Splenic Mononuclear Cells Research Articles

Intracerebroventricular Aβ-Induced Neuroinflammation Alters Peripheral Immune Responses in Rats.

An important marker in Alzheimer disease (AD) is the abnormal production and accumulation of β-amyloid peptide (Aß) in brain. It produces oxidative damage in neurons and inflammation due to its neurotoxic properties. The present study was designed to investigate neuroinflammation (hippocampal levels of ROS, nitrite, TNF-α, and IL-1β), neurodegeneration (plaques andchromatolysisin hippocampus), and memory impairments (working memory and reference memory) and the effect of this neuroinflammation on some peripheral immunological parameters such as phagocytic activity of blood WBC and splenic polymorphonuclear (PMN)cells, leucocyte adhesion inhibition index (LAI), and cytotoxicity of splenic mononuclear cells (MNC) after the intracebroventricular injection of aggregated Aβ(1-42)in a4week study. The results showed that the hippocampal and serum levels of ROS, nitrite, TNF-α, and IL-1β were significantly higher in the AD animals along with increased chromatolysis and impairments of memory. There was also a significant increase in the phagocytic activity of splenic PMN and cytotoxicity of splenic MNC and a decrease in the phagocytic activity of blood WBC and LAI of splenic MNC in the Aβ(1-42)-injected AD rats compared to that of control and sham-operated rats. The results indicate that the increased levels of inflammatory markers in the hippocampus may provide signals to the periphery and can alter the systemic immune responses.

Immunomodulatory effects of mesenchymal stem cell-derived exosomes on experimental type-1 autoimmune diabetes.

Exosomes derived from adipose tissue-derived mesenchymal stem cells (AD-MSCs) have immunomodulatory effects of T-cell inflammatory response and reduction of clinical symptoms on streptozotocin-induced of the type-1 diabetes mellitus (T1DM). Beside control group and untreated T1DM mice, a group of T1DM mice was treated with intraperitoneal injections of characterized exosomes derived from autologous AD-MSCs. Body weight and blood glucose levels were measured during the procedure. Histopathology and immunohistochemistry were used for evaluation of pancreatic islets using hemotoxylin and eosin (H&E) staining and anti-insulin antibody. Isolated splenic mononuclear cells (MNCs) were subjected to splenocytes proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, immunophenotyping of regulatory T cells and cytokines. A significant increase in the levels of interleukin-4 (IL-4), IL-10, and transforming growth factor-β, and a decrease in the levels of IL-17 and interferon-γ in concordance with the significant increase in the Treg cell ratio in splenic MNCs (P < 0.05) was shown in T1DM mice treated with AD-MSC's exosomes as compared to T1DM untreated mice. This amelioration of autoimmune reaction after treatment of T1DM mice with the AD-MSC exosomes was confirmed with a significant increase in islets using H&E staining and Immunohistochemistry analyses. As expected, body weight, blood glucose levels in a survival of T1DM mice treated with AD-MSC's exosomes were maintained stable in comparison to untreated T1DM mice. It can be concluded that AD-MSC's exosomes exert ameliorative effects on autoimmune T1DM through increasing regulatory T-cell population and their products without a change in the proliferation index of lymphocytes, which makes them more effective and practical candidates.

Role of Lactobacillus paracasei prtP gene in regulating mouse intestinal immunity

