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1
Marongiu, Laura, Francesca Mingozzi, Clara Cigni, Roberta Marzi, Marco Di Gioia, Massimiliano Garrè, Dario Parazzoli, et al. "Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells." Science Signaling 14, no. 676 (March 30, 2021): eaaz2120. http://dx.doi.org/10.1126/scisignal.aaz2120.
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Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane–localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.
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Palucka, Karolina A., Nicolas Taquet, Francoise Sanchez-Chapuis, and Jean Claude Gluckman. "Dendritic Cells as the Terminal Stage of Monocyte Differentiation." Journal of Immunology 160, no. 9 (May 1, 1998): 4587–95. http://dx.doi.org/10.4049/jimmunol.160.9.4587.
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Abstract Monocytes (MO) cultured for ≥5 days with either macrophage-CSF (M-CSF) or granulocyte macrophage (GM)-CSF and IL-4 differentiated without concomitant proliferation into CD14+ macrophages (Mφ) or CD1a+ dendritic cells (DC), respectively. When adherent and nonadherent CD14high Mφ from M-CSF cultures were separated and cultured further in cytokine-free medium or with GM-CSF/IL-4, most cells from both fractions that were exposed to GM-CSF/IL-4 acquired CD1a expression and DC morphology and function. Conversely, GM-CSF/IL-4 withdrawal at day 5 and additional culture of sorted CD1a+ DC for 2 to 7 days in cytokine-free medium led to cells rapidly becoming adherent CD1a−CD14+ Mφ. Replacing GM-CSF/IL-4 with M-CSF hastened the conversion of DC to Mφ without increasing cell numbers. CD1a+CD14−CD83+ mature DC were induced by a ≥2-day exposure to MO-conditioned medium, LPS, or TNF-α/IL-1β. Upon cytokine removal or culture with M-CSF, DC that had been pushed to maturation by conditioned medium or LPS remained stable or died in the new environment. TNF-α/IL-1β-driven DC displayed heterogeneous CD83 expression and could thus be sorted into CD83high and CD83low/− cells; in cytokine-free medium or in M-CSF, most CD83low/− cells converted to Mφ, whereas most CD83high cells remained nonadherent CD1a+CD14− or died and thus appeared truly terminally differentiated. Hence, MO are precursors of Mφ as well as of DC, with each cell type having the capability to convert into the other until late in the differentiation/maturation process. Accordingly, the cytokine environment and the presence of differentiation and/or other stimulatory signals may be the “final decision-making factors” determining whether these cells will acquire DC or Mφ characteristics and function.
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Verhasselt, V., C. Buelens, F. Willems, D. De Groote, N. Haeffner-Cavaillon, and M. Goldman. "Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway." Journal of Immunology 158, no. 6 (March 15, 1997): 2919–25. http://dx.doi.org/10.4049/jimmunol.158.6.2919.
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Abstract To investigate the responses of dendritic cells (DC) during Gram-negative infections, we analyzed the effects of graded doses of LPS on the cytokine profile, phenotype, and allostimulatory potential of human DC generated by culturing plastic-adherent PBMC in presence of IL-4 and granulocyte-macrophage-CSF. First, we found that LPS stimulates the production of high levels of TNF-alpha, IL-6, IL-8, IL-12 by DC and up-regulates their expression of HLA-DR, B7-1, B7-2, and CD40. The effects of LPS were dose dependent, with a significant stimulatory effect already observed at a concentration of 0.1 ng/ml and a plateau being reached at 10 ng/ml. These phenotypic changes correlated with increased allostimulatory properties of LPS-activated DC because DC treated with LPS were significantly more efficient than untreated DC in eliciting IL-2 and IFN-gamma synthesis by alloreactive T cells and stimulating their proliferation. Experiments using neutralizing anti-IL-12 mAb indicated that LPS-induced IL-12 is responsible for the increased production of IFN-gamma but not for the increased proliferation during MLR. Finally, we observed that the DC responses to low levels of LPS (1 ng/ml) were dramatically inhibited by a blocking anti-CD14 mAb, although DC do not express CD14 molecules on their membrane. Experiments using serum depleted of soluble CD14 (sCD14) and sCD14 either purified from human serum or in recombinant form further established that DC respond to LPS via a soluble CD14-dependent pathway.
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Ziros, Panos, Ilina Micheva, Ioannis Habeos, Athanasios Papavasiliou, and Nicholas Zoumbos. "Expression and Activation of the Farnsesoid X Receptor in Human Dendritic Cells." Blood 106, no. 11 (November 16, 2005): 2220. http://dx.doi.org/10.1182/blood.v106.11.2220.2220.
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Abstract Baground: The farnesoid X receptor/bile acid receptor (FXR, NR1H4) is a member of the nuclear hormone superfamily that regulates bile acid as well as lipoprotein and glucose metabolism after heterodimerization with retinoid X receptor. Dendritic cells (DCs) are potent antigen presenting cells capable of regulating immune responses. It has previously been reported that spleen, a tissue rich in DCs, expresses FXR. Taking this into account, we wanted to investigate the possible involvement of FXR in DCs biology. Material and Methods: Immature DCs were generated from peripheral human blood monocytes (CD14+ cells) by culturing them with GM-CSF and IL4 for 7 days. For maturation, immature DCs were cultured with the addition of TNFα, and LPS for another 24 hours. The expression and activity of FXR in DCs was assessed with RT-PCR, western immunoblotting, immunofluoresence and EMSA analysis. Results and Discusion:We have shown with RT-PCR that FXR is marginally expressed in human monocytes (CD14+ cells) and its expression is elevated during the differentiation process of these to dendritic cells. Moreover, using specific primers we have found that only FXR -alpha and not FXR-beta is expressed in DCs. Western blot analysis with a specific anti-FXR antibody confirmed the FXR expression in DCs. Immunofluoresence microscopy with a specific anti-FXR antibody showed that FXR, in contrast with the classical FXR-expressing tissues (liver, kidney), has strong cytoplasmic and perinuclear localization and weak nuclear localization in DCs. The nuclear localization is potentiated upon treatment of the immature dendritic cells with LPS or TNFα. Finally, using a DNA probe that contains consensus FXR binding sites, we have shown with EMSA analysis that FXR is transcriptional active in immature DCs but not in CD14 monocytes, and is strongly activated upon LPS or TNFα treatment for 48 h of immature DCs. Overall our study reveals, for the first time, that FXR, a nuclear hormone receptor with a limited tissue expression, might have a new role in DCs biology, since it is not only expressed in human dendritic cells but moreover its transcriptional activity is affected by signals (TNFα, LPS) that influence the function of DCs.
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Borriello, Francesco, Raffaella Iannone, Sarah Di Somma, Viviana Vastolo, Giuseppe Petrosino, Feliciano Visconte, Maddalena Raia, et al. "LPS-elicited TSLPR expression enriches a functionally discrete subset of human CD14+ CD1c+ monocytes." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 75.1. http://dx.doi.org/10.4049/jimmunol.198.supp.75.1.
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Abstract Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor complex, a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLP receptor complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is ill-defined. We demonstrate that TSLP enhances human CD14+ monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14+ CD16− monocytes (TSLPR+ mono) expresses TSLP receptor complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional and transcriptomic analysis revealed specific features of TSLPR+ mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e. GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR+ mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14+ CD1c+ cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared to healthy controls. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14+ CD16− monocytes and prompt further ontogenetic and functional analysis of CD14+ CD1c+ and LPS-activated CD14+ CD1c+ TSLPR+ monocytes.
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Kurochkina, Y., A. Sizikov, E. Chernykh, and T. Tyrinova. "AB0072 GLUCOCORTICOIDS MODIFY NOT ONLY THE PROPERTIES OF DENDRITIC CELLS, BUT ALSO PROGENITOR CELLS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1168.2–1168. http://dx.doi.org/10.1136/annrheumdis-2022-eular.2571.
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BackgroundDendritic cells (DCs) are universal antigen-presenting cells that can have both tolerant and immunosuppressive properties. Tolerogenic properties of DCs are mediated by activation T-regulatory cells, production of anti-inflammatory cytokines and activation T cell apoptosis. Currently, DC is an attractive object of study as a new treatment for rheumatoid arthritis. It is known that glucocorticoids make it possible to control properties of DCs to the tolerogenic side.ObjectivesThe aim of the study is to investigate the effect of pulse therapy by glucocorticodes on the properties of DCs and peripheral blood monocytes from which DCs were generated.MethodsTwenty five patients with rheumatoid arthritis were recruited in this study. All patients follow ACR/EULAR criteria (2010). All studies were performed after receiving informed consent. All patients received conventional synthetic DMARDs. DCs were generated from blood monocytes culturing for 5 days with GM-CSF and IFN-α, LPS as maturation stimuli was added on fourth day. The functions of DCs were evaluated by allostimulatory activity in mixed lymphocyte culture. The properties of DC were studied before pulse therapy (methylprednisolone 500mg №3) and 4 days after pulse therapy.The relative number of monocytes (classical CD14++CD16+, intermediate CD14+CD16+ and nonclassical CD14+CD16++) before and after pulse therapy was also studied in comparison with healthy donors.ResultsPrior to pulse therapy, the DCs from RA patients were characterized by a high ability to stimulate the proliferation of allogenic T-lymphocytes about 20000 cpm. After pulse therapy, the stimulating ability of DCs significantly decreased (9800 vs. 2000 cpm, p=0.04). Evaluation of the relative content of classical (CD14++CD16-), intermediate (CD14++CD16+) and nonclassical (CD14+CD16++) monocytes of healthy donors (n=18) and RA patients (n=25) revealed a decrease in the relative content of CD14++CD16- cells in patients (Me 78 vs. 90%; p=0.02) and an increase in the proportion of CD14++CD16+ (Me 4.0 vs. 2.0%; p=0.034) and CD14+ CD16++ monocytes (Me 5.0 vs. 1.5%; p=0.02). After the end of pulse therapy in RA patients, the relative content of CD14++CD16-cells increased (89 vs. 78%), and the proportion of CD14+CD16++ monocytes decreased (1 vs. 5%), and RA patients no longer significantly differed in these indicators from healthy donors. Thus, a decrease in the content of CD14+CD16++ cells in the monocytes population after pulse therapy was associated with a decrease in the effectiveness of DCs generated to stimulate the proliferation of allogenic T cells.ConclusionThe effect of pulse therapy of glucocorticoids is associated not only with the ability of glucocorticoids to inhibit the maturation of IFN-DC and induce their tolerant phenotype at the stage of differentiation of monocytes into DCs, but also with the effect on the subpopulation of circulating monocytes that are precursors of DC.Disclosure of InterestsNone declared
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Brosbøl-Ravnborg, Anne, Bettina Bundgaard, and Per Höllsberg. "Synergy between Vitamin D3and Toll-Like Receptor Agonists Regulates Human Dendritic Cell Response during Maturation." Clinical and Developmental Immunology 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/807971.
