Induced Macrophage Activation Research Articles

Contamination of bacterial extracellular vesicles (bEVs) in human urinary extracellular vesicles (uEVs) samples and their effects on uEVs study

AbstractBacterial overgrowth is common for improperly stored urine. However, its effects on human urinary extracellular vesicles (uEVs) study had not been previously examined nor documented. This study investigated the presence of bacterial EVs (bEVs) contaminated in uEVs samples and their effects on uEVs study. Nanoscale uEVs were isolated from normal human urine immediately after collection (0‐h) or after 25°C‐storage with/without preservative (10 mM NaN3) for up to 24‐h. Turbidity, bacterial count and total uEVs proteins abnormally increased in the 8‐h and 24‐h‐stored urine without NaN3. NanoLC‐ESI‐LTQ‐Orbitrap MS/MS identified 6–13 bacterial proteins in these contaminated uEVs samples. PCR also detected bacterial DNAs in these contaminated uEVs samples. Besides, uEVs derived from 8‐h and 24‐h urine without NaN3 induced macrophage activation (CD11b and phagocytosis) and secretion of cytokines (IFN‐α, IL‐8, and TGF‐β) from macrophages and renal cells (HEK‐293, HK‐2, and MDCK). All of these effects induced by bacterial contamination were partially/completely prevented by NaN3. Interestingly, macrophage activation and cytokine secretion were also induced by bEVs purified from Escherichia coli. This study clearly shows evidence of bEVs contamination and their effects on human uEVs study when the urine samples were inappropriately stored, whereas NaN3 can partially/completely prevent such effects from the contaminated bEVs.

Immunomodulatory Effects of an Aqueous Extract of Black Radish on Mouse Macrophages via the TLR2/4-Mediated Signaling Pathway.

Here, we determined the immunostimulatory effects of black radish (Raphanus sativus ver niger) hot water extract (BRHE) on a mouse macrophage cell line (RAW 264.7) and mouse peritoneal macrophages. We found that BRHE treatment increased cell proliferation, phagocytic activity, nitric oxide (NO) levels, cytokine production, and reactive oxygen species synthesis. Moreover, BRHE increased the expression of the following immunomodulators in RAW 264.7 cells and peritoneal macrophages: pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), iNOS, and COX-2. BRHE treatment significantly up-regulated the phosphorylation of components of the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), Akt, and STAT3 signaling pathways. Further, the effects of BRHE on macrophages were significantly diminished after the cells were treated with the TLR2 antagonist C29 or the TLR4 antagonist TAK-242. Therefore, BRHE-induced immunostimulatory phenotypes in mouse macrophages were reversed by multiple inhibitors, such as TLR antagonist, MAPK inhibitor, and Akt inhibitor indicating that BRHE induced macrophage activation through the TLR2/4-MAPK-NFκB-Akt-STAT3 signaling pathway. These results indicate that BRHE may serve as a potential immunomodulatory factor or functional food and provide the scientific basis for the comprehensive utilization and evaluation of black radish in future applications.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1β (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

Human dermal fibroblast-derived exosomes induce macrophage activation in systemic sclerosis.

Prior work demonstrates that co-cultured macrophages and fibroblasts from patients with SSc engage in reciprocal activation. However, the mechanism by which these cell types communicate and contribute to fibrosis and inflammation in SSc is unknown. Fibroblasts were isolated from skin biopsies obtained from 7 SSc patients or 6 healthy age and gender-matched control subjects following written informed consent. Human donor-derived macrophages were cultured with exosomes isolated from control or SSc fibroblasts for an additional 48 h. Macrophages were immunophenotyped using flow cytometry, qRT-PCR and multiplex. For mutual activation studies, exosome-activated macrophages were co-cultured with SSc or healthy fibroblasts using Transwells. Macrophages activated with dermal fibroblast-derived exosomes from SSc patients upregulated surface expression of CD163, CD206, MHC Class II and CD16 and secreted increased levels of IL-6, IL-10, IL-12p40 and TNF compared with macrophages incubated with healthy control fibroblasts (n = 7, P < 0.05). Exosome-stimulated macrophages and SSc fibroblasts engaged in reciprocal activation, as production of collagen and fibronectin was significantly increased in SSc fibroblasts receiving signals from SSc exosome-stimulated macrophages (n = 7, P < 0.05). In this work, we demonstrate for the first time that human SSc dermal fibroblasts mediate macrophage activation through exosomes. Our findings suggest that macrophages and fibroblasts engage in cross-talk in SSc skin, resulting in mutual activation, inflammation, and extracellular matrix (ECM) deposition. Collectively, these studies implicate macrophages and fibroblasts as cooperative mediators of fibrosis in SSc and suggest therapeutic targeting of both cell types may provide maximal benefit in ameliorating disease in SSc patients.