Objective To study the role of serine protease encoding gene prtP carried by Lactobacillus paracasei (Lp) in regulating mouse intestinal immunity. Methods Female BALB/c mice were randomly divided into seven groups and respectively administered with PBS, medium (5×108 CFU/ml) and high dose (1×109 CFU/ml) of Lp, Lp△prtP and ReLp△prtP for 21 consecutive days by intragastric gavage. Parameters including immune organ index, splenic lymphocyte transformation rate, energy metabolism in celiac macrophages, phagocytic ability of celiac macrophages and cytokines were detected. Quantitative changes in CD11c+ CD80+ cells in culture supernatants of splenic mononuclear cells were analyzed by flow cytometry. Results Compared with Lp△prtP, Lp could significantly promote the natural transformation of splenic lymphocytes, increase the level of energy metabolism in celiac macrophages, enhance the phagocytic ability of celiac macrophages, and up-regulate the expression of IFN-α, IL-10 and IFN-γ and the percentage of CD11c+ CD80+ cells. Conclusion Serine protease encoding gene prtP in Lp can regulate the mucosal immune system in mice. Key words: Lactobacillus paracasei; prtP gene; Intestinal immunity

Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa.

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-κB, NFAT, and AP-1) to various extents in murine RAW 264.7 macrophages, even though similar pathways are activated. The agonists also elicit differential expression of maturation markers in bone marrow-derived dendritic cells, as well as differential cytokine secretion from these cells and from splenic mononuclear cells. In vivo assays also show that agonists of Dectin-1 and Dectin-2, but not Mincle, induce heavy IgA secretion in intestinal mucosa even when delivered parenterally. Strikingly, this effect appears to be formulation-independent. Collectively, the data suggest that adjuvants based on Dectin-1 and Dectin-2 agonists may significantly improve the efficacy of parenteral vaccines by inducing robust local immune reactions in intestinal mucosa.

The inhibition of CB1 receptor accelerates the onset and development of EAE possibly by regulating microglia/macrophages polarization

Cannabinoid 1 receptor (CB1R) regulates the neuro-inflammatory and neurodegenerative damages of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R inhibition exerts inflammatory effects is still unclear. Here, we explored the cellular and molecular mechanisms of CB1R in the treatment of EAE by using a specific and selective CB1R antagonist SR141716A. Our study demonstrated that SR141716A accelerated the clinical onset and development of EAE, accompanied by body weight loss. SR141716A significantly up-regulated the expression of toll like receptor-4 (TLR-4) and nuclear factor-kappaB/p65 (NF-κB/p65) on microglia/macrophages of EAE mice as well as levels of inflammatory factors (TNF-α, IL-1β, IL-6) and chemokines (MCP-1, CX3CL1), accompanied by the shifts of cytokines from Th2 (IL-4, IL-10) to Th1 (IFN-γ)/Th17 (IL-17) in the spinal cords of EAE mice. Similar changes happened on splenic mononuclear cells (MNCs) except chemokine CX3CL1. Consistently, SR141716A promoted BV-2 microglia to release inflammatory factors (TNF-α, IL-1β, IL-6) while inhibited the production of IL-10 and chemokines (MCP-1, CX3CL1). Furthermore, when splenic CD4+ T cells co-cultured with SR141716A-administered BV-2 microglia, the levels of IL-4 and IL-10 were decreased while production of IL-17 and IFN-γ increased significantly. Our research indicated that inhibition of CB1R induced M1 phenotype-Th17 axis changed of microglia/macrophages through TLR-4 and NF-κB/p65 which accelerated the onset and development of EAE. Therefore, CB1R may be a promising target for the treatment of MS/EAE, but its complexity remains to be carefully considered and studied in further clinical application.

Effect of docosahexenoic acid microalgae oil supplementation during pregnancy and lactation on levels of serum polyunsaturated fatty acid and tolerance to ovalbumin in suckling mice

