Comparative effects of aspirin and NO-releasing aspirins on differentiation, maturation and function of human monocyte-derived dendritic cells in vitro

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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Differentiation and function of human monocyte-derived dendritic cells under the influence of leflunomide

Archives of Biological Sciences ◽

10.2298/abs1102353s ◽

2011 ◽

Vol 63 (2) ◽

pp. 353-364

Author(s):

Z. Stojic-Vukanic ◽

M. Colic ◽

A. Backovic ◽

J. Antic-Stankovic ◽

B. Bufan ◽

...

Keyword(s):

Dendritic Cells ◽

Flow Cytometric Analysis ◽

Human Monocyte ◽

Surface Expression ◽

Immunosuppressive Drugs ◽

Experimental Models ◽

Immunosuppressive Drug ◽

Adhesive Molecules ◽

And Function

Leflunomide is an immunosuppressive drug effective in experimental models of transplantation and autoimmune diseases and in the treatment of active rheumatoid arthritis (RA). Having in mind that it has been shown that some other immunosuppressive drugs (glucocorticoids, mycophenolate mofetil, sirolimus etc.) impair dendritic cell (DC) phenotype and function, we investigated the effect of A77 1726, an active metabolite of leflunomide, on the differentiation and function of human monocyte-derived dendritic cells (MDDC) in vitro. Immature MDDC were generated by cultivating monocytes in medium supplemented with GM-CSF and IL-4. To induce maturation, immature MDDC were cultured for 2 additional days with LPS. A77 1726 (100 ?M) was added at the beginning of cultivation. Flow cytometric analysis showed that MDDC differentiated in the presence of A77 1726 exhibited an altered phenotype, with a down-regulated surface expression of CD80, CD86, CD54 and CD40 molecules. Furthermore, the continuous presence of A77 1726 during differentiation and maturation prevented successful maturation, judging by the decreased expression of maturation marker CD83, costimulatory and adhesive molecules on A77 1726-treated mature MDDC. In addition, A77 1726-pretreated MDDC exhibited a poor stimulatory capacity of the allogeneic T cells and a low production of IL-10 and IL-18. These data suggest that leflunomide impairs the differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

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Human Monocyte-Derived Dendritic Cells Are Tolerogenic through Both IDO1 and IDO2

Blood ◽

10.1182/blood.v116.21.4298.4298 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4298-4298

Author(s):

Sara Trabanelli ◽

Antonio Curti ◽

Darina Očadlíková ◽

Cecilia Evangelisti ◽

Valentina Salvestrini ◽

...

Keyword(s):

Cell Proliferation ◽

Dendritic Cells ◽

T Cell ◽

Critical Role ◽

Human Monocyte ◽

Kynurenine Pathway ◽

T Cell Proliferation ◽

Indoleamine 2,3 Dioxygenase ◽

And Function

Abstract Abstract 4298 Indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like (IDO2) are enzymes involved in the tryptophan catabolism along the kynurenine pathway. While it is established that IDO1-expressing dendritic cells (DCs) contribute to tolerance in a number of biological settings, little is known about the expression and function of IDO2 in DCs. Human DCs can be generated in vitro to obtain immunogenic antigen-presenting cells (APC), used as cellular vaccines. In the clinical setting, DCs are commonly matured with a cytokine cocktail (CC) which includes TNF-a, IL-1b, IL-6 and PGE2. In particular, PGE2 enhances APC function of DCs by increasing IL-12 production and facilitating DC migration to lymph nodes. However, PGE2 is also a strong IDO1 inducer, which by this route can also limit the anti-tumor activity of DC-based immunotherapies. Thus, understanding the roles of IDO1 and IDO2 in DCs may impact the development of vaccines or DC-based immunotherapies. In the present study, we fully characterized IDO1 and IDO2 expression and function in human monocyte-derived dendritic cells (Mo-DCs). Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-g and the CC. We observed that immature Mo-DCs had little if any expression of both IDO1 and IDO2, whereas mature Mo-DCs exhibited upregulation of both enzymes. Among the different maturation stimuli, CC was the most effective in upregulating IDO1 and IDO2, both at the message and protein levels. This effect was associated also with the highest kynurenine production. By means of IDO1 and IDO2 expression, mature Mo-DCs were inhibited in stimulating allogeneic T cell proliferation and generated a population of CD4+CD25+FOXP3+ Tregs which highly suppressed allogeneic and autologous T-cell proliferation. On the basis of evidence that IDO1 is preferentially inhibited by the L-isoform of 1 methyl-tryptophan (1-MT) and IDO2 by the D-isoform, we performed functional enzyme tests in presence of both isoforms. Notably, both isoforms exhibited inhibitory effects, although we observed a stronger effect of L-1-MT than with D-1-MT suggesting a greater contribution of IDO1 than IDO2. These results offer direct evidence that Mo-DCs express functional IDO1 and IDO2 proteins. During the maturation phase, Mo-DCs enhance their tolerogenic qualities, and in particular the capacity to induce Tregs, through the upregulation of both IDO1 and IDO2. Beside the critical role of IDO1 in enhancing the immunosuppressive capacity of DCs, we show, for the first time, that IDO2 is involved also. Our findings imply that, from a clinical standpoint, to improve the efficacy of DC-based vaccines mature DCs should be combined with molecules that can inhibit the activity of both IDO1 and IDO2. Disclosures: Metz: NewLink Genetics: Employment. Prendergast:New Link Genetics Corp: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Proteasome Inhibitors Affect the Function of Human Dendritic Cells and Induce Caspase-Mediated Apoptosis.