Involvement of Histone Lysine Crotonylation in the Regulation of Nerve-Injury-Induced Neuropathic Pain.

Histone lysine crotonylation (KCR), a novel epigenetic modification, is important in regulating a broad spectrum of biological processes and various diseases. However, whether KCR is involved in neuropathic pain remains to be elucidated. We found KCR occurs in macrophages, sensory neurons, and satellite glial cells of trigeminal ganglia (TG), neurons, astrocytes, and microglia of the medulla oblongata. KCR in TG was detected mainly in small and medium sensory neurons, to a lesser extent in large neurons. Peripheral nerve injury elevated KCR levels in macrophages in the trigeminal and dorsal root ganglia and microglia in the medulla oblongata but reduced KCR levels in sensory neurons. Inhibition of histone crotonyltransferases (p300) by intra-TG or intrathecal administration of C646 significantly alleviated partial infraorbital nerve transection (pIONT)- or spinal nerve ligation (SNL)-induced mechanical allodynia and thermal hyperalgesia. Intra-TG or intrathecal administration of Crotonyl coenzyme A trilithium salt to upregulate KCR dose-dependently induced mechanical allodynia and thermal hyperalgesia in mice. Mechanismly, inhibition of p300 alleviated pIONT-induced macrophage activation and reduced the expression of pain-related inflammatory cytokines Tnfα, Il1β and chemokines Ccl2 and Cxcl10. Correspondingly, exogenous crotonyl-CoA induced macrophage activation and the expression of Tnfα, Il1β, Il6, Ccl2 and Ccl7 in TG, which C646 can repress. These findings suggest that histone crotonylation might be functionally involved in neuropathic pain and neuroinflammation regulation.

Spermidine inhibits LPS-induced pro-inflammatory activation of macrophages by acting on Nrf2 signaling but not autophagy

This study was designed to investigate the roles of spermidine on lipopolysaccharide (LPS)-induced macrophage activation and underlying mechanisms. Spermidine was found to induce a moderate activation of autophagy but robust activation of nuclear factor erythroid 2-related factor 2 (Nrf-2) in a murine macrophage cell line RAW264.7 cells. Spermidine blunted LPS-driven proinflammatory activation of RAW264.7 cells evidenced by the reduced release of NO, TNF-α, and reactive oxygen species (ROS). The effects of spermidine were not affected by the autophagy inhibitor, while the autophagy inducers could not block the activation of macrophages induced by LPS. Interestingly, Nrf-2 inducers significantly suppressed the secretion of NO and TNF-α in RAW264.7 cells exposed to LPS, while the roles of spermidine were significantly blunted by the knockout of Nrf-2. Furthermore, spermidine suppressed the activation of NF-κB and the NLRP3 inflammasome in mouse liver. Collectively, spermidine could suppress LPS-induced macrophages activation possibly by activating Nrf-2 in macrophages.

Tetrahydroxylated bile acids improve cholestatic liver and bile duct injury in the Mdr2-/- mouse model of sclerosing cholangitis via immunomodulatory effects.

