Dysregulation of miR-223, miR-146a, and miR-193a Expression Profile in Acute and Chronic Phases of Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice.

Multiple sclerosis (MS) is a chronic autoimmune disease with an unknown etiology. The purpose of this research was to assess miR-223, miR-146a, and miR-193a in acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) mice to consider the possible role of these genes in the pathogenesis of MS. EAE induction was given by myelin oligodendrocyte glycoprotein peptide on female C57BL/6 mice. Clinical scores and other criteria were followed daily until day 21 for the acute group and day 77 for the chronic group. At the end of the course, inflammation and demyelination of the central nervous system (CNS) were assessed by histological analysis. MicroRNA expression levels were assessed by real-time PCR. EAE development attenuated in the chronic group, and histological analysis showed less infiltration and demyelination in the chronic group compared to the acute group. The upper expression of miR-223 is demonstrated in the acute phase of EAE. Moreover, the expression levels of miR-146a and miR-193a decreased in the chronic phase of EAE. MiR-223 showed a highly coordinated elevation in the acute phase both in vivo and in vitro. MiR-146a shares a pathway with miR-223 through effecting IL-6 expression. Further studies are needed to reveal their impact on EAE and possible applications as drug targets and biomarkers.

Poly (I:C) increases the expression of galectin 1, 3, 9 and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells.

Background: Mesenchymal stem cells (MSCs) are commonly employed as a powerful tool for the treatment of immune-mediated problems owing to their capacity to regulate the immune system and differentiate into different tissues. Researchers use mesenchymal stem cell products given the limitations associated with the application of MSCs. Exosomes are nanometer vesicles derived from MSCs that are used in cell-free therapy. Inflammatory environmental conditions, such as stimulation of Toll-like receptor 3 (TLR-3), has the ability to adjust the immune-regulating properties and anti-inflammatory function of mesenchymal stem cells and their exosomes. Galectins and hepatocyte growth factor (HGF) are known as immunomodulatory factors in mesenchymal stem cells. This study was designed to examine the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) after stimulation with Poly (I:C) (Polyinosinic:polycytidylic acid sodium salt). Methods: To begin, the explant technique was used to extract mesenchymal stem cells from human umbilical cord Wharton's jelly. Then, the stem cells were stimulated using Poly (I:C) at three time intervals of 12, 24 and 48 h. Exosomes secreted from the supernatant of cells were extracted and exosome confirmation tests, including Scanning electron microscopy (SEM), Dynamic light scattering (DLS) and Flow cytometry were performed. Finally, the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes was evaluated by Real-Time PCR at three time intervals of 12, 24 and 48 h after stimulation. Results: The findings of our study indicated that following stimulation with Poly (I:C), the expression of galectin-9 and HGF (P < 0.05) genes was markedly higher than in the control group after 12 h. After 24 h, the expression of galectin-9 (P < 0.01), galectin-3 and HGF (P < 0.05) increased; the expression of galectin-1, galectin-3, (P < 0.05), galectin-9 and HGF genes (p < 0.01) significantly increased compared to the control group after 48 h. Conclusion: TLR3 stimulation can increase the expression of galectins and HGF genes in exosomes derived from hWJ-MSCs and may be improve the immunosuppressive abilities of exosomes.

Amelioration of inflammation through reduction of oxidative stress in rheumatoid arthritis by treating fibroblast-like synoviocytes (FLS) with DMF-loaded PLGA nanoparticles.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory condition, and Dimethyl fumarate (DMF) is known for inducing antioxidant enzymes and reducing reactive oxygen species (ROS). Fibroblast-like synoviocytes (FLS) contribute to joint damage by releasing interleukins (IL-1ß, IL-6, and IL-8) in response to ROS. Given ROS's impact on FLS acquiring an invasive phenotype, our study explored the effects of poly lactic-co-glycolic acid (PLGA) nanoparticles containing DMF on the expression of the HO-1 enzyme and the inflammatory cytokines IL-1ß, IL-6, and IL-8 in FLS cells. METHODS: In this study, we evaluated and compared the impact of Free-DMF and PLGA-DMF, on the gene expression of the HO-1 and inflammatory cytokines (IL-1ß, IL-6, and IL-8) in FLS cells derived from 13 patients with rheumatoid arthritis. qRT-PCR method was used to quantify the gene expression levels. RESULTS: PLGA-DMF nanoparticles demonstrated a significant increase in HO-1 expression and a significant decrease in IL-1ß gene expression. Also, a significant decrease in IL-6 gene expression was seen under the effect of Free-DMF. These results indicate the potential effectiveness of PLGA-DMF nanoparticles in reducing inflammation and improving rheumatoid arthritis symptoms. DISCUSSION: According to the findings, PLGA-DMF nanoparticles are expected to be effective in reducing inflammation and improving the symptoms of rheumatoid arthritis. Also, further studies on other factors affected by oxidative stress such as cell invasion factors and survival factors after the effect of PLGA-DMF nanoparticle are recommended.