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Human dendritic cells (DC) can be differentiated from blood monocytes in the presence of GM-CSF and IL-4 and matured by lipopolysaccharide (LPS). Vitamin D3inhibits the maturation of human DC measured by changes in surface expression of HLA-DR, CD14, CD40, CD80, CD83, and CD86. We here examine the function of vitamin D3during DC maturation. One of the earliest changes to LPS-induced maturation was an increase in CD83 expression. Vitamin D3inhibited the increase in expression of HLA-DR, CD40, CD80, CD83, and CD86 and the decrease in expression of CD14, which was paralleled morphologically by vitamin D3-induced inhibition of dendritic cell differentiation. Vitamin D3acted in synergy with the TLR agonists LPS and peptidoglycan (PGN) in inducing IL-6, IL-8, and IL-10, whereas vitamin D3completely inhibited LPS-induced secretion of IL-12. The synergy occurred at concentrations where neither vitamin D3nor the TLR agonists alone induced measurable cytokine secretion. Both LPS and PGN enhanced the level of the vitamin D3receptor (VDR). Taken together, these data demonstrated that vitamin D3and TLR agonists acted in synergy to alter secretion of cytokines from human DC in a direction that may provide an anti-inflammatory environment.
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Ruiz-Jiménez, Caleb, Daiana Celias, Leonardo Silvane, Laura Cervi, and Ana M. Espino. "Fasciola hepatica Fatty Acid Binding Protein (Fh12) inhibits the activation of murine dendritic cells." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 68.15. http://dx.doi.org/10.4049/jimmunol.198.supp.68.15.
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Abstract Fasciola hepatica, is a helminth that excretes-secretes different proteins that modulate cells of the immune system, generating anti-inflammatory responses. It has been demonstrated that Fh12 achieves this anti-inflammatory effect by inhibiting the expression of TLR4 induced by LPS in macrophages. Fh12 target the CD14 co-receptor, thus blocking the LPS-CD14 binding, which stop the entire TLR4 activation cascade from the beginning of the LPS-stimuli. Concurrently, Fh12 suppress the phosphorylation of various kinases (p38, JNK and ERK) downstream the TLR4 signaling cascade and induce the activation of macrophages by an alternative pathway. Dendritic cells (DCs) are antigen-presenting cells that play a key role at early phase of innate immunity and that are essential in the development of adaptive immune response. The main objective of the current study was to investigate the effect of Fh12 on the activation of murine DCs matured in the presence of LPS. DCs were isolated from bone marrow of naïve C57BL/6 mice, cultured, differentiated and stimulated in vitro with Fh12 1h prior to stimulation with LPS for 18 hrs. ELISA was used to quantify the amount of a panel of secreted cytokines in culture supernatant. FACS was used to determine the activation of MHCII and co-stimulatory molecules. Results demonstrated that Fh12 significantly inhibits the production of IL-12 and IL-6 and reduced the expression of MHCII and co-stimulatory molecules like CD80 on DCs surface. All assays have a significant P value < 0.05. These results suggest that Fh12 exerts a strong suppressive effect on activation of DC cells, which could have relevant implications in the subsequent development of adaptive immune response to microbial pathogens.
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Stec, Malgorzata, Bozena Mytar, Kazimierz Weglarczyk, Irena Ruggiero, and Marek Zembala. "Characterization of Monocyte Subpopulations (CD14+CD16− and CD14++CD16+) generated from Cord Blood Haematopoietic Progenitor CD34+." Blood 112, no. 11 (November 16, 2008): 3551. http://dx.doi.org/10.1182/blood.v112.11.3551.3551.
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Abstract Introduction. In the past decade significant advances in ex vivo expansion of haematopoietic stem cells were made. Protocols of differentiating CD34+ cells into cells from hematopoietic linages, especially dendritic cells, were described. However, little is known about in vitro differentiation of CD34+ cells to monocytes. In peripheral blood there are two main populations of monocytes CD14++CD16− and CD14+CD16+. They represent about 10 percent of total blood monocytes. Recently we have showed that expansion and differentiation of cord blood CD34+ cells to monocytes/macrophages leads to generation of CD14+CD16− and CD14++CD16+ monocyte subpopulations (Stec et al, J Leukoc Biol. 2007). Aim of study. To compare two monocyte subpopulation: CD14+CD16− and CD14++CD16+ obtained from our expansion and differentiation protocol of CD34+ cord blood cells. Materials and methods. After 3–10 days expansion in X-VIVO10 medium with FBS, SCF, IL-3, FLT-3L, TPO and then 7–10 days differentiation in IMDM medium with FBS, M-CSF, FLT-3L, IL-3, SCF, CD14+CD16− and CD14++CD16+ subpopulations were isolated by FACS sorting (FACSVantage). Release of IL-6, IL8, IL-12, IP-10, MIP1 α, MIP-1 β, RANTES, VEGF and FGF after LPS/INFγ or cancer cells stimulation were analysed using CBA (Cytometric Bead Array) method. Cytotoxic activity against cancer cells were assessed by MTT test and migration capacity was assesed with uncoated porous filters (8 μm) in a 24-well Boyden chamber. The adhesion capacity of subpopulations on human umbilical vein endothelial cells (HUVEC) were assessed with 5(6)-CFDA, SE (Molecular Probes) application and fluorescent microscope. NOD-SCID mice were used to evaluate the influence of subpopulations on angiogenesis and tumour growth. Results. CD14++CD16+ monocytes released more IL-8, IL-12, IP-10, MIP1-α, MIP1-β, and RANTES than CD14+CD16− monocytes after LPS/INFγ or cancer cells stimulation. After 24 hours culture in medium without cytokines and than stimulation with LPS/INFγ or cancer cells, CD14++CD16+ subpopulation released more RANTES, TNF, MIP1-α, IL10 than CD14+CD16−cells. CD14+CD16− subpopulation exhibited greater chemotaxis to SDF-1 and MIP1-α than CD14++CD16+ (12,2%±5,5% versus 2,2%±1%). At the same time CD14++CD16+ monocytes showed significantly higher cytotoxic activity against tumour cells in vitro than CD14+CD16− (34,04±17,45% versus 15,56±13,66%). CD14++16+ population showed increased adhesion to HUVEC than CD14+16− population (79±20 cells versus 30±13 cells). In in vivo tests in NODSCID mice both subpopulations together inhibited angiogenesis, but CD14+CD16− subpopulation inhibited stronger tumour growth than CD14++CD16+ subpopulation. Conclusions. Using our expansion protocol it was possible to obtain two subpopulations of monocytes: CD14+CD16− and CD14++CD16+ which showed the differences in the release of cytokines/chemokines following stimulation with LPS/INFγ or tumour cells, chemotactic and antiangiogenic activity and cytotoxicity and are clearly distinct from known main subpopulations of blood monocytes CD14+CD16+ and CD14++. Additional tests, which are still in progress in our lab will provide evidence if both populations are functional monocytes.
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Tsan, Min-Fu, and Baochong Gao. "Cytokine function of heat shock proteins." American Journal of Physiology-Cell Physiology 286, no. 4 (April 2004): C739—C744. http://dx.doi.org/10.1152/ajpcell.00364.2003.
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Extensive work in the last 10 years has suggested that heat shock proteins (HSPs) may be potent activators of the innate immune system. It has been reported that Hsp60, Hsp70, Hsp90, and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation and maturation of dendritic cells (antigen-presenting cells) in a manner similar to the effects of lipopolysaccharide (LPS) and bacterial lipoprotein, e.g., via CD14/Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be due to the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) as being responsible for the reported HSP cytokine effects include failure to use highly purified, low-LPS preparations of HSPs; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Thus it is essential that efforts should be directed to conclusively determine whether the reported HSP cytokine effects are due to HSPs or to contaminant(s) present in the HSP preparations before further exploring the implication and therapeutic potential of the putative cytokine function of HSPs.
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Pugin, Jérôme, Sabine Stern-Voeffray, Bruno Daubeuf, Michael A. Matthay, Greg Elson, and Irène Dunn-Siegrist. "Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock." Blood 104, no. 13 (December 15, 2004): 4071–79. http://dx.doi.org/10.1182/blood-2003-04-1290.
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Abstract In this paper, we show that plasma from patients with severe sepsis and septic shock but not normal plasma supports lipopolysaccharide (LPS) activation of epithelial cells expressing Toll-like receptor 4 (TLR4). Recombinant soluble myeloid differentiation protein-2 (MD-2) complemented normal plasma and allowed LPS activation of epithelial cells to levels measured with “septic” plasma, whereas soluble MD-2-depleted plasma lost its effects. The same “MD-2 activity” was found in urine from a patient with septic shock and in lung edema fluids from patients with adult respiratory distress syndrome (ARDS). Recombinant soluble MD-2 enabled LPS-dependent activation of epithelial cells bearing TLR4. LPS-binding protein (LBP) and soluble CD14 increased the sensitivity of TLR4-expressing epithelial cells to LPS but were not able to mediate LPS activation of these cells in the absence of soluble MD-2. An anti-MD-2 monoclonal antibody blocked LPS activation of TLR4-expressing cells only in the presence of septic plasma or septic urine. These results suggest that septic plasma containing soluble MD-2 leaking into the extravascular space supports LPS activation of TLR4-expressing epithelial cells. We therefore propose that soluble MD-2 is an important mediator of organ inflammation during sepsis. (Blood. 2004;104:4071-4079)
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Dutertre, Charles-Antoine, Sonia Amraoui, Annalisa DeRosa, Jean-Pierre Jourdain, Lene Vimeux, Matthieu Goguet, Séverine Degrelle, et al. "Pivotal role of M-DC8+ monocytes from viremic HIV-infected patients in TNFα overproduction in response to microbial products." Blood 120, no. 11 (September 13, 2012): 2259–68. http://dx.doi.org/10.1182/blood-2012-03-418681.
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Abstract HIV infects activated CD4+ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)+ and CD1c (BDCA-1)+ dendritic cell counts were reduced. Conversely, CD14+CD16++ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14++CD16−M-DC8− monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8+ monocytes were mostly responsible for this overproduction. Moreover, M-DC8+ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patient's plasma. This M-DC8+ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.
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Maiti, George, Jihane Frikeche, Carly Yuen-Man Lam, Asim Biswas, Vishal Shinde, Marie Samanovic, Jonathan C. Kagan, Mark J. Mulligan, and Shukti Chakravarti. "Matrix lumican endocytosed by immune cells controls receptor ligand trafficking to promote TLR4 and restrict TLR9 in sepsis." Proceedings of the National Academy of Sciences 118, no. 27 (July 2, 2021): e2100999118. http://dx.doi.org/10.1073/pnas.2100999118.
