Functional expression of indoleamine 2,3-dioxygenase by murine CD8 alpha(+) dendritic cells.

Immunoregulatory antigen-presenting cells (APC) play an important role in maintaining T cell homeostasis and self-tolerance. In particular, recent evidence demonstrates a role for inhibition of T cell proliferation by macrophage tryptophan catabolism involving the activity of the enzyme indoleamine 2,3-dioxygenase (IDO). Dendritic cells (DC) have also been shown to exert immunoregulatory effects mediated by tryptophan catabolism and to cause T cell apoptosis. In the present study, we have comparatively analyzed the expression of IDO activity by murine macrophages and splenic DC. By means of PCR, Western blotting and measurements of enzyme functional activity, we obtained evidence that, different from macrophages, DC constitutively express IDO. Following activation by IFN-gamma, the latter cells, in particular the CD8 alpha(+) subset, exhibit high functional activity and, unlike macrophages, mediate apoptosis of T(h) cells in vitro. Therefore, in the mouse, CD8 alpha(+) DC may be unique APC capable of fully expressing the IDO mechanism functionally.     

Inhibition of indoleamine 2,3-dioxygenase activity enhances the anti-tumour effects of a Toll-like receptor 7 agonist in an established cancer model

Toll-like receptor (TLR) agonists have been shown to have anti-tumour activity in basic research and clinical studies. However, TLR agonist monotherapy does not sufficiently eliminate tumours. Activation of the innate immune response by TLR agonists is effective at driving adaptive immunity via interleukin-12 (IL-12) or IL-1, but is counteracted by the simultaneous induction of immunosuppressive cytokines and other molecules, including IL-10, transforming growth factor-β, and indoleamine 2,3-dioxygenase (IDO). In the present study, we evaluated the anti-cancer effect of the TLR7 agonist, imiquimod (IMQ), in the absence of IDO activity. The administration of IMQ in IDO knockout (KO) mice inoculated with tumour cells significantly suppressed tumour progression compared with that in wild-type (WT) mice, and improved the survival rate. Moreover, injection with IMQ enhanced the tumour antigen-specific T helper type 1 response in IDO-KO mice with tumours. Combination therapy with IMQ and an IDO inhibitor also significantly inhibited tumour growth. Our results indicated that the enhancement of IDO expression with TLR agonists in cancer treatment might impair host anti-tumour immunity while the inhibition of IDO could enhance the therapeutic efficacy of TLR agonists via the increase of T helper type 1 immune response.     

Ambivalent effects of dendritic cells displaying prostaglandin E2-induced indoleamine 2,3-dioxygenase

Prostaglandin E2 (PGE(2)), an abundantly produced lipid messenger in mammalian organisms, has been attributed to possess potent albeit ambivalent immunological functions. Recently, PGE(2) has been reported to stimulate the commonly believed immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway in human dendritic cells (DCs), but without promoting DC immunosuppressive activity. Here, we report that PGE(2) used as a DC maturation agent apparently has more diverse functions. PGE(2)-matured DCs acquired powerful IDO activity, which was sustained even after removing PGE(2). These IDO-competent DCs were able to stimulate allogeneic T-cell proliferation, but achieved inhibitory activity as their content in DC/T-cell co-cultures increased. The DC inhibitory activity was reversed upon blockade of IDO activity, confirming that the suppressive effect was in fact mediated by IDO and occurred in a dose-dependent fashion. IDO-mediated T-cell suppression was restored upon re-stimulation of T cells in the absence of IDO activity, confirming its reversibility. T cells stimulated by PGE(2)-matured IDO-competent DCs were sensitized to produce multiple cytokines, comprising Th1, Th2, and Th17 phenotypes. Collectively, these data suggest that T cells stimulated by PGE(2)-matured DCs are not terminally differentiated and their ultimate type of response may be formed by microenvironmental conditions.     

