CD83+CCR7+ NK cells induced by interleukin 18 by dendritic cells promote experimental autoimmune uveitis

Natural killer (NK) cells have been reported to play a pathological role in autoimmune uveitis. However, the mechanisms regarding NK cells in uveitis and factors that affect NK‐cell activation in this condition remain unclear. Here, we report that the number of CD3^‐NK1.1⁺CD83⁺CCR7⁺ cells is increased in the inflamed eyes within a mouse model of experimental autoimmune uveitis (EAU), and these cells express elevated levels of NKG2D, CD69 and IFN‐γ. Adoptively transferring CD83⁺CCR7⁺NK cells aggravates EAU symptoms and increases the number of CD4⁺IFN‐γ⁺T cells and dendritic cells (DCs) within the eye. These CD83⁺CCR7⁺NK cells then promote the maturation of DCs and IFN‐γ expression within T cells as demonstrated in vitro. Furthermore, IL‐18, as primarily secreted by DCs in the eyes, is detected to induce CD83⁺CCR7⁺NK cells. In EAU mice, anti‐IL‐18R antibody treatment also decreases retinal tissue damage, as well as the number of infiltrating CD83⁺CCR7⁺NK cells, T cells and DCs in the inflamed eyes and spleens of EAU mice. These results suggest that CD83⁺CCR7⁺NK cells, as induced by IL‐18 that primarily secreted by DCs, play a critical pathological role in EAU. Anti‐IL‐18R antibody might serve as a potential therapeutic agent for uveitis through its capacity to inhibit CD83⁺CCR7⁺NK cells infiltration.     

Mycobacterium abscessus MAB2560 induces maturation of dendritic cells via Toll-like receptor 4 and drives Th1 immune response

In this study, we showed that Mycobacterium abscessus MAB2560 induces the maturation of dendritic cells (DCs), which are representative antigen-presenting cells (APCs). M. abscessus MAB2560 stimulate the production of pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-12p70] and reduce the endocytic capacity and maturation of DCs. Using TLR4^-/- DCs, we found that MAB2560 mediated DC maturation via Toll-like receptor 4 (TLR4). MAB2560 also activated the MAPK signaling pathway, which was essential for DC maturation. Furthermore, MAB2560-treated DCs induced the transformation of naïve T cells to polarized CD4⁺ and CD8⁺ T cells, which would be crucial for Th1 polarization of the immune response. Taken together, our results indicate that MAB2560 could potentially regulate the host immune response to M. abscessus and may have critical implications for the manipulation of DC functions for developing DC-based immunotherapy. [BMB Reports 2014;47(9): 512-517]     

Metabolic Effects of Cholecystectomy: Gallbladder Ablation Increases Basal Metabolic Rate through G-Protein Coupled Bile Acid Receptor Gpbar1-Dependent Mechanisms in Mice

Background & Aims

Bile acids (BAs) regulate energy expenditure by activating G-protein Coupled Bile Acid Receptor Gpbar1/TGR5 by cAMP-dependent mechanisms. Cholecystectomy (XGB) increases BAs recirculation rates resulting in increased tissue exposure to BAs during the light phase of the diurnal cycle in mice. We aimed to determine: 1) the effects of XGB on basal metabolic rate (BMR) and 2) the roles of TGR5 on XGB-dependent changes in BMR.

Methods

BMR was determined by indirect calorimetry in wild type and Tgr5 deficient (Tgr5^-/-) male mice. Bile flow and BAs secretion rates were measured by surgical diversion of biliary duct. Biliary BAs and cholesterol were quantified by enzymatic methods. BAs serum concentration and specific composition was determined by liquid chromatography/tandem mass spectrometry. Gene expression was determined by qPCR analysis.

Results

XGB increased biliary BAs and cholesterol secretion rates, and elevated serum BAs concentration in wild type and Tgr5^-/- mice during the light phase of the diurnal cycle. BMR was ~25% higher in cholecystectomized wild type mice (p <0.02), whereas no changes were detected in cholecystectomized Tgr5^-/- mice compared to wild-type animals.

Conclusion

XGB increases BMR by TGR5-dependent mechanisms in mice.     

