Splenic stroma-educated regulatory dendritic cells induce apoptosis of activated CD4 T cells via Fas ligand-enhanced IFN-γ and nitric oxide

Stromal microenvironments of bone marrow, lymph nodes, and spleen have been shown to be able to regulate immune cell differentiation and function. Our previous studies demonstrate that splenic stroma could drive mature dendritic cells (DC) to further proliferate and differentiate into regulatory DC subset that could inhibit T cell response via NO. However, how splenic stroma-educated regulatory DC release NO and whether other molecules are involved in the suppression of T cell response remain unclear. In this study, we show that splenic stroma educates regulatory DC to express high level of Fas ligand (FasL) by TGF-β via ERK activation. The findings, that inhibition of CD4 T cell proliferation by regulatory DC required cell-to-cell contact and FasL deficiency impaired inhibitory effect of regulatory DC, indicate that regulatory DC inhibit CD4 T cell proliferation via FasL. Then, regulatory DC have been found to be able to induce apoptosis of activated CD4 T cells via FasL in caspase 8- and caspase 3-dependent manner. Interestingly, FasL on regulatory DC enhanced IFN-γ production from activated CD4 T cells, and in turn T cell-derived IFN-γ induced NO production from regulatory DC, working jointly to induce apoptosis of activated CD4 T cells. Blockade of IFN-γ and NO could reduce the apoptosis induction. Therefore, our results demonstrated that splenic stroma-educated regulatory DC induced T cell apoptosis via FasL-enhanced T cell IFN-γ and DC NO production, thus outlining a new way for negative regulation of T cell responses and maintenance of immune homeostasis by regulatory DC and splenic stromal microenvironment.     

Monophosphoryl lipid A plus IFNγ maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNγ generates mature DCs that produce IL-12 and polarize CD4⁺ T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNγ induce superior tumour antigen-specific CD8⁺ CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNγ DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNγ DCs. The high amounts of induced CTLs are functional as they produce IFNγ and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNγ DCs are very promising for the use in future cancer immunotherapy.     

IL-6 and IFN-α from dsRNA-stimulated dendritic cells control expansion of regulatory T cells

Foxp3⁺CD4⁺ regulatory T cells (Treg) control not only autoimmunity but also the effective immune response against RNA virus infections, which produces virus-derived double-stranded RNA (dsRNA). To induce effective anti-viral immunity, it is a key issue to learn how Treg respond to dsRNA in vitro and in vivo. We here showed that synthetic dsRNA, polyI:C, caused peripheral expansion of functional Treg in a TICAM-1- and IL-6-dependent manner in vivo. PolyI:C did not expand Treg directly, but promoted the expansion of naturally occurring Treg indirectly through IL-6 produced from dendritic cells (DCs). In addition, the expansion of Treg by IL-6 was inhibited by IFN-α from polyI:C-stimulated DCs. These data suggest that the balance of IL-6 and IFN-α in the region of RNA virus infection may determine the number of peripheral Treg, which affects the effective immune responses against viruses.     

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36 ± 3 μM in CCD841 cells and 0.5 ± 0.1 μM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.     

Atg5 but not Atg7 in dendritic cells enhances IL-2 and IFN-γ production by Toxoplasma gondii-reactive CD4+ T cells

The autophagy proteins (Atg) modulate not only innate but also adaptive immunity against pathogens. We examined the role of dendritic cell Atg5 and Atg7 in the production of IL-2 and IFN-γ by Toxoplasma gondii-reactive CD4⁺ T cells. T. gondii-reactive mouse CD4⁺ T cells exhibited unimpaired production of IL-2 and IFN-γ when stimulated with Atg7-deficient mouse dendritic cells that were infected with T. gondii or pulsed with T. gondii lysate antigens. In marked contrast, dendritic cells deficient in Atg5 induced diminished CD4⁺ T cell production of IL-2 and IFN-γ. This defect was not accompanied by changes in costimulatory ligand expression on dendritic cells or impaired production of IL-12 p70, IL-1β or TNF-α. Knockdown of Irg6a in dendritic cells did not affect CD4⁺ T cell cytokine production. These results indicate that Atg5 and Atg7 in dendritic cells play differential roles in the modulation of IL-2 and IFN-γ production by T. gondii-reactive CD4⁺ T cells.     