Objective: To study the tolerance to ovalbumin (OVA) in suckling mice whose mothers had different doses of docosahexenoic acid (DHA) microalgae oil (DMO) supplementation during pregnancy and lactation. Method: According to different doses of DMO fed to mother mice during pregnancy and lactation, 66 suckling mice were divided into four groups. Suckling mice whose mothers were fed with 0.7% DMO were designated as low dose group (group L) (n=16), 2.1% DMO as middle dose group (group M) (n=16), 3.5% DMO as high dose group (group H) (n=17) and no DMO as control group (n=17). Before exposing to OVA, 8 suckling mice were killed in each group at 21-day-old. Remaining suckling mice were killed at 59-day-old after repeated OVA exposure. The serum polyunsaturated fatty acid (PUFA) levels of suckling mice were analyzed by high performance liquid chromatography (HPLC) at the age of 21- and 59-day.Histological examinations of jejunum were performed by HE staining and the mast cells in jejunum were observed by toluidine blue staining. OVA-IgE in serum, total IgA and OVA-IgA in the feces and IL-4 and IFN-γ in the supernatants of splenic mononuclear cells (SMC) were measured by ELISA. Real time PCR was performed to identify the gene expression of IL-10, TGF-β1 mRNA in SMC. Differences among groups were compared by one-way AVOVA and that between each group were compared by LSD. Result: In group M and H, the serum levels of n-3DHA (108±29)μg/ml; (102±34)μg/ml vs.(40±19)μg/ml (F=12.052, P=0.000)and n-3 eicosapentaenoic acid (6.7±2.3)μg/ml; (7.7±2.0)μg/ml vs. (3.9±1.1)μg/ml(F=9.573, P=0.000) were significantly higher than that in control group at the age of 21-day. The serum levels of n-3DHA were higher in group H (17.1±2.9)μg/ml than that in control group (5.9±3.3) μg/ml after repeated OVA exposure at the age of 59-day (F=10.339, P<0.000). Compared with control group (53±12) pg/ml, the levels of IL-4 in SMC in group H (42±9)pg/ml were lower (F=2.484, P<0.05). Conclusion: The serum levels of DHA in baby mice, whose mothers was fed with DMO during pregnancy and lactation, were significantly increased till adulthood. However, the effect on tolerance to OVA was limited.

Enhanced humoural and cellular immune responses to influenza H7N9 antigen HA1\u20132 fused with flagellin in chickens

BackgroundSudden increases in the number of human A (H7N9) cases reported during December and January have been observed in previous years. Most reported infection cases are due to prior exposure to live poultry or potentially contaminated environments. Low pathogenicity of influenza A (H7N9) virus in avian species complicates timely discovery of infected birds. Therefore, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry to reduce the risk of human infection and prevent the emergence of novel mutated strains. In addition to a good antigen, an effective vaccine also requires an appropriate adjuvant to enhance its immunogenicity. Previously, we generated an H7N9 influenza recombinant subunit vaccine (HA1–2-fliC), in which haemagglutinin globular head domain (HA1–2) was fused with flagellin (fliC), a potent TLR5 ligand, and demonstrated that HA1–2-fliC elicited effective HA1–2-specific immune responses in mice.ResultsIn this study, we determined flagellin-induced expression profiles of cytokines and chemokines in different types of avian immune cells in vitro and ex vivo. We found that flagellin significantly increased the expression levels of CXCL inflammatory chemokines (CXCLi1 and CXCLi2) and CCL chemokines (MIP-1β and MCP-3) in avian macrophage HD11 cells. In addition, HA1–2-fliC induced significant upregulation of cytokines (IL-1β, IL-6, IL-18 and IFN-γ) and chemokines (CXCLi1, CXCLi2 and MIP-1β) in ex vivo splenic lymphocytes and peripheral blood mononuclear cells (PBMCs), suggesting that flagellin promoted immune responses of avian cells in vitro. We also evaluated specific humoural and cellular immune responses induced by HA1–2-fliC and found that chickens immunised intramuscularly with HA1–2-fliC showed significantly higher HA1–2-specific immunoglobulin (Ig)G titers in serum. Furthermore, HA1–2-fliC potentiated cellular immune responses, as reflected by an increase in CD4+ and CD8+ T cells and proliferation of PBMCs. Significantly higher levels of IFN-γ and IL-4 in PBMCs from chickens vaccinated with HA1–2-fliC further indicated that HA1–2-fliC promoted a balanced Th1/Th2 immune response.ConclusionsWe demonstrated that the use of the flagellin as an adjuvant potentiated immunogenicity of influenza subunit vaccine HA1–2 in vitro and in vivo. These findings provide a basis for the development of H7N9 influenza HA1–2 subunit vaccines for chickens.

