Short-term lipopolysaccharide stimulation induces differentiation of murine bone marrow-derived dendritic cells into a tolerogenic phenotype

Dendritic cells (DCs) are professional, antigen-presenting cells, which induce and regulate T cell reactivity. DCs are crucial in innate and adaptive immune responses, and are also involved in central and peripheral tolerance induction. Tolerance can be mediated by immature and semi-mature DCs expressing low levels of co-stimulator and major histocompatibility complex (MHC) molecules. The aim of this study was to investigate the ability of short-term lipopolysaccharide (LPS) stimulation to modulate the stage of differentiation of bone marrow-derived DCs. For this purpose, DCs obtained from DBA1/lacJ mice were stimulated for four (4hLPS/DCs) or 24 (24hLPS/DCs) hours with LPS, using DCs without stimulation (0hLPS/DCs) as a control. Flow cytometry analysis of 4hLPS/DCs showed intermediate CD40 and MHC class II expression, lower than that of 24hLPS/DCs (fully mature), and greater than that of 0hLPS/DCs (immature). A functional assay showed that 4hLPS/DCs displayed increased endocytotic ability compared to 24hLPS/DCs, indicating a semi-mature state. 4hLPS/DCs were greater producers of IL-10 protein and TGFbeta1 mRNA than 24hLPS/DCs and immature DCs, displaying a cytokine production pattern that is characteristic of tolerogenic DCs. An assay for antigen-presenting capacity demonstrated that 4hLPS/DCs induced secretion of IL-2 from an OTH4 T cell hybridoma, indicating a functional presenting activity. Finally, the tolerogenic phenotype of 4hLPS/DCs was demonstrated by their ability to interfere with the progression of bovine type II collagen (bII)-induced arthritis (CIA) when they were loaded with bCII antigen and injected into mice with established CIA. We conclude that the stimulation of murine bone marrow-derived DCs with LPS for four hours generates semi-mature DCs with tolerogenic capability.     

Midkine inhibits inducible regulatory T cell differentiation by suppressing the development of tolerogenic dendritic cells

Midkine (MK), a heparin-binding growth factor, reportedly contributes to inflammatory diseases, including Crohn's disease and rheumatoid arthritis. We previously showed that MK aggravates experimental autoimmune encephalomyelitis (EAE) by decreasing regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs), a population that regulates the development of autoimmune responses, although the precise mechanism remains uncertain. In this article, we show that MK produced in inflammatory conditions suppresses the development of tolerogenic dendritic cells (DCregs), which drive the development of inducible Treg. MK suppressed DCreg-mediated expansion of the CD4(+)CD25(+)Foxp3(+) Treg population. DCregs expressed significantly higher levels of CD45RB and produced significantly less IL-12 compared with conventional dendritic cells. However, MK downregulated CD45RB expression and induced IL-12 production by reducing phosphorylated STAT3 levels via src homology region 2 domain-containing phosphatase-2 in DCreg. Inhibiting MK activity with anti-MK RNA aptamers, which bind to the targeted protein to suppress the function of the protein, increased the numbers of CD11c(low)CD45RB(+) dendritic cells and Tregs in the draining lymph nodes and suppressed the severity of EAE, an animal model of multiple sclerosis. Our results also demonstrated that MK was produced by inflammatory cells, in particular, CD4(+) T cells under inflammatory conditions. Taken together, these results suggest that MK aggravates EAE by suppressing DCreg development, thereby impairing the Treg population. Thus, MK is a promising therapeutic target for various autoimmune diseases.     

Myosin IIa activation is crucial in breast cancer derived galectin-1 mediated tolerogenic dendritic cell differentiation

Background

Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.

Methods

Circulating CD14⁺CD16⁺ of breast cancer patients and induced CD14⁺CD16⁺ DCs were identified as tDCs by treating CD14⁺ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.

Results

The CD14⁺CD16⁺ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c⁺ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c⁺/Ly6G⁺/IL-10⁺ in the BALB/c mice.

Conclusions

Galectin-1 can induce tDCs after internalizing into CD14⁺ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.

General significance

Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.     

Toll-like receptor 6 drives differentiation of tolerogenic dendritic cells and contributes to LcrV-mediated plague pathogenesis

Educating dendritic cells (DC) to become tolerogenic DC, which promote regulatory IL-10 immune responses, represents an effective immune evasion strategy for pathogens. Yersinia pestis virulence factor LcrV is reported to induce IL-10 production via interaction with Toll-like receptor (TLR) 2. However, TLR2-/- mice are not protected against subcutaneous plague infection. Using complementary in vitro and in vivo approaches and LcrV as a model, we show that TLR6 associates with TLR2 to induce tolerogenic DC and regulatory type-1 T cells selectively secreting IL-10. In contrast, TLR1 heterodimerizes with TLR2 to promote proinflammatory IL-12p40 cytokine, producing DC and inflammatory T cell differentiation. LcrV specifically hijacks the TLR2/6 pathway to stimulate IL-10 production, which blocks host protective inflammatory responses. These results explain why TLR2 can mediate both pro- and anti-inflammatory responses and identify TLR6 as a distinct receptor driving regulatory IL-10 responses.     

IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor alpha-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-alphabeta, and in purified murine splenic DCs treated with IFN-alphabeta in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-gamma by DC and an enhanced ability of DCs to stimulate Ag-specific CD8(+) T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-alphabeta receptor, suggesting a role for IFN-alphabeta in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-alphabeta.     

