Inhibition Of Dendritic Cells Maturation Research Articles

Bicyclic eremophilane-type petasite sesquiterpenes potentiate peroxisome proliferator-activated receptor γ activator-mediated inhibition of dendritic cells.

Dendritic cell (DC) activation induces expression of co-stimulatory surface molecules, as well as migration into secondary lymphoid organs, where they activate naïve T-cells. A family of plant derivatives, eremophilane-type petasite sesquiterpenes, can regulate the immune system through DC targeting due to their anti-inflammatory effects. Peroxisome proliferator–activated receptor gamma (PPARγ) is involved in inhibition of inflammatory responses and induction of DCs to acquire a mucosal phenotype. Since mucosal DCs are central in innate immune responses, we hypothesized that eremophilane-type petasite sesquiterpenes exerted their anti-inflammatory effects by inhibiting DC maturation and activation through PPARγ. This study assessed the bicyclic eremophilane-type petasite sesquiterpene compounds Fukinone and 10βH-8α,12-Epidioxyeremophil-7(11)-en-8β-ol (ZYFDC21 and ZYFDC22) in the maturation and activation of mouse DC. We measured surface expression of co-stimulatory molecules by flow cytometry and cell-free supernatant cytokine production upon lipopolysaccharide stimulation by enzyme-linked immunosorbent assays (ELISAs) in the presence or absence of PPARγ agonists. DCs were generated from C57BL/6 mice bone marrow cells and harvested. Cells were exposed to bicyclic eremophilane-type petasite sesquiterpenes ZYFDC21 or ZYFDC22 in the presence or absence of synthetic PPARγ agonists (GW1929 and TGZ) or the natural PPARγ ligand 15d-PGJ2, followed by overnight activation with LPS. We observed differences in the upregulation of surface expression of CD86, along with TNF, IL-6, and IL-12p70 released by DCs stimulated with LPS, when using combinations of bicyclic eremophilane-type petasite sesquiterpenes ZYFDC21 or ZYFDC22, and PPARγ agonists, in particular the PPARγ ligand 15d-PGJ2. Our results indicate that bicyclic eremophilane-type petasite sesquiterpenes ZYFDC21 or ZYFDC22 inhibit maturation and activation of DC, and this activity is augmented upon PPARγ activation.

Delayed Ischemic Preconditioning Attenuated Renal Ischemia-Reperfusion Injury by Inhibiting Dendritic Cell Maturation

Background/Aims: Even though delayed ischemic preconditioning (DIPC) has been reported to produce renal protection, the underlying mechanism remains poorly understood. We reported that a 15-minute renal ischemic preconditioning (IPC) 4 days before subsequent ischemia-reperfusion attenuated renal injury Kidney dendritic cells (DCs) are abundant in the renal tubulointerstitium and, depending on their status, can induce immune activation or tolerance. The aim of the present study was to investigate the role of DCs in IPC of the kidney. Methods: Mouse kidneys were challenged by transient brief episodes of sublethal ischemia followed by subsequent prolonged ischemia. DC abundance and maturation in the spleen and kidney were measured by flow cytometry and immunohistochemical staining. To confirm the function of mature DCs in the renoprotective effect of IPC on renal ischemia-reperfusion injury the A2 adenosine receptor (A2AR) antagonist SCH58261 was administered to stimulate DC maturation prior to assessment of renal functional and histological injury and the inflammatory reaction. Results: Compared with sham-operated animals, preconditioned mice had a reduced injury with less CD11c⁺ cells, lower levels of the pro-inflammatory cytokine IL-17 and reduced expression of the mature DC marker CCR7. Preconditioned mice also produced more of the anti-inflammatory cytokine IL-10. Both renal cells and splenocytes from these mice had more DCs (CD45⁺/CD11c⁺/F4/80^-), but fewer of these DCs were mature (CD45⁺/CD11c⁺/ F4/80^-/MHC-II⁺/CD80⁺) compared with those from sham-treated animals, suggesting that the immunomodulatory effect of renal ischemic preconditioning is both local and systemic. Additionally, injection of the A2AR antagonist SCH58261 reversed IPC-induced inhibition of DC maturation and mitigated the protective effect of preconditioning, suggesting that DC maturation contributes to immune cell-mediated ischemic preconditioning. Conclusion: Our results show that DIPC of the kidney provides local and systemic immunosuppression by inhibiting DC maturation and hence mediates a renal protective effect.

