Evaluation of β-Catenin Inhibition of Axitinib and Nitazoxanide in Human Monocyte-Derived Dendritic Cells.
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ABSTRACT:
SUBMITTER: Azeem W
PROVIDER: S-EPMC8391762 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This study is to examine the monocyte-derived dendritic cell (DC) response to hepatitis C virus (HCV) in a cell culture system. Adherence-derived DCs were incubated with various titres of JFH-1 (HCV genotype 2a), generated from transfected Huh 7.5 cells or co-incubated with Newcastle disease virus (NDV). Infection and the type 1 interferon (IFN) response were assessed by real-time reverse transcriptase-polymerase chain reaction, morphology by light microscopy and immunophenotype by flow cytometry. Our data demonstrated no viral replication or particle release from DC after HCV infection. Morphologically, monocytes showed a tendency to shift to immature DCs when cultured with HCV, when compared with control monocytes. This shift was confirmed by flow cytometry and appeared to be related to viral titres. There was also an increase in immature DC numbers. HCV infection induced IFN? expression in DCs, and the amount seemed to be inversely correlated with viral titres indicating that HCV has the capacity to negatively regulate such cells. However, IFN? does not appear to be affected by direct contact with the virus. A strong IFN? signal induced by NDV in DC was substantially diminished by HCV. HCV negatively affects the maturation of DCs and suppresses the type 1 IFN response of DC. Our results suggest a mechanism of viral evasion of host immunity.
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Project description:Inflammatory monocyte-derived dendritic cells (Mo-DCs) have been described in several chronic inflammatory disorders, such as rheumatoid arthritis (RA), and are suspected to play a detrimental role by fueling inflammation and skewing adaptive immune responses. However, the characterization of their phenotype is still limited, as well as the comprehension of the factors that govern their differentiation. Here, we show that inflammatory Mo-DCs generated in vitro expressed a large and atypical panel of C-type lectin receptors, including isoforms of CD209 and CD206, CD303 and CD207, as well as intracellular proteins at their surfaces such as the lysosomal protein CD208. Combination of these markers allowed us to identify cells in the synovial fluid of RA patients with a close phenotype of inflammatory Mo-DCs generated in vitro. Finally, we found in coculture experiments that RA synoviocytes critically affected the phenotypic differentiation of monocytes into Mo-DCs, suggesting that the crosstalk between infiltrating monocytes and local mesenchymal cells is decisive for Mo-DCs generation.
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Project description:The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.
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Project description:Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2-12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-α, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells "foamy DCs" and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A.
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Project description:Dendritic cells (DCs) are highly specific antigen presenting cells, which link innate and adaptive immune responses and participate in protecting hosts from invading pathogens. DCs can be generated in vitro by culturing human monocytes with GM-CSF and IL-4 followed by LPS induced DC maturation. We set out to study the suppressor of cytokine signaling (SOCS) proteins during maturation and activation of human monocyte-derived DCs from peripheral blood in vitro. We found that the expression of SOCS2 mRNA and protein is dramatically up-regulated during DC maturation. Silencing of SOCS2 using siRNA, inhibited DC maturation as evidenced by a decreased expression of maturation markers such as CD83, co-stimulatory molecules CD40, CD86 and HLA-DR. Furthermore, silencing of SOCS2 decreased LPS induced activation of MAP kinases (SAKP/JNK, p38, ERK), IRF3, decreased the translocation of the NF-kappaB transcription factor and reduced downstream gene mRNA expression. These results suggest a role for SOCS2 in the MyD88-dependent and -independent TLR4 signaling pathways. In conclusion, our results demonstrate that SOCS2 is required for appropriate TLR4 signaling in maturating human DCs via both the MyD88-dependent and -independent signaling pathway.
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Project description:In colon cancer, wingless (Wnt)/β-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/β-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and β-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/β-catenin/glycogen synthase kinase-3β (GSK-3β) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/β-catenin/GSK-3β signaling.
| S-EPMC8156814 | biostudies-literature
Project description:BackgroundDendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). This generates high cell numbers and allows instructing DCs to guarantee effective antitumor responses. However, the impact of the monocyte isolation step in the antitumor effectiveness of the generated MoDCs is still unknown. Here, we compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL.ResultsMACS technology allowed a higher monocyte yield and purity and, by flow cytometry, monocytes displayed higher size and lower granularity. In the resting state, EasySep_MoDCs showed a higher basal expression of HLA-DR, and no significant response to stimulation by LPS and TNF-α. When stimulated with whole tumor cells lysates, both MoDCs expressed similar levels of maturation and co-stimulatory markers. However, when cultured with autologous T cells, MACS_MoDCs induced significantly higher IFN-γ secretion than EasySep_MoDCs, indicating a stronger induction of Th1 cell response profile. Concordantly, T cells induced by MACS_MoDCs also showed a higher release of cytotoxic granules when in contact with tumor cells.ConclusionsOverall, both the MACS and the EasySep isolation immunomagnetic technologies provide monocytes that differentiate into viable and functional MoDCs. In our experimental settings, resting EasySep_MoDCs showed a higher basal level of maturation but show less responsivity to stimuli. On the other hand, MACS_MoDCs, when stimulated with tumor antigens, showed better ability to stimulate Th1 responses and to induce T cell cytotoxicity against tumor cells. Thus, monocyte isolation techniques crucially affect MoDCs' function and, therefore, should be carefully selected to obtain the desired functionality.
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Project description:The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.
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Project description:The recombinant canarypox virus ALVAC is being extensively studied as vaccine vector for the development of new vaccine strategies against chronic infectious diseases and cancer. However, the mechanisms by which ALVAC initiates the immune response have not been completely elucidated. In order to determine the type of innate immunity triggered by ALVAC, we characterized the gene expression profile of human monocyte derived dendritic cells (MDDCs) upon ALVAC infection. These cells are permissive to poxvirus infection and play a key role in the initiation of immune responses. The majority of the genes that were up-regulated by ALVAC belong to the type I interferon signaling pathway including IRF7, STAT1, RIG-1, and MDA-5. Genes involved in the NF-kappaB pathway were not up-regulated. The gene encoding for the chemokine CXCL10, a direct target of the transcription factor IRF3 was among those up-regulated and DC secretion of CXCL10 following exposure to ALVAC was confirmed by ELISA. Many downstream type I interferon activated genes with anti-viral activity (PKR, Mx, ISG15 and OAS among others) were also up-regulated in response to ALVAC. Among these, ISG15 expression in its unconjugated form by Western blot analysis was demonstrated. In view of these results we propose that ALVAC induces type I interferon anti-viral innate immunity via a cytosolic pattern-recognition-receptor (PRR) sensing double-stranded DNA, through activation of IRF3 and IRF7. These findings may aid in the design of more effective ALVAC-vectored vaccines.
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Project description:Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-?-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-?. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(low)HLADR(low) CD1a(+) MCL1(+), did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high) HLADR(low)) pre-M2 (CCL22(+) CD206(+) CD163(+) IL1RN(+) IL-10(-) CXCL10(-) IL-12(-)) mixed (CD1a(+) CD14+ CD68(+)) macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-?B, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic cells, blocking IL-17A, may be either dangerous or beneficial for cancer outcomes, thus contributing to the apparent controversy around the role of IL-17A in cancer.
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