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.

Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells

The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and DIAPH3 silencing evokes a transition to an amoeboid tumor phenotype in multiple cell backgrounds. This amoeboid transformation is accompanied by increased tumor cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the formation of atypically large (>1 μm) membrane blebs that can be shed as extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of nano-sized EV (e.g., exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused EV shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of EV production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells. DU145 EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV produced as a result of DIAPH3 loss or growth factor stimulation may condition the tumor microenvironment through multiple mechanisms, including the proliferation of cancer cells and suppression of tumor-infiltrating immune cells.

Meprin-β regulates production of pro-inflammatory factors via a disintegrin and metalloproteinase-10 (ADAM-10) dependent pathway in macrophages

Inflammatory response plays an important role not only in the normal physiology but also in the pathology such as atherosclerosis. Meprin, an astacin metalloproteinase, has exhibited proinflammatory effects in vivo and in vitro studies. Here, we tried to further investigate the proinflammatory potential of meprin-β and the possible underlying mechanisms in primary human peripheral blood macrophages. In our current study, ELISA assay revealed that meprin-β increased the production of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-18 and interleukin-6 (IL-6) in macrophages. However, meprin-β shows no effects on the level of ligands of epidermal growth factor receptor (EGFR), and the activation of EGFR. The molecular mechanism was associated with activation of a disintegrin and metalloproteinase 10 (ADAM10) and the phosphorylation of IκB. Further analysis of upstream mechanisms showed that activation of NF-κB by meprin-β was mediated by inhibiting ADAM10-downstream extracellular signal regulated kinase (ERK1/2) pathway. Taken together, these results indicated that meprin-β exhibited pro-inflammatory effects by targeting activating ADAM10, leading to ERK1/2-mediated activation of NF-κB in macrophages, and this would make meprin-β a strong candidate for further study as proinflammatory target.

Unbiased Screening of Marine Sponge Extracts for Anti-inflammatory Agents Combined with Chemical Genomics Identifies Girolline as an Inhibitor of Protein Synthesis

Toll-like receptors (TLRs) play a critical role in innate immunity, but activation of TLR signaling pathways is also associated with many harmful inflammatory diseases. Identification of novel anti-inflammatory molecules targeting TLR signaling pathways is central to the development of new treatment approaches for acute and chronic inflammation. We performed high-throughput screening from crude marine sponge extracts on TLR5 signaling and identified girolline. We demonstrated that girolline inhibits signaling through both MyD88-dependent and -independent TLRs (i.e., TLR2, 3, 4, 5, and 7) and reduces cytokine (IL-6 and IL-8) production in human peripheral blood mononuclear cells and macrophages. Using a chemical genomics approach, we identified Elongation Factor 2 as the molecular target of girolline, which inhibits protein synthesis at the elongation step. Together these data identify the sponge natural product girolline as a potential anti-inflammatory agent acting through inhibition of protein synthesis.

Effect of Porphyromonas gingivalis on Citrullination of Proteins by Macrophages In Vitro

Citrullination of proteins within inflamed periodontal tissues may provide an important link between periodontitis and rheumatoid arthritis. The aim of this study is to determine whether the presence of Porphyromonas gingivalis peptidylarginine deiminase (PPAD) can influence citrullination of proteins by either increasing the amount of local citrullinated protein or influencing the peptidylarginine deiminase (PAD) enzymes found in the monocyte/macrophage population. Human peripheral blood monocytes and macrophages were incubated in the presence of live or heat-killed P. gingivalis. Expression of PAD2 and PAD4, PPAD, and citrullinated proteins were assessed by either a combination of real-time polymerase chain reaction, Western blotting, or a colorimetric assay. PPAD was detected only in mononuclear cells incubated in the presence of live P. gingivalis and resulted in increased extracellular citrullination. Endogenous PAD (mRNA and protein) expression was detected in monocytes and macrophages but was not affected by P. gingivalis. Although P. gingivalis produces a PAD that can citrullinate extracellular proteins and may contribute to the citrullinated protein load in gingival tissues, it does not appear to affect PAD expression or citrullination by host monocytes or macrophages.