Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells

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.

Download Full-text

A Basis for Distinguishing Cultured Dendritic Cells and Macrophages in Cytospins and Fixed Sections

Pediatric and Developmental Pathology ◽

10.1007/s10024-004-5045-2 ◽

2005 ◽

Vol 8 (1) ◽

pp. 43-51 ◽

Cited By ~ 17

Author(s):

Jukka Vakkila ◽

Michael T. Lotze ◽

Connie Riga ◽

Ronald Jaffe

Keyword(s):

Dendritic Cells ◽

Cell Types ◽

Fixed Tissue ◽

Reliable Identification ◽

Formalin Fixed Tissue ◽

Tissue Sections ◽

Hla Dr ◽

Formalin Fixed

There is a burgeoning literature on the contrasting role of intratumoral dendritic cells (DCs) and tumor-associated macrophages, making reliable identification of both cell types in clinical and experimental tissue sections important. However, because these cell types are closely related and share several differentiation antigens, their absolute distinction in tissue sections is difficult. We differentiated DCs and macrophages from monocytes in vitro, prepared cytospins and paraffin-embedded sections of the various cell populations, and tested a variety of antibodies that purportedly recognize monocytes and DCs for their capacity to react and distinguish cells after conventional formalin fixation. Cultured DCs but not macrophages were detected by fascin, DC-LAMP, and CD83 with a predictable increase in staining that paralleled their maturation. Staining by CD1a was found on immature DCs but was weak and absent on mature DCs and macrophages, respectively. CD14 and CD163 were characteristic for macrophages and absent on DCs. CD68, HLA-DR, and S100 did not discriminate between DCs and macrophages. We conclude that antigens such as HLA-DR and S100 are not in themselves sufficient for identification of DCs in formalin-fixed tissue sections, but that additional macrophage-specific (CD14, CD163) and DC-specific (CD1a, CD83, fascin, DC-LAMP) antigens should be used to distinguish cell types from each other and to provide information on their state of maturation.

Download Full-text

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.

Download Full-text

GSK-3 mediates differentiation and activation of proinflammatory dendritic cells

Blood ◽

10.1182/blood-2006-06-028951 ◽

2006 ◽

Vol 109 (4) ◽

pp. 1584-1592 ◽

Cited By ~ 82

Author(s):

Elena Rodionova ◽

Michael Conzelmann ◽

Eugene Maraskovsky ◽

Michael Hess ◽

Michael Kirsch ◽

...

Keyword(s):

Dendritic Cells ◽

In Vitro Model ◽

Glycogen Synthase ◽

Human Monocyte ◽

Glycogen Synthase Kinase 3 ◽

Inhibitory Effects ◽

Tnf Α ◽

Vitro Model

Abstract The key components of the intracellular molecular network required for the expression of a specific function of dendritic cells (DCs) are as yet undefined. Using an in vitro model of human monocyte-derived DC differentiation, this study investigates the role of glycogen synthase kinase 3 (GSK-3), a multifunctional enzyme critical for cellular differentiation, apoptosis, self-renewal, and motility, in this context. We demonstrate that GSK-3 (1) inhibits macrophage development during differentiation of DCs, (2) is constitutively active in immature DCs and suppresses spontaneous maturation, and (3) acquires a proinflammatory functional status mediating high levels of IL-12, IL-6, and TNF-α secretion, and partially inhibits IL-10 in the context of DC activation. In particular, GSK-3 enhances IL-12p35 mRNA expression and thus the production of the proinflammatory cytokine IL-12p70 by integrating the activities of other kinases priming GSK-3 targets and the inhibitory effects of Akt-1. GSK-3 may therefore act as a key integrator of activating and inhibitory pathways involved in proinflammatory DC differentiation and activation.

Download Full-text

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.

Download Full-text

IFN Regulatory Factor-1 Modulates the Function of Dendritic Cells in Patients with Acute Coronary Syndrome

Cellular Physiology and Biochemistry ◽

10.1159/000430123 ◽

2015 ◽

Vol 36 (2) ◽

pp. 599-610 ◽

Cited By ~ 4

Author(s):

Min Guo ◽

Rui Yan ◽

Caixia Wang ◽

Hongtao Shi ◽

Meng Sun ◽

...

Keyword(s):

T Cells ◽

Acute Coronary Syndrome ◽

Dendritic Cells ◽

Mapk Pathway ◽

Human Monocyte ◽

Regulatory Factor ◽

Coronary Syndrome ◽

Artery Disease ◽

And Function

Background: Atherosclerosis is widely recognized as a complex inflammatory disease involving pathogenic immune response of T cells and antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Accumulating evidence has revealed that mature DCs play critical roles in the differentiation of effector T cells into CD4+ T cells, which effectively participate in the onset of acute coronary syndrome (ACS). IFN regulatory factor (IRF)-1 has been shown to be involved in various immune processes. The role of IRF-1 in DCs in the pathogenesis of ACS has not been investigated. Methods and Results: We examined the relative mRNA and protein expression of IRF-1 in human monocyte-derived DCs in patients with coronary artery disease (CAD). The overexpression or silencing of IRF-1 expression in DCs in patients with ACS was performed to explore the possible role of IRF-1 in the maturation and function of DCs involved in ACS. The results showed that the relative expression of IRF-1 in DCs is obviously increased in patients with ACS. The overexpression or silencing of IRF-1 expression could effectively promote or attenuate the maturation and function of DCs. In addition, we revealed that the MAPK pathway (phosphorylation of JNK, p38 and ERK1/2) might be downstream of IRF-1 signalling pathway in activation of circulating DCs in ACS patients. Conclusion: The present data demonstrate that IRF-1 could effectively promote the immune maturation and function of DCs in ACS patients.