Indoleamine 2,3-dioxygenase and regulatory dendritic cells contribute to the allograft protection induced by infusion of donor-specific splenic stromal cells

It has been reported that splenic stromal cells (SSCs) are capable of directly supporting the development of CD11c(lo)CD45RB(+ )IL-10-producing dendritic cells (DCs) from lineage-negative c-kit(+) progenitor cells in the absence of exogenous cytokines. In vitro, DCs that differentiate on stromal cells suppress mixed leukocyte reaction responses and induce primary alloreactive CD4(+) T cells to differentiate into IL-10-producing Tr1 cells. However, the precise mechanisms by which these SSCs exert their regulatory functions in vivo remain undefined. Furthermore, their possible contribution to the development of allograft transplantation tolerance has yet to be examined. Here, we have used both murine skin and cardiac allograft transplantation models to explore whether in vivo alloresponses can be regulated by infusion with donor-derived SSCs and to investigate the possible mechanisms by which SSCs exert regulatory effects to prevent allograft rejection. We show that intravenous SSC infusion prolonged murine skin allograft survival. The prolonged graft survival is associated with augmentation of the generation of regulatory DC subsets and CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), as well as upregulation of the production of suppressive cytokines IL-10 and transforming growth factor (TGF)-β. Moreover, we found that indoleamine 2,3-dioxygenase and SSC-derived regulatory DCs contribute to allograft protection by infusion of donor-specific SSCs. Our data suggest that donor-derived SSCs could be used as a therapeutic target to promote transplantation tolerance.     

A subset of dendritic cells express joining chain (J-chain) protein

Joining chain (J-chain) is well known as an integrated component of dimeric immunoglobulin A (IgA) and pentameric IgM. We show here that the J-chain protein is also expressed in a subset of CD11c+ dendritic cells (DC) in C57BL/6 mice. J-chain knockout mice (J-/- mice) had a reduced fraction of CD4-/CD8alpha+ and mPDCA-1+ DC in the spleen. J-/- mice also had reduced levels of RNA for the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in the spleen. Furthermore, in lymph nodes from C57BL/6 mice the majority of J-chain-expressing CD11c+ cells also expressed IDO, while the number of IDO-expressing cells in lymph nodes and the amount of IDO protein in splenic CD11c+ cells were reduced in J-/- mice. Also, J-/- mice had a lower ratio of kynurenine/tryptophan in serum compared to C57BL/6 mice, indicating a lower overall IDO activity in J-/- mice. We also show that J-/- mice are less susceptible to tolerance induction than C57BL/6 mice. In conclusion, our data show that J-chain protein is expressed outside the B-cell compartment in a subset of immunoregulatory DC that are compromised in animals that cannot express J-chain.     

IDO/TTS色氨酸代谢途径对类风湿关节炎滑膜T细胞增殖的影响

自身反应性T细胞在类风湿关节炎(RA)疾病的发生发展中发挥着极其重要的作用,抑制这群细胞是RA治疗的关键.表达吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)的树突状细胞(DC)能够通过此代谢通路剥夺环境中的色氨酸,并生成促凋亡因子犬尿氨酸等,使T细胞的增殖和功能受抑制,最终诱导...     

不同Toll样受体介导的树突状细胞因子表达

目的研究树突状细胞在不同Toll样受体(TLR)配体刺激下的早期反应。方法将鼠骨髓细胞诱导分化得到DC,利用TLR配体刺激,采用芯片分析方法研究其免疫相关基因在9h的表达;采用细胞因子分析系统,检测了12、48h细胞培养上清中细胞因子、趋化因子的产生。结果在给予LPS、PolyI:C、R848、CpG和anti-CD40刺激9h后,IL-1、IL-2rg、IL-13、CCL2、CCL4和CCL7的基因表达明显升高,CCL3、CCL5和CXCL9、CXCL10、CXCL11的表达增加最为显著(P<0.01);而IL-10(PolyI:C除外),CCL-6的表达降低(P<0.05)。培养12h后,IL-1、IL-6、IL-10、IL-12(p40)、IL-12(p70)、TNFa、G-CSF、GM-CSF、KC和MIP-1a(CCL3)产生在5种TLR配体刺激下均显著增加(P<0.01);培养48h后,IL-1、IL-10、IL-12(p40)、IL-12(p70)、TNFa、IFN-γ、G-CSF和MIP-1a(CCL3)产生也同样显著增加(P<0.01)。相对于其它TLR配体,CpG刺激时MIP-1a(CCL...     