Role of Nuclear Factor (Erythroid-Derived 2)-Like 2 Signaling for Effects of Fumaric Acid Esters on Dendritic Cells.

To date, the intracellular signaling pathways involved in dendritic cell (DC) function are poorly understood. The antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been shown to affect maturation, function, and subsequent DC-mediated T cell responses of murine and human DCs. In experimental autoimmune encephalomyelitis (EAE), as prototype animal model for a T helper cell-mediated autoimmune disease, antigen presentation, cytokine production, and costimulation by DCs play a major role. We explore the role of Nrf2 in DC function, and DC-mediated T cell responses during T cell-mediated autoimmunity of the central nervous system using genetic ablation and pharmacological activation in mice and men to corroborate our data in a translational setting. In murine and human DCs, monomethyl fumarate induced Nrf2 signaling inhibits DC maturation and DC-mediated T cell proliferation by reducing inflammatory cytokine production and expression of costimulatory molecules. In contrast, Nrf2-deficient DCs generate more activated T helper cells (Th1/Th17) but fewer regulatory T cells and foster T cell proliferation. Transfer of DCs with Nrf2 activation during active EAE reduces disease severity and T cell infiltration. Our data demonstrate that Nrf2 signaling modulates autoimmunity in murine and human systems via inhibiting DC maturation and function thus shedding further light on the mechanism of action of antioxidative stress pathways in antigen-presenting cells.

The role of regulatory B cells in allergen immunotherapy.

Allergen immunotherapy (AIT) is currently the only curative treatment available for allergic diseases, and has been used in clinical practice for over a century. Induction and maintenance of immune tolerance to nonhazardous environmental and self-antigens is essential to maintain homeostasis and prevent chronic inflammation. Regulatory B (BREG) cells are immunoregulatory cells that protect against chronic inflammatory responses primarily through production of anti-inflammatory cytokines such as IL-10, transforming growth factor-β, and IL-35. The importance of BREG cells has been extensively demonstrated in the context of autoimmune diseases. Data showing their role in the regulation of allergic responses are slowly accumulating. This review summarizes recent findings relevant to the topic of BREG cells and their potential role in AIT. BREG cells support AIT in models of allergic airway inflammation and intestinal inflammation through induction of regulatory T (TREG) cells. In humans BREG frequency increases during venom immunotherapy while the phenotype of allergen-specific B cells changes. Mechanisms of BREG-mediated tolerance to allergens include IL-10-mediated suppression of effector T cell, including TH2 responses, induction of TREG cells, IL-10-mediated inhibition of Dendritic cell maturation, modulation of T follicular helper responses, and production of anti-inflammatory IgG4 antibodies. Current evidence supports a potential role for BREG cells in induction and maintenance of allergen tolerance during AIT. A better understanding of the role of B cells and BREG cells in AIT could open potential new windows for developing targeted therapies specifically focused on promoting BREG responses during AIT.

Association of pre-allogeneic hematopoietic stem cell transplant 25-hydroxy-vitamin D level and development of acute skin graft-versus-host-disease: A retrospective analysis of 154 patients.

