Diacerein mitigates renal ischemia/reperfusion injury via inhibition of renal inflammation, dendritic cells maturation and apoptosis: the role of TLR4/NF-kB/NLRP3/IL-1beta signaling pathway / La diacereína mitiga la lesión por isquemia/reperfusión renal a través de la inhibición de la inflamación renal, la maduración de las células dendríticas y la apoptosis: el papel de la vía de señalización TLR4/NF-kB/NLRP3/IL-1beta

SUMMARY: One of the reasons for acute kidney damage is renal ischemia. Nevertheless, there are limited protective and therapeutic approaches for this problem. Diacerein is an anti-inflammatory drug characterized by numerous biological activities. We aimed to determine the ameliorative impact of diacerein on renal ischemia/reperfusion injury (I/R) condition, exploring the underlying mechanisms. Twenty-four male rats were allotted into four groups (n= 6): sham group; Diacerein (DIA) group; I/R group, in which a non-crushing clamp occluded the left renal pedicle for 45 min, and the right kidney was nephrectomized for 5 min before the reperfusion process; I/R + diacerein group, injected intraperitoneally with 50 mg diacerein/kg i.m 30 minutes prior to I/R operation. Ischemia/ reperfusion was found to affect renal function and induce histopathological alterations. The flow cytometry analysis demonstrated an elevated expression of innate and mature dendritic cells in I/R renal tissues. Moreover, upregulation in the expression of the inflammatory genes (TLR4, Myd88, and NLRP3), and overexpression of the pro-inflammatory cytokines (IL-1β), apoptotic (caspase-3) and pyroptotic (caspase-1) markers were observed in I/R-experienced animals. The aforementioned deteriorations were mitigated by pre-I/R diacerein treatment. Diacerein alleviated I/R-induced inflammation and apoptosis. Thus, it could be a promising protective agent against I/R.

La isquemia renal es una de los motivos del daño renal agudo. Sin embargo, los enfoques protectores y terapéuticos para este problema son limitados. La diacereína es un fármaco antiinflamatorio caracterizado por numerosas actividades biológicas. Nuestro objetivo fue determinar el impacto de mejora de la diacereína en la condición de lesión por isquemia/ reperfusión renal (I/R), explorando los mecanismos subyacentes. Veinticuatro ratas macho se distribuyeron en cuatro grupos (n= 6): grupo simulado; grupo de diacereína (DIA); grupo I/R, en el que una pinza no aplastante ocluyó el pedículo renal izquierdo durante 45 min, y el riñón derecho fue nefrectomizado durante 5 min antes del proceso de reperfusión; Grupo I/R + diacereína, inyectado por vía intraperitoneal con 50 mg de diacereína/kg i.m. 30 min antes de la operación I/R. Se encontró que la isquemia/ reperfusión afecta la función renal e induce alteraciones histopatológicas. El análisis de citometría de flujo demostró una expresión elevada de células dendríticas innatas y maduras en tejidos renales I/R. Además, se observó una regulación positiva en la expresión de los genes inflamatorios (TLR4, Myd88 y NLRP3) y una sobreexpresión de las citoquinas proinflamatorias (IL-1β), marcadores apoptóticos (caspasa-3) y piroptóticos (caspasa-1) en animales con experiencia en I/R. Los deterioros antes mencionados fueron mitigados por el tratamiento previo a la diacereína I/R. La diacereína alivió la inflamación y la apoptosis inducidas por I/R. Por lo tanto, podría ser un agente protector prometedor contra I/R.

Oral Administration of Incense Resin (Styrax benzoin) Extract Enhances Spatial Learning, Memory, and Dendrite Complexity of Mice

Abstract Hippocampus is a part of the brain that has a major role in spatial learning and memory which can be affected by herbal extracts. Incense resin (Styrax benzoin) has been used by local communities to improve intelligence. However, there is no scientific evidence of the functions of Styrax benzoin for regulating hippocampal function. The aim of this study was intended to analyze and investigate the effect of incense resin on learning, memory, and dendrite complexity of mice. Three months old male Deutch Democratic Yokohama (DDY) mice were injected orally with graded doses of 100, 150, and 200 mg/kg of incense resin aqueous extract daily for 30 days. Spatial learning and memory performance levels were tested with Y-maze alternation, novel object recognition, and Morris water maze. The branches and maximum dendritic span in the dentate gyrus were observed by the Golgi-Cox staining. Overall, our results showed that incense resin extract increased learning and memory ability, and the number of dendrite branching in the dentate gyrus.