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Infections and inflammation are profoundly influenced by the extracellular matrix (ECM), but their molecular underpinnings are ill defined. Here, we demonstrate that lumican, an ECM protein normally associated with collagens, is elevated in sepsis patients’ blood, while lumican-null mice resolve polymicrobial sepsis poorly, with reduced bacterial clearance and greater body weight loss. Secreted by activated fibroblasts, lumican promotes Toll-like receptor (TLR) 4 response to bacterial lipopolysaccharides (LPS) but restricts nucleic acid–specific TLR9 in macrophages and dendritic cells. The underlying mechanism involves lumican attachment to the common TLR coreceptor CD14 and caveolin 1 (Cav1) in lipid rafts on immune cell surfaces via two epitopes, which may be cryptic in collagen-associated lumican. The Cav1 binding epitope alone is sufficient for cell surface enrichment of Cav1, while both are required for lumican to increase cell surface TLR4, CD14, and proinflammatory cytokines in response to LPS. Endocytosed lumican colocalizes with TLR4 and LPS and promotes endosomal induction of type I interferons. Lumican-null macrophages show elevated TLR9 in signal-permissive endolysosomes and increased response, while wild types show lumican colocalization with CpG DNA but not TLR9, consistent with a ligand sequestering, restrictive role for lumican in TLR9 signaling. In vitro, lumican competes with CD14 to bind CpG DNA; biglycan, a lumican paralog, also binds CpG DNA and suppresses TLR9 response. Thus, lumican and other ECM proteins, synthesized de novo or released from collagen association during ECM remodeling, may be internalized by immune cells to regulate their transcriptional programs and effector responses that may be harnessed in future therapeutics.
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Engering, Anneke, Sandra J. van Vliet, Teunis B. H. Geijtenbeek, and Yvette van Kooyk. "Subset of DC-SIGN+ dendritic cells in human blood transmits HIV-1 to T lymphocytes." Blood 100, no. 5 (September 1, 2002): 1780–86. http://dx.doi.org/10.1182/blood-2001-12-0179.
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The dendritic cell (DC)–specific molecule DC-SIGN is a receptor for the HIV-1 envelope glycoprotein gp120 and is essential for the dissemination of HIV-1. DC-SIGN is expressed by DCs, both monocyte-derived DCs and DCs in several tissues, including mucosa and lymph nodes. To identify a DC-SIGN+ DC in blood that may be involved in HIV-1 infection through blood, we have analyzed the expression of DC-SIGN in human blood cells. Here we describe the characterization of a subset of DCs in human blood, isolated from T-/NK-/B-cell–depleted peripheral blood mononuclear cells (PBMCs) on the basis of expression of DC-SIGN. This subset coexpresses CD14, CD16, and CD33 and is thus of myeloid origin. In contrast to CD14+ monocytes, DC-SIGN+ blood cells display a DC-like morphology and express markers of antigen-presenting cells, including CD1c, CD11b, CD11c, CD86, and high levels of major histocompatibility complex (MHC) class I and II molecules. This DC population differs from other described CD14−blood DC subsets. Functionally, DC-SIGN+ blood DCs are able to stimulate proliferation of allogeneic T cells and can produce tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) upon activation with lipopolysaccharide (LPS). When they encounter HIV-1, low amounts of these blood DC-SIGN+ DCs enhance infection of T lymphocytes in trans, whereas blood monocytes and CD14−blood DCs are not capable of transmitting HIV-1. Therefore DC-SIGN+ blood DCs can be the first target for HIV-1 upon transmission via blood; they can capture minute amounts of HIV-1 through DC-SIGN and transfer HIV-1 to infect target T cells in trans.
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Ghosh, Mallika, Jaganathan Subramani, Mamunur Rahman, and Linda Shapiro. "CD13 is a novel regulator of TLR4 endocytosis in dendritic cells (CAM5P.241)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 180.12. http://dx.doi.org/10.4049/jimmunol.192.supp.180.12.
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Abstract Toll like receptors (TLRs) recognize antigens produced by infection or injury to induce innate immunity via cell surface or endocytic pathways. TLR4 sequentially triggers both pathways, leading to TLR4 activation of the pro-inflammatory NF-kB pathway from the cell surface followed by dynamin-mediated TLR4 endocytosis, activation of TRIF and production of anti-inflammatory IRF (Interferon Regulatory Factor)3-regulated genes. We found that myeloid cell surface peptidase CD13 regulates TLR4 signal transduction by controlling dynamin-dependent endocytosis. In response to LPS, CD13KO DCs showed enhanced activation of IRF3, increase in its target gene IFN-β and inducible nitric oxide activity in a dynamin dependent manner. Endogenous TLR4 ligands produced in response to ischemic injury provoked hyperactivation of pIRF3 and its target cytokines in CD13KO animals. CD13KO DCs internalize higher levels of cell-associated TLR4 ligands released by dying cells or the TLR4 ligand LPS, indicating that CD13 regulates inflammatory TLR4 responses in vivo by modulating DC antigen uptake. Our results further suggest that CD13 and the LPS binding protein, CD14, may potentially act as opposing regulators of TLR4 endocytosis in inflammation. We propose that in response to injury or infection, CD13 serves to balance the immune response by modulating TLR4 induced pro- and anti-inflammatory cytokine profiles and maintain inflammatory equilibrium that is critical to proper wound healing.
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Kurochkina, Y., E. Chernykh, and A. Sizikov. "POS0673 TOLEROGENIC DENDRITIC CELLS IN RHEUMATOID ARTHRITIS PATIENTS: NEWS AND PROMISES." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 581.2–581. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2261.
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Background:Dendritic cells (DCs) are known to contribute to the pathogenesis of rheumatoid arthritis (RA) through presentation of cartilage glycoprotein, production of proinflammatory cytokines and activation of Th1/Th17 responses. Along with stimulating activity, DCs may exhibit suppressive functions via capacity to induce T cell apoptosis/anergy and to generate regulatory T cells. Since these DCs have potential to control autoreactive T-lymphocytes, the enhancing of tolerogenic properties of DCs seems to be a new important strategy in treatment of RA. Dexamethasone is widely used in clinical practice and can be used as a tolerogenic substance. Therefore, the properties of DCs generated in presence of dexamethasone are of great clinical interests.Objectives:The aim of our study is to describe the properties of tolerogenic DCs, generated with dexamethasone in patients with RA and their influence on autologous T-cells.Methods:Sixty five patients with RA with high and moderate activity of disease were recruited in this study. All patients follow ACR/EULAR criteria (2010). All studies were performed after receiving informed consent. All patients received conventional synthetic DMARDs. DCs were generated from blood monocytes culturing for 5 days with GM-CSF and IFN-α in the presence dexamethasone (dexDCS), applied on third day. LPS as maturation stimuli was added on fourth day. The expression of CD14, CD83, HLA-DR, TLR-2 on the surface of DCs was measured by flow cytometry. The functions of DCs were evaluated by measuring cytokine production and DCs allostimulatory activity in mixed lymphocyte culture. Mature DCs generated in absence of dexamethasone used as control.Results:We revealed that dexDCs are characterized by enhanced expression of CD14+cells and decreased number of CD83+cells but percent of HLA-DR+cells were constant (about 85). DexDCs show high expression of TLR-2 is seen as tolerogenic molecule (75%vs51%, p=0.05 compared to control). DexDCs also have marked prominent increase of TNFα/IL-10 ratio in contrast to control (0.59 vs 1.8, p=0.03). DexDCs suppressed proliferation of allogenic T-cells (2005 vs 7980 cpm, p=0.0002). To assess the stability of the DC in the proinfflamatory micro-environment after assessing stimulatory activity dexDCs were then cultivated with LPS and allostimulatory activity were evaluated one more. The stimulation activity dexDCs after incubation with LPS were not increase (4692 vs 6053 cpm, p=0.7). Also earlier we showed possibility of dexDCs induse apoptosis of autologous T-cells, activation of CD4+IL10+Tr1 and possession of antigen-specific suppression.Conclusion:The data obtained indicate that dexDCs from RA patients have the main tolerogenic features and stable in inflammatory environment that proves their potential in the treatment of rheumatoid arthritis.Disclosure of Interests:None declared
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Jun, Shi, Kazuma Ikeda, Nobuhara Fujii, Kinuyo Kasumoto, Mitune Tanimoto, Li Xiao, and Pu Quan. "The Direct Cytotoxicity of Activated Human Umbilical Cord Blood Dendritic Cells to Tumor Cell." Blood 104, no. 11 (November 16, 2004): 3812. http://dx.doi.org/10.1182/blood.v104.11.3812.3812.
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Abstract Besides their role as antigen presenting cells, human peripheral blood mononuclear cell and CD34+ cell-derived dendritic cells (DCs), now have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumor-cell killing activity by human cord blood cell-derived dendritic cells (CBDCs). We report here that human cord blood monocyte-derived DCs aquire the ability to kill tumor cells after activation with lipopolysaccharide (LPS) or interferon-γ(IFN-γ), associated with the enhanced TNF-α-related apoptosis-inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14-positive cells collected from cord blood from healthy volunteers were cultured with interleukin-4 and granulocyte-machrophage colony- stimulating factor for seven days to induce CBDCs, which showed no cytotoxicity. However, after activation with IFN-γ for additional 12 hours, CBDCs exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS for 12 hours induced cytotoxicity against Daudi and Jurkat cells as we detected in human peripheral blood monocytes- drived DCs (PBDCs). IFN-γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface Fas Ligand as analyzed by flow cytometry. Our results suggest that activated CBDCs can serve as immunological effectors against tumor cells, through TRAIL- dependent and Fas-independent mechanisms.
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Kurt, Robert A., Chiquita Palha De Sousa, Christopher Blum, and Erica Sgroe. "Murine mammary carcinoma cells and CD11c+ dendritic cells elicit distinct responses to lipopolysaccharide and exhibit differential expression of genes required for TLR4 signaling (40.4)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 40.4. http://dx.doi.org/10.4049/jimmunol.182.supp.40.4.
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Abstract Particular attention has been given to Toll-like receptors (TLR) on dendritic cells (DC) because of their ability to bridge innate and adaptive defenses. Since TLR are also expressed by epithelial cells, and because the majority of cancers are carcinomas, and thus of epithelial origin, we were interested in comparing responsiveness of a carcinoma and DC to a TLR agonist. For this purpose we used the mammary carcinoma 4T1 and CD11c+ DC. Both 4T1 and DC expressed genes encoding multiple TLR and secreted the proinflammatory chemokines CCL2 and CXCL1 in response to the TLR4 agonist lipopolysaccharide (LPS). However, DC, but not 4T1 secreted IL-1beta, TNF-alpha, and upregulated CD80 and CD86 expression following LPS treatment. Gene arrays were used to delineate potential reasons for the differential responsiveness of the cells to LPS. The arrays showed that genes encoding TLR4, CD14, myeloid differentiation primary response gene 88 (Myd88), and Toll-like receptor adaptor molecule 2 (TICAM-2) were expressed at greater levels by the DC; results which were verified by quantitative RT-PCR. These data demonstrate that 4T1 and CD11c+ DC are distinctly responsive to LPS, and that this may be a consequence of differential expression of genes encoding proteins important for TLR signaling.
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Maldonado-Arocho, Francisco J., and Kenneth A. Bradley. "Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3β." Infection and Immunity 77, no. 5 (March 9, 2009): 2036–42. http://dx.doi.org/10.1128/iai.01329-08.
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ABSTRACT Bacillus anthracis secretes two bipartite toxins, edema toxin (ET) and lethal toxin (LT), which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor, the catalytic subunit of ET, is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here, we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN, a marker of immature DCs, and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3β, like LPS-matured DCs. Interestingly, cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET.