Bile salt export pump (Bsep) (Abcb11) −/− mice are protected from acquired cholestatic injury due to metabolic preconditioning with a hydrophilic bile acid (BA) pool with formation of tetrahydroxylated bile acids (THBAs). We aimed to explore whether loss of Bsep and subsequent elevation of THBA levels may have immunomodulatory effects, thus improving liver injury in the multidrug resistance protein 2 (Mdr2) (Abcb4) −/− mouse. Cholestatic liver injury in Mdr2 −/− Bsep −/− double knockout (DKO), Mdr2 −/− , Bsep −/− , and wild‐type mice was studied for comparison. Mdr2 −/− mice were treated with a THBA (3α,6α,7α,12α‐Tetrahydroxycholanoic acid). RNA/protein expression of inflammatory/fibrotic markers were investigated. Serum BA‐profiling was assessed by ultra‐performance liquid chromatography tandem mass spectrometry. Hepatic immune cell profile was quantified by flow cytometric analysis (FACS). In vitro, the THBA effect on chenodeoxycholic acid (CDCA)–induced inflammatory signaling in hepatocyte and cholangiocytes as well as lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ)–induced macrophage activation was analyzed. In contrast to Mdr2 −/− , DKO mice showed no features of sclerosing cholangitis. Sixty‐seven percent of serum BAs in DKO mice were polyhydroxylated (mostly THBAs), whereas Mdr2 −/− mice did not have these BAs. Compared with Mdr2 −/−, DKO animals were protected from hepatic inflammation/fibrosis. THBA feeding in Mdr2 −/− mice improved liver injury. FACS analysis in DKO and Mdr2 −/− THBA‐fed mice showed changes of the hepatic immune cell profile towards an anti‐inflammatory pattern. Early growth response 1 (EGR1) protein expression was reduced in DKO and in Mdr2 −/− THBA‐fed mice compared with Mdr2 −/− control mice. In vitro, THBA‐reduced CDCA induced EGR1 protein and mRNA expression of inflammatory markers in hepatocytes and cholangiocytes. LPS/IFN‐γ–induced macrophage activation was ameliorated by THBA. THBAs repress EGR1‐related key pro‐inflammatory pathways. Conclusion: THBA and their downstream targets may represent a potential treatment strategy for cholestatic liver diseases.

Structural characterization and immunomodulatory activity of a mannan from Helvella leucopus

A new polysaccharide fraction HLP-1 (2.55 × 105 Da) was obtained from the fruiting bodies of Helvella leucopus. Structural characterization of HLP-1 was elucidated by infrared spectroscopy, monosaccharide composition analysis, methylation analysis, nuclear magnetic resonance spectroscopy, scanning electron microscopy and Congo red assay. HLP-1 was a mannan with a backbone of →6)-α-D-Manp(1 → 4)- α-D-Manp(1 →6)-α-D-Manp(1 → 3)-α-D-Manp(1 → 4)-α-D-Manp(1 → 3)-α-D-Manp(1→, which branched at the O-6 position and terminated with T-β-D-Manp. Moreover, HLP-1 could significantly improve the proliferation and neutral red phagocytosis of RAW264.7. Besides, HLP-1 could stimulate the production of nitric oxide (NO), ROS, tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6). HLP-1 induced macrophage activation via NF-κB signal pathway. These findings indicated that HLP-1 was a potential immune enhancement agent applied in functional foods.

Sotatercept analog suppresses inflammation to reverse experimental pulmonary arterial hypertension

Sotatercept is an activin receptor type IIA-Fc (ActRIIA-Fc) fusion protein that improves cardiopulmonary function in patients with pulmonary arterial hypertension (PAH) by selectively trapping activins and growth differentiation factors. However, the cellular and molecular mechanisms of ActRIIA-Fc action are incompletely understood. Here, we determined through genome-wide expression profiling that inflammatory and immune responses are prominently upregulated in the lungs of a Sugen-hypoxia rat model of severe angio-obliterative PAH, concordant with profiles observed in PAH patients. Therapeutic treatment with ActRIIA-Fc—but not with a vasodilator—strikingly reversed proinflammatory and proliferative gene expression profiles and normalized macrophage infiltration in diseased rodent lungs. Furthermore, ActRIIA-Fc normalized pulmonary macrophage infiltration and corrected cardiopulmonary structure and function in Bmpr2 haploinsufficient mice subjected to hypoxia, a model of heritable PAH. Three high-affinity ligands of ActRIIA-Fc each induced macrophage activation in vitro, and their combined immunoneutralization in PAH rats produced cardiopulmonary benefits comparable to those elicited by ActRIIA-Fc. Our results in complementary experimental and genetic models of PAH reveal therapeutic anti-inflammatory activities of ActRIIA-Fc that, together with its known anti-proliferative effects on vascular cell types, could underlie clinical activity of sotatercept as either monotherapy or add-on to current PAH therapies.

Treponema pallidum (Syphilis) Antigen TpF1 Induces Activation of Macrophages and Accelerates P2X7R-Induced NLRP3-Dependent Release of IL-1β.