Evaluation of immunostimulatory activity of Chyawanprash using in vitro assays.

Chyawanprash is an ayurvedic formulation used in Indian traditional medicinal system for its beneficial effect on human health. We investigated the immunostimulatory effects of Chyawanprash (CHY) using in vitro assays evaluating the secretion of cytokines such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1beta (IL-1β) and Macrophage Inflammatory Protein-1-alpha (MIP-1-α) from murine bone marrow derived Dendritic Cells (DC) which play pivotal role in immunostimulation. The effects of CHY on phagocytosis in murine macrophages (RAW264.7) and Natural Killer (NK) cell activity were also investigated. At non-cytotoxic concentrations (20–500 µg/ml), CHY enhanced the secretion of all the three cytokines from DC. CHY also stimulated both, macrophage (RAW264.7) as well as NK cell activity, in vitro. In conclusion, the data substantiates the immunoprotective role of CHY at cellular level mediated by immunostimulation in key immune cells viz. dendritic Cells, macrophages and NK cells.

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

BACKGROUND: In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1-4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA). RESULTS: The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC50 values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 µg/mL. Four highly pure steroid derivatives (1-4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S)5α-cholestane-3ß,4ß,6α,7α,8ß,15α,16ß,26-octol (3) on the production of IL-12 p40 and IL-6 (IC50s = 3.11 ± 0.08 and 1.35 ± 0.03 µM), and for (25S) 5α-cholestane-3ß,6α,8ß,15α,16ß,26-hexol (1) and (25S)5α-cholestane-3ß,6α,7α,8ß,15α,16ß,26-heptol (2) on the production of IL-12 p40 (IC50s = 0.01 ± 0.00 and and 1.02 ± 0.01 µM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production. CONCLUSION: This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.

Regulation of anti-inflammatory cytokines IL-10 and TGF-beta in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-beta production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-beta significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-beta. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-beta through activation of extracellular signal-regulated kinase 1/2.

Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4.

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.

Human Leukocyte Antigen-G Expression on Dendritic Cells Induced by Transforming Growth Factor-beta1 and CD4[+] T Cells Proliferation

During antigen capture and processing, mature dendritic cells [DC] express large amounts of peptide-MHC complexes and accessory molecules on their surface. DC are antigen-presenting cells that have an important role in tolerance and autoimmunity. The transforming growth factor-beta 1 [TGF-beta1] cytokine has a regulatory role on the immune and non-immune cells. The aim of this study is to evaluate the effect of TGF-beta1 on the induction of human leukocyte antigen-G [HLA-G] expression on the DC which is derived from monocyte. Methods: In this study, we evaluated the effect of TGF-beta1 in induction HLA-G expression on the monocyte-derived DC by flowcytometry and then CD4[+] T cell proliferative responses in the presence of DC-treated TGF-[beta1] was studied. Results: The results of this study showed that DC bearing HLA-G down-regulated activation of CD4[+] T cells and production of IL-6 and IL-17 in comparison with control [P<0.05]. Conclusion: It is concluded that TGF-beta1 has an important regulatory role in CD4[+] T cell proliferation by increasing HLA-G on DC and these cells can probably prevent unexpected immune responses in vivo