e18539 Background: The pathophysiology of acute skin graft versus host disease (asGVHD) is thought to be initiated when donor T cells encounter recipient dendritic cells (DC) and become activated. Studies have shown that vitamin D (VD) inhibits DC maturation, polarizes T cell populations toward Th2 cytokine phenotype, And biases toward tolerating T cell populations. We analyzed pre-SCT serum vitamin D levels and sought to determine an association with development of acute skin GVHD. Methods: 154 patients, with matched SCT at UTSW Medical Center from December 2007 to December 2014, and who had serum samples banked prior to conditioning were included in the analysis. Pertinent data were extracted from the UTSW BMT database and EMR. Serum samples collected 1-3 months prior to SCT were used to detect 25-hydroxyvitamin D (VD) concentrations. Cumulative incidence (CI) of asGVHD grade II-IV were assessed within 100 days post-SCT. A significantl difference (p-value = 0.026, Breslow-Day test) in the associations between Vit D and asGVHD for patients under RIC and ABLgroups was present, so we analyzed the primary endpoint, time to asGVHD since SCT, separately among two these 2 groups. CI functions were constructed with death and relapse as competing risks. Results: Out of 154 patients, 93 were male, mean age was 51, range 20-72 and median VD was 21 ng/ml (range 7.1-63.3). There were 112 patients with VD < 25 ng/mL (VD < 25) and 41 with VD ≥ 25 ng/mL (VD≥25). Age, conditioning regimen, donor type, cell source, ABO compatibility, GVHD prophylaxis were similar in both groups. Overall CI asGVHD was 24%. CI of asGVHD was 23.8% in VD≥25 and 24.1% in VD < 25. Patients were then analyzed separately with respect to type of conditioning regimen they received (Ablative (ABL) vs Reducing intensity (RIC)). Figure 1A shows that in the ABL group the CI of asGVHD appears increased in patient with VitD≥25 (40% vs 27.1%, p = 0.27). In contrast, Figure 1B shows that in RIC group the CI of asGVHD is higher for those with VD < 25 (4.8% vs 24.1% , p = 0.05). Conclusions: After accounting for competing factors, KM analysis in the RIC group showed that patients with VD level < 25 ng/mL were at a higher risk of developing asGVHD. Further studies to clarify the association between VD status and development of asGVHD are warranted.

Dendritic cells with single Ig interleukin-IR-related molecule gene overexpression exhibit immature properties and prolong allograft survival

Objective To study the effect of single Ig interleukin (IL)-IR-related molecule (SIGIRR) on maturation of dendritic cells (DCs) and rejection of allografts in mice. Methods Murine bone marrow cells were extracted and induced into DCs. Adenoviral vector coding SIGIRR (Ad-SIGIRR) was constructed to increase SIGIRR gene expression in bone marrow-derived DCs. The phenotype and cytokine secretion of DCs transfected with Ad-SIGIRR (DC-SIGIRR) and control group in the absence and the presence of lipopolysaccharides (LPS) were analyzed. In vivo, C57BL/6 mice, which were pretreated with DC-SIGIRR or control treatment via intravenous injection, received islet transplantation. Intraperitoneal glucose tolerance test (IPGTT), graft survival time and histological changes were observed. Results DC-SIGIRR remained relative immature state after LPS stimulation. DC-SIGIRR expressed lower levels of costimulatory (CD40, CD80, CD86) and major histocompatibility complex (MHC) molecules, secreted lower levels of IL-12 [(547.80±84.67) pg/ml, P=0.026] and higher levels of IL-10 [(410.40±36.11) pg/ml, P=0.031] than in the control group. IPGTT suggested the blood glucose peak in SIGIRR group was statistically lower than in the other groups [(19.0±1.0) mmol/L, P=0.041]. Histological examination revealed less immune damage of the islet grafts, higher secretion activity and significantly longer allograft survival time [(14.3±3.6) days, P=0.036] in DC-SIGIRR pretreatment group. Conclusion SIGIRR inhibits DC maturation and DC transfected with SIGIRR prolongs allografts survival, which suggests therapeutic potential to prevent rejection in organ transplantation. Key words: Single Ig interleukin-IR-related molecule; Gene transfect; Dendritic cell; Islet transplantation; Rejection

Association of Pre-Allogeneic Hematopoietic Stem Cell Transplant 25-Hydroxy-Vitamin D Level and Development of Acute Skin Graft-Versus-Host-Disease: A Retrospective Analysis of 154 Patients