Interleukin-1 receptor associated kinase 1 is a potential therapeutic target of anti-inflammatory therapy for systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease and currently has no effective therapy. The genome-wide analyses indicate that interleukin-1 receptor associated kinase 1 (IRAK1) is associated with the susceptibility of SLE in humans. In the present study, we identified that IRAK1 was overexpressed and hyper-activated in splenic mononuclear cells from B6.MRL-Faslpr/Nju (B6.lpr) mice and peripheral blood mononuclear cells (PBMCs) from SLE patients. Intraperitoneal treatment with a small molecular inhibitor of IRAK1 (IRAK1/4 inhibitor or IRAK-Inh) significantly mitigated inflammatory responses and renal injury in B6.lpr mice. IRAK-Inh treatment or knockdown of IRAK1 by specific siRNA decreased the relative levels of NF-κBp65 phosphorylation in human PBMCs from SLE patients. Therefore, IRAK1 may be a potential target for anti-inflammatory therapy for SLE and other inflammatory diseases.

Aloperine Protects Mice against DSS-Induced Colitis by PP2A-Mediated PI3K/Akt/mTOR Signaling Suppression.

Colitis is a major form of inflammatory bowel disease which involved mucosal immune dysfunction. Aloperine is an alkaloid isolated from the shrub Sophora alopecuroides L. and has been recognized as an effective treatment for inflammatory and allergic diseases. The present study aimed to examine the molecular mechanisms underlying aloperine-mediated colitis protection. We found that aloperine treatment improved colitis induced by dextran sodium sulfate (DSS) based on body weight, disease activity index, colonic length, and spleen index. Aloperine also effectively attenuated DSS-induced intestinal inflammation based on the pathological score and myeloperoxidase expression and activity in colon tissues. In addition, aloperine regulated T-cell proportions and promoted Foxp3 expression in the spleens and mesenteric lymph nodes of DSS-induced colitis mice and in the spleens of the Foxp3GFP mice. Aloperine inhibited Jurkat and mouse naïve T-cell apoptosis. Furthermore, aloperine inhibited PI3K/Akt/mTOR signaling and upregulated PP2A expression in the DSS-induced colitis mice and in Jurkat cells, but LB-100 (PP2A inhibitor) resulted in an elevated Akt activity in Jurkat cells, activated T-cells, and human splenic mononuclear cells. Aloperine inhibited T-cell and lymphocyte proliferation, but LB-100 reverse these effects. In conclusion, aloperine regulates inflammatory responses in colitis by inhibiting the PI3K/Akt/mTOR signaling in a PP2A-dependent manner.

Therapeutic effect of ursolic acid in experimental visceral leishmaniasis

Leishmaniasis is an important neglected tropical disease, affecting more than 12 million people worldwide. The available treatments are not well tolerated and present diverse side effects in patients, justifying the search for new therapeutic compounds. In the present study, the therapeutic potential and toxicity of ursolic acid (UA), isolated from the leaves of Baccharis uncinella C. DC. (Asteraceae), were evaluated in experimental visceral leishmaniasis. To evaluate the therapeutic potential of UA, hamsters infected with L. (L.) infantum were treated daily during 15 days with 1.0 or 2.0 mg UA/kg body weight, or with 5.0 mg amphotericin B/kg body weight by intraperitoneal route. Fifteen days after the last dose, the parasitism of the spleen and liver was stimated and the main histopathological alterations were recorded. The proliferation of splenic mononuclear cells was evaluated and IFN-γ, IL-4, and IL-10 gene expressions were analyzed in spleen fragments. The toxicity of UA and amphotericin B were evaluated in healthy golden hamsters by histological analysis and biochemical parameters. Animals treated with UA had less parasites in the spleen and liver when compared with the infected control group, and they also showed preservation of white and red pulps, which correlate with a high rate of proliferation of splenic mononuclear cells, IFN-γ mRNA and iNOS production. Moreover, animals treated with UA did not present alterations in the levels of AST, ALT, creatinine and urea. Taken together, these findings indicate that UA is an interesting natural compound that should be considered for the development of prototype drugs against visceral leishmaniasis.