Functional changes of dendritic cells in hypersensivity reactions to amoxicillin

A better understanding of dendritic cell (DC) involvement in responses to haptenic drugs is needed, because it represents a possible approach to the development of an in vitro test, which could identify patients prone to drug allergies. There are two main DC subsets: plasmacytoid DC (pDC) and myeloid DC (mDC). β-lactams form hapten-carrier conjugates and may provide a suitable model to study DC behavior in drug allergy reactions. It has been demonstrated that drugs interact differently with DC in drug allergic and non-allergic patients, but there are no studies regarding these subsets. Our aim was to assess the functional changes of mDC and pDC harvested from an amoxicillin-hypersensitive 32-year-old woman who experienced a severe maculopapular exanthema as reflected in interleukin-6 (IL-6) production after stimulation with this drug and penicillin. We also aim to demonstrate, for the first time, the feasibility of this method for dendritic cell isolation followed by in vitro stimulation for studies of drug allergy physiopathology. DC were harvested using a double Percoll density gradient, which generates a basophil-depleted cell (BDC) suspension. Further, pDC were isolated by blood DC antigen 4-positive magnetic selection and gravity filtration through magnetized columns. After stimulation with amoxicillin, penicillin and positive and negative controls, IL-6 production was measured by ELISA. A positive dose-response curve for IL-6 after stimulation with amoxicillin and penicillin was observed for pDC, but not for mDC or BDC suspension. These preliminary results demonstrate the feasibility of this methodology to expand the knowledge of the effect of dendritic cell activation by drug allergens.

Effect of suicide gene therapy in combination with immunotherapy on antitumour immune response & tumour regression in a xenograft mouse model for head & neck squamous cell carcinoma.

Background & objectives: Prodrug activation strategy as well as immunotherapy have been widely used for cancer gene therapy. In the present study, using a head and neck squamous cell carcinoma (HNSCC) xenograft nude mouse model, we have investigated whether the two therapies in combination could improve tumour cell kill. We also investigated induction of immune effector cells viz., NK (DX5+) and DC (CD11c+) in vivo, post-combination gene therapy. Methods: A retroviral vector producing cell line (PLTK47.1 VPC) carrying Herpes simplex virus thymidine kinase gene (HSVtk) was used for intratumoural injection into NT8e xenograft tumours followed by the prodrug ganciclovir (GCV). IL-2 plasmid DNA was injected intramuscularly. Immune cells were analyzed by flow-cytometry. Non parametric ANOVA was performed with Kruskal Wallis test. Results: IL-2 could induce proliferation of both NK cells (DX5+) and dendritic cells (CD11c+) in vivo. Apoptosis was higher in combination therapy group as compared to HSVtk/GCV alone or IL-2 alone and was mediated through caspase-3 dependent pathway. Significant reduction in tumour volume was seen in all 3 treatment arms as compared to controls. Interpretation & conclusions: Combination of suicide gene therapy and immunotherapy leads to successful tumour regression in a HNSCC xenograft mouse model. Immunotherapy could help in a systemic long lived anti-tumour immune response which would prove powerful for the treatment of metastatic cancers, and also for minimal residual disease. The results of this study may form the basis for Phase 1 clinical trials.

Cyclosporin A-treated Dendritic Cells may affect the outcome of organ transplantation by decreasing CD4+CD25+ regulatory T cell proliferation

One of the mechanisms for generation of tolerance involves immature dendritic cells (DCs) and a subpopulation of regulatory CD4+ CD25+ T lymphocytes (T REG). The purpose of this work was to analyze how Cyclosporine A (CsA), a widely used immunosuppressive drug, may affect T REG proliferation. Purified and activated murine DCs obtained from bone marrow precursors differentiated with rGMCSF were co-cultured with purified CFSE-labeled T REG from OTII mice, and their phenotype and proliferation analyzed by flow cytometry. Our data indicate that DCs differentiated in the presence of CsA show an altered phenotype, with a lower expression of MHC-II and a lower activating capacity. Additionally, these CsA-treated DCs show decreased production of IL-2 and IL-12 and increased IL-10 secretion when stimulated with LPS, indicating an effect on the polarization of the immune response. Interestingly, CsA-treated DCs show an anti-tolerogenic effect since they reduce the proliferation of T REG cells from 72 to 47 percent. Further inhibition to a 24 percent of T REG proliferation was obtained as a direct effect of CsA on T REG. In conclusion, the anti-tolerogenic effect of CsA should be considered in the planning of immunosuppression in the context of clinical transplantation.