Monocyte-produced prostaglandin induces Fcγ receptor expression on human T cells

Incubation of human T cells for 18 hr with prostaglandin E₂ (PGE₂ 3 × 10^?6M) causes a slight but significant increase in the percentage of Tγ cells and a reduction in Tμ cells. When PGE was added to “non-Tγ” cells, the increase in the percentage of Tγ cells was more marked (from 1.5% Tγ without PGE to 11% Tγ with PGE₂, P < 0.001). Supernates from cultures of human monocytes also caused an increase in Tγ cells (10% Tγ without supernate to 18% with supernate, P < 0.01), and this increase was blocked if the monocytes were cultured with indomethacin, a prostaglandin synthetase inhibitor (9% Tγ cells). Thus, monocytes may regulate Fcγ receptors on T cells via PGE₂ production.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

In vitro sensitization with HSV-lnfected cells induces Tμ cells to become cytotoxic Tγ cells which are theophylline resistant

Following in vitro sensitization with HSV-infected cells, T_γ cells comprise most of the cytotoxic effector cell population. However, whereas freshly obtained T_γ cells exhibit theophylline sensitivity in the sheep erythrocyte rosette assay, presensitized T_γ cells are theophylline resistant. Similarly, when T cells are fractionated according to their theophylline sensitivity before the sensitization culture, theophylline-resistant T_μ cells appear as the precursors of T_γ cytotoxic effector cells, the T_μ-T_γ switch occurring with a transitory eclipse of Fc receptors, and maintenance of theophylline resistance.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

Thymic stromal lymphopoietin induces tight junction protein claudin-7 via NF-κB in dendritic cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell (DC)-mediated Th2-type inflammatory responses. The activated DCs can penetrate the epithelium to directly take up antigen without compromising the barrier function. Although it is reported that DCs express tight junction molecules and can establish tight junction-like structures with adjacent epithelial cells to preserve the epithelial barrier, the regulation of expression of tight junction molecules in DCs remains unknown. In the present study, to investigate the mechanical regulation of expression of tight junction molecules in DCs, XS52 DCs that was a long-term DC line established from the epidermis of a newborn BALB/c mouse, were treated with TSLP or toll-like receptor (TLR) ligands. In XS52 cells, tight junction molecules claudin-1, -3, -4, -6, -7, -8, and occludin were detected. mRNA expression of TSLP receptor and all these tight junction molecules was significantly increased in activated XS52 cells after treatment with TSLP. In addition, expression of claudin-7 protein was increased in dose- and time-dependent manner. In XS52 cells, which express TLR2, TLR3, TLR4, and TLR7, but not TLR9, expression of claudin-7 protein was also increased after treatment with ligands of TLR2, TLR4 or TLR7/8, Pam₃Cys-Ser-(Lys)₄, LPS, or CL097. The NF-κB inhibitor IMD-0354 prevented upregulation of claudin-7 after treatment with TSLP or TLR ligands. These findings indicate that TSLP induces expression of tight junction protein claudin-7 in DCs via NF-κB as well as via TLRs and may control tight junctions of DCs to preserve the epithelial barrier during allergic inflammation.     

25-OCH3-PPD induces the apoptosis of activated t-HSC/Cl-6 cells via c-FLIP-mediated NF-κB activation