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation

Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Hydrophobic bile acids suppress expression of AE2 in biliary epithelial cells and induce bile duct inflammation in primary biliary cholangitis

Understanding the mechanisms of chronic inflammation in primary biliary cholangitis (PBC) is essential for successful treatment. Earlier work has demonstrated that patients with PBC have reduced expression of the anion exchanger 2 (AE2) on biliary epithelial cells (BEC) and deletion of AE2 gene has led to a PBC-like disorder in mice. To directly address the role of AE2 in preventing PBC pathogenesis, we took advantage of our ability to isolate human BEC and autologous splenic mononuclear cells (SMC). We studied the influence of hydrophobic bile acids, in particular, glycochenodeoxycholic acid (GCDC), on AE2 expression in BEC and the subsequent impact on the phenotypes of BEC and local inflammatory responses. We demonstrate herein that GCDC reduces AE2 expression in BEC through induction of reactive oxygen species (ROS), which enhances senescence of BEC. In addition, a reduction of AE2 levels by either GCDC or another AE2 inhibitor upregulates expression of CD40 and HLA-DR as well as production of IL-6, IL-8 and CXCL10 from BEC in response to toll like receptor ligands, an effect suppressed by inhibition of ROS. Importantly, reduced AE2 expression enhances the migration of autologous splenic mononuclear cells (SMC) towards BEC. In conclusion, our data highlight a key functional role of AE2 in the maintenance of the normal physiology of BEC and the pathogenic consequences of reduced AE2 expression, including abnormal intrinsic characteristics of BEC and their production of signal molecules that lead to the chronic inflammatory responses in small bile ducts.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4+ mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172+ MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis.

Apolipoprotein A-I gene transfer exerts immunomodulatory effects and reduces vascular inflammation and fibrosis in ob/ob mice.

BackgroundObesity is associated with vascular inflammation, fibrosis and reduced high-density lipoproteins (HDL)-cholesterol. We aimed to investigate whether adenoviral gene transfer with human apolipoprotein (apo) A-I (Ad.A-I), the main apo of HDL, could exert immunomodulatory effects and counteract vascular inflammation and fibrosis in ob/ob mice.MethodsAd.A-I transfer was performed in 8 weeks (w) old ob/ob mice, which were sacrificed 7 w later. The aorta was excised for mRNA analysis and the spleen for splenocyte isolation for subsequent flow cytometry and co-culture with murine fibroblasts. HDL was added to mononuclear cells (MNC) and fibroblasts to assess their impact on adhesion capacity and collagen deposition, respectively.ResultsAd.A-I led to a 1.8-fold (p < 0.05) increase in HDL-cholesterol versus control ob/ob mice at the day of sacrifice, which was paralleled by a decrease in aortic TNF-α and VCAM-1 mRNA expression. Pre-culture of MNC with HDL decreased their adhesion to TNF-α-activated HAEC. Ad.A-I exerted immunomodulatory effects as evidenced by a downregulation of aortic NOD2 and NLRP3 mRNA expression and by a 12 %, 6.9 %, and 15 % decrease of the induced proliferation/activity of total splenic MNC, CD4+, and CD8+ cells in ob/ob Ad.A-I versus control ob/ob mice, respectively (p < 0.05). Ad.A-I further reduced aortic collagen I and III mRNA expression by 62 % and 66 %, respectively (p < 0.0005), and abrogated the potential of ob/ob splenocytes to induce the collagen content in murine fibroblasts upon co-culture. Finally, HDL decreased the TGF-ß1-induced collagen deposition of murine fibroblasts in vitro.ConclusionsApo A-I transfer counteracts vascular inflammation and fibrosis in ob/ob mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-016-0131-6) contains supplementary material, which is available to authorized users.