PRP as a modulator of inflammation in FLS of RA patients by regulation of galectins and TGF-ß1.

An autoimmune and inflammatory condition known as rheumatoid arthritis is characterized by joint inflammation and an aggressive fibroblast-like synoviocytes. (FLS) One of the most significant immunological regulators are the galectins. Platelet-rich plasma are probably effective in immunomodulation. The aim of the present work is to investigate the role of platelet rich plasma (PRP) as a modulation of inflammation, which affects the expression of galectins and TGF-ß in FLS from Rheumatoid arthritis (RA) patients. Methods: Human FLS cells from RA patients' synovial fluid were cultured in DMEM-F12 medium, characterized by flowcytometry, treated with PRP alone, TNF-α+PRP, SF + PRP, TNF-α alone, and untreated control groups. Expression of Galectin-1, Galectin-3, Galectin-9, and TGF-ß1 genes was assessed by Real-Time PCR. Results: In SF + PRP, TNF + PRP, and PRP groups, the gene expression of Galectin-3 was considerably reduced (P > 0.05). Galectin-1 and TGF-ß1 expression levels were also lowered (P > 0.05) in the TNF + PRP groups. Galectin-9 expression increased significantly in the PRP group (P > 0.05). Galectin-3 expression was markedly and extensively reduced in multiple study groups after treatment of FLS cells with 10 % PRP. Galectin-3 expression was considerably reduced when FLS were exposed to TNF- and synovial fluid in conjunction with PRP to simulate localized body inflammation. Conclusion: Our results showed that PRP may be useful in lowering FLS-induced inflammation in RA patients' joints, particularly when Galectin-3 is involved. In the future, inflammatory illnesses like RA may be treated locally using PRP or its derivatives, which will have a larger immune modulation role and more likely pathways.

The effect of the anti-leukemia inhibitory factor on the immune system in the Balb/c mice bearing breast cancer induced with 4T1 cells.

BACKGROUND: Breast cancer is one of the most common cancers. Leukemia inhibitory factor (LIF) is considered as one of the effective factors in the growth of breast cancer, and anti-leukemia inhibitory factor antibody is considered as one of the treatment options for this type of cancer. METHODS: Mice models of breast cancer were made with 4T1 cell line and were randomly divided into four groups. The first group included the mice that received anti-LIF (Anti LIF group). The mice in the second group received anti-LIF and doxorubicin (Anti LIF & DOX). The mice in the third group received only doxorubicin (DOX). Finally, the mice in the fourth group did not receive any intervention. 22 days after tumor induction, some of the mice were killed, and their tumor tissues, lymph nodes, and spleens were separated for evaluating P53, Caspase-3, TIM-3, LAG-3, CTLA-4, and PD-1 genes expression. The percentage of regulatory T cells and level of interferon gamma (IFN-γ) and transforming growth factor-beta (TGF-ß) were evaluated. The rest of the mice were kept to check the tumor size and their survival rate. RESULTS: The proposed intervention did not have any significant effect on the tumor growth and the survival rate. However, the expression of P53 gene and Caspase-3 in the tumor tissue of the Anti LIF group had a significant enhancement. In tumor tissues and lymph nodes, the expression of T-bet, PD-1, TIM-3, and LAG-3 genes in the Anti LIF group showed a significant increase. There was no significant difference between groups in the percentage of regulatory T cells and level of IFN-γ and TGF-ß. CONCLUSIONS: The proposed interventions were able to have a direct effect on tumors, but no significant effect was observed on the immune system.

Switch off inflammation in spleen cells with CD40-targeted PLGA nanoparticles containing dimethyl fumarate.