Syphilis is a chronic infectious disease caused by Treponema pallidum (Tp) infection, which causes local inflammation in the host. TpF1 is an oligomeric protein expressed by the Tp-infected host that can induce the host immune response. There are few studies regarding the role of TpF1 in macrophage activation and the subsequent release of cytokines. The objective of this study is to elucidate the effects of TpF1 on the pathological process of Syphilis. In addition, we explored how purinergic 2X7 (P2X7R) induced NOD-like receptor family protein 3 (NLRP3) -dependent release of interleukin-1β (IL-1β) and the underlying mechanisms. We explored the influence of TpF1 on cytokine release by macrophages using qRT-PCR and ELISA. The specific phenotype of activated macrophages was determined by flow cytometry. TpF1 was able to activate macrophages and induce the M1 macrophage phenotype. Moreover, TpF1 activated the NLRP3 inflammasome in macrophages, which was mediated by P2X7R. The Tp-induced protein TpF1 is able to induce macrophage activation and P2X7R-induced NLRP3-dependent release of IL-1β. Our findings provide a theoretical basis for clarifying the clinical symptoms and pathogenesis of syphilis.

Structural characteristics and immune-enhancing activity of fractionated polysaccharides from Athyrium Multidentatum (Doll.) Ching.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1β, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

Evaluation of the Immunological Activity of Gryllus bimaculatus Water Extract.

Edible insects are commonly consumed across the world because of their size, availability, and nutritional benefits. They have also been recommended as a potential solution to food shortage because of their high nutritional value. In this study, we demonstrated the immunological effects of Gryllus bimaculatus on RAW 264.7 cells and splenocytes obtained from mouse. This is the first study to evaluate the immunological effects of G. bimaculatus water extract. Innate and adaptive immunity were evaluated and measured in RAW 264.7 cells and/or mouse splenocytes using a cell viability assay; changes in cytokine abundance, nitric oxide production, and cell surface molecule abundance were determined using flow cytometry; and western blotting analysis was performed for various immune signaling pathways. G. bimaculatus water extract showed no cytotoxicity in cells, and the results suggest that treatment with G. bimaculatus water extract can induce macrophage activation through mitogen-activated protein kinase and nuclear factor-κB signaling, induction of proinflammatory cytokines [interleukin (IL)-6, IL-1β, and tumor necrosis factor-α] and activation of the expression of cell surface molecules [cluster of differentiation (CD)80, CD86, major histocompatibility complex (MHC) class I, and MHC class II]. Treatment with G. bimaculatus water extract increased the production of cytokines (IL-2, IL-4, and interferon-γ) in splenocytes. The results indicate that G. bimaculatus water extract can regulate innate and adaptive immunity via modulation macrophages and splenocytes activation and can serve as an immunological agent. We inferred that G. bimaculatus is a safe and efficient natural material that enhances immunological activity.

LPS Activated Macrophages Induced Hepatocyte Pyroptosis via P2X7R Activation of NLRP3 in Mice.

Pyroptosis is a programmed cell death related to caspase-1, accompanied by the secretion of pro-inflammatory cytokines. To explore the effects of LPS on the P2X7R/NLRP3 pathway in macrophages, and hepatocytes pyroptosis in mice. LPS was used to establish an animal model of the acute liver injury. The macrophage RAW264.7 was induced by LPS to establish a cell model. The P2X7R inhibitor A438079 and agonist BZATP were added. RAW264.7 was co-cultured with AML-12 cells. Pyroptosis and the ratio of CD11b+CD86+/CD11b+CD206+ were analyzed by flow cytometry. ELISA, WB, and qRT-PCR were applied to analyze factors involved in the P2X7R/NLRP3 pathway. LPS induced liver damage in mice, promoted cell pyroptosis and increased the levels of IL-18, IL-1β, ALT, AST, and TBIL. P2X7R, GSDMD, and GSDMD-N expressions also increased in the LPS group. LPS induced macrophage activation in vivo. NLRP3, ASC, P2X7R, and caspase-1 expressions in vitro promoted. ELISA confirmed that the IL-1β and IL-18 levels repressed in the BZATP (P2X7R agonist) group, while the trend was opposite in the A438079 (P2X7R inhibitor) group. LPS activated the P2X7R/NLRP3 pathway in macrophages. After RAW264.7 was co-cultured with AML-12 cells, the pyroptosis of AML-12 cells promoted but the proliferation decreased in the BZATP group. GSDMD and GSDMD-N expressions promoted in the BZATP group, while the trend was opposite in the A438079 group. LPS activated macrophages via P2X7R activation of NLRP3 and induced hepatocyte pyroptosis, which provided novel potential targets for the liver injury treatment.