Exopolysaccharide Produced by Lactobacillus Plantarum Induces Maturation of Dendritic Cells in BALB/c Mice

Lactobacillus plantarum (L. plantarum) exopolysaccharide (EPS) is an important bioactive component in fermented functional foods. However, there is a lack of data concerning the effects of L. plantarum EPS on maturation of mouse dendritic cells (DCs). In this study, we purified L. plantarum EPS and examined its effects on cytokines production by dendritic cells in serum and intestinal fluid of BALB/c mice, then investigated its effects on phenotypic and functional maturation of mouse bone marrow-derived dendritic cells (BMDCs). Cytokines (nitric oxide, IL-12p70, IL-10 and RANTES) in serum and intestinal fluid were analyzed by enzyme linked immunosorbent assay (ELISA) after the mice received EPS for 2, 5 and 7 days, respectively. DCs derived from bone marrow of BALB/c mouse were treated with EPS, then the phenotypic maturation of BMDCs was analyzed using flow cytometer and the functional maturation of BMDCs was analyzed by ELISA, and, lastly, mixed lymphocyte proliferation was performed. We found the molecular weight of purified EPS was approximately 2.4×10⁶ Da and it was composed of ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2:1:1:10:4:205:215. We observed that L. plantarum EPS enriched production of nitric oxide, IL-12p70 and RANTES, and decreased the secretion of IL-10 in the serum or intestinal fluid as well as in the supernatant of DCs treated with the EPS. The EPS also up-regulated the expression of MHC II and CD86 on DCs surface and promoted T cells to proliferate in vitro. Our data provide direct evidence to suggest that L. plantarum EPS can effectively induce maturation of DCs in mice.     

Impaired pro-inflammatory cytokine production and increased Th2-biasing ability of dendritic cells exposed to Taenia excreted/secreted antigens: A critical role for carbohydrates but not for STAT6 signaling

In cysticercosis, a parasitic disease caused by cestodes, the details of early interactions between parasite antigens and innate cells from the host are not well understood. In this study, the role of cestode-conditioned dendritic cells (DCs) in priming Th1 versus Th2 responses to bystander antigen was examined by using CD11c⁺ DCs as antigen-presenting cells and naive CD4⁺ DO11.10 lymphocytes specific to ovalbumin (OVA) as responding cells. No conventional maturation was induced in DCs exposed to Taenia crassiceps excreted/secreted antigens (TcES). The ability of TcES to affect Toll-like receptor (TLR)-mediated maturation and the pro-inflammatory response was analyzed by co-pulsing DCs with TcES and TLR ligands. DCs exposed to TcES blocked TLR4, TLR9 and Toxoplasma soluble antigen-induced phenotypic maturation. TcES-exposed DCs also blocked secretion of pro-inflammatory cytokines and alloreactive T cell proliferation, while preserving IL-10 production. DCs pulsed with TcES + OVA suppressed IFN-γ, whereas they induced greater IL-4 production by CD4⁺ DO11.10 cells. TcES with chemically-altered glycans failed to modulate TLR-mediated activation of DCs and their Th1-inhibitng ability, which was STAT6-independent. Our results reflect the capacity of TcES glyco-antigens to modulate Th1-type and inflammatory responses mediated through DC activation.     

Dectin-1/TLR2 and NOD2 Agonists Render Dendritic Cells Susceptible to Infection by X4-Using HIV-1 and Promote cis-Infection of CD4+ T Cells

HIV-1 pathogenesis is intimately linked with microbial infections and innate immunity during all stages of the disease. While the impact of microbial-derived products in transmission of R5-using virus to CD4⁺ T cells by dendritic cells (DCs) has been addressed before, very limited data are available on the effect of such compounds on DC-mediated dissemination of X4-tropic variant. Here, we provide evidence that treatment of DCs with dectin-1/TLR2 and NOD2 ligands increases cis-infection of autologous CD4⁺ T cells by X4-using virus. This phenomenon is most likely associated with an enhanced permissiveness of DCs to productive infection with X4 virus, which is linked to increased surface expression of CXCR4 and the acquisition of a maturation profile by DCs. The ensuing DC maturation enhances susceptibility of CD4⁺ T cells to productive infection with HIV-1. This study highlights the crucial role of DCs at different stages of HIV-1 infection and particularly in spreading of viral strains displaying a X4 phenotype.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

The Formylpeptide Receptor 2 (Fpr2) and Its Endogenous Ligand Cathelin-related Antimicrobial Peptide (CRAMP) Promote Dendritic Cell Maturation

Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2^−/−) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2^−/− mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2^−/− mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2^−/− DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP^−/− mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.     