The purpose of this study was designing and synthesizing a PLGA formulation targeted with anti-CD40 monoclonal antibody, which has suitable physicochemical properties as a dimethyl fumarate (DMF) drug delivery system having minimal cytotoxicity. Therefore, this research was performed to determine the effect of anti-CD40mAb-DMF-NPs on the expression of IL-1ß, IL-6 and TNF-α cytokine genes in mouse splenocytes. The toxicity of different groups, namely free PLGA, free DMF, DMF-containing PLGA, anti-CD40mAb-DMF-NPs, was evaluated by MTT assay. PLGA formulations conjugated with mAbCD40 were loaded with DMF drug that showed little cytotoxic effect against mouse splenocytes. QRT-PCR method was subsequently used to assess the effect of the mentioned groups on the expression of IL-1ß, TNF-α and IL-6 genes. After treatment of the cells with DMF alone or with polymer carriers, the expression of IL-1ß, IL-6 and TNF-α cytokine genes was significantly reduced. The decrease in expression was markedly higher in the antibody-targeted nanoparticles group relative to other treatment groups. Our results in this area are promising and provide a good basis for further future studies in this regard.

Preventive cancer vaccination with P5 HER-2/neo-derived peptide-pulsed peripheral blood mononuclear cells in a mouse model of breast cancer.

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+ IFN-γ+, CD8+ IFN-γ+, and CD8+ granzyme B+ T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

IRAK1 Gene Polymorphism in Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. The present study intends to specify rs1059703, rs4810485, and rs1883832 gene polymorphisms of interleukin-1 receptor-associated kinase (IRAK1) and cluster of differentiation 40 (CD40) in RA. IRAK1 is a serine/threonine kinase and CD40 is a tumor necrosis factor receptor, both of which are involved in RA. There are conflicting results on functional effects of these polymorphisms, so we performed this research for a more accurate estimation on rheumatoid arthritis risk. Methods: Two-hundred RA patients diagnosed according to ACR criteria and 200 normal controls participated in this case-control study. DNA Purification kit (Gene Transfer Pioneers, GTP) was used for genomic DNA extraction and three SNPs, including IRAK1 rs1059703 (C/T), CD40 rs1883832 (C/T) and rs4810485 (G/T), were genotyped by PCR-RFLP. The genotypes and allele frequencies of SNPs were analyzed by chi-square test to detect their contribution to RA. Results: A significant correlation was found between rs1059703 T allele (OR = 2.36, 95% CI = 1.7-3.1, p = .0001) and TT and CT genotypes (TT genotype, OR = 2.54, 95%CI = 1.2-3.3, P = .0078, CT genotype; OR = 2.18 95%CI = 1.4-3.2P = .0002) of rs1059703 C/T polymorphism in terms of susceptibility to RA in recessive and over-dominant models. Alleles and genotypes of CD40 SNPs were not significantly different between RA cases and controls. The findings showed significant differences in rs1059703 IRAK1 genotypes with medical and laboratory features of patients. Conclusion: Our results showed that the rs1059703 T allele (risk allele) of IRAK1 gene increases the risk of RA and the severity of disease, affecting the onset age of RA in Iranian patients.

Identification of a FAS/FASL haplotype associated with endometriosis in Iranian patients.

Risk factors for ovarian cancer include a number of genetic variants as well as endometriosis. The FAS-FASL system is one of the apoptotic pathways that play an essential role in the apoptotic process within the endometrium. Here, we evaluate the correlation between FAS-FASL polymorphisms with the risk of endometriosis in Iranian patients and healthy controls. We extracted DNA from whole blood samples using a DNA Purification Kit. Using the PCR-RFLP method, three SNPs, including FAS (-670 A/G) and FASL (-844 C/T and _124G/A) genes, were genotyped in 112 patients with endometriosis as well as 110 healthy controls. The frequency of genotypes and the alleles of these SNPs were analyzed by the chi-squared test for the significant association. Haplotype analysis was done by the PLINK software. The frequency distribution of haplotypes was significant between SNPs so that the ACG haplotype was more frequent in the cases than in the controls (p = .017). These results indicate that haplotype analysis can be useful for SNP analysis. The ACG haplotypes in FAS-670A/G, FASL-844C/T, and _124G/A genes may be correlated with the progression of endometriosis.