IntroductionAcute graft versus host disease (aGVHD) is a leading cause of morbidity and mortality in patients who undergo allogeneic hematopoietic stem cell transplant (SCT). aGVHD is thought to be initiated when donor T cells encounter recipient antigen presenting cells (APC) such as dendritic cells (DC) and become activated. Preclinical studies have shown that vitamin D (VD) inhibits DC maturation biasing T cell proliferation towards tolerating rather than alloreactive T cell populations [Rosenblatt et al. BMT 2010]. A study of 53 patients who underwent myeloablative (ABL) SCT showed pre-SCT VD <25 ng/mL was associated with increased cumulative incidence (CI) of chronic GVHD but not for aGVHD [Glotzbecke et al. BMT.2013]. Another study evaluating day-30 VD levels post-SCT in 54 patients showed that VD < 20 ng/mL was associated with increased risk of acute skin GVHD, but only in patients who underwent reduced intensity conditioning (RIC) [Ganetsky et al. Abstract # 416. BBMT. 2014]. Given the conflicting data of the effect of VD on the incidence of aGVHD, and small sample size of prior studies, we performed a retrospective analysis to evaluate whether pre-HSCT serum vitamin D levels were associated with acute skin GVHD in patients undergoing both ABL and RIC SCT.MethodsOne hundred fifty-four patients at the University of Texas Southwestern (UTSW) Medical Center who had serum samples banked prior to RIC or ABL conditioning and then underwent matched related (MRD) or unrelated (MUD) SCT from December 2007 to December 2014, were included in the analysis. CI of acute skin GVHD grade II-IV (asGVHD) was assessed within 100 days post-SCT. VD cutoff of 25 ng/mL was chosen based on receiver operating curve (ROC) analysis from a prior study [Glotzbecke et al. BMT.2013].The shape and distribution of continuous variables was examined. Mean and standard deviations were calculated for continuous variables and percent’s calculated for categorical data. The χ2-test and Fisher’s exact test were used to compare categorical variables and Student’s t-test was used to compare parametric data and calculate p values. Stratified analysis was performed to evaluate for presence of interaction between dichotomous VD variable and other covariates. Statistically significant interaction was found between VD levels and ABL vs. RIC regimens. We therefore stratified analysis of our primary endpoint, CI of grade II-IV asGVDH, into RIC and ABL groups. Kaplan-Meir (KM) curves for CI of asGVHD were constructed with death and relapse and competing factors. Difference between CI curves was tested using the Gray method.ResultsOut of 154 patients, 93 (60.3%) were male, mean age was 51 (range 20-72) and mean VD was 22.6 (range 7.6-63.6). Overall CI of asGVHD was 24%. Most patients (112, 72.7%) had with VD <25 ng/mL (VD<25). On bivariate analysis, conditioning regimen (ABL vs RIC), donor type (MRD vs MUD), cell source (peripheral blood vs bone marrow), age at SCT, and ABO compatibility were similar among those with VD levels <25 and ≥25. CI of asGVHD was 22.0% and 25.0% in VD≥25 and VD<25 groups, respectively (p=0.696).Patients were then analyzed separately with respect to type of conditioning regimen they received (ABL and RIC). The ABL group had 79 patients with CI of asGVHD of 29.1% versus 18.6% in the 75 patients of the RIC group (p=0.12). In the ABL group, CI of asGVHD with VD≥25 was 40% versus 27.1% in VD<25 (p=0.4). In RIC group, CI of asGVHD with VD≥25 was 4.8% versus 24.1% with VD <25 (p=0.095).Figure 1A shows that in the ABL group the CI of asGVHD appears increased in patient with VitD≥25 (40% vs 27.1%, p=0.27). In contrast, Figure 1B shows that in the RIC group, the CI of asGVHD is higher for those with VD<25 (4.8% vs 24.1%, p=0.05).ConclusionPre-SCT VD level was not associated with asGVHD, however there was a trend toward significance in patients who underwent RIC with a VD<25mg/mL. After accounting for competing factors, KM analysis showed that in patients who undergo RIC, VD level < 25 ng/mL is associated with a statistically significant increased risk of developing asGVHD. Interestingly, VD > 25 may be associated with increased risk of asGVHD in patients who underdo ALB conditioning, although these results were not statistically significant. This is the largest study analyzing the association of peri-SCT VD levels and GVHD to date. Further studies to clarify the interaction between VD status, conditioning regimen and development of asGVHD are warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

VEGF Requires the Receptor NRP-1 To Inhibit Lipopolysaccharide-Dependent Dendritic Cell Maturation.