内皮源性吲哚胺2,3-双加氧酶抑制周细胞迁移及收缩蛋白表达

 目的: 探讨内皮源性吲哚胺2,3-双加氧酶(IDO)对周细胞迁移及收缩蛋白表达的影响。方法: 体外培养人肺动脉内皮细胞(HPAECs)及大鼠脑微血管周细胞。建立过表达IDO的HPAECs模型(IDO-HPAECs)。实验分为3组:对照组,以HPAECs条件培养基干预周细胞;处理组,以IDO-HPAECs条件培养基干预周细胞;抑制组,以含1-甲基色氨酸(1-mT)的IDO-HPAECs条件培养基干预周细胞。测定共培养体系一氧化氮(NO)、色氨酸及犬尿氨酸浓度。观察周细胞活性、迁移及收缩蛋白表达情况。结果: IDO-HPAECs条件培养基干预周细胞6～48 h对其活性无显著影响。处理组的周细胞迁移能力显著低于对照组(P < 0.01),抑制组显著高于处理组(P < 0.01)。共培养体系的NO浓度在各组间差异无统计学意义(P > 0.05)。处理组的色氨酸浓度显著低于对照组(P < 0.01),而抑制组显著高于处理组(P < 0.01)。处理组的犬尿氨酸浓度显著高于对照组(P < 0.01),而抑制组显著低于处理组(P < 0.01)。处理组的α-平滑肌肌动蛋白与结蛋白表达水平显著低于对照组(P < 0.01),抑制组显著高于处理组(P < 0.01)。结论: 内皮源性IDO抑制周细胞迁移及收缩蛋白表达,可能参与机体微血管功能调控。     

Tolerogenic dendritic cells transferring hyporesponsiveness and synergizing T regulatory cells in transplant tolerance

Dendritic cells are among the most potent antigen-presenting cells and are important in the development of both immunity and tolerance. Tolerogenic dendritic cell (Tol-DC) is a key factor in the induction and maintenance of tolerance during transplantation. However, the precise mechanism and direct evidence of in vivo immune modulation remain unclear. In the present study, we identified critical roles of immune modulation on transplant tolerance through interactions between Tol-DCs and regulatory T (Treg) cells. Tol-DCs remained in an immature state and were insensitive to maturation stimuli. Tol-DCs in tolerant recipients heightened the expression of indoleamine 2,3-dioxygenase (IDO) that induced allogeneic T-cell apoptosis. Adoptive transfer of Tol-DCs isolated from primary tolerant recipients resulted in augmentation of CD4(+)CD25(+)CTLA4(+) Treg cells and prolonged graft survival in secondary allogeneic heart transplantation and synergized with Treg cells to induce tolerance in secondary recipients. This study indicates that Tol-DC offers two functions during the process of tolerogenesis: suppression of anti-donor T-cell responses through production of IDO and interaction with Treg cells, which provides a framework for future research into tolerance induction.     

Abatement of cyclophosphamide-induced splenic immunosuppressive indoleamine 2, 3-dioxygenase and altered hematological indices in Wister rats by dietary quercetin

The immunotoxicity mediated by cyclophosphamide (CYP) was earlier reported. Quercetin, due to its anti-oxidative/inflammatory properties elicits a plethora of health benefits. However, the influence of quercetin on the splenic/immunotoxicity linked with CYP-induced indoleamine 2,3-dioxygenase (IDO) is unavailable in the literature. We investigated the effects of quercetin on the splenic immunosuppressive IDO and hematological indices of immune response in rats. Animals were treated with CYP (100 mg/kg) alone or co-treated (CYP + quercetin [100 mg/kg + 50 mg/kg respectively]) at days 1, 3, 5, and 7. Results revealed that CYP treatment alone significantly provoked an oxidative-inflammatory response, increased serum kynurenine concentration, and concomitantly increased immunosuppressive IDO and tryptophan 2, 3-dioxygenase (TDO), in the spleen as well as altering hematological indices. Quercetin co-treatment enhanced activities of antioxidant enzymes, inhibited myeloperoxidase (MPO) activity, lowered levels of nitric oxide, interferon-Υ (IFN-Υ), and interleukin-6 (IL-6), and reduced kynurenine concentration as well as IDO/TDO activities. Quercetin co-treatment augmented white blood cell (WBC), CD4-T cells, and other hematological indices of the immune response. In conclusion, quercetin prevented CYP-induced alterations in immune response in rats by lowering the activities of immunosuppressive IDO and TDO, inhibiting oxidative-inflammatory stress, diminution of kynurenine concentrations, and augmenting hematological parameters.     