PTPN22 1858 C/T polymorphism is associated with alteration of cytokine profiles as a potential pathogenic mechanism in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is one of the most common prevalent autoimmune diseases. The 1858 C/T (rs2476601) single nucleotide polymorphism (SNP) within the PTPN22 gene has been associated with susceptibility to inflammatory based diseases in several populations. It is implicated that altered cytokine production has a potential pathogenic role in the development of RA. The aim of this work was to analyze the association of 1858 C/T PTPN22 polymorphism in RA patients with cytokine profiles. MATERIALS AND METHODS: This study was performed on 120 RA patients who were referred to the Rheumatology Research Centre, Shariati Hospital (Tehran, Iran), and 120 healthy controls. Genomic DNA was extracted and genotyped for 1858 C/T PTPN22 gene SNP using the PCR-RFLP technique. Serum levels of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ as well as Anti-CCP and RF was measured by ELISA method. RESULTS: Results showed that 1858 C/T PTPN22 SNP significantly (P =  0.007, OR = 2.321, 95% CI = 1.063-5.067) associated with RA. The 1858 T allele frequency was also significantly increased in RA patients in comparison to the controls (P =  0.008, OR = 3.583, 95% CI = 1.3-9.878). Our data demonstrated a significant reduction of IL-4 and IL-10 in PTPN22 1858C/T compared to 1858C/C RA patients. In addition, upregulation of IL-6, IFN-γ, and TNF-α was observed in PTPN22 1858C/T vs. 1858C/C RA patients. DISCUSSION: Our findings implicate altered cytokine profiles as a possible pathogenic mechanism by which the 1858 T allele may contribute to the progress of RA.

RGD-Modified Nano-Liposomes Encapsulated Eptifibatide with Proper Hemocompatibility and Cytotoxicity Effect.

BACKGROUND: Eptifibatide (Integrilin®) is a hepta-peptide drug which specifically prevents the aggregation of activated platelets. The peptide drugs are encapsulated into nanolipisomes in order to decreasing their side effects and improving their half-life and bioavailability. OBJECTIVES: In this study, the in vitro cytotoxicity and hemocompatibility of RGD-modified nano-liposomes (RGD-MNL) encapsulated a highly potent antiplatelet drug (eptifibatide) was investigated. MATERIAL AND METHODS: RGD-MNL encapsulated eptifibatide was prepared using lipid film hydration and freeze/thawing method. The morphology and size distribution (about 90 nm) of RGD-MNL were characterized using transmission electron microscopy (TEM). The in-vitro cytotoxicity of nano-liposomes was examined using the MTT, LDH release and reactive oxygen species (ROS) generation assays. The effect of RGD-MNL on red blood cells (RBC) was investigated using hemolysis and LDH release assays. RESULTS: The results revealed that RGD-MNL had no significant cytotoxic effect on HeLa and HUVEC cell lines, and also no ROS generation increase in the cells. In addition, the adverse effect of RGD-MNL on LDH release and membrane integrity of RBC was not observed. CONCLUSIONS: In conclusion, the recommended RGD-MNL formulations have not any significant cytotoxicity on normal cells or RBC and have potential for protecting and enhancing the activity of antiplatelet drugs.

Implications of immunotherapy with high-dose glatiramer acetate in acute phase of spinal cord injury in rats.

Objective: Recently, many researches with different viewpoints have focused on application of immunotherapy agents in treatment of spinal cord injury (SCI) according to neuroprotective results in some neurodegenerative disease. Glatiramer acetate (GA) is the most commonly used drug for Multiple sclerosis (MS) patients that exerts an immunomodulatory effect against Myelin basic protein (MBP) antigen. Materials and methods: High-dose (2mg/kg) treatment of GA for 28 consecutive days after SCI was compared with its low-dose (0.5 mg/kg) treatment, SCI control and Sham control rat groups. Results: High-dose GA group had significantly worsened outcome in standard functional recovery evaluation test (BBB) 12 weeks after SCI compared to SCI control and low-dose GA groups, which was confirmed by augmented spinal cavity volume and reduced ventral horn motor neurons in high-dose GA group; however, there was no significant difference between low-dose GA and control SCI group. In addition, proliferation test performed on lymphocytes from spleen and lymph nodes one week after SCI showed that high-dose GA injection has more significant effect on Division Index (DI) in response to MBP stimulation compared to low-dose GA and control SCI groups, which was associated with significant increase in IFN-γ, IL-4, and IL-17A secretion. Conclusion: Along with confirmation of deleterious aspects of autoimmunity resulting from autoreactive lymphocytes against myelin antigens in SCI, this study has shown that high-dose immunotherapy using GA, especially in acute phase after SCI, overwhelms any neuroprotective effect of adoptive immune system.