In vivo effects of calcitriol on phenotypic and functional properties of dendritic cells

Dendritic cells [DCs] play a central role in the initiation and expansion of T cell mediated immune responses with potential immuniotherapy application. The compounds which have the ability to induce immunomodulatory effects on CDs may be employed for the treatment of immunopathologic conditions such as autoimmune diseases. The aim of this study was to investigate the in vivo effects of calcitriol pactive form of vitamin D3] on DCs. 0.1 microgram calcitriol was injected intra-peritoneally into C57BL/6 mice every other day within 3 weeks, and spleen DCs were extracted by magnetic beads. The phenotypic and functional properties of DCs were studied by flow cytometry and mixed lymphocyte reaction [MLR], respectively. The expression of CD86 and MHC II, as maturation markers and costimulatory molecules were significantly decreased [p=0.028 and p=0.047, respectively] while CD11b expression, as a marker of mice myeloid DCs which mostly induces Th2 cytokine profile, was significantly increased [p=0.011]. Allogeneic T cell stimulation in MLR was also significantly inhibited in comparison with the control groups [p<0.05]. Our data indicate that in vivo calcitriol administration inhibits maturation and activation of DCs in the same manner as in vitro conditions

Enhancement of antitumor effect using dendritic cells activated with natural killer cells in the presence of Toll-like receptor agonist

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.

Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-alpha, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40-mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPSstimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-alpha and IL-1beta, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.

The Comparison of Cytotoxic T-Lymphocyte Effects of Dendritic Cells Stimulated by the Folate Binding Protein Peptide Cultured with IL-15 and IL-2 in Solid Tumor

The current modalities for treating cancer employ not only single but multiple approaches involving surgery, radiotherapy and chemotherapy. Unfortunately, the survival outcome is not promising even with these approaches. Alternative approaches for cancer therapy are now emerging. Immunotherapy is aiming at both increasing the power, and in redirecting the specificity of the patients' immune system to attack the tumor cells. Recently, many studies using tumor associated lymphocytes (TAL) isolated from malignant ascites cultured in a media containing interleukin-2 exhibit antitumor responses. IL-2 is a lymphokine produced by T-cells. It facilitates activation, sustained growth and rescue from apoptosis. Lately, newly developed IL-15 has also exhibited antitumor activity similar to IL-2. IL-15 is a newly described cytokine produced from monocytes-marcrophages and T-cells. It has a different molecular structure but it functions like IL-2 by binding to the IL-2R beta and gamma c chain. These antitumor responses are mediated by the cytotoxic T lymphocytes (CTL) that recognize the antigen in the context of the MHC molecules using the T cell receptors. CD8+-CTL recognize the peptide epitopes that are processed from the cellular proteins in the context of the MHC class I molecules. These peptides have a restricted length of 8-11 amino acids. The folate binding protein (FBP) is overexpressed in over 90% of ovarian and 20-50% in breast cancers. The FBP is the source of the antigenic peptides that are recognized by a number of these CTL-TAL, and is antigenic to both ovarian and breast cancer in vivo. To define the antitumor response of IL-15 and its' FBP immunogenicity, a peptide defining epitope E39 and E75 were presented by the PMBC derived dendritic cells (DC) from healthy donors isolated by the CD14 method to ovarian and breast CTL-TAL. Stimulating both ovarian and breast CTL- TAL by E39 or E75 pulsed DC (DC-E39, DC-E75), in the presence of IL-15 and IL-2 can rapidly enhance or induce the E39 or E75 specific CTL activity. The antitumor activities were measured by a chromium release assay for the tumor specific lysis activity using the ovarian and breast cancer cell lines. The tumor specific lysis activity for the ovarian TALs for IL-15 vs IL-2 were 28.6+/-3.9% and 30.3+/-3.2%, respectively and in the breast TALs, they were 14.8+/-3.1% vs 13.5+/-2.9%, respectively. Using autologous tumor cells, a slightly higher tumor specific lysis activity was obtained for the ovarian TALs cultured in IL-15 compared to IL-2 (72.0+/-8.2% vs 68.5+/-3.6%). However, for the breast TALs, they were 39.5+/-4.2% vs 41.5+/-3.3%, respectively. IL-15 is a newly developed cytokine that shows promising antitumor activity similar to IL-2. However, it requires lower dosage and is less toxic. Therefore, IL-15 might be a potential anticancer immunotherapeutic agent.