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κβ, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.

EFFECTS OF IN VITRO 1,25 DIHYDROXYVITAMIN D ON MATURATION OF DENDRITIC CELLS IN GRAVES’ DISEASE PATIENTS

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.

Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis.

Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

Inhibition of neddylation regulates dendritic cell functions via Deptor accumulation driven mTOR inactivation

Neddylation, a newly identified post-translational modification, is significant for the activity and stability of target proteins. The exact role of neddylation in the pathogenesis of inflammatory bowel disease, specifically those mediated by dendritic cells (DCs), was still rarely reported. Here, we showed that inhibition of neddylation protected mice from mucosal inflammation. Targeting neddylation also inhibited DC maturation characterized by reduced cytokine production, down-regulated costimulatory molecules and suppressed capacity in allogeneic T cell stimulation. Additionally, inactivation of neddylation promotes caspase dependent apoptosis of DCs. These phenomena were attributed to the inactivation of mTOR, which was caused by Cullin-1 deneddylation induced Deptor accumulation. Together, our findings revealed that neddylation inhibition suppressed DC functions through mTOR signaling pathway and provided a potential therapeutic opportunity in inflammatory bowel diseases.

84. Alpha 1 Antitrypsin Protein & Gene Therapies for the Treatment of Lupus in Animal Models

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disorder. Although the pathogenesis of SLE remains elusive, over-reactive dendritic cells (DCs), which promote B cell activation and autoantibodies secretion, play critical roles in the disease development. Alpha-1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory, cytoprotective and immunoregulatory properties. We has shown that AAT has protective effect in autoimmune disease models including type 1 diabetes and rheumatoid arthritis. In this study, we tested therapeutic potentials of AAT in controlling DC and B cells function and the development of SLE in a spontaneous lupus mouse model. Using bone marrow cells from normal (C57BL/6) and lupus prone (B6. TC, MRL/lpr) mice, we showed that AAT treatment significantly inhibited lipopolysaccharide (LPS) (TLR4 agonist) or CpG (TLR9 agonist) induced DC (cDC and pDC) maturation. In this in vitro system, AAT significantly inhibited the production of IFN-I, IL-1β, IL-6 and TNF-α from DCs. AAT treated DC also have significantly lower potential in stimulating B cell proliferation and functions. Based on these results, performed following experiments using spontaneous lupus mouse models. In order to test this therapeutic potential, we treated female MRL/lpr mice (at 7-weeks of age, n=10) with clinical grade of AAT (2 mg/mouse, every 3 days) or PBS for 11 weeks. Consistent with our in vitro observations, results from this experiment showed that AAT treatment significantly inhibited DC maturation and reduced DC susceptibility to LPS stimulation. Importantly, AAT treatment significantly lowered serum antibody (anti-dsDNA IgG and anti-nuclear antibodies, ANAs) levels and urine albumin levels. Detailed pathological examinations showed that AAT treatment prevented kidney disease development. To confirm this observations and test the long-term effect of AAT treatment, we performed second experiment using both AAT protein and gene therapies. In this experiment, we treated adult female NZM2410 (another commonly used spontaneous lupus model) mice with AAT protein and rAAV8-CB-AAT vector (1×1011 vg/mouse, single IP injection). While 100% mice in control group develop lupus and died at 46 week of age, 50% mice in AAT protein treatment group remain lupus free at 55 weeks of age. Intriguingly, AAT gene therapy (rAAV8-CB-AAT) significantly prevent lupus development (70% mice remain lupus free at 63 weeks of age, P=0.0059). These results clearly demonstrated the therapeutic effect of AAT protein and gene therapy in lupus mouse model. Our results also indicated the advantage of rAAV8 mediated gene therapy for the chronic autoimmune diseases. In summary, we showed AAT inhibited the activation and function of DCs and B cells in vitro and in vivo. We also showed AAT treatment (protein therapy and gene therapy) prevent lupus development in spontaneous lupus mouse models. Considering the safety profile of AAT and rAAV, our results may be translated into clinical application and lead to a novel (safe and effective) therapy for SLE in humans. This work was supported by grants from University of Florida and Grifols, Inc.