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Evrard, B., D. Balestrino, A. Dosgilbert, J. L. J. Bouya-Gachancard, N. Charbonnel, C. Forestier, and A. Tridon. "Roles of Capsule and Lipopolysaccharide O Antigen in Interactions of Human Monocyte-Derived Dendritic Cells and Klebsiella pneumoniae." Infection and Immunity 78, no. 1 (October 19, 2009): 210–19. http://dx.doi.org/10.1128/iai.00864-09.
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ABSTRACT In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.
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Zhang, Guag-Xian, Fang Zhou, Bogoljub Ciric, Hongmei Li, Yaping Yan, Ke Li, Melissa Cullimore, et al. "Ability of LPS to regulate expression of tolerance-related molecules on dendritic cells is blocked by IL-10 deficiency (48.18)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 48.18. http://dx.doi.org/10.4049/jimmunol.188.supp.48.18.
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Abstract Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays an important role in regulating the local inflammatory immune response, but regulatory mechanisms of this cytokine have not been fully elucidated. Here we demonstrate that IL-10 deficiency renders LPS treatment ineffective in regulating expression of CD40, CD80, CD86, B7-H2 and B7-DC on dendritic cells (DCs) and blocks up-regulation of IL-27. This inability to respond to LPS was found in both IL-10-/- bone marrow-derived and splenic DCs. Compared to wild type DCs, IL-10-/- DCs expressed similar levels of TLR4 and CD14, but produced less LPS binding protein (LBP). The deficiency in LBP production may explain the failure of IL-10-/- DCs to respond normally to LPS. Moreover, lack of IL-10 modulated the proportions of CD11c+CD8+ and CD11c+B220+ DCs, which play an important role in local inflammatory responses and tolerance. IL-10 deficiency also blocked expression of galectin-1, CD205 and CD103, which are necessary for central and peripheral tolerance. While they did not respond to LPS, IL-10-/- DCs produced increased levels of IL-6 and CCL4 after TNF-α treatment. Together, our results demonstrate that IL-10 deficiency affects the immune functions of DCs, which may contribute to the increased severity of autoimmune diseases seen in IL-10-/- mice.
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Mohamadzadeh, Mansour, Frederic Berard, Gregory Essert, Cecile Chalouni, Bali Pulendran, Jean Davoust, George Bridges, A. Karolina Palucka, and Jacques Banchereau. "Interleukin 15 Skews Monocyte Differentiation into Dendritic Cells with Features of Langerhans Cells." Journal of Experimental Medicine 194, no. 7 (October 1, 2001): 1013–20. http://dx.doi.org/10.1084/jem.194.7.1013.
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Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rγ chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a+HLA-DR+CD14−DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)α, and CD40L induce maturation of IL15-DCs to CD83+, DC-LAMP+ cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3α/CCL20. However, IL15-DCs cannot be qualified as “genuine” Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.
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Clark, E. A., K. H. Grabstein, and G. L. Shu. "Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes." Journal of Immunology 148, no. 11 (June 1, 1992): 3327–35. http://dx.doi.org/10.4049/jimmunol.148.11.3327.
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Abstract Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.
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Martins, Priscilla da Costa, Hugo Amorim dos Santos de Souza, Carolina Moreira Blanco, Luana Santos-de-Oliveira, Lilian Rose Pratt-Riccio, Cláudio Tadeu Daniel-Ribeiro, and Paulo Renato Rivas Totino. "Modulation of Signal Regulatory Protein α (SIRPα) by Plasmodium Antigenic Extract: A Preliminary In Vitro Study on Peripheral Blood Mononuclear Cells." Microorganisms 10, no. 5 (April 26, 2022): 903. http://dx.doi.org/10.3390/microorganisms10050903.
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Signal regulatory protein α (SIRPα) is an immunoreceptor expressed in myeloid innate immune cells that signals for inhibition of both phagocytosis and inflammatory response. Malaria parasites have evolutionarily selected multiple mechanisms that allow them to evade host immune defenses, including the modulation of cells belonging to innate immunity. Notwithstanding, little attention has been given to SIRPα in the context of immunosuppressive states induced by malaria. The present study attempted to investigate if malaria parasites are endowed with the capacity of modulating the expression of SIRPα on cells of innate immune system. Human peripheral blood mononuclear cells (PBMC) from healthy individuals were incubated in the presence of lipopolysaccharide (LPS) or crude extracts of P. falciparum or P. vivax and then, the expression of SIRPα was evaluated by flow cytometry. As expected, LPS showed an inhibitory effect on the expression of SIRPα in the population of monocytes, characterized by cell morphology in flow cytometry analysis, while Plasmodium extracts induced a significant positive modulation. Additional phenotyping of cells revealed that the modulatory potential of Plasmodium antigens on SIRPα expression was restricted to the population of monocytes (CD14+CD11c+), as no effect on myeloid dendritic cells (CD14−CD11c+) was observed. We hypothesize that malaria parasites explore inhibitory signaling of SIRPα to suppress antiparasitic immune responses contributing to the establishment of infection. Nevertheless, further studies are still required to better understand the role of SIRPα modulation in malaria immunity and pathogenesis.
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Martins, Priscilla da Costa, Hugo Amorim dos Santos de Souza, Carolina Moreira Blanco, Luana Santos-de-Oliveira, Lilian Rose Pratt-Riccio, Cláudio Tadeu Daniel-Ribeiro, and Paulo Renato Rivas Totino. "Modulation of Signal Regulatory Protein α (SIRPα) by Plasmodium Antigenic Extract: A Preliminary In Vitro Study on Peripheral Blood Mononuclear Cells." Microorganisms 10, no. 5 (April 26, 2022): 903. http://dx.doi.org/10.3390/microorganisms10050903.
Full textAbstract:
Signal regulatory protein α (SIRPα) is an immunoreceptor expressed in myeloid innate immune cells that signals for inhibition of both phagocytosis and inflammatory response. Malaria parasites have evolutionarily selected multiple mechanisms that allow them to evade host immune defenses, including the modulation of cells belonging to innate immunity. Notwithstanding, little attention has been given to SIRPα in the context of immunosuppressive states induced by malaria. The present study attempted to investigate if malaria parasites are endowed with the capacity of modulating the expression of SIRPα on cells of innate immune system. Human peripheral blood mononuclear cells (PBMC) from healthy individuals were incubated in the presence of lipopolysaccharide (LPS) or crude extracts of P. falciparum or P. vivax and then, the expression of SIRPα was evaluated by flow cytometry. As expected, LPS showed an inhibitory effect on the expression of SIRPα in the population of monocytes, characterized by cell morphology in flow cytometry analysis, while Plasmodium extracts induced a significant positive modulation. Additional phenotyping of cells revealed that the modulatory potential of Plasmodium antigens on SIRPα expression was restricted to the population of monocytes (CD14+CD11c+), as no effect on myeloid dendritic cells (CD14−CD11c+) was observed. We hypothesize that malaria parasites explore inhibitory signaling of SIRPα to suppress antiparasitic immune responses contributing to the establishment of infection. Nevertheless, further studies are still required to better understand the role of SIRPα modulation in malaria immunity and pathogenesis.
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Xiao, Yang, and Leqin Zhang. "Study On Immune Mechanism of Human Marrow Mesenchymal Stem Cells Adjusting Dendritic Cells for Treatment of Aplastic Anemia." Blood 116, no. 21 (November 19, 2010): 5137. http://dx.doi.org/10.1182/blood.v116.21.5137.5137.
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Abstract Abstract 5137 Objective (1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs. Materials and Methods (1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell.(3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS, the phenotype analysis of DCs before and after culture was examined by flow cytometry, respectively. Results (1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p < 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p < 0.05). Meanwhile, ELISA analysis indicated that the concentration of IL-2 and IFN-γ were significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L, respectively (p < 0.05). However, IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p < 0.05).(2) After the induction of immature DCs by LPS, flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC, while that of CD14+ decreased from 83.6% to 3.5% (p < 0.05).(3) After the co-culture of mature DCs and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a, CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%, respectively. Conclusions Our study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus suggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation. Disclosures: No relevant conflicts of interest to declare.
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Ostanin, A. A., O. Y. Leplina, E. A. Burakova, T. V. Tyrinova, A. A. Fokina, A. S. Proskurina, S. S. Bogachev, D. A. Stetsenko, and E. R. Chernykh. "Phosphate-modified CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells." Vavilov Journal of Genetics and Breeding 24, no. 6 (October 28, 2020): 653–60. http://dx.doi.org/10.18699/vj20.659.
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Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D-SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.
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Sakhno, Ludmila V., Ekaterina Ya Shevela, Marina A. Tikhonova, Sergey D. Nikonov, Alexandr A. Ostanin, and Elena R. Chernykh. "Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis." Journal of Immunology Research 2015 (2015): 1–14. http://dx.doi.org/10.1155/2015/793292.
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The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generatedin vitromacrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity toM. tuberculosisantigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which werein vitrogenerated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γcoupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generatedin vitrofrom peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γproduction in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response toM. tuberculosiswas discussed.
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Takezako, Naoki, Naohiro Sekiguchi, Akihisa Nagata, Takeshi Hagino, Satoshi Noto, and Akiyoshi Miwa. "ST2 Utilizes Distinct Pathways to Suppress Inflammatory Cytokine Production and Modulate Rapid Differentiation of Human Monocytes Into CD83+ Dendritic Cells." Blood 120, no. 21 (November 16, 2012): 2133. http://dx.doi.org/10.1182/blood.v120.21.2133.2133.
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Abstract Abstract 2133 Introduction and Aim: Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that initiate and mediate immune responses. DCs are equipped with the biochemical machinery to process and present peptide fragments of protein antigens on major MHC molecules. Both myelomonocytic precursor cells and differentiated monocytes have been shown to be capable of differentiating into peripheral DCs. Peripheral blood monocytes can give rise to macrophages, but also develop into DCs under specific conditions. However, the factors and/or conditions that regulate differentiation along the DC pathway are not completely understood. ST2 is a member of the interleukin-1 (IL-1) receptor family and is expressed by type-2 helper T (Th2) cells. Two forms of ST2, a soluble secreted form (ST2) and a transmembrane form (ST2L), are generated by alternative splicing. A recent study showed that IL-33 is a specific ligand of ST2L and induces the production of Th2 cytokines. However, the counter-receptor for ST2 has not yet been identified. We previously demonstrated that ST2 was able to bind to the surface of a human monocytic leukemia cell line (THP-1 cells) and that it decreased binding of nuclear factor (NF)-κB to the IL-6 promoter. This time, we tested the ability of ST2 to influence the differentiation of normal human peripheral blood monocytes into DCs when cultured under serum-free conditions. We found that ST2 acts through distinct mechanisms to suppress the differentiation of CD14+ monocytes into DCs as well as inhibiting their secretion of the inflammatory cytokines IL-6, IL-1β, and TNF-α. Methods: The FACS protocol employed specific mAbs for human CD14, CD83, CD80, CD40, CD33, and HLA-DR, as well as isotype-matched control mAbs, and has been reported previously. Flow cytometry studies were done with a FACS Calibur flow cytometer (BD Biosciences) and WinMDI software. Human CD14+ peripheral blood monocytes were purchased from Lonza Walkersville, Inc., and were plated at a density of 2.5×106 cells/well in RepCell tissue culture plates (CellSeed) containing macrophage serum-free medium (SFM) (Invitrogen Life Technologies) supplemented with 50 ng/ml human rGM-CSF (Amgen) to maintain cell viability, as described previously. Cells were cultured overnight and differentiation was induced by addition of 50 ng/ml LPS derived from Escherichia coli strain O26:B6 (Sigma-Aldrich). In some experiments, cultures were supplemented at time zero with 50 ng/ml hST2-V5 or LacZ-V5, as described previously. In addition, cultures were supplemented with IL-33 at time zero. ELISA kits (R&D Systems) were used to quantify several cytokines. Results: ST2 altered cytokine production by LPS-treated monocytes, and inhibited the secretion of IL-6, IL-1β, and TNF-α. We evaluated the influence of ST2 on the percentage of monocytes adopting DC characteristics and their function, revealing a distinct effect on LPS-induced secretion of IL-6 and on members of the NF-κB family of transcription factors. Furthermore, IL-33 did not affect the percentage or function of monocytes adopting DC characteristics. Conclusion: These studies identified ST2 as a potent regulatory factor that suppresses the differentiation of DCs via distinct pathways. Disclosures: No relevant conflicts of interest to declare.