TmPSMA-02: A CD2 endodomain containing double armored PSMA CAR T with enhanced efficacy and lower immune toxicity.

158 Background: Early clinical responses to anti-PSMA Chimeric Antigen Receptor T cells (CARs) have been observed but much like the leukemia experience, significant toxicity from macrophage activation (MAS) has also been observed Building on our experience with a first in-human armoured CAR (TmPSMA-01) targeting PSMA (with a 41BB and CD3 endodomain) coupled with a dominant-negative TGFBR2 module, where we observed anti-tumor effects but also immune-mediated toxicity, we designed a CAR with additional armour, a PD1-CD28 switch, to overcome the immune suppressive microenvironment and further improve efficacy. We successfully built tricistronic constructs that expresses the CAR, a dnTGFBR2 module and a switch receptor for PD1 and CD28. We compared the 41BB endodomain and the CD2 domain, which has shown preliminary effects reducing cytokine release. We hypothesized that these changes would increase efficacy in immune-suppressive solid tumors while reducing excessive cytokine release and untoward immune toxicity. Methods: CAR constructs were synthesized into a self-inactivating lentiviral backbone and CAR T cells manufactured comprising a control TmPSMA-01 construct and two primary constructs: PSMA-CD2z:dnTGFBR:PD1-CD28 and PSMA-41BBz:dnTGFBR:PD1-CD28. Expression of the CAR as well as T cell markers were determined by flow cytometry, and CAR affinity for target by Lumicks. CAR activation and cytotoxicity were assessed by ELISPOT, xCelligence and Celligo standard assays. A co-culture macrophage activation system was applied to each construct to assess the potential of each construct to induce MAS-like responses. Results: A significant difference was seen in the MFI of the CAR, with the CD2 based CAR consistently ̃50% lower than the 41BB and this correlated to a faster off-rate from target cells measured by Lumicks. There was no difference in exhaustion or memory markers, and cytotoxicity across a range of E:T ratios was equivalent between the CD2 and 41BB constructs. Preliminary data indicated the CD2 endodomain preserved T cell activation but with lower cytokine release. The CD2 constructs consistently produced significantly less total IL2 and IFNg after exposure to targets, yet had similar numbers of ELISPOT positive cells for IL2 and IFNg indicating less cytokine production per kill. This also resulted in significantly less macrophage based cytokines such as IL6 and IL8 in the coculture model. Using a cell line knocked out for PDL1/2 and then reengineered for constitutive PDL1/2 expression, we demonstrated improved speed of killing in the xCelligence system dependent on the PD1:CD28 switch but no increase in cytokine release. Conclusions: A CD2 based endodomain tricistronic CAR targeting PSMA (TmPSMA-02) maintained cytotoxicity, memory profile and activation while having a significantly reduced risk of inducing macrophage activation.

A Novel Polysaccharide Isolated From Fresh Longan (Dimocarpus longan Lour.) Activates Macrophage via TLR2/4-Mediated PI3/AKT and MyD88/TRAF6 Pathways.

A novel immunomodulatory polysaccharide (LP4) with a molecular weight 6.31 × 104 g/mol was purified from fresh longan pulp. It was composed of mannose, glucose, glucuronic acid, galactose, xylose, arabinose, galacturonic acid, fucose, and rhamnose in a molar percentage of 36:31:10:7:4:4:3:2:2, and mainly linked by (1→6)-β-Man, (1→4)-β-Glc and (1→6)-α-Glc. LP4 can obviously enhance the phagocytosis of macrophages and promote the proliferation of lymphocytes. After treating macrophages with LP4 (12.5–50 μg/ml), the production of IL-1β and TNF-α was significantly increased. These increases of cytokines were suppressed when the TLR2/TLR4 receptors were inhibited by anti-TLR2 and/or anti-TLR4 antibodies. Moreover, the mRNA expression of INOS, AKT, PI3K, TRAF6 and MyD88 was significantly suppressed by TLR2/TLR4 antibodies. These results indicated that LP4 induced macrophage activation mainly via the TLR2 and TLR4-induced PI3K/AKT and MyD88/TRAF6 pathways.