Download Full-text

Resveratrol pretreatment mitigates LPS-induced acute lung injury by regulating conventional dendritic cells’ maturation and function

Open Life Sciences ◽

10.1515/biol-2021-0110 ◽

2021 ◽

Vol 16 (1) ◽

pp. 1064-1081

Author(s):

Bingnan Guo ◽

Yigen Peng ◽

Yuting Gu ◽

Yi Zhong ◽

Chenglei Su ◽

...

Keyword(s):

Dendritic Cells ◽

Acute Lung Injury ◽

Lung Injury ◽

Enhanced Recovery ◽

Lavage Fluid ◽

Protective Role ◽

High Morbidity ◽

And Function

Abstract Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a severe syndrome lacking efficient therapy and resulting in high morbidity and mortality. Although resveratrol (RES), a natural phytoalexin, has been reported to protect the ALI by suppressing the inflammatory response, the detailed mechanism of how RES affected the immune system is poorly studied. Pulmonary conventional dendritic cells (cDCs) are critically involved in the pathogenesis of inflammatory lung diseases including ALI. In this study, we aimed to investigate the protective role of RES via pulmonary cDCs in lipopolysaccharide (LPS)-induced ALI mice. Murine ALI model was established by intratracheally challenging with 5 mg/kg LPS. We found that RES pretreatment could mitigate LPS-induced ALI. Additionally, proinflammatory-skewed cytokines decreased whereas anti-inflammatory-related cytokines increased in bronchoalveolar lavage fluid by RES pretreatment. Mechanistically, RES regulated pulmonary cDCs’ maturation and function, exhibiting lower level of CD80, CD86, major histocompatibility complex (MHC) II expression, and IL-10 secretion in ALI mice. Furthermore, RES modulated the balance between proinflammation and anti-inflammation of cDCs. Moreover, in vitro RES pretreatment regulated the maturation and function of bone marrow derived dendritic cells (BMDCs). Finally, the adoptive transfer of RES-pretreated BMDCs enhanced recovery of ALI. Thus, these data might further extend our understanding of a protective role of RES in regulating pulmonary cDCs against ALI.

Download Full-text

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

International Immunopharmacology ◽

10.1016/j.intimp.2009.03.016 ◽

2009 ◽

Vol 9 (7-8) ◽

pp. 910-917 ◽

Cited By ~ 14

Author(s):

Biljana Bufan ◽

Slavko Mojsilović ◽

Dragana Vučićević ◽

Dragana Vučević ◽

Saša Vasilijić ◽

...

Keyword(s):

Dendritic Cells ◽

Human Monocyte ◽

And Function

Download Full-text

GITRL on dendritic cells aggravates house dust mite-induced airway inflammation and airway hyperresponsiveness by modulating CD4+ T cell differentiation

Respiratory Research ◽

10.1186/s12931-020-01583-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yaping Wang ◽

Kou Liao ◽

Bo Liu ◽

Chao Niu ◽

Wenjing Zou ◽

...

Keyword(s):

Dendritic Cells ◽

Cell Differentiation ◽

House Dust ◽

House Dust Mite ◽

Airway Hyperresponsiveness ◽

T Cell Differentiation ◽

Asthmatic Children ◽

Dust Mite

Abstract Background Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) plays an important role in tumors, autoimmunity and inflammation. However, GITRL is not known to modulate the pathogenesis of allergic asthma. In this study, we investigated whether regulating GITRL expressed on dendritic cells (DCs) can prevent asthma and to elucidate its mechanism of action. Methods In vivo, the role of GITRL in modulating house dust mite (HDM)-induced asthma was assessed in adeno-associated virus (AAV)-shGITRL mice. In vitro, the role of GITRL expression by DCs was evaluated in LV-shGITRL bone marrow dendritic cells (BMDCs) under HDM stimulation. And the direct effect of GITRL was observed by stimulating splenocytes with GITRL protein. The effect of regulating GITRL on CD4+ T cell differentiation was detected. Further, GITRL mRNA in the peripheral blood of asthmatic children was tested. Results GITRL was significantly increased in HDM-challenged mice. In GITRL knockdown mice, allergen-induced airway inflammation, serum total IgE levels and airway hyperresponsiveness (AHR) were reduced. In vitro, GITRL expression on BMDCs was increased after HDM stimulation. Further, knocking down GITRL on DCs partially restored the balance of Th1/Th2 and Th17/Treg cells. Moreover, GITRL stimulation in vitro inhibited Treg cell differentiation and promoted Th2 and Th17 cell differentiation. Similarly, GITRL mRNA expression was increased in the peripheral blood from asthmatic children. Conclusions This study identified a novel role for GITRL expressed by DCs as a positive regulator of CD4+ T cells responses in asthma, which implicates that GITRL inhibitors may be a potential immunotherapy for asthma.

Download Full-text

Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

Infection and Immunity ◽

10.1128/iai.00524-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 845-853 ◽

Cited By ~ 58

Author(s):

Wafa Khamri ◽

Marjorie M. Walker ◽

Peter Clark ◽

John C. Atherton ◽

Mark R. Thursz ◽

...

Keyword(s):

T Cells ◽

Helicobacter Pylori ◽

Dendritic Cells ◽

Virulence Factors ◽

Human Monocyte ◽

Interleukin 17 ◽

Th17 Response ◽

H Pylori

ABSTRACT Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4+ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.

Download Full-text

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.

Download Full-text