25-OCH(3)-PPD is a dammarane-type triterpene sapogenin isolated from the roots, leaves and seeds of Panax notoginseng, which has shown anti-tumor effects in several human cancer lines. In this study, we evaluated the effects of 25-OCH(3)-PPD on apoptosis of activated t-HSC/Cl-6 cells induced by tumor necrosis factor-α (TNF-α). The inhibitory effects of eleven compounds isolated from Panax ginseng and P. notoginseng were detected in activated t-HSC/Cl-6 cells. 25-OCH(3)-PPD produced a significant inhibitory effect on activated t-HSC/Cl-6 cells. However, 25-OCH(3)-PPD showed almost no effect on the cell viability of Chang liver cells, a type of normal human hepatic cell line. Therefore, we aimed to determine the anti-fibrotic potential of 25-OCH(3)-PPD and to characterize the signal transduction pathways involved in activated HSCs. 25-OCH(3)-PPD decreased the fibrosis markers, including α-smooth muscle actin (α-SMA), transforming growth factor β-1 (TGF-β1) and tissue inhibitors of metalloproteinases-1 (TIMP-1). 25-OCH(3)-PPD elevated the level of cellular GSH in activated HSCs, which demonstrated that 25-OCH(3)-PPD might inhibit HSC activation by its antioxidant capacity. Further analyses revealed that 25-OCH(3)-PPD increased the levels of cleaved caspase-3, decreased the ratio of Bcl-2/Bax and the expression of survivin via c-FLIP-mediated NF-κB activation and shed light on the regulation of apoptosis. Therefore, 25-OCH(3)-PPD may prove to be an excellent candidate agent for the therapy of hepatic fibrosis.     

Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Dichotomous effects of IFN-γ on dendritic cell function determine the extent of IL-12-driven antitumor T cell immunity

Sustained intratumoral delivery of IL-12 and GM-CSF can overcome tumor immune suppression and promote T cell-dependent eradication of established disease in murine tumor models. However, the antitumor effector response is transient and rapidly followed by a T suppressor cell rebound. The mechanisms that control the switch from an effector to a regulatory response in this model have not been defined. Because dendritic cells (DC) can mediate both effector and suppressor T cell priming, DC activity was monitored in the tumors and the tumor-draining lymph nodes (TDLN) of IL-12/GM-CSF-treated mice. The studies demonstrated that therapy promoted the recruitment of immunogenic DC (iDC) to tumors with subsequent migration to the TDLN within 24-48 h of treatment. Longer-term monitoring revealed that iDC converted to an IDO-positive tolerogenic phenotype in the TDLN between days 2 and 7. Specifically, day 7 DC lost the ability to prime CD8(+) T cells but preferentially induced CD4(+)Foxp3(+) T cells. The functional switch was reversible, as inhibition of IDO with 1-methyl tryptophan restored immunogenic function to tolerogenic DC. All posttherapy immunological activity was strictly associated with conventional myeloid DC, and no functional changes were observed in the plasmacytoid DC subset throughout treatment. Importantly, the initial recruitment and activation of iDC as well as the subsequent switch to tolerogenic activity were both driven by IFN-γ, revealing the dichotomous role of this cytokine in regulating IL-12-mediated antitumor T cell immunity.     

Nicotine modulates the immunological function of dendritic cells through peroxisome proliferator-activated receptor-γ upregulation

We examined the effects of nicotine on differentiation and function of monocyte-derived human dendritic cells (DCs). NiDCs, which were the DCs differentiated in the presence of nicotine, showed lower levels of CD1a. Secretion of IL-12 and TNF-α by lipopolysaccharide (LPS)-stimulated NiDCs was significantly suppressed compared to monocyte-derived DCs grown without nicotine. NiDCs displayed a diminished capacity to induce allogeneic T cell proliferation with a reduced production of IFN-γ, and maintained/enhanced LPS-mediated expression of coinhibitory molecules. Interestingly, NiDCs enhanced the expression of nuclear receptor peroxisome proliferator-activated receptors γ (PPAR γ), which has immunomodulatory properties. Expression of PPAR γ and PPAR γ-target genes was significantly inhibited by pretreatment with d-tubocurarine, antagonist of non-selective nicotinic acetylcholine receptors (nAChR). In addition, reduction of Th1 responses was inhibited after blocking nAChR-mediated signal. These data suggest the effect of nicotine on altering DC immunogenicity by impeding Th1 immunity is partially mediated by upregulation of PPAR γ.     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.     

α-Mangostin induces apoptosis and suppresses differentiation of 3T3-L1 cells via inhibiting fatty acid synthase

α-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC 2.3.1.85). Our studies showed that the cytotoxicity of α-mangostin with IC(50) value of 20 μM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (ΔΨm) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 μM or 100 μM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by α-mangostin was via inhibition of FAS. Futhermore, α-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of α-mangostin than mature adipocytes. Further studies showed that α-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that α-mangostin might be useful for preventing or treating obesity.     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.