Evaluation of Anticancer Potential of Flavones from Rhamnus alaternus against B16F10 Melanoma Cells

Melanoma has become an important health problem and new treatment have become an imperative medical need. Therefore, the finding and identification of natural product with less toxic effects, capable of promoting melanoma cell death have become an important goal of research in oncotherapy. In this study, we want to investigate the anticancer activity of an enriched total oligomers flavonoids (TOF) extract of R. alaternus in melanoma cells. First, TOF was exhibited to be rich in flavones. We revealed that this extract reduced proliferation and increased of sub-G1 and S phase cells built-up in B16-F10 cells in a dose-related manner. Moreover, In Vivo, TOF reduced tumor volume and weight with percentages of inhibition of 92.4% and 92.9%, respectively. R. alaternus was also found to be effective in reducing the level of pro-inflammatory cytokine IL-6 during metastasis. Level of TH1 cytokine, such as IL-2, was significantly enhanced by TOF treatment. Indeed, the histological examination of the tumor revealed the absence of mitoses and the presence of numerous melanin pigmented macrophage cells in the R. alaternus extract-treated group that could be explained by the induction of macrophage activation and by the arrest of the cell cycle in the Sub-G1 and S phases.

Eicosapentaenoic acid inhibits lipopolysaccharide (LPS)-induced macrophage activation in a manner that is enhanced with colchicine

Background and Aims: Activated macrophages, characterized by increased inducible nitric oxide synthase (iNOS) expression, participate in atherosclerosis and its complications. Cyclooxygenase (COX) regulates iNOS activity. As both a substrate for and potential inhibitor of COX, the omega-3 fatty acid (ω-3) eicosapentaenoic acid (EPA) may reduce iNOS activity. Colchicine has broad anti-inflammatory effects through inhibition of microtubule polymerization. We compared the separate and combined effects of EPA and colchicine on iNOS activity in lipopolysaccharide (LPS)-activated macrophages.

Differential effects of omega-3 fatty acids on lipopolysaccharide (LPS)-induced macrophage activation in combination with cox inhibition

Background and Aims: Macrophages contribute to chronic diseases by promoting inflammation. Macrophage activation results in increased inducible nitric oxide synthase (iNOS) expression which can be modulated by cyclooxygenase (COX) activity. As both a substrate and potential inhibitor of COX activity, the omega-3 fatty acid EPA may modulate iNOS activity. We investigated the effect of EPA and DHA on iNOS activity in LPS-activated macrophages.

Abstract 2751: Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization

Abstract Head and neck cancers (HNC) can persist despite infiltration of immune cells into the tumor microenvironment. One mechanism that may contribute to failed immune clearance of tumors stems from the conditioning of tumor infiltrating macrophages into a cell type that supports tumor survival rather than a cell with pro-inflammatory characteristics. To understand mechanisms that lead to conditioning of macrophages by tumors, we assessed the effects of HNC cell lines on macrophage maturation. Macrophages were matured by incubating purified human monocytes for 3-4 days in medium alone or in medium supplemented with supernatants from HNC cell lines (FADU and SCC9) or a human leukemia monocytic cell line (THP-1). Macrophages were recovered and examined by flow cytometry for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, and IL-15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1) and intracellular chemokine expression (IP-10). Relative to unconditioned macrophages or cells exposed to THP-1 supernatants, cells incubated with FADU or SCC9 supernatants displayed enhanced survival, downregulation of cell surface HLA-DR, CD86, IL-15R, CD36 and intracellular IP-10 expression. In contrast, the expression of PD-L1, CD14, and CD206 were increased by HNC cell line supernatants. To assess function, matured macrophages were stimulated overnight with the TLR4 agonist, LPS. Induction of macrophage activation was measured by increased expression of HLA-DR and IL-15R. Despite sustained the expression of TLR4 and CD14, macrophages matured in HNC supernatants failed to upregulate HLA-DR and IL-15R expression when stimulated with the canonical TLR4 agonist, LPS. These phenotypic and functional changes were accompanied by a decrease in intracellular phospho-p38 expression and a robust induction of WIP-1 phosphatase. Next, we considered the hypothesis that fatty acids released from tumors might contribute to these phenotypes. To test this, we added Etomoxir, an inhibitor of fatty acid oxidation, into cell cultures during exposure to tumor supernatants. The addition of Etomoxir reversed a number of these phenotypic changes induced by tumor supernatants with the exception of PD-L1 induction. Moreover, depletion of fatty acids from tumor supernatants with lipid removal agent, mitigated the effects of supernatants on macrophage maturation. Additionally, we found that macrophages incubated with either palmitic acid or oleic acid for 3-4 days developed similar phenotypes as cells incubated in tumor supernatants. Together, our data suggest that fatty acids are both necessary in tumor supernatants and sufficient on their own to mediate maturation of macrophages into a cell type with limited pro-inflammatory characteristics (M2-like). Approaches that modify the expression or effects of fatty acids in tumor microenvironment may be useful in HNC. Citation Format: Marwah M. Albakri, Stanley C. Huang, Hammad Tashkandi, Scott F. Sieg. Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2751.