The effects of 1,25-dihydroxyvitamin D3 on markers related to the differentiation and maturation of bone marrow-derived dendritic cells from control and obese mice

Vitamin D has been reported to regulate the maturation and function of dendritic cells (DCs). Obesity was shown to be associated with the dysregulation of vitamin D metabolism and malfunction of DCs. We investigated the effects of in vitro 1,25(OH)₂D₃ treatment (0, 1, or 10 nM) on phenotype and expression of genes related to function of bone marrow-derived DCs (BMDCs) from control and obese mice. C57BL/6 N mice were fed a control or high-fat (10% or 45% kcal fat: CON or HFD) diets for 15 weeks. Differentiation toward DCs was induced with GM-CSF (20 ng/ml) and maturation was induced by LPS (50 ng/ml); 10 nM 1,25(OH)₂D₃ treatment inhibited BMDC differentiation (CD11c⁺) and decreased the percentage of mature DCs (MHCII^highCD11c⁺ and CD86^highCD11c⁺) in both CON and HFD groups. The Il10 expression in stimulated BMDCs from the CON group increased with the 10 nM 1,25(OH)₂D₃ treatment, but not in those from the HFD group. The Il12b mRNA levels in stimulated BMDCs were lower in the HFD group than in the CON group. In conclusion, lower levels of Cd 40, Cd83 and Il12 mRNA in LPS-stimulated BMDCs from obese mice suggest malfunction of DCs as antigen presenting cells. 1,25(OH)₂D₃ treatment inhibited the differentiation and maturation of BMDCs in both control and obese mice. Differential effects of 1,25(OH)₂D₃ on the expression of Il10 between control and obese mice suggest that regulation of immune response by vitamin D could be influenced by obesity.     

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8⁺ T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.     

Hepatitis B virus surface antigen can activate human monocyte-derived dendritic cells by nuclear factor kappa B and p38 mitogen-activated protein kinase mediated signaling

Hepatitis B virus Ag (HBsAg), a major antigen of hepatitis B virus (HBV), is also a vaccine component for prevention of HBV infection. Dendritic cells (DCs) of HBV carriers reportedly exhibit functional impairment. In this study, the aim was to investigate the effect of HBsAg on activation of human monocyte-derived dendritic cells (MD-DCs), and the subsequent signal transduction pathway. Treatment of MD-DCs with HBsAg resulted in enhanced cell surface expression of cluster of differentiation 80, CD83, CD86 and major histocompatibility complex class II, and increased interleukin (IL)-12 p40, IL-12p70, and IL-10 production. Furthermore, HBsAg treatment of MD-DCs with HBsAg resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon and IL-10. The pathway of MD-DCs activation by HBsAg was further investigated in the present study. Inhibition of nuclear factor (NF)-kappa B (κB) by helenalin and p38 mitogen-activated protein kinase (MAPK) by SB203580 prevented production of IL-12 p40, IL-12 p70, and IL-10. HBsAg also augmented MAPK phosphorylation. Thus, cytokine secretion of human MD-DCs by HBsAg is blocked by inhibition of the NF-κB and p38 MAPK pathways. Likewise, decreased inhibition of kappa B alpha concentrations and MAPK phosphorylation are critical for MD-DC maturation by HBsAg. These findings may provide a strategy for improving the prophylactic and therapeutic efficacy of vaccines and tumor therapies that utilize these pathways.     