Blood ◽

10.1182/blood.v106.11.2229.2229 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2229-2229

Author(s):

Karin von Schwarzenberg ◽

Alessio Nencioni ◽

Anita Bringmann ◽

Lothar Kanz ◽

Franco Patrone ◽

...

Keyword(s):

Dendritic Cells ◽

Proteasome Inhibitors ◽

Human Monocyte ◽

Proteasome Inhibition ◽

Parp Cleavage ◽

Induced Apoptosis ◽

And Function ◽

Human Dendritic Cells

Abstract Proteasome inhibitors (PI) show antitumor activity against a broad spectrum of human malignancies both in vitro and in vivo. Yet, the consequences of exposure to these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effect of the proteasome inhibitors on human monocyte-derived dendritic cells (DCs). Exposure to PI results in a reduced activation induced DC maturation and cytokine production, inhibition of their migratory capacity and impaired ability to stimulate T-cell responses. These functional and phenotypic alterations were paralleled by a decreased phosphorylation of the MAP kinase member ERK1/2 while not affecting p38. Furthermore, incubation of DC with bortezomib inhibited the expression of nuclear localized transcription factors that were shown to be important for DC differentiation and function like IRF3, Rel-b and c-rel. Addition of PI to culture medium induced apoptosis of differentiated DCs and strongly reduced the yield of viable DCs when given to monocytes before differentiation to DCs was induced. DC apoptosis was accompanied by caspase activation as detected by caspase-3 and PARP cleavage. Cytochrome c cytosolic relocalization was detectable following exposure to bortezomib and was not prevented by caspase inhibition. This points to the mitochondrial damage as to an upstream event in DC apoptosis via proteasome inhibition. While not affecting Bcl-2 levels, bortezomib was found to promote Bax upregulation in DCs, thus providing a possible explanation for mitochondria dysfunction in response to this compound. In conclusion, this study shows that besides the inhibition of Nf-kB bortezomib is affecting several other pivotal signal transduction pathways in human cells and suggests that inhibition of DC function and induction of apoptosis in DCs may represent a mechanism by which bortezomib can affect the immune responses in patients treated with this compound.

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Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro

Viruses ◽

10.3390/v10110617 ◽

2018 ◽

Vol 10 (11) ◽

pp. 617 ◽

Cited By ~ 5

Author(s):

Helen Freyberger ◽

Yunxiu He ◽

Amanda Roth ◽

Mikeljon Nikolich ◽

Andrey Filippov

Keyword(s):

Staphylococcus Aureus ◽

Dendritic Cells ◽

Inflammatory Response ◽

Adaptive Immune Response ◽

Human Monocyte ◽

Activation Markers ◽

Mhc I ◽

Adaptive Immune ◽

Human Dendritic Cells

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.

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IN LABORATORY GENERATION AND MATURATION OF HUMAN MONOCYTE-DERIVED DENDRITIC CELLS FOR CANCER IMMUNOTHERAPY

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2020.v12s4.40104 ◽

2020 ◽

pp. 46-52

Author(s):

KANCHAN K. MISHRA ◽

SUMIT BHARADVA ◽

MEGHNAD G. JOSHI ◽

ARVIND GULBAKE

Keyword(s):

Dendritic Cells ◽

Immune Responses ◽

Gradient Centrifugation ◽

Critical Role ◽

Human Monocyte ◽

Blood Monocytes ◽

Adaptive Immune ◽

Immunodeficiency Virus ◽

Adaptive Immune Systems

Dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, furthermore they act as a bridge between the innate and the adaptive immune systems they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. The first reported trial using DCs in 1995, since they have been used in trials all over the world for several of indications, including cancer and human immunodeficiency virus infection. Generally, for in vitro experiments or for DCs vaccination monocyte-derived dendritic cells (moDCs) were generated from purified monocytes that isolated from peripheral blood by density gradient centrifugation. A variety of methods can be used for enrichment of monocytes for generation of clinical-grade DCs. Herein we summarized up to date understanding of systems and inputs used in procedures to differentiate DCs from blood monocytes in vitro.

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Nonsteroidal anti-estrogens inhibit the functional differentiation of human monocyte-derived dendritic cells

Blood ◽

10.1182/blood.v95.9.2875.009k12_2875_2882 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2875-2882 ◽

Cited By ~ 90

Author(s):

Janne Komi ◽

Olli Lassila

Keyword(s):

Dendritic Cells ◽

Human Monocyte ◽

Functional Differentiation ◽

Cd40 Ligation ◽

Treatment And Prevention ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Antigen Presenting ◽

Stimulating Factor

Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and regulate immune responses. Immature CD1a+ DC can be cultured from CD14+monocytes in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in vitro. Results of this study show that the nonsteroidal anti-estrogens toremifene and tamoxifen inhibit this differentiation. In the presence of anti-estrogens the cells lose CD14 expression, but remain CD1a− and clearly have less dendritic processes than immature DC. Functionally, anti-estrogen-treated cells are inferior to immature DC in inducing proliferation of allogeneic T cells and in producing IL-12 p70 protein after CD40 ligation. The expression of the costimulatory molecules CD80 and CD86 is differentially regulated by anti-estrogens during DC differentiation. Furthermore, anti-estrogens are also able to inhibit the terminal maturation of DC. By inhibiting the functional differentiation of DC, anti-estrogens may have a role in the treatment and prevention of autoimmune diseases.

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