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling.

Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.

Cardiac Tissue Injury and Remodeling Is Dependent Upon MR Regulation of Activation Pathways in Cardiac Tissue Macrophages.

Macrophage mineralocorticoid receptor (MR) signaling is an important mediator of cardiac tissue inflammation and fibrosis. The goal of the present study was to determine the cellular mechanisms of MR signaling in macrophages that promote cardiac tissue injury and remodeling. We sought to identify specific markers of MR signaling in isolated tissue macrophages (cardiac, aortic) vs splenic mononuclear cells from wild-type and myeloid MR-null mice given vehicle/salt or deoxycorticosterone (DOC)/salt for 8 weeks. Cardiac tissue fibrosis in response to 8 weeks of DOC/salt treatment was found in the hearts from wild-type but not myeloid MR-null mice. This was associated with an increased expression of the profibrotic markers TGF-β1 and matrix metalloproteinase-12 and type 1 inflammatory markers TNFα and chemokine (C-X-C motif) ligand-9 in cardiac macrophages. Differential expression of immunomodulatory M2-like markers (eg, arginase-1, macrophage scavenger receptor 1) was dependent on the tissue location of wild-type and MR-null macrophages. Finally, intact MR signaling is required for the phosphorylation of c-Jun NH2-terminal kinase in response to a proinflammatory stimulus in bone marrow monocytes/macrophages in culture. These data suggest that the activation of the c-Jun NH2-terminal kinase pathway in macrophages after a tissue injury and inflammatory stimuli in the DOC/salt model is MR dependent and regulates the transcription of downstream profibrotic factors, which may represent potential therapeutic targets in heart failure patients.

Activation of Interferon Regulatory Factor 3 (IRF3) is Required in Both T cells and APCs for Optimal Expression of IFN-γ, IL-17, and Granzyme B during T cell responses.

Abstract Recently we saw a link between activation of Interferon Regulatory Factor (IRF)-3 and expression of IFN-γ, as well as Granzyme B (GrB) from T cells during viral infections in C57Bl/6 (B6) and IRF3KO mice. To investigate this further, we stimulated splenic mononuclear cells (SMC) from B6 and IRF3KO mice in vitro with anti-CD3±anti-CD28± poly I:C, a TLR3/IRF3 agonist. We show during responses with anti-CD3/anti-CD28 that IRF3 is activated within 24 h in B6 SMC and expression of IFN-γ, IL-17 and GrB is impaired in IRF3KO SMC. Adding poly I:C with anti-CD3/anti-CD28 accelerated IRF3 activation and significantly increased IFN-γ, but not IL-17 or GrB, from IRF3KO SMC. Interestingly, Foxp3 expression and percentage of Foxp3+ T cells were significantly lower in unstimulated IRF3KO SMC compared with B6 SMC. To determine the contribution of IRF3 activation in T cells vs APCs, B6 and IRF3KO bone marrow dendritic cells (DCs; 3×104) were mixed in various combinations with WT or IRF3KO purified T cells (3×105), as well as anti-CD3±anti-CD28± poly I:C. After 48 h, IFN-γ, IL-17, and GrB expression was greatest in cultures where both B6 DCs and B6 T cells were mixed. IFN-γ, IL-17 and GrB decreased when either IRF3KO DCs or IRF3KO T cells were part of the mixture. Least production of IFN-γ, IL-17, and GrB occurred when DCs and T cells were from IRF3KO mice. These results suggest that IRF3 in both APCs and T cells is required to achieve optimum production of IFN-γ, IL-17, and GrB during T cell responses. Moreover, the data unexpectedly show that IRF3 is activated during T cell immune responses without a TLR3 agonist. The factor(s) responsible for IRF3 activation during APC/T cell responses is unclear but appears to be a soluble factor whose expression is stimulated through CD28.