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Ricklin, Meret, and Artur Summerfield. "DC-sign as a marker for inflammatory skin dendritic cells in the pig (P6074)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 141.5. http://dx.doi.org/10.4049/jimmunol.190.supp.141.5.
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Abstract The objective of this study was to phenotypically characterize dendritic cells (DC) associated with inflammatory skin diseases in the pig. To further study dermal DC, the expression profile of DC-sign, a C-type lectin and human DC-marker was characterized on various populations of DC. To this end, we isolated peripheral blood mononuclear cells (PBMCs), analysed blood monocytes, conventional blood DCs (cDC) and plasmacytoid DCs (pDC). Furthermore monocytes were differentiated to monocyte-derived DC (MoDCs) or monocyte-derived macrophages (MMθ). In addition, DC were isolated from separated epidermis and dermis of lesional and non-lesional skin. In the blood unstimulated pDCs, cDC and monocytes were negative for DC-sign expression. After LPS stimulation a slight upregulation could be observed. MoDC expressed higher levels of DC-sign than MMθ. Langerhans cells as well as dermal DC from normal skin were DC-sign-. However, after induction of an inflammation the newly immigrated DC were DCsign+. In contrast to Mθ these were CD14-CD163-. These results indicate that in the pig DC sign represents a marker for monocyte-derived inflammatory DC of use to study the pathogenesis of inflammatory skin diseases.
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Purnamasari, Dyah, Samsuridjal Djauzi, Siti Setiati, Alida Harahap, Tjokorda Gde Dalem Pemayun, Joedo Prihartono, and Pradana Soewondo. "EFFECTS OF IN VITRO 1,25 DIHYDROXYVITAMIN D ON MATURATION OF DENDRITIC CELLS IN GRAVES’ DISEASE PATIENTS." Asian Journal of Pharmaceutical and Clinical Research 9, no. 5 (September 1, 2016): 221. http://dx.doi.org/10.22159/ajpcr.2016.v9i5.13287.
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ABSTRACTObjective: The autoimmune reaction in Graves’ disease (GD) is induced by self-antigen, which is presented by dendritic cells (DCs). DCs in GD have more active immune responses than those in healthy subjects. The ability of DC as antigen-presenting cell is determined by its maturity level. InGD, vitamin D level is inversely proportional to antibody titer and proportionally associated with remission status. Studies on healthy subjects andautoimmune patients (systemic lupus erythematosus (SLE), multiple sclerosis (MS), and Crohn’s disease) have demonstrated immunoregulatoryeffects of vitamin D, mainly through inhibition of DC maturation, which may decrease the DC’s immunogenic profile. This study aims to identify theeffect of 1,25-D3 in vitro on DC maturation in patients with GD.Methods: This is an experimental study, which was conducted in 12 GD patients with thyrotoxicosis. Monocyte-derived DC of GD patients wascultured, with or without 1,25-D3 in vitro at monocytic phase. The DC maturation was then stimulated by lipopolysaccharide (LPS) and evaluatedbased on the expression of DC markers (human leukocyte antigen-D-related [HLA-DR], CD80, CD40, CD83, CD14, and CD206) and the ratio of cytokineinterleukin-12 (IL-12)/IL-10 levels in the supernatants.Results: Following the LPS stimulation, DC with 1,25-D3 showed lower expressions of HLA-DR, CD80, CD40, and CD83, and higher expressions ofCD14 and CD206 compared to DC without 1,25-D3. DC with 1,25-D3 had lower ratio of IL-12/IL-10 levels than those without 1,25-D3.Conclusion: In vitro 1,25-D3 supplementation inhibits DC maturation in patients with GD.Keywords: Vitamin D, Graves’ disease, Dendritic cells.
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Hudec, Michael, Kamila Riegerová, Jan Pala, Viera Kútna, Marie Černá, and Valerie Bríd O´Leary. "Celiac Disease Defined by Over-Sensitivity to Gliadin Activation and Superior Antigen Presentation of Dendritic Cells." International Journal of Molecular Sciences 22, no. 18 (September 15, 2021): 9982. http://dx.doi.org/10.3390/ijms22189982.
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The autoimmune condition, Celiac Disease (CeD), displays broad clinical symptoms due to gluten exposure. Its genetic association with DQ variants in the human leukocyte antigen (HLA) system has been recognised. Monocyte-derived mature dendritic cells (MoDCs) present gluten peptides through HLA-DQ and co-stimulatory molecules to T lymphocytes, eliciting a cytokine-rich microenvironment. Having access to CeD associated families prevalent in the Czech Republic, this study utilised an in vitro model to investigate their differential monocyte profile. The higher monocyte yields isolated from PBMCs of CeD patients versus control individuals also reflected the greater proportion of dendritic cells derived from these sources following lipopolysaccharide (LPS)/ peptic-tryptic-gliadin (PTG) fragment stimulation. Cell surface markers of CeD monocytes and MoDCs were subsequently profiled. This foremost study identified a novel bio-profile characterised by elevated CD64 and reduced CD33 levels, unique to CD14++ monocytes of CeD patients. Normalisation to LPS stimulation revealed the increased sensitivity of CeD-MoDCs to PTG, as shown by CD86 and HLA-DQ flow cytometric readouts. Enhanced CD86 and HLA-DQ expression in CeD-MoDCs were revealed by confocal microscopy. Analysis highlighted their dominance at the CeD-MoDC membrane in comparison to controls, reflective of superior antigen presentation ability. In conclusion, this investigative study deciphered the monocytes and MoDCs of CeD patients with the identification of a novel bio-profile marker of potential diagnostic value for clinical interpretation. Herein, the characterisation of CD86 and HLA-DQ as activators to stimulants, along with robust membrane assembly reflective of efficient antigen presentation, offers CeD targeted therapeutic avenues worth further exploration.
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Okamoto, Masato, Go Oh-e, Tetsuya Oshikawa, Sachiko Furuichi, Tomoyuki Tano, Sharif U. Ahmed, Sachiko Akashi, et al. "Toll-Like Receptor 4 Mediates the Antitumor Host Response Induced by a 55-Kilodalton Protein Isolated from Aeginetia indica L., a Parasitic Plant." Clinical Diagnostic Laboratory Immunology 11, no. 3 (May 2004): 483–95. http://dx.doi.org/10.1128/cdli.11.3.483-495.2004.
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ABSTRACT A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-κB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.
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Smith, Rosemary E., Vanshree Patel, Sandra D. Seatter, Maureen R. Deehan, Marion H. Brown, Gareth P. Brooke, Helen S. Goodridge, et al. "A novel MyD-1 (SIRP-1α) signaling pathway that inhibits LPS-induced TNFα production by monocytes." Blood 102, no. 7 (October 1, 2003): 2532–40. http://dx.doi.org/10.1182/blood-2002-11-3596.
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Abstract MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFα) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFα secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFα secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFα production and consequent inflammatory disease. (Blood. 2003;102:2532-2540)
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Curti, Antonio, Sara Trabanelli, Chiara Onofri, Sergio Rutella, Raimondo De Cristofaro, Michele Baccarani, and Roberto M. Lemoli. "Functional IDO Is Expressed on CD34+- and Monocyte-Derived Dendritic Cells According to Differentiation Status." Blood 112, no. 11 (November 16, 2008): 1552. http://dx.doi.org/10.1182/blood.v112.11.1552.1552.
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Abstract Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34+-derived 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-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4+CD25+FOXP3+ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34+ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34−, CD14+, CD1a+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3+ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34+-derived DCs. Differentiation of CD34+ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.
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Kelly, Erin, Anthony Lawrenz, Alan Wong, Erin M. Harberts, Robert K. Ernst, Afshin Shirazian, and Qin Chen. "A donor-specific primary monocyte-derived dendritic cell maturation model for high throughput screening of immunostimulatory compounds." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 73.15. http://dx.doi.org/10.4049/jimmunol.198.supp.73.15.
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Abstract Dendritic cell (DC)-based immunotherapy is a promising treatment alternative for drug-resistant malignancies. Identifying candidate immunostimulatory compounds that can reliably facilitate DC maturation in the context of antigen-specific therapies is a significant hurdle in the development of such immunotherapies. In this study, we present a high throughput in vitro primary cell assay to evaluate dose-dependent DC maturation in a panel of healthy donor primary cells. CD14+ monocytes from four healthy donors were isolated from leukapheresis (Leuko Pak)-derived peripheral blood mononuclear cells (PBMC), differentiated by culture with GM-CSF and IL-4 cytokines, and then stimulated with lipopolysaccharide (LPS), CpG oligonucleotides, or a panel of novel Toll-like receptor 4 (TLR4) ligands. To quantify the resultant maturation, flow cytometry was used to measure the expression of cell surface biomarkers MHC-II, CD40, CD80, CD83, and CD86. While monocytes from all donors responded to the ligands tested, the intensity of activation marker expression varied between donors. LPS-induction of CD83 expression ranged from 27 to 82% of DCs amongst the 4 donors tested, highlighting the importance of multi-donor screening in interpreting quantitative results. In conclusion, a high throughput in vitro primary monocyte-derived DC differentiation assay reveals donor to donor variations that may go unobserved in screening experiments completed in cell lines or single donor primary cells sources. This assay can be used to assist researchers in lead generation, lead optimization, and mechanism of action studies of immunostimulatory compounds.
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Jiang, Hong, Mary Brigid Bradley, Carmella van de Ven, Prakash Satwani, Laxmi Baxi, and Mitchell S. Cairo. "Differential Expression of Genes Associated with Toll-Like Receptor-4 (TLR4) Signal Transduction Pathway in Lipopolysaccharide (LPS)-Activated Cord Blood (CB) vs. Adult Peripheral Blood (APB) Monocyte (Mo)-Derived Dendritic Cells (DC)." Blood 104, no. 11 (November 16, 2004): 3444. http://dx.doi.org/10.1182/blood.v104.11.3444.3444.