Tumor Cells from EBV-Positive T- or NK-Cell Neoplasms Induce Differentiation and Activation of Macrophages By Secreting Humoral Factors

Introduction: Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms (EBV-T/NK neoplasms), such as extranodal NK-/T-cell lymphoma, aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV) are often accompanied with severe systemic inflammation. During their progression, hemophagocytic lymphohistiocytosis (HLH) may develop, which is a lethal condition.To clarify the mechanisms of HLH development in EBV-T/NK neoplasms, we investigated the direct effects of EBV-T/NK cells on the differentiation and activation of macrophages.Methods: The following EBV-T/NK-cell lines established from EBV-T/NK neoplasms were used: SNT8, SNT15, and SNT16 as T-cell lines (SNT cells), and SNK1, SNK6, and SNK10 as NK-cell lines (SNK cells). For investigating the effects of EBV-positive T or NK cells on the differentiation and activation of macrophages, the culture supernatant of these EBV-positive cells was used. U937 and THP1 cells, human monocytic cell lines, were cultured with the supernatant, and morphologic examination and flow cytometry (FCM) were used to examine their differentiation into the macrophage. Phorbol 12-myristate 13-acetate was used as a positive control for induction of the differentiation. FCM was used to determine the phagocytic activity based on the uptake of fluorescent beads. For detecting the cytokine production, the mRNA expression and cytokine concentrations were examined using TaqMan® Gene Expression Assays and enzyme-linked immunosorbent assay, respectively. The EBV-derived microRNA expression was investigated using the next-generation sequencer (MiSeq®)and TaqMan® Gene Expression Assays.Results: After culturing with the supernatant from the EBV-positive T or NK cells, the macrophage-like morphological change was observed in the THP1 and U937 cells. Interestingly, the effects were clear in the cells treated with the supernatant from the SNK cells, whereas the effects of the supernatant from the SNT cells were minimal. Consistent with the morphological changes, the co-culture with the SNK-cell supernatant significantly enhanced the CD11b expression, which is a marker for macrophages, on the THP1 and U937 cells. Next, the macrophage-like function in the differentiated cells was examined. We found that phagocytosis was significantly enhanced after the co-culture with the SNK-cell supernatant. In addition, the mRNA expression of the inflammatory cytokines IL-6, IL-8, and TNF-α was induced after the co-culture. These results indicated that the SNK-cell supernatant induced the differentiation of THP1 and U937 cells into the M1-macrophages and their activation. Finally, supernatant factors responsible for the differentiation and activation were also examined. The high concentration of TNF-α, which is known to induce macrophage activation, was determined in the SNK-cell culture supernatant, whereas it was not detected in the SNT-cell supernatant. In addition, the expression of several miR-BARTs that was detected in the SNK cells was reduced in the SNT cells. Interestingly, miR-BARTs were confirmed in the U937 cells cultured with the SNK-cell supernatant.Conclusions: The findings of this study indicate that TNF-α and miR-BARTs secreted from EBV-positive SNK cells may induce the differentiation and activation of macrophages. They may be utilized as molecular targets in the treatment of HLH with EBV-positive T- or NK-cell neoplasms. DisclosuresNo relevant conflicts of interest to declare.