Altered expression patterns of complement factor H and miR-146a genes in acute-chronic phases in experimental autoimmune encephalomyelitis mouse.

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.

Polymorphism of Foxp3 gene affects the frequency of regulatory T cells and disease activity in patients with rheumatoid arthritis in Iranian population.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that mainly affects joints and characterized by chronic joint inflammation and infiltration of various immune cells in the synovium. Forkhead box P3 (Foxp3)-expressing regulatory T cells (Tregs) play a crucial role in preventing autoimmunity and undesirable T cell responses. However, there are controversial reports regarding the defective function or frequency of these cells in various studies, which may be in part related to different polymorphisms of FoxP3 and influence of ethnicity on these differences. Therefore, the main subject of this study was to evaluate the association of Foxp3 gene polymorphism and Treg frequency in Iranian patients with RA. Accordingly, 240 RA patients diagnosed according to American college of rheumatology 2010 criteria and 240 normal subjects were recruited for this study. Genomic DNA was genotyped for -3279 C/A Foxp3 gene SNP using the PCR-RFLP. The frequency of Tregs and serum levels of interleukin (IL)-10, transforming growth factor (TGF)-ß, anti-cyclic citrullinated peptide (CCP) and rheumatoid factor (RF) were determined by flow cytometry and ELISA methods, respectively. The results showed a significant association of Foxp3 -3279 A allele with augmented risk of RA in Iranian patients compared to wild-type allele. While the frequencies of CA and AA genotypes were significantly higher in patients, RA patients with AA genotype had a significant lower frequency of Tregs compared to patients with CC and CA genotypes. Consistently, TGF-ß and IL-10 significantly diminished in patients with AA genotype compared to patients with CA and CC genotypes. Our findings indicated that the AA genotype of Foxp3 in RA patients is associated with downregulation of Tregs and susceptibility to RA in the Iranian population.

Evaluation of AD-MSC (adipose-derived mesenchymal stem cells) as a vehicle for IFN-ß delivery in experimental autoimmune encephalomyelitis.

Interferon-ß (IFN-ß) is commonly used as a disease modifying drug for the treatment of relapse-remitting multiple sclerosis (RR-MS). However, the underlying mechanism by which IFN-ß mediate this immunosuppressive effect is still unknown. In this study, we analyzed the effects of genetically modified adipose-derived mesenchymal stem cells (AD-MSCs) expressing murine interferon beta (MSCs-VP/IFN-ß) on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Lymph node mononuclear cells and serum were examined by using RT-PCR and ELISA methods to measure the production of IL-10 and IL-17 gene and protein expression, respectively. Our results indicated that in the MSCs-VP/IFN-ß treated group induction of Tregs and IL-10 and reduction of IL-17 were significant. Taken together, we showed that using AD-MSCs expressing IFN-ß as an anti-inflammatory agent, offer evidence supporting that the stem cell therapies in EAE conceivably will improve the valuable effects of IFN-ß in this autoimmune disease.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Effects of DKK-3, a Wnt signaling inhibitor, on dendritic cell phenotype and T cell polarization.

Wnt signaling plays crucial roles in regulation of a wide range of processes in different cell types including immune cells and, in particular, dendritic cells and T cells. Growing indications point out that Wnt pathway components modulate the both innate and adaptive immune responses through regulating DC functions. We investigated the effects of recombinant DKK-3 protein on the phenotype and biological functions of bone marrow-derived DCs (BM-DCs) as well as T cell polarization. The phenotype and the cytokine production of BM-derived DCs in the presence DKK-3 were analyzed using flow cytometry and ELISA, respectively. Also, capability of DCs to activate T cells was evaluated by CFSE-labeled splenocytes. Regulatory T cell induction, T cell polarization, and cytokine secretion were assessed by flow cytometry and ELISA in splenocytes cultured in the presence of DKK-3. Our results showed that the expression of CD86 and CD40 increased in the DKK-3-treated DC, while the expression of PDL-1 and PDL-2 diminished. Furthermore, the presence of DKK-3 decreased IL-10 and IL-4 production and increased IFN-gamma production by treated DCs.DKK-3. Moreover, DKK-3 shifted naive CD4 T cells towards TH1 cells through up-regulation of T-bet and down-regulation of GATA-3. Our results, therefore, suggest that DKK-3 protein has the ability to promote the generation of Th1-immunostimulatory DCs from its precursors.

