Effect of High Dietary Manganese on the Immune Responses of Broilers Following Oral Salmonella typhimurium Inoculation

Manganese (Mn) is an essential nutrient for both host and pathogen. Recent studies have demonstrated the nutritional immunity of Mn against Salmonella infection in mammals. To investigate the effect of high dietary Mn on immune responses of broilers following Salmonella challenge, 144 1-day-old male broilers were fed a basal diet (containing 20.04 mg Mn/kg) plus an additional 40 (the control group) or 400 mg Mn/kg (the H-Mn group) for 7 days. The 72 broilers in each group were then orally inoculated with 5 × 10⁷ CFUs of Salmonella typhimurium (ATCC#14028) or phosphate-buffered saline. Peripheral blood, spleens, cecal tonsils, and bursa of Fabricius were collected from Salmonella-inoculated and Salmonella-noninoculated broilers (n = 6) at 2 days post inoculation (2 DPI) and 7 days post inoculation (7 DPI). Peripheral blood lymphocyte subpopulations were determined by flow cytometry. The messenger RNA (mRNA) abundance of genes was determined by quantitative real-time polymerase chain reaction. Salmonella counts were higher (P < 0.05) in the H-Mn group than that in the control group at 2 DPI in the cecal contents of Salmonella-inoculated broilers. High dietary Mn increased CD3⁺CD4⁺ and CD3⁺CD8⁺ percentages in the peripheral blood of Salmonella-inoculated broilers at 2 DPI. Salmonella inoculation increased interleukin (IL)-6 mRNA expression in spleens and bursa of Fabricius at 2 DPI and increased IL-1β and IL-6 mRNA expression in cecal tonsils at 7 DPI in the H-Mn group. These changes were not observed in the control group. High dietary Mn increased interferon-γ (IFN-γ) in spleens and decreased IFN-γ and IL-12 mRNA expression in cecal tonsils of Salmonella-inoculated broilers at 2 DPI. High dietary Mn decreased IL-17 mRNA expression in the bursa of Fabricius at 7 DPI, but increased this expression in cecal tonsils at 2 and 7 DPI in Salmonella-inoculated broilers. These results suggested that dietary Mn level affected T helper (Th) 1-cytokine reaction in spleens and cecal tonsils, and Th17-mediated immunity in cecal tonsils and the bursa of Fabricius of broilers when challenged with Salmonella.

     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from <Emphasis Type="Italic">Salmonella</Emphasis>-susceptible and -resistant chickens

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire and are coupled to downstream production of cytokines, chemokines, and antimicrobial peptides by TLR adaptor proteins. Our laboratory previously demonstrated a role for TLR function in the differential innate response of two lines of chickens to bacterial infections. The aim of the present study was to elucidate the role of TLRs in the differential innate responsiveness by measuring differences between lines A (resistant) and B (susceptible) in heterophil mRNA expression of selected TLRs (TLRs 4, 5, and 15) and TLR adaptor proteins (MyD88, TRIF, and TIRAP) in response to stimulation with Salmonella enterica serovar Enteritidis (SE). Although heterophils from both lines had significantly increased expression of TLR 15 mRNA in response to stimulation with SE, heterophils from chickens resistant to infection with SE had significantly greater levels of TLR 15 mRNA expression prior to and following stimulation with SE than heterophils from chickens susceptible to infection with SE. No significant differences were noted between lines in nonstimulated levels of TIRAP, but upon SE stimulation, line A birds had higher levels of expression than B birds. No significant differences were found in heterophils between lines for mRNA expression of TLRs 4 and 5 nor MyD88 and TRIF. These data indicate that differences in the gene expression of TLR 15 by heterophils likely accounts for some of the observed differences between the lines in their susceptibility to infection.     

Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar enteritidis infection of chickens

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14(-) strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.     

CD39-mediated effect of human bone marrow-derived mesenchymal stem cells on the human Th17 cell function

This study investigated the immune-modulatory effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on human Th17 cell function through the CD39-mediated adenosine-producing pathway. The suppressive effects of hBMSCs were evaluated by assessing their effects on the proliferation of Th17 cells and the secretion of interferon (IFN)-γ and interleukin (IL)-17A by Th17 cells with or without anti-CD39 treatment. Changes in CD39 and CD73 expression on the T cells with or without co-culture of hBMSCs were evaluated by flow cytometry. hBMSCs effectively suppressed the proliferation of Th17 cells and the secretion of both IL-17A and IFN-γ from Th17 cells using by both flow cytometry and ELISA, while anti-CD39 treatment significantly reduced the inhibitory effects of hBMSCs on the proliferation and secretion of the Th17 cells. The hBMSCs induced increased expression of the CD39 and CD73 on T cells correlated with the suppressive function of hBMSCs, which was accompanied by increased adenosine production. Our data suggests that hBMSCs can effectively suppress immune responses of the Th17 cells via the CD39-CD73-mediated adenosine-producing pathway.     

Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing glycoprotein B of infectious laryngotracheitis virus and chicken IL-18

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes severe and economically significant respiratory disease in poultry worldwide. Herein, the immunogenicity of two recombinant fowlpox viruses (rFPV-gB and rFPV-gB/IL18) containing ILTV glycoprotein B (gB) and chicken interleukin-18 (IL-18) were investigated in a challenge model. One-day-old specific-pathogen-free chickens were vaccinated by wing-web puncture with the two rFPVs and challenged with the virulent ILTV CG strain. There were differences in antibody levels elicited by either rFPV-gB/IL18 or rFPV-gB as determined using ELISA. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-gB/IL18 were higher (P < 0.05) than in those immunized with rFPV-gB, and the level of proliferative response of the T cells in the rFPV-gB/IL18-vaccinated group was higher (P < 0.05) than that in the rFPV-gB group. All chickens immunized with rFPV-gB/IL18 were protected (10/10), whereas only eight of 10 of the chickens immunized with the rFPV-gB were protected. The results showed that the protective efficacy of the rFPV-gB vaccine could be enhanced by simultaneous expression of chicken IL-18.     

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.     

Mesenchymal stromal cells but not cardiac fibroblasts exert beneficial systemic immunomodulatory effects in experimental myocarditis

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.     

The Adverse Effects of Se Toxicity on Inflammatory and Immune Responses in Chicken Spleens

Selenium (Se) is an essential trace element, but excessive intake of Se could induce Se poisoning, and result in various health problems. NF-κB regulated many molecules of the immune response and the inflammatory response, and Th1/Th2 balance played a key in the regulation of immune response. The aim of this study is to investigate the role of NF-κB pathway and Th1/Th2 imbalance in the adverse influence of Se poisoning on chicken spleens. In the current study, 90 chickens were randomly divided into two groups (n?=?45 per group). The chickens were maintained either on a basal diet (the control group) containing 0.2 mg/kg Se or a high supplemented diet (the Se group) containing 15 mg/kg Se for 45 days. Then, we observed the pathohistology of spleen cells and detected NO content, iNOS activity, and the expression of NF-κB, iNOS, COX-2, PTGE, IL-6, TNF-α, Foxp3, IL-4, and IFN-γ in chicken spleens. In chicken spleens of the Se group, the result showed typical characteristics of inflammation: the content of NO and the activity of iNOS were increased, and the expression of NF-κB, iNOS, COX-2, PTGE, IL-6, TNF-α, and IL-4 was enhanced and that of Foxp3 and IFN-γ was decreased. Our study showed that Se toxicity could promote inflammation via NF-κB pathway, impairing the immune function, and changing Th1/Th2 balance in the chicken spleens.     

PVAX-MAGE-1基因疫苗延缓小鼠肝癌细胞H22腹水瘤和实体瘤的生长

为观察重组基因疫苗PVAX-MAGE-1的抑瘤效应,构建黑色素瘤抗原-1(melanoma antigen-1,MAGE-1)真核基因表达载体--PVAX-MAGE-1.以重组质粒免疫C57BL/6小鼠后,ELISA法检测表明,与对照鼠(PVAX-1和生理盐水注射小鼠)比较,免疫小鼠脾淋巴细胞上清液中的细胞因子IL-2和IFN-γ明显升高(P0.05);淋巴细胞-肿瘤细胞混合培养证明,免疫小鼠外周血CD8+T细胞对靶细胞的特异性杀伤作用明显增强(P0.05).体内实验证明,PVAX-MAGE-1免疫C57BL/6小鼠,可显著延缓移植性H22腹水瘤及实体瘤在小鼠体内的生长.实验结果提示,重组基因疫苗PVAX-MAGE-1有明显的延缓肿瘤生长的作用,其抑瘤作用与提高T淋巴细胞IL和IFN表达,增强对肿瘤杀伤作用直接相关.     