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Abstract LPS activates immature DC via TLR4 and induces maturation of DC for initiating antigen presenting activity (Medzhitov; Nat Rev Immunol 2001). We have previously demonstrated decreased gene expression and protein production of IL-12, IL-15, IL-18 in activated CB MNC and decreased DC MLR (Lee/Cairo, Blood 1996; Qian/Cairo, Blood 1997; Wu/Cairo, Blood 100:3668 p51b 2002). Recently, we have identified differential gene expression patterns including differential immunoregulatory and chemokine genes in LPS-CB vs APB Mo by microarray (Jiang/Cairo, J. Immunol 2004). Since the myeloid lineage DC is derived from Mo, we sought to determine in LPS activated CB vs. APB DC, differential expressed genes that associate with TLR4-mediated signaling pathway. Briefly, Mo were purified from fresh CB or APB and cultured for 7 days with GM-CSF & IL-4 [immature DC (iDC)] and LPS [mature DC (mDC)]. Aliquots from iDC and mDC were analyzed for DC immunophenotype, morphology and DC allogeneic antigen activity. mRNA was isolated, reverse transcripted to cDNA, labeled & hybridized to oligonucleotides (Affymetrix, U133A). Data was analyzed by MAS 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Several genes were analyzed by RT-PCR (One-Step SuperScript, Invitrogen) and protein expression was analyzed by Western Blot (Bio-Rad). Inverted microscopy demonstrated DC mature morphology at day 8 and flow cytometry demonstrated decreased CD14 and increased CD83 expression in CB & APB mDC. We also demonstrated significant increase in the allogeneic stimulatory effects on CD4+ T cells in APB vs. CB mDC. The microarray analysis demonstrated a significant decreased gene expression of TLR4 [3 fold (F)] and CD14 (2.1 F) (p<0.05) in CB vs APB-DC. We further identified LPS significantly induced increased expression of TLR4 downstream signaling molecular genes such as MAPKKK, NF-kB and TANK in APB compared to CB mDC (3–8 F) (p<0.05). There were also significant amplifications of a variety of other gene categories in LPS activated APB vs CB mDC (p<0.05) including cell surface molecule CD80 (3.7F) and IL-2Ra (5.3 F), cytokine IL-23 (3.5F) & IL-12 (13 F), signal transduction STAT1 (3.4F) & IRF-7 (7.7 F), and immunoregulatory TNFSF10 (12F) & ISG20 (39F). Gene expression of NF-kB1, TRAF1 & IRF-7 by RT-PCR demonstrated an increased expression in LPS-APB vs CB mDC and were compatible with microarray. Moreover, Western analysis of IRF-7 demonstrated increased protein expression in LPS-APB vs CB mDC. In summary, we have identified decreased gene expression patterns in LPS-CB vs APB DC, especially those in the TLR4 signal transduction pathway (MAP3K, TRAF, TANK & NF-kB), and suggest these differentially expressed genes may enhance the activation of TLR4 pathway in LPS-APB vs CB DC, resulting in differential regulation of CB vs APB DC antigen presentation capacities. Furthermore, these decreased expressed genes in other molecular categories (e.g.IL-23, IFNg, IL6, CD80, STAT1, IRF-7, SOCS3) in LPS-CB vs APB DC may be partially responsible for differential innate and adaptive immune function of CB vs APB. Moreover, the differential regulated expression of genes may in part help to explain reduced incidence of severe aGVHD, delay in immune reconstitution and/or increased infectious mortality following HLA disparate UCBT.
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Modra, Courtney J., Lubomira Jamriska, Min Rao, Georgina J. Clark, and Derek N. J. Hart. "MMRI-23: A Potential Role in AML Antibody-Mediated Therapy." Blood 108, no. 11 (November 16, 2006): 4581. http://dx.doi.org/10.1182/blood.v108.11.4581.4581.
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Abstract Novel targets expressed on early AML precursor cell populations, with restricted expression on normal tissues, provide attractive options for antibody based therapeutics. We identified the CD300 family as Ig superfamily members with the capacity, through signaling motifs, to induce triggering and inhibitory signals to the leukocytes on which they are expressed. Here we report on an inhibitory member of this family, 35-L5, as a potential target for antibody-mediated therapy in AML. To assess its potential, we analyzed 35-L5 transcript and cell surface expression on normal blood cells, cell lines, and AML cells using quantitative RT-PCR and a monoclonal antibody (MMRI-23) made against a recombinant 35-L5 Ig fusion protein. RT-PCR showed its expression in normal PBMC was restricted to CD14+ monocytes, CD11c+ dendritic cells (myeloid DCs), monocyte derived dendritic cells (MoDC), with limited expression by B cells. MMRI-23 bound CD14+ monocytes, myeloid DC and MoDC, but not CD19+ B, CD4+ T, CD56+ NK cells and CD11c− (plasmacytoid) DC. Interestingly, MMRI-23 binding varied between donors and 35-L5 expression was upregulated on mature MoDC, when compared to immature MoDC from the same donor (3 from 6 donors). However, MMRI-23 binding to PBMC remained stable when cultured with activation agents PMA and ionomycin, LPS, or PHA for 24 and 48 hours. 35-L5 transcript and surface protein expression was evident in the monocytic derived U937, leukemic derived HL60, erythroleukemic derived HEL, CML derived K562, and B lymphoblast derived Daudi cell lines, but was limited in the KMH2 and HDLM2 cell lines and absent in the Jurkat, Raji, L428 and Mann cell lines. Preliminary studies in AML using quantitative RT-PCR showed that 35-L5 transcript was increased compared to control normal CD14 cell expression (n=9). Furthermore, MMRI-23 bound to 6 of 8 AML samples of the FAB M1, M2 and M5 subtypes. MMRI-23 crosslinking induced neither cell apoptosis, nor cell differentiation and change of CD14, CD33, CD64, CD16, CD11b expression. Defining the role of 35-L5 on normal and AML progenitor cells is necessary to evaluate its potential as a possible target for antibody-mediated therapy. MMRI-23 may have potential for drug conjugated antibody-mediated therapy, but 35-L5’s inhibitory function may also be exploited to develop a novel mechanism for controlling myeloid leukemias.
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Rodewohl, Anja, Johanna Scholbach, Anna Leichsenring, Margarethe Köberle, and Franziska Lange. "Age-dependent cellular reactions of the human immune system of humanized NOD scid gamma mice on LPS stimulus." Innate Immunity 23, no. 3 (February 5, 2017): 258–75. http://dx.doi.org/10.1177/1753425917690814.
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Despite sepsis being a life-threatening disease, targeted drugs that improve the therapy of affected patients are still lacking. Infants and adults differ in the maturity level of their immune system and this results in distinct reactions to Gram-negative bacteria. To study reactions of human immune cells in vivo, we used NOD scid gamma mice transplanted with human CD34+ stem cells to engraft a functional human immune system. Human cells undergo differentiation and maturation in these mice after transplantation and, accordingly, animals were divided into two groups: 8–13 wk and 15–22 wk after transplantation. Endotoxemia was induced by injecting LPS. Six h later, mice were euthanized. In both groups, LPS stimulation induced a decrease of CD14+ monocytes in peripheral blood, an up-regulation of activation markers on different cell subsets such as myeloid dendritic cells, and a release of the human cytokines TNF-α, IL-6 and IL-10. However, significant differences were detected with regard to the amounts of released cytokines, and 8–13-wk-old mice produced more IL-6, while PTX3 was mainly released by 15–22-wk-old animals. Thus, here we provide a potential model for preclinical research of sepsis in infants and adults.
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Yuan, Ning, Benoit G. Guilbault, Graeme T. Milton, Jodie Fadum, Jackie E. Damen, Allen C. Eaves, Terry E. Thomas, and Albertus W. Wognum. "A method for rapid isolation of highly purified human monocytes using fully automated negative cell selection (36.25)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S17. http://dx.doi.org/10.4049/jimmunol.178.supp.36.25.
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Abstract The preparation of highly purified monocytes for experimentation has traditionally been difficult, requiring multiple steps and many hours of work. We report the successful development of a rapid negative selection method that enables the preparation of highly purified monocytes from human peripheral blood using EasySep® column-free immunomagnetic cell separation technology. A cocktail of monoclonal antibodies incorporated into tetrameric antibody complexes was used to crosslink unwanted cells in the sample to EasySep® magnetic particles. The tube containing the labelled cell suspension was then placed in an EasySep® magnet for 2.5 minutes. Unlabelled cells were recovered by pouring off the cell suspension while labelled unwanted cells were held to the walls of the tube by the magnetic field. The whole procedure was completed in 30 minutes and yielded CD14+CD16− monocyte fractions that were on average 90% pure with an average recovery above 60%. Stimulation of purified monocytes with GM-CSF, IL-4 and LPS led to efficient differentiation into dendritic cells (DC) as identified by the expression of the DC markers CD1a and CD83, and the loss of the monocyte marker CD14. Differentiated DC induced robust allogeneic CD4+ T cell proliferation, confirming that the isolated monocytes were fully competent to differentiate into functional DCs. This method was also fully automated using the RoboSep® cell separator.
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Held, Stefanie AE, Annkristin Heine, Julia Wolf, Solveig Daecke, Anita Bringmann, and Peter Brossart. "Generation of Immunosuppressive Antigen Presenting Cells with the Phenotype and Function of Myeloid Derived Suppressor Cells." Blood 120, no. 21 (November 16, 2012): 1032. http://dx.doi.org/10.1182/blood.v120.21.1032.1032.
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Abstract Abstract 1032 Myeloid derived suppressor cells (MDSC) play an important role in the regulation of immune responses by suppressing the function of antigen presenting cells and T cells. In humans several populations of MDSC with different phenotypes were characterized due to their distinct immunosuppressive function. However, the origin and development of these cells is not well understood. We observed that incubation of peripheral blood monocytes with IL-10 during their differentiation to dendritic cells (DC) in the presence of GM-CSF and IL-4 results in the generation of an APC population with dramatically reduced stimulatory capacity and a CD14+ and HLA-DR low phenotype (IL-10-APC) similar to the recently described human MDSC subpopulation. In coincubation experiments we found that the addition of these cells to immature or LPS activated DC generated from peripheral blood monocytes resulted in a cell number dependent inhibition of their stimulatory capacity in the mixed lymphocyte reaction assay. Furthermore, these IL-10-APCs reduced the expression of CD1a and costimulatory molecules on DC as well as their activation by LPS characterized by diminished expression of maturation markers including CD83, CD80, CD86 or CD40. IL-10-APC almost completely abolished the secretion of cytokines and chemokines by mature and immature DC involved in T cell stimulation and migration such as TNF-a, IL-6, MIP-1a or Rantes. These effects were not due to induction of IL-10 production and were not observed when purified CD14+ monocytes were used as a control in the experiments. In order to analyze the possible mechanisms and molecules involved in these inhibitory effects we found that IL-10-APC expressed higher levels of PD-1, GITR, GITRL and osteoactivin, an immunosuppressive molecule that was shown to inhibit the function of T cells. The effects mediated by these molecules were further confirmed by utilizing blocking antibodies. Interestingly, addition of IL-10-APC to mature or immature DC induced an increased expression of osteoactivin and its corresponding receptor syndecan 4 on DC thus demonstrating that osteoactivin mediates its effects by upregulating its own receptor. In the next set of experiments we isolated the CD14+ HLA-DR low cell population from buffy coats of healthy donors. We found, that these cells similar to the IL-10-APCs express high levels of osteoactivin and syndecan-4 that can be further upregulated by the addition of IL-10. Our results demonstrate that osteoactivin mediates its inhibitory effects by induction of its cognate receptor syndecan 4. In addition, cells with the phenotype and function of MDSC can differentiate from human peripheral blood monocytes in the presence of GM-CSF, IL-4 and IL-10. This immunosuppressive cell population can easily be generated in vitro and might be applied for the treatment of patients with GVHD or autoimmune disorders. Disclosures: No relevant conflicts of interest to declare.