Decline in Immunological Responses Mediated by Dendritic Cells in Mice Treated with 18α-Glycyrrhetinic Acid

ABSTRACTObjective: 18α-Glycyrrhetinic acid (18α-GA), a bioactive component of Glycyrrhiza glabra, has been shown in vitro immunomodulatory effects on dendritic cells (DCs). The aim of the present study is to evaluate the in vivo effect of 18α-GA on DCs and T cell responses.Methods: 18α-GA was intraperitoneally administered to mice and splenic DCs were evaluated for expression of co-stimulatory molecules using flow cytometry. Isolated DCs were added to mixed lymphocyte reaction (MLR) and the proliferation of T cells was measured using BrdU assay. The level of IFN-γ in the MLR supernatant was determined by enzyme-linked immunosorbent assay. The in vivo effect of isolated DCs on antigen-specific delayed type hypersensitivity (DTH) response, and the number of regulatory T (Treg) cells in mice spleen by flow cytometry, were investigated.Results: DCs isolated from 18α-GA-treated mice expressed lower levels of CD40 (p < 0.05) and MHC II (p < 0.01) compared to those of control group. In MLR assay isolated DCs decreased T cell proliferation to 83.54% ± 4.3% of control (p < 0.05). The level of IFN-γ in the MLR supernatant was declined to 25.2% ± 6.8% of control. In DTH test, DCs isolated from 18α-GA-treated mice significantly suppressed antigen-specific cell mediated immune response (3.3 ± 1 mm in test group versus 6.5 ± 1.2 mm in control group, ρ < 0.01). The percentage of Treg cells in spleen of 18α-GA-treated mice (6.37% ± 2.3%) was lower than that of control group (13.85% ± 0.4%, ρ < 0.05).Conclusions: In vivo administration of 18α-GA resulted in inhibition of DCs maturation and T cell-mediated responses, the effects that may candidate this compound for its possible benefits in immune-mediated diseases.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

ID: 143: Characterization of IL-6-type cytokine receptor expression during DC maturation and activation

Interleukin-6 (IL-6) is a regulator of cell growth and differentiation as well as hematopoiesis, immune response and inflammation. The classical IL-6 receptor complex consists of the membrane-bound, non-signal transducing IL-6 receptor alpha chain (IL-6R α ) and the transmembrane, signal transducing subunit glycoprotein 130 (gp130). Gp130 is ubiquitously expressed; the IL-6R α expression is limited to immune cells and hepatocytes. Dendritic cells (DCs) are key players of immune responses and express both gp130 and IL-6R α . As professional antigen-presenting cells they stimulate naive T cells upon activation by microbial stimuli or tissue destruction. DC maturation and activation is regulated by numerous cytokines and transcription factors, including IL-6 and STAT3. Experiments with IL-6 knock-out mice as well as gp130 knock-in mice revealed that IL-6 inhibits DC maturation and further, plays an important role to keep DC in an immature state in the absence of infection. Studies in bone marrow-derived DCs (BMDCs) also provide evidence that IL-6 signaling can directly inhibit LPS-induced BMDC maturation/activation via STAT3 activation. Although these results suggest that IL-6 is an important regulator of the DC lineage, little is known about the expression/regulation of its receptor subunits during DC maturation/activation. In the present study, we found that IL-6R α and gp130 expression increase during maturation of BMDCs from MHCIIlowCD11clow to MHCIIhighCD11chigh cells. Furthermore, we describe that gp130 cell surface expression is differentially regulated upon BMDC activation using alternative stimuli and that under steady-state conditions different DC subtypes of the spleen exhibit different cell surface expression levels of gp130 and IL-6R α .