Toll-like receptors 3, 7, 8, and 9 in gastric cancer

Toll-like receptors (TLRs) have been shown to have anti-tumor, pro-tumor, or even dual effects in cancer, and are thus potential prognostic biomarkers and immunotherapeutic targets. The present study aimed to evaluate associations between endosomal TLRs, namely TLR3, TLR7, TLR8, and TLR9, expression and clinicopathological variables and survival in gastric cancer. A total of 564 gastric adenocarcinoma patients were included in this retrospective cohort study. Samples and clinicopathological data were retrieved and organized into tissue microarray blocks. Protein expressions were detected by immunohistochemical staining. The patients were divided into low expression and high expression groups by median values of expression. Cox regression provided hazard ratios (HR) with 95% confidence intervals (CI), adjusted for confounders. Patients with high nuclear TLR3 expression had significantly poorer 5-year survival than the low nuclear TLR3 expression group in the univariable analysis (crude HR 1.31, 95% CI 1.07–1.60). With radically resected patients, poor prognosis was also seen in the multivariable analysis (adjusted HR 1.38, 95% CI 1.08–1.77). Cytoplasmic TLR3, TLR7, TLR8, and TLR9 were not associated with 5-year survival. In conclusion, high nuclear TLR3 expression seems to have prognostic impact in gastric cancer, while TLR7, TLR8, and TLR9 do not.     

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.     

Toll-like receptor 7/8 agonists stimulate plasmacytoid dendritic cells to initiate TH17-deviated acute contact dermatitis in human subjects

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Human Langerhans' cells and dermal-type dendritic cells generated from CD34 stem cells express different toll-like receptors and secrete different cytokines in response to toll-like receptor ligands

Langerhans' cells (LC) and dermal dendritic cells (dDC) are located in the superficial and deeper layers of the skin respectively and represent the main dendritic cell (DC) populations of the skin. LC-like and dDC-like DC can be generated from CD34 stem cells and this system is widely used as a model for investigating these cells and in therapeutic vaccination. Here we report toll-like receptor (TLR) expression in human LC and dDC derived from CD34 stem cells. In vitro-generated DC expressed TLR-1, 2, 4, 6, 8 and 10. LC, but not dDC, expressed TLR-5, whereas only dDC expressed TLR-3. Maturation of LC was mediated by TLR-2, 4 and 5 ligands, but not by a TLR-3 ligand. dDC maturation was induced by TLR-3 and -4, but not with TLR-5 ligand and only weakly by a TLR-2 ligand. Stimulated LC secreted interleukin (IL)-1beta, low levels of tumour necrosis factor-alpha (TNF-alpha) and IL-8, but not IL-6 or IL-10. dDC secreted TNF-alpha, IL-6, IL-8 and IL-10, but little IL-1beta. IL-12p70 was not produced by ligand-stimulated dDC or LC, but was secreted by monocyte-derived DC (mdDC) stimulated with lipopolysaccharide (LPS). Thus, in vitro-generated LC and dDC detect different pathogen-associated molecules and show different cytokine-secretion profiles in response to TLR ligands.     

Expression and function analysis of indoleamine 2 and 3-dioxygenase in bladder urothelial carcinoma

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for tryptophan metabolism inducing immune tolerance of tumors. The purpose of this study is to investigate IDO expression and its prognostic significance in bladder urothelial carcinoma (BUC). In this study, immunohistochemical staining for IDO expression in BUC tissues (n = 84) and normal bladder tissues (n = 22) was performed. The mRNA expression levels of IDO in BUC and normal bladder were analyzed by quantitative RT-PCR. Survival analysis was performed for the correlation of IDO expression and clinicopathological factors with disease-free survival. Positive expression of IDO was found in 48 of 84 cases in BUC tissues and was significantly correlated with histological classification, histological grade and TNM stage. While IDO expression in normal bladder tissues was expressed in only 4 of 22 (18.2%) cases. Moreover, IDO mRNA levels of BUC were significantly higher than that of normal bladder. We also found that IDO, histological grade and TNM stage were closely associated with DFS. These results indicated that IDO was related to the progression of BUC and might be one of the crucial prognostic factors for BUC.     

The role of indoleamine 2, 3-dioxygenase in lepromatous leprosy immunosuppression

To elucidate further the possible role of the tryptophan, rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) in leprosy, the distribution of IDO-positive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC-lineage IDO(+) cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO-positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) and increased IDO expression in M. leprae-stimulated CD14(+) cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae-induced IDO-protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up-regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)-γ synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN-γ and M. leprae was abrogated by 1-methyltryptophan (1-MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.     