Transfer of regulatory properties from tolerogenic to proinflammatory dendritic cells via induced autoreactive regulatory T cells

Infectious tolerance is a term generally assigned to the process through which regulatory T cells (Tregs) transfer immunoregulatory properties to other T cells. In this study, we demonstrated that a similar process applies to human dendritic cells (DCs), albeit through a different mechanism. We induced and cloned proinsulin-specific Tregs using tolerogenic DCs and investigated mechanisms by which induced Ag-specific regulatory T cells (iaTregs) endorse the suppressive effects. iaTregs expressed FOXP3, programmed death-1, and membrane-bound TGF-β and upregulated IL-10 and CTLA-4 after stimulation with the cognate Ag. The iaTregs suppressed effector T cells only when both encountered the cognate Ags on the same APCs (linked suppression). This occurred independently of IL-10, TGF-β, programmed death-1, or CTLA-4. Instead, iaTregs used a granzyme B-mediated mechanism to kill B cells and monocytes, whereas proinflammatory DCs that resisted being killed were induced to upregulate the inhibitory receptors B7 (family) homolog 3 and ICOS ligand. These re-educated mature monocyte-derived dendritic cells (mDCs) suppressed effector T cells and induced IL-10-producing cells from the naive T cell pool. Our data indicated that human tolerogenic DCs confer infectious tolerance by inducing Ag-specific Tregs, which, in turn, re-educate proinflammatory mature DCs into DCs with regulatory properties.     

Identification of an S-adenosylhomocysteine hydrolase-like transcript induced during dendritic cell differentiation

Dendritic cells (DC) are the professional antigen-presenting cells that initiate immune responses. While DC take up antigen, migrate to lymph nodes and present processed antigen to T lymphocytes, little is known of the intracellular biochemical pathways controlling these events. Using the differential display technique, employing the activated blood DC-like cell line L428, we isolated a cDNA induced during DC differentiation likely to have a regulatory function. This cDNA encoded a putative 530-amino-acid (aa) protein consisting of a unique hydrophilic domain (106 aa) and a domain (424 aa) similar to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (AHCY). Therefore, this molecule was termed DC-expressed AHCY-like molecule (DCAL). DCAL mRNA was expressed moderately in fresh blood DC, but was not detectable in other peripheral blood mononuclear cells. DCAL mRNA increased markedly during activation of blood and skin DC (Langerhans cells), but was diminished in terminally differentiated tonsil DC. Cultured monocytes expressed little DCAL mRNA, but levels increased markedly when differentiated into DC by cytokines GM-CSF and IL-4. The DCAL gene [Chromosome (Chr) 1] and another previously identified DCAL-like molecule KIAA0828 (Chr 7) differed from the AHCY gene (Chr 20) in gene organization. Thus, DCAL may have a role in controlling critical events in DC differentiation and belong to a novel family of AHCY-like molecules.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.     

1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.     

zVAD-fmk对未成熟树突状细胞诱导初始性CD4＋T细胞分化为调节性T细胞影响的体外研究

目的 研究 Caspase 通路在未成熟树突状细胞（imDC）诱导同种异体 CD4＋ T 细胞转化为调节性 T 细胞（Treg）中的作用及探讨免疫耐受机制建立可能的分子机制.方法 将人外周血分离、培养出的imDC 与健康胎儿脐血中分离CD4＋ T 细胞混合培养,同时加入zVAD-fmk,以流式细胞仪检测CD4＋CD25＋ T 细胞（Treg 细胞）转化率.结果 （1）imDC 的鉴定：外周血经诱导后分离的imDC,以流式细胞仪检测细胞表面分子,imDC 表面分子表达的结果：CD80（7.27 ± 0.13）、CD83（3.53 ± 0.35）、CD1a（4.29 ± 0.27）;（2）混合培养后CD4＋CD25＋T细胞的转化率结果为：空白组（1.78 ± 0.11）﹪、对照组（22.23 ± 0.77）﹪、低浓度zVAD-fmk 组（21.63 ± 0.82）﹪、中浓度zVAD-fmk 组（12.24 ± 0.54）﹪、高浓度zVAD-fmk 组（12.20 ± 0.96）﹪,结果对照组和低浓度组、中浓度组和高浓度组间比较,P 〉 0.05,其余各组间比较,P 〈 0.05.加入zVAD-fmk 并与imDC 细胞混合培养的T 细胞转化率相对于未加入阻断剂的T 细胞较低,同时Caspase 信号通路对zVAD-fmk 无浓度依赖性.结论 imDC 可以诱导同种异体初始性CD4＋ T 细胞分化为Treg.Caspase 信号通路特异性的阻断剂zVAD-fmk 可以部分抑制Treg 的转化,说明Caspase 信号通路在诱导免疫耐受中可能起了较为重要的作用.     

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.     

Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro

Background

Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action.

Methodology/Principal Findings

We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro.

Conclusions

This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.     

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.     

Involvement of eicosanoids in the hypothermic response to lipopolysaccharide during endotoxemia in rats

Hypothermia is one of the prominent features of the acute phase response to endotoxin (LPS). This study was undertaken to elucidate the effects of the COX-inhibitor Indomethacin (INDO) and the selective FLAP inhibitor MK-886 on LPS-induced hypothermia, mortality and increase in production of hypothalamic prostaglandin E(2) (PGE(2)) and leukotriene during endotoxemia.It has been demonstrated that INDO and MK-886 significantly attenuate the hypothermia induced by LPS, but MK-886 has a lesser (protective) effect than INDO. Only INDO was found to attenuate significantly the hyperthermic response to LPS. Furthermore, INDO significantly reduced the elevation in hypothalamic PGE(2) levels. MK-886 significantly reduced the elevation in hypothalamic leukotriene production only when LPS was given in a dose of 1mg/kg. Both drugs failed to reduce the elevation in plasma TNF-alpha and mortality induced by LPS.We conclude that in rats, febrile response to endotoxin involves many inflammatory mediators. However, it seems that PGE(2) and leukotrienes do not have a pivotal role in the mechanism of LPS-induced mortality.