Immunoregulative effect of Fasudil on encephalomyelitic T cells in experimental autoimmune encephalomyelitis mice

To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms. C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed. Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-β (TGF-β) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05). Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.

PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis

Dogs infected with Leishmania infantum have a reduced number of T lymphocytes. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family that is expressed by immune cells, and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces the deactivation or apoptosis of T cells. This study aimed to evaluate the expression of PD-1 and its ligands, as well as blocking in the induction of apoptosis in T lymphocytes, TNF-α, IL-4 and nitric oxide production by leucokocytes from PBMC and spleen and the parasite load in dogs with visceral leishmaniasis (VL). Our results showed that the expression of PD1 and its ligands was increased in CD3+ T cells and CD21+ B lymphocytes within the peripheral blood and splenic mononuclear cells of dogs with VL. In peripheral blood monocytes, only PD-1 ligands exhibited increased expression; however, in spleen macrophages, increased expression of both PD-1 and its ligands was observed. Levels of apoptosis in peripheral blood and splenic T lymphocytes were higher in dogs with VL compared to healthy dogs. Blocking monoclonal antibodies to PD-1 and its ligands in the culture of mononuclear cells from the peripheral blood and spleen decreased the amount of CD3+ T lymphocyte apoptosis. The concentration of nitric oxide, TNF-α and IL-4 increased in the culture supernatants of peripheral blood mononuclear cells treated with a blocking monoclonal antibody against PD-1. The TNF-α concentration increased in the culture supernatants of splenic cells following all treatments with antibodies blocking PD-1 and its ligands; however, the amount of IL-4 increased only in the presence of a PD-1 blocking agent. Treatment with a PD-1 blocking monoclonal antibody in the mononuclear peripheral blood of dogs with VL reduced the parasite burden while increased TNF-α. We conclude that in canine visceral leishmaniasis, PD-1 and its ligands are involved in the induction of T lymphocyte apoptosis and in regulating the production of nitric oxide, TNF-α, and IL-4, as well as the parasitic load.

Effects of stimulation of muscarinic acetylcholine receptors in medial septum on some immune responses in rats

ObjectivesThough the immunomodulatory role of medial septum (MS) has been indicated, but the contribution of the muscarinic acetylcholine (mAch) receptors presented in the internal network of the neurons of MS in this regard is not known. The aim of the present study is to assess the contribution of mAch receptors of MS on some immunological parameters. MethodsDifferent immunological parameters i.e. phagocytic activity of peripheral leukocytes, adhesibility and cytotoxicity of splenic mononuclear cells (MNC), delayed type of hypersensitivity (DTH) responses, Total Count (TC) and Differential Count (DC) of WBC with serum corticosterone (CORT) concentration have been measured after stimulation and blocking of mAch receptors of MS in rats. Ach or atropine has been micro infused into MS of separate groups of rats for stimulating or blocking of the mAch receptors respectively. ResultsIn Ach or atropine microinfused rats, the TC of WBC remained unaltered in the present study. The percentage of neutrophil has increased and the percentage of lymphocyte has decreased in Ach microinfused rats, but these parameters remain unaltered in atropine microinfused rats. The observed immunological parameters have increased after microinfusion of 0.60μM and 0.12μM of Ach and serum CORT concentration has decreased in those animals. These immunological parameters have decreased and serum CORT has increased after microinfusion of atropine (2 and 4mM) in MS of rats. ConclusionIt appears that the Ach receptors in MS can modulate the observed immunological parameters, and serum CORT plays an important role for these immune changes.