沉默吲哚胺2,3-双加氧酶2基因对黑色素瘤细胞B16-BL6增殖、迁移和侵袭的影响

目的:探讨沉默吲哚胺2,3-双加氧酶2(IDO2)基因对小鼠黑色素瘤B16-BL6细胞增殖、迁移及侵袭等生物学行为的影响。方法:IDO2-siRNA转染体外培养的黑色素瘤细胞B16-BL6,应用real-time PCR和Western blot检测IDO2和IDO1基因的表达;平板集落形成实验检测IDO2基因沉默对肿瘤细胞增殖的影响;细胞划痕实验和Transwell小室细胞迁移实验观察IDO2对肿瘤细胞迁移的影响;Transwell小室侵袭实验观察肿瘤细胞侵袭能力。结果:沉默B16-BL6细胞中IDO2基因能使细胞单集落形成密度降低,划痕迁移变慢,Transwell小室细胞迁移数减少,侵袭细胞数减少。结论:沉默IDO2可以影响黑色素瘤细胞B16-BL6的增殖、迁移和侵袭能力。     

Regulatory role of tryptophan degradation pathway in HLA-G expression by human monocyte-derived dendritic cells

Dendritic cells (DC) are strong inducers of immunity but they can also be tolerogenic. During monocyte differentiation to DC the immunosuppressive indoleamine-2,3-dioxygenase (IDO) is induced. IDO degrades Trp to kynurenine, which is further metabolized to 3-hydroxyanthranilic acid. DC can also express mRNA and protein of the tolerogenic molecule HLA-G, but there is no surface expression. We studied the effect of the Trp degrading pathway on HLA-G expression by DC. When monocytes were differentiated to immature DC in presence of either Trp or its metabolites kynurenine or 3-hydroxyanthranilic acid they expressed cell surface HLA-G, and Trp also increased shedding of HLA-G1. Trp induced HLA-G cell surface expression when present during maturation with IFN-γ + LPS, but not with TNF-. Kynurenine increased HLA-G expression in both TNF- and IFN-γ + LPS matured DC, and 3-hydroxyanthranilic acid had a very weak effect on HLA-G cell surface expression when present during maturation. Shedding of HLA-G1 was more pronounced in IFN-γ + LPS-matured DC than in immatured DC. Maturation with IFN-γ + LPS in presence of kynurenine also increased HLA-G5 secretion. The mechanism involved seems to be post-translational as mRNA and cellular HLA-G protein content was not increased with Trp, kynurenine or 3-hydroxyanthranilic acid treatments. Finally, immature DC preincubated with Trp, kynurenine and 3-hydroxyanthranilic acid have after a decreased capacity to stimulate T cells in mixed lymphocyte reaction. In IFN-γ + LPS-matured DC this decreased capacity was obtained with kynurenine and 3-hydroxyanthranilic acid. These results suggest that IDO can induce HLA-G cell surface expression in DC, and that these two molecules can cooperate in the immune suppression.     

低氧对人成熟树突状细胞MMP9/TIMP1及CCR7 表达的影响

目的： 观察低氧对人单核细胞来源成熟树突状细胞（mDCs）趋化因子受体（CCR7）,基质金属蛋白酶-9（MMP-9）及其组织抑制剂（TIMP-1）表达的影响,以期为改进DCs疫苗体内迁移能力低下提供新策略。方法： 分离制备人外周血单核细胞（PBMCs）,采用体外常规培养体系 ［粒-巨噬细胞集落刺激因子（GM-CSF）与白细胞介素-4（IL-4）共同刺激］ 诱导DCs,于诱导后第5 d加入TNF-α促成熟,48 h后将mDCs置低氧环境下（1％ O2、5％ CO2、94％ N2）分别继续培养6 h、12 h,设常氧对照组（21% O2、5% CO2）,收获细胞及培养上清;采用RT-PCR技术检测MMP-9、TIMP-1及CCR7的表达;明胶酶谱法检测培养上清中MMP-9的水平与活性,流式细胞术检测mDCs表面CCR7的表达。结果： RT-PCR及酶谱实验结果显示,低氧6 h、12 h处理组mDCs MMP-9水平显著下降;与对照组比较,各低氧处理组mDCs均未检测到TIMP-1 mRNA表达的变化;转录及细胞表面表达分析结果显示,与常氧对照组比较,低氧6 h处理组趋化因子受体CCR7表达显著降低,而12 h处理组其表达显著回升至近常氧组水平。结论： 低氧下调mDCs MMP-9表达,破坏MMP-9/TIMP-1平衡,可能是DCs疫苗体内迁移能力低下的主要因素之一。趋化因子受体CCR7在mDCs对低氧应答中可能起重要作用。