Tolerogenic Dendritic Cells That Inhibit Autoimmune Arthritis Can Be Induced by a Combination of Carvacrol and Thermal Stress

Tolerogenic dendritic cells (DCs) can induce regulatory T cells and dampen pathogenic T cell responses. Therefore, they are possible therapeutic targets in autoimmune diseases. In this study we investigated whether mouse tolerogenic DCs are induced by the phytonutrient carvacrol, a molecule with known anti-inflammatory properties, in combination with a physiological stress. We show that treatment of DCs with carvacrol and thermal stress led to the mRNA expression of both pro- and anti-inflammatory mediators. Interestingly, treated DCs with this mixed gene expression profile had a reduced ability to activate pro-inflammatory T cells. Furthermore, these DCs increased the proportion of FoxP3⁺ regulatory T cells. In vivo, prophylactic injection of carvacrol-thermal stress treated DCs pulsed with the disease inducing antigen was able to suppress disease in a mouse model of arthritis. These findings suggest that treatment of mouse bone marrow derived DCs with carvacrol and thermal stress induce a functionally tolerogenic DC that can suppress autoimmune arthritis. Herewith carvacrol seems to offer novel opportunities for the development of a dietary based intervention in chronic inflammatory diseases.     

MIF synergizes with Trypanosoma cruzi antigens to promote efficient dendritic cell maturation and IL-12 production via p38 MAPK

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of the MIF-dependent responses to Trypanosoma cruzi, we investigated host resistance in MIF-/- mice (on the BALB/c background) during an intraperitoneal infection. We focused on the potential involvement of MIF in dendritic cell (DC) maturation and cytokine production. Following a challenge with 5 x 10³ T. cruzi parasites, wild type (WT) mice developed a strong IL-12 response and adequate maturation of the draining mesenteric lymph node DCs and were resistant to infection. In contrast, similarly infected MIF^-/- mice mounted a weak IL-12 response, displayed immature DCs in the early phases of infection and rapidly succumbed to T. cruzi infection. The lack of maturation and IL-12 production by the DCs in response to total T. cruzi antigen (TcAg) was confirmed by in vitro studies. These effects were reversed following treatment with recombinant MIF. Interestingly, TcAg-stimulated bone marrow-derived DCs from both WT and MIF^-/- mice had increased ERK1/2 MAPK phosphorylation. In contrast, p38 phosphorylation was only upregulated in WT DCs. Reconstitution of MIF to MIF^-/- DCs upregulated p38 phosphorylation. The MIF-p38 pathway affected MHC-II and CD86 expression as well as IL-12 production. These findings demonstrate that the MIF-induced early DC maturation and IL-12 production mediates resistance to T. cruzi infection, probably by activating the p38 pathway.     

Effect of double-stranded DNA on maturation of dendritic cells in vitro

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5 μg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.     

25(OH)D3 Affects the Maturation and Function of Mouse Bone Marrow-Derived Dendritic Cells Stimulated by Mycobacterium Bovis BCG

It has been shown that vitamin D deficiency increases an individual’s susceptibility to tuberculosis (TB). However, very little is known about the effect of vitamin D on the immune response to Mycobacterium tuberculosis (M. tb) in dendritic cells (DCs). Because DCs play an important role in TB infection, we investigated the phenotypic characteristics and functional capabilities of mouse bone marrow-derived dendritic cells (BMDCs) after stimulation with Bacillus Calmette-Guérin (BCG) in the presence or absence of 25(OH)D₃(100 nM). Bone marrow cells from mice were cultured with GM-CSF (20 ng/ml) and were then treated with 25(OH)D₃ for 7 days. On day 6, 5 µg/ml of BCG (≥1.0×10⁶ CFU/mg) was added to the cells for 24 hours, and on day 7, the non-adherent cells were harvested for phenotypic and functional analyses. After incubation with 25(OH)D₃, the expression levels of MHC-II and CD86 on the surface of the dendritic cells (DCs) and the ability of the DCs to stimulate proliferation of allogeneic mixed lymphocytes were lower than control cells (p<0.05). Furthermore, the level of Interleukin (IL) -4 secreted by the BMDCs in the 25(OH)D₃ culture was lower than that in the control culture (p<0.01). However, the BMDCs cultured with 25(OH)D₃ produced significantly higher levels of IL-2, IL-6, IL-10 and interferon gamma(IFN-γ) than those in the control culture (p<0.05). These findings suggest that 25(OH)D₃ modulates the immune response during mycobacterial infection by affecting the maturation and function of DCs.     

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4CD25Foxp3 regulatory T cells and down-regulates cardiac allograft rejection

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4⁺CD25^highFoxp3⁺ regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.