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Monkley, Susan, Jayendra Kumar Krishnaswamy, Melker Göransson, Maryam Clausen, Johan Meuller, Kristofer Thörn, Ryan Hicks, Stephen Delaney, and Louise Stjernborg. "Optimised generation of iPSC-derived macrophages and dendritic cells that are functionally and transcriptionally similar to their primary counterparts." PLOS ONE 15, no. 12 (December 17, 2020): e0243807. http://dx.doi.org/10.1371/journal.pone.0243807.
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Induced pluripotent stem cells (iPSC) offer the possibility to generate diverse disease-relevant cell types, from any genetic background with the use of cellular reprogramming and directed differentiation. This provides a powerful platform for disease modeling, drug screening and cell therapeutics. The critical question is how the differentiated iPSC-derived cells translate to their primary counterparts. Our refinement of a published differentiation protocol produces a CD14+ monocytic lineage at a higher yield, in a smaller format and at a lower cost. These iPSC-derived monocytes can be further differentiated into macrophages or dendritic cells (DC), both with similar morphological and functional profiles as compared to their primary counterparts. Transcriptomic analysis of iPSC-derived cells at different stages of differentiation as well as comparison to their blood-derived counterparts demonstrates a complete switch of iPSCs to cells expressing a monocyte, macrophage or DC specific gene profile. iPSC-derived macrophages respond to LPS treatment by inducing expression of classic macrophage pro-inflammatory response markers. Interestingly, though iPSC-derived DC show similarities to monocyte derived DC, they are more similar transcriptionally to a newly described subpopulation of AXL+ DC. Thus, our study provides a detailed and accurate profile of iPSC-derived monocytic lineage cells.
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Naqvi, Afsar, and Imran Ahmad. "LncRNAs modulate immune response in myeloid cells challenged with periodontal pathogens." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 227.26. http://dx.doi.org/10.4049/jimmunol.204.supp.227.26.
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Abstract Objective Long noncoding RNAs (lncRNA) are an emerging class of regulatory RNAs. LncRNAs have been implicated in differentiation and regulation of various immune cells. However, the role of lncRNA in myeloid cell regulation, in the context of periodontal inflammation remains poorly studied. Here we examined lncRNA-mediated regulation of myeloid cells upon challenge with periopathic bacteria. Method CD14+ monocytes were purified from PBMCs and were differentiated into macrophage (Mϕ) or dendritic cells (DC). Monocytes were differentiated to M1, M2 Mϕ, or DC in presence of GM-CSF, M-CSF or GM-CSF+IL-4 respectively for 7 days. Mϕ and DC were challenged A. actinomycetemcomitans (Aa), P. gingivalis (Pg) or treated with LPS from Aa and Pg. Total RNA isolated from periopathogen treated cells was used for qPCR. LncRNA knockdown was achieved by siRNA transfection using Lipofectamine 2000 and cell surface markers, cytokine analysis, antigen uptake/processing and phagocytosis assays were performed. Result DC challenged with Pg LPS exhibit differential expression of various lncRNAs (19 were up, 2 were down). Expression of selected lncRNAs in periopathogen challenged Mϕ/DC show that lncRNA are sensitive to periopathogen challenge. Cells transfected with lncRNA targeting siRNA showed skewing of M1 or M2 Mϕ polarization markers and altered secretion of pro- or anti-inflammatory cytokines upon periopathogen challenge. Finally, lncRNA knockdown resulted in functional impairment of Mϕ and DC function as reflected by decreased antigen presentation and phagocytosis. Conclusion We have functionally characterized lncRNAs in myeloid cells challenged with periopathogens and demonstrated their regulatory role in shaping immune response.
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Timganova, V. P., M. S. Bochkova, S. V. Uzhviyuk, K. Yu Shardina, S. A. Zamorina, and M. B. Rayev. "Generation of human myeloid suppressor cells in the in vitro experimental model." Russian Journal of Immunology 23, no. 2 (April 15, 2020): 157–62. http://dx.doi.org/10.46235/1028-7221-352-goh.
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Myeloid suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that generally differentiate into macrophages, granulocytes, and dendritic cells. However, in pathology, these cells acquire a suppressor phenotype, blocking immune response. In particular, MDSC levels increase in many pathological conditions, including inflammation, sepsis, traumatic shock, autoimmune diseases, cancer, and pregnancy. Over the past 12 years, an interest in examining this cell population has been steadily increased [PUBMED: 2008 (65 articles); 2020 ( 650 entries)] that will expand our understanding of immune system functioning. In humans, MDSCs are characterized by HLA-DR-CD33+CD11b+ phenotype, in turn being subdivided into CD15+ or CD66+ granulocytic (G-MDSC), CD14+ monocytic (M-MDSC), and early (e-MDSC) MDSC bearing HLA-DR-CD11b+CD33+CD14-CD66b- phenotype. This work was aimed to develop an adequate experimental model allowing to evaluate cytokine-driven differentiation of human MDSCs from peripheral blood mononuclear cells in long-term in vitro culture system. For this, peripheral blood mononuclear cells were isolated from healthy donors induced to express MDSC phenotype with GM-CSF and IL-6 (40 or 20 ng/ml) cultured for 7, 14, 21 days. In several experiments, LPS (100 ng/ml) was added to the cultured cells 24 hours before immunophenotyping. The percentage of living Zombie Aquanegative cells in cultures (gated on cells according to FSC/SSC) ranged from 90.5-93.9%. No significant differences were observed between cultured cells. In our experimental conditions, the mean percentage of total MDSC subpopulation reached 2-2.3% of total living cells, exceeding that one by 9-10-fold found in freshly isolated mononuclear cells from healthy subjects. Based on the results of our experimental study, we found that induction of e-MDSC derived from human peripheral blood mononuclear cells requires two weeks of co-culture with 40 ng/ml IL-6 and 40 ng/ml GM-CSF. “Mature” MDSCs (M-MDSC + G-MDSC) yield was peaked in the following conditions: co-culture for 3 weeks with 20 ng/ml IL-6 and 20 ng/ml GM-CSF added with 100 ng/ml LPS 24 hours before completing protocol. Overall, further examining factors modulating MDSC differentiation will reveal conditions necessary for generating this suppressor cell subset potentially used in clinical practice.
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Marzaioli, V., A. Floudas, M. Canavan, S. Wade, K. Murray, R. Mullan, D. Veale, and U. Fearon. "OP0025 CD209+/CD14+ DENDRITIC CELLS ARE ENRICHED AND ACTIVATED AT THE SITE OF INFLAMMATION AND ARE MODULATED BY JAK/STAT SIGNALLING." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 14.1–14. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2122.
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Background:Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells which are at the interface between innate and adaptive immunity. A specific subset of DCs is known to derive from monocyte and has a key role in inflammation and infection.Objectives:This study aimed to characterize the phenotype and function of a distinct CD209+/CD14+ DC subset in the periphery and at the site of inflammation in patients with rheumatoid (RA) and psoriatic arthritic (PsA), in addition to examining the effect Tofacitinib and TNF inhibitor on their development.Methods:Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) were isolated by Ficoll density gradient from healthy subject (HC), and patients with RA and PsA. Single cell synovial tissue suspension (ST) from RA and PsA patients were also established using enzymatic/mechanical digestion. PBMC, SFMC and ST were analysed by flow cytometry to identify the CD209+/CD14+ DC subset, its frequency and the cell surface expression of chemokines receptors (CCR6, CCR7, CXCR3, CXCR4 and CXCR5) and activation markers (CD40 and CD80). In addition, PBMC were stimulated with different TLR (LPS, CPG, R848, Poly I:C) and intracellular staining for IL12, TNFα, IL1β and IL6 was performed by flow cytometry. Lineage negative cells (CD3/CD19/CD56-) were stimulate with GMCSF/IL4 in the presence or absence of the JAK/STAT inhibitor Tofacitinib or the TNF inhibitor Humira, and the CD209+/CD14+ DC development was evaluated by flow cytometry.Results:We identified, for the first time, a distinct CD209+/CD14+ DC population in PBMC of patients with RA and PsA, with similar frequency across the groups. However, when PBMC were stimulated with TLRs, an increase of IL12 and TNFα was observed in RA and PsA PBMC when compared to HC. Interestingly, this distinct DC population was significantly enriched at the site of inflammation, in both SFMC and ST, displaying a more mature phenotype, evident by the observed significant increase in CD40 and CD80 expression. SPICE analysis further identified differential expression and co-expression of chemokine receptors at the periphery of RA and PsA patients, when compared to the HC. Furthermore synovial tissue single cell analysis from RA/PsA demonstrated a unique chemokines receptors profile demonstrating increased single expression and co-expression of CXCR3 and CXCR5 compared to periphery. Finally, we have previously observed that JAK/STAT is involved in monocyte-derived dendritic cells population development (1,2), therefore we performed CD3, CD19 and CD56 depletion of RA/PsA PBMC followed by stimulation with GMCSF/IL4, to spike the Mo-DC population, in the presence of Tofacitinib or Humira. Interestingly, we observed that JAK/STAT inhibition, but not TNF inhibitor, reduced the generation and development of CD209+/CD14+ DC.Conclusion:We identify for the first time a distinct monocyte-derived DC population characterized as CD209+/CD14+ in the periphery of RA and PsA patients. This population was enriched at the site of inflammation and displayed a unique chemokine receptor profile and activation markers, suggesting that these cells are already activated in the periphery of IA patients, and are recruited and further activated in the inflamed joint. In addition, we showed that the CD209+/CD14+ DC development is regulated by JAK/STAT signalling, but not TNF inhibition.References:[1]Marzaioli V, Canavan M, Floudas A, et al. Monocyte-Derived Dendritic Cell Differentiation in Inflammatory Arthritis Is Regulated by the JAK/STAT Axis via NADPH Oxidase Regulation. Front. Immunol. 2020;11:1406.[2]Marzaioli V, Hurtado-Nedelec M, Pintard C, et al. NOX5 and p22phox are 2 novel regulators of human monocytic differentiation into dendritic cells. Blood. 2017;130(15):1734–1745.Acknowledgements:The authors also wish to thank all the patients who volunteered to participate into this study and the fundingDisclosure of Interests:Viviana Marzaioli: None declared, Achilleas Floudas: None declared, Mary Canavan: None declared, Siobhan Wade: None declared, Kieran Murray: None declared, Ronan Mullan: None declared, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Consultant of: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Grant/research support from: Janssen, Abbvie, Pfizer, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Janssen, Abbvie, Pfizer, UCB
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Torosantucci, Antonella, Giulia Romagnoli, Paola Chiani, Annarita Stringaro, Pasqualina Crateri, Sabrina Mariotti, Raffaela Teloni, Giuseppe Arancia, Antonio Cassone, and Roberto Nisini. "Candida albicans Yeast and Germ Tube Forms Interfere Differently with Human Monocyte Differentiation into Dendritic Cells: a Novel Dimorphism-Dependent Mechanism To Escape the Host's Immune Response." Infection and Immunity 72, no. 2 (February 2004): 833–43. http://dx.doi.org/10.1128/iai.72.2.833-843.2004.