HIV结构蛋白与痘苗病毒共表达产物的免疫原性研究

目的 将艾滋病病毒外膜蛋白(env)与干扰素(IFNα-2b)融合基因表达的融合蛋白作为免疫原免疫小鼠,观察小鼠的免疫功能状态和细胞免疫应答.方法 将IFNα-2b基因片段插入到env基因的下游,经脂质体转染,筛选重组痘苗病毒,经SDS-PAGE和Western blot鉴定表达产物.用重组病毒vJ1 6env/IFNα-2b免疫小鼠,以生理盐水和野生型痘苗病毒作为对照,检测小鼠脾淋巴细胞对ConA、LPS及IgG的反应性;用流式细胞仪测定小鼠脾淋巴细胞CD4 、CD8T细胞计数与CTL.结果 与对照组比较,实验组脾淋巴细胞对ConA、LPS及IgG的反应性显著增高(P＜0.05);CD4T淋巴细胞计数和CTL活性也显著增高(P＜0.05);CD8T淋巴细胞计数呈增高趋势,但未达到显著意义的程度.结论 重组痘苗病毒v J16env/IFNα-2b能增强小鼠的免疫功能和诱导细胞免疫.     

Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: Interferon-gamma is a key factor in the protection of Marek's disease by vaccination

Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-γ induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-γ could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.     

Short-term protection conferred by Leishvacin® against experimental Leishmania amazonensis infection in C57BL/6 mice

To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.     

Successful vaccination of BALB/c mice against human hookworm (Necator americanus): the immunological phenotype of the protective response

In this murine (BALB/c) model of necatoriasis, high levels of protection against challenge infection by Necator americanus larvae (n=300) were afforded by successive vaccinations at 14-day intervals, either subcutaneously or percutaneously, with gamma-irradiated N. americanus larvae (n=300). Percutaneous vaccination was significantly more effective than the subcutaneous route, with pulmonary larval burdens at 3 days post-infection being reduced by 97.8 vs. 89.3%, respectively, after three immunisations (P<0.05). No worms were recovered from the intestines of thrice vaccinated mice. Two percutaneous vaccinations also reduced worm burdens, by 57% in the lungs and 98% in the intestines; P<0.05. In vaccinated animals, lung pathology (mainly haemorrhage) following infection was greatly reduced compared with non-vaccinated animals. In vaccinated mice (but not in non-vaccinated mice) mast cells accumulated in the skin and were degranulated. RT-PCR analyses of mRNAs in the skin of vaccinated animals indicated increased expression of interleukin (IL)-4 relative to gamma-interferon (gamma-IFN). Lymphocytes from the axillary (skin-draining) lymph nodes of vaccinated mice, stimulated in vitro with concanavalin A, exhibited enhanced secretion of IL-4 protein and a higher IL-4/gamma-IFN protein ratio than lymphocytes from non-vaccinated animals. In vaccinated mice, levels of IgG1 and IgG3 (directed against larval excretory/secretory products) were elevated for the most part compared with those in non-vaccinated animals. These data demonstrate the successful vaccination of BALB/c mice against human hookworm infection and suggest that a localised Th2 response may be important for conferring protection against necatoriasis.     

Micro RNA-19a suppresses IL-10 in peripheral B cells from patients with atherosclerosis

Background and aimsThe interleukin (IL)-10-production B cells play an important role in the pathogenesis of atherosclerosis (Asro) with unknown mechanism. Micro RNA (miR)-17-92 cluster has strong immune regulatory activities. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells of Asro patients.MethodsPatients with Asro were recruited into this study. Peripheral blood samples were collected from the patients. B cells were isolated from the blood samples and analyzed to elucidate the role of miR-17-92 in the regulation of IL-10 expression.ResultsPeripheral B cells from patients with Asro show lower levels of IL-10 than that from healthy subjects. The IL-10 expression in the B cells is negatively correlated with the expression of miR-19a in the B cells. The serum levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4 in Asro patients were higher than healthy subjects. Exposure to TNF-α or IFN-γ or IL-4 suppressed IL-10 expression in B cells via increasing the expression of miR-19a in B cells, which could be abolished by Inhibition of miR-19a.ConclusionsTNF-α or IFN-γ or IL-4 suppresses IL-10 in B cells via up regulating miR-19a expression.     

Immunogenicity and protective capacity of a virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.