EMT in immuno-resistance.

EMT in immuno-resistance.

Immunosuppressive effect of zhankuic acid C from Taiwanofungus camphoratus on dendritic cell activation and the contact hypersensitivity response.

Some ergostane triterpenoids from Taiwanofungus camphoratus have been shown to exhibit anti-inflammatory activity in vitro. However, the effect of ergostane triterpenoids on the immune response remains unknown. In this study, we elucidated that ergostane triterpenoids significantly decreased the cytokines and chemokine release by dendritic cells (DC) and that, in the case of zhankuic acid C (ZAC), the decrease was dose-dependent and inhibited DC maturation. ZAC inhibited the contact hypersensitivity response and infiltrative T cells in the ears of DNFB-stimulated mice. Thus, we demonstrate for the first time that ZAC exhibits an immunosuppressive effect on DC activation and the contact hypersensitivity response. It is suggested that ZAC can potentially be used for treating chronic inflammation and autoimmune diseases.

Role of growth hormone in maturation and activation of dendritic cells via miR-200a and the Keap1/Nrf2 pathway.

Dendritic cells (DCs) are antigen-presenting cells that participate in the immune response; recently, it has been reported that growth hormone (GH) promotes their maturation. The aim of this study was to investigate mechanisms by which GH acts on DC maturation and activation. Human peripheral blood monocytes (HPBMs) were induced to become immature DCs and treated with GH to obtain mature DCs. An osteosarcoma mouse model was established by injection of LM8 cells to investigate anti-tumour effect of GH-induced DCs invivo. After administration of GH, DCs reduced miR-200a expression and nuclear Nrf2 accumulation; miR-200a down-regulation inhibited DC maturation. Nrf2 ubiquitination level was increased by Keap1 overexpression in murine bone marrow derived dendritic cells (BMDCs), which was cancelled by miR-200a in GH exposed cells. In vivo, tumour volume was significantly reduced by GH-treated DCs and the effect was reversed by overexpression of miR-200a. GH promoted maturation and activation of DCs, and regulation of miR-200a played a part in this process by modulation of the Keap1/Nrf2 pathway.

Captopril inhibits maturation of dendritic cells and maintains their tolerogenic property in atherosclerotic rats

Atherosclerosis (AS) is a systemic disease of the immune system featuring hyperactive dendritic cells (DCs) in atherosclerotic plaques and organs. Captopril, a representative medicine of angiotensin-converting enzyme inhibitors, has been demonstrated to be effective in treating AS. However, captopril's anti-atherosclerotic mechanism is still poorly understood. Therefore, this study was primarily performed to investigate the effects of captopril on the function of DCs in vivo. AS in rats was induced by feeding them with atherogenic diets, and it was evaluated by the levels of plasma lipids and aortic cholesterol. DCs' activity was appraised by endocytic activity, mixed lymphocyte reactions and cytokine secretion. The markers of DCs (CD103, CD80, CD86 and MHC-II) and Treg (CD4+, CD25+ and Foxp3+) were assayed by western blotting analysis and flow cytometry. Cytokine level was measured by an enzyme-linked immunosorbent assay. The results showed that captopril treatment (10, 20mg/kg/d) obviously improved dyslipidemia and reduced the levels of aortic cholesterol. Captopril significantly reduced CD103, CD80, CD86 and MHC-II protein expression while increasing that of Foxp3 in aortic tissue. Further study indicated oral administration of captopril up-regulated endocytic activity and reduced the immunostimulatory function of splenic DCs. Captopril treatment also promoted IL-10 & TGF-β production while decreasing that of IL-6 & IL-12 in splenic DCs. Finally, the results of flow cytometry indicated that captopril obviously inhibited DC maturation and promoted Treg polarization. Captopril treatment was able to inhibit DC maturation and maintain their tolerogenic property, which is closely associated with DC anti-atherosclerosis activity.

Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.