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ABSTRACT The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence. We show here that human monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) after phagocytosis of Y forms did not differentiate into dendritic cells (DCs); they retained CD14, did not acquire CD1a, and were unable to express the maturation markers CD83 and CCR7. Moreover, they did not produce IL-12p70 but secreted IL-10. In addition, they spontaneously expressed high levels of tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 mRNA transcripts and were able to induce proliferation of alloreactive memory but not naïve T lymphocytes. Conversely, monocytes that had phagocytosed GT forms differentiated into mature CD83+ and CCR7+ DCs; however, there was no up-regulation of CD40, CD80, and major histocompatibility complex class II, irrespective of lipopolysaccharide (LPS) treatment. In addition, these cells were unable to produce IL-12 even after LPS stimulation, but they were not functionally exhausted, as shown by their capacity to express TNF-α and IL-8 mRNA transcripts. These cells were able to prime naïve T cells but not to induce their functional polarization into effector cells. These data indicate that phagocytosis of Y and GT forms has profound and distinct effects on the differentiation pathway of monocytes. Thus, the differentiation of human monocytes into DCs appears to be tunable and exploitable by C. albicans to elude immune surveillance.
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Trabanelli, Sara, Antonio Curti, Darina Očadlíková, Cecilia Evangelisti, Valentina Salvestrini, Richard Metz, Lisa Laury-Kleintop, George Prendergast, and Roberto M. Lemoli. "Human Monocyte-Derived Dendritic Cells Are Tolerogenic through Both IDO1 and IDO2." Blood 116, no. 21 (November 19, 2010): 4298. http://dx.doi.org/10.1182/blood.v116.21.4298.4298.
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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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Sebelin, Kathrin, Antje Meier, Carola Beier, Bernd Dörken, Antonio Pezzutto, and Marion Subklewe. "Human BDCA-1 Positive Blood Dendritic Cells in Immunosuppressed Patients: Phenotype, Function and Maturation." Blood 106, no. 11 (November 16, 2005): 2231. http://dx.doi.org/10.1182/blood.v106.11.2231.2231.
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Abstract Immunosuppressive drugs used in patients (pts) after stem cell / organ transplantation (Tx) as well as in pts with autoimmune disease are known to impair the cellular immune response. This results in an increased incidence of viral infections and viral associated malignancies which has been ascribed to the effect of immunosuppressive drugs on lymphocytes. However, in vitro data indicate that immunosuppressive drugs also target Dendritic Cells (DCs), the most potent antigen-presenting cells and initiators of lymphocyte responses. So far, most studies are based on in vitro data obtained with DC culture in the presence of different concentrations of single immunosuppressive drugs. To investigate the effect of immunosuppression on DC phenotype and function in vivo, we quantitatively and qualitatively analyzed freshly isolated human BDCA-1(CD1c) positive DCs from 15 solid organ transplant (SOT) recipients under immunosuppressive treatment. The percentage of BDCA-1 positive cells among total PBMCs was not statistically different in pts vs ctrls (0,52 vs 0,65, p<0,18). BDCA-1 positive DCs were analyzed for expression of HLA class I and II, CD14, costimmulatory molecules and chemokine expression. Interestingly, CD14 was found to be significantly higher expressed on pt-DCs vs ctrl-DCs suggesting a more immature DC-phenotype. We observed a trend toward a reduced expression of HLA-DR and CD86 on pts-DCs as compared to ctrls-DCs (p=0,059). Surface profile of BDCA-1 positive DCs was also analyzed after 48h of LPS and CD40L stimulation. Here we found a marked upregulation of HLA-DR and CD86 in pts- DCs as well as ctrl-DCs. Supernatant of stimulated DCs was analyzed with cytokine capture beads for secretion of inflammatory cytokines. High secretion of IL-6, IL-1 beta and partially of TNF-alpha by stimulated DCs was observed in both groups. Other Th2 type cytokines (IL-10, IL-4, IL-5) and Th1 type cytokines like IFN-gamma and Il-2 were not significantly secreted. We additionally addressed the question if mature and functionally competent DCs could be generated ex vivo from this pts cohort. After 9 days of culture with GM-CSF, IL-4, IL-1, IL-6, TNF-alpha and PGE2 fully mature DCs could be generated. Co-culture of EBV-peptide-pulsed DCs with autologous T-cells resulted in significant expansion of EBV-specific T cells in pts and ctrls. These T cells were fully functional as shown by IFN-γ secretion detected by ELISPOT. In summary, this is the first analysis of freshly isolated BDCA-1 positive DCs from immunosuppressed pts. Our data support the notion that immunosuppressive drugs target DCs and contribute to a maturation defect of circulating blood DCs which may help to understand the mechanism of impaired cellular immune responses in immunosuppressed pts. However, ex vivo generated DCs from immunosuppressed pts do not show an impairment in phenotype and function, suggesting that they could be efficiently be used in immunotherapeutic strategies.
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Yokoyama, Ayumi, Miwako Narita, Naoko Sato, Asuka Sekiguchi, Anri Saito, Norihiro Watanabe, and Masuhiro Takahashi. "Enhancing Effects of CCL19 on Antigen Uptake and Antigen Presenting Ability of Human Mature Dendritic Cells (DCs)." Blood 104, no. 11 (November 16, 2004): 3830. http://dx.doi.org/10.1182/blood.v104.11.3830.3830.
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Abstract Recently, it has been demonstrated that CCL19 induces not only chemotaxis but also the extension of dendrites and endocytosis by mature DCs derived from mouse splenocytes. There are some reports presenting CCL19-associated inhibition on the generation of cytotoxic T lymphocytes towards allogeneic DCs, but another reports presenting the inhibition of allogeneic response by the antagonist of CCL19. In the present study, we tried to induce CCR7 expression on human monocyte-derived mature DCs using cytokines and examined CCL19 effects on the functions of these mature DCs. In this present study, we demonstrated that CCL19 could promote the ability of endocytosis and allogeneic antigen presentation of monocyte-derived mature DCs. CD14+ cells were separated from human PB-MNCs using anti-CD14 conjugated magnetic microbeads. The cells were seeded in tissue culture dish and cultured in RPMI medium containing GM-CSF and IL-4. On day 4, a pro-inflammatory cytokine cocktail (TNF-α, IL-1α, IL-6 and INF-γ) and PGE2 ware added to this dish as the DC maturation stimuli and the cultures were continued for more one day. Surface phenotypes such as CD1a, CD14, CD80, CD83, CD86, HLA DR and CCR7 were analyzed by flow cytometry. To analyze endocytosis, immature and mature DCs were incubated with FITC-dextran at 37°C and 4°C in the presence or absence of CCL19 for 5 minutes. Cells were washed with ice-cold PBS then incubated for 10 minutes on ice. After washing, the cells were re-suspended in PBS and the fluorescence intensity of the cells was analyzed by flow cytometry. Incubation of cells with the FITC-dextran on ice was used as a background control. Antigen presenting ability was analyzed by 3H-thymidin incorporation assay in the presence or absence of CCL19, in which autologous and allogeneic lymphocytes were used as responder cells. After the culture using TNF-α, IL-1α, IL-6, INF-γ and PGE2 (TIIIP) as maturation stimuli, DCs highly expressed CD83, CD80, CD86 and HLA-DR and moderately expressed CCR7 in almost the same manner of mature DCs cultured with LPS. About the internalization of FITC-dextran in immature DCs, approximately 50% of the cells rapidly became FITC-dextran positive after 5–10 minutes of incubation without the addition of CCL19. Mature TIIIP-induced mature DCs, however, scarcely internalized FITC-dextran at 5–10 minutes; the proportion of FITC+ cells was less than 5% of the total cells. But the addition of CCL19 increased the uptake by TIIIP-induced mature DCs. In the presence of CCL19, the proportion of mature DCs positive for FITC-dextran was about 30% after 5 minutes of incubation. In allogeneic MLC, 3H-thymidin uptake of lymphocytes stimulated with TIIIP-induced mature DCs was definitely increased by CCL19 addition, while there were no effects of CCL19 in immature DCs. We recognized the role of CCL19 in the positive regulation of endocytosis in human mature DCs. In addition to CCL19-associated enhancement of endocytosis of antigens by mature DCs, CCL19 has also been demonstrated to promote the antigen presenting ability of mature DCs. These findings suggested that the regulation of CCR7-CCL19 interaction could be usefully applied in generating efficient DCs with possessing both active phagocytosis and potent antigen presenting ability for DC-based immunotherapy in various disorders.
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Naqvi, Raza Ali, Imran Ahmad, Araceli Maria Valverde Estepa, and Afsar Naqvi. "Modulation of myeloid cell functions by long noncoding RNAs RN7SK and HCG11." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 111.18. http://dx.doi.org/10.4049/jimmunol.208.supp.111.18.
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Abstract Objective Expressional dynamics of long noncoding RNAs (lncRNA)” regulates a gamut of biological processes including immunity. Role of lncRNA in response to oral pathogens and their regulatory impact on the innate immune functions of myeloid cells remain poorly explored. Therefore, we hypothesize that periodontal pathogens may cause the alteration of lncRNA profiles in myeloid cells and modulate their innate immune functions. Method CD14+ monocytes sorted from human PBMCs were differentiated into macrophages (MΦ) or dendritic cells (DC), and challenged with periopathic bacteria (A. actinomycetemcomitans [Aa] and P. gingivalis [Pg] for 4, 12 or 24 h. lncRNA expression was done at these time points by RT-qPCR array. Differentially expressed lncRNAs were assessed for: 1) cell migration , 2) phagocytosis, and 3) antigen uptake/processing in . Cell surface markers were analysed by flow-cytometry. Result Challenge of DCs with Pg, Pg LPS, Aa, and Aa LPS separately result in the differential expression of 21 lncRNAs (19 up, 2 down). Knockdown of LncRNA, SNHG11, NUTM2A-AS1, MCM3AP-AS1, JPX, and HCG11 (upregulated) and RN7SK (downregulated) were used to evaluate myleoid cell functions. HCG11 knockdown revealed attenuation of cell migration, while RN7SK significantly enhanced antigen uptake/processing in APCs . Importantly, RN7SK knockdown results in downregulation of M2 Mϕ surface markers (CD163, CD206 or Dectin) and concomitant increase in M1 Mϕ markers (MHC II or CD32) suggesting its critical role in macrophage polarization. Conclusion Our results show that periodontal pathogens alter lncRNA profiles and impair innate immune functions in myeloid cells, thereby suggesting critical roles of lncRNAs in periodontopathogenesis. Supported by NIH/NIDCR R03 DE027147, R01DE027980, and R21DE026259 to AN.