TNF-α modulates the immunosuppressive effects of MSCs on dendritic cells and T cells.

Mesenchymal stem cells are progenitor cells that have capabilities to differentiate different cell types. Also, MSCs possess immune suppressive effects on DC differentiation and T cell activation through a wide range of soluble factors and receptors. The properties of MSCs change through activation of cytokines particularly IFN-γ and TNF-α. The DC phenotypes and functions including the expression of co-stimulatory and co-inhibitory molecules and capabilities of DCs to induce allogeneic activation of CFSE-labeled splenocytes as well as cytokine production when they were differentiated in the presence of MSCs, TNF-α activated MSCs, IFN-γ activated MSCs and IFN-γ & TNF-α activated MSCs were analyzed. Treg population and T cell polarization were investigated using flowcytometry and real-time PCR respectively. Here, we showed that IFN-γ slightly enhances immunosuppressive effects of MSCs on immune system through induction tolerogenic DCs with elevated expression of IDO and increasing Treg population. Conversely, TNF-α decreases immunomodulation properties of MSCs on immune cells through the enhancement of co-stimulatory molecules such as ICOSL and HLA-DR, reduction of PDL-1 and PDL-2 expression and decrease of TGF-ß and IL-10 in DCs as well as inhibition of T cell polarization into TH2 and Treg. Taken together, these data showed crucial effects of microenvironments on MSC behaviors indicating that functions of MSCs differentially altered in the presence of different cytokines.

Investigation of immunomodulatory properties of human Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction.

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are considered as an alternative for bone-marrow-derived MSCs. These cells have immunosuppressive properties. It was unclear whether the WJ-MSCs would sustain their immunomodulatory characteristics after lentiviral transduction or not. In this study, we evaluated immunomodulatory properties of WJ-MSCs after lentiviral transduction. HWJ-MSCs were transduced with lentiviral particles. Expression of transduced and un-transduced hWJ-MSCs surface molecules and secretion of IL-10, HGF, VEGF and TGF-ß was analyzed. Cell proliferation and frequency of CD4(+)CD25(+) CD127(low/neg) Foxp3(+) T regulatory cells was measured. There was no difference between the surface markers and secretion of IL-10, HGF, VEGF and TGF-ß in transduced and un-transduced hWJ-MSCs. Both cells inhibited the proliferation of PHA stimulated PBMCs, and improved the frequency of T regulatory cells. These findings suggest that lentiviral transduction does not alter the immunomodulatory function of hWJ-MSCs. However, lentiviral transduction may have a wide range of applications in gene therapy.

Fibroblasts feeder niche and Flt3 Ligand as a novel inducer of plasmacytoid dendritic cells development in vitro.

Plasmacytoid dendritic cell (pDC), plays central role in antiviral immunity. The aim of this study was to assess the effect of Flt3 ligand (FL) alone or with L929 fibroblast feeder or L929 conditioned media on differentiation of mouse bone marrow (BM) cells into pDC in vitro. Murine BM cells were cultured with FL or with L929 or conditioned media for 9days. The differentiated cells were analyzed using flow cytometry for PDCA-1, B220 and CXCR4. The relative expression of Stat3, CXCR4, CXCR7, IFN-ß, TGF-ß and Runx2 in differentiated cells determined by real time PCR. The development of pDC showed up to 19% increase after co-culture of BM cells with fibroblast feeder. Upregulation of Stat3, Runx2 and CXCR4 due to the presence of fibroblast feeder with FL in culture results in improved pDC development. Furthermore, 30% L929 supernatant along with Flt3 ligand was able to derive pDC up to 8.9% in comparison with FL alone, which was 6.6% in vitro. Thus, for the first time we introduced L929 fibroblast feeder as a niche producer of M-CSF and probably other growth factors and chemokines, which promotes the development of pDC in vitro along with FL, similar to in vivo niche.