Classical swine fever virus suppresses maturation and modulates functions of monocyte-derived dendritic cells without activating nuclear factor kappa B

Classical swine fever virus (CSFV) compromises the host immune system, causing the severe disease of pigs. Dendritic cells (DCs) are the most potent inducers of immune responses. In the present study, we investigated the functional properties of porcine monocyte-derived DCs (Mo-DCs) affected by CSFV. Results showed that the expression of surface markers of DCs such as major histocompatibility complex class II (MHC-II), CD80, CD83 and CD86 were unimpaired, but an obviously increased expression of CD172a in DCs was noticed 48 h after CSFV infection. The expression profiles of cytokines were detected in cultured Mo-DCs after various treatments for 48 h by Q-RT-PCR. The findings suggested that CSFV infection significantly increased the mRNA expression of IL-10 and TNF-α, and inhibited IL-12 expression, with little effect on IFN-α and IFN-γ expression. We further demonstrated that CSFV was incapable of activating the nuclear factor kappa B (NF-κB) in infected DCs, which was characterized by an unvaried DNA binding activity of NF-κB, the lack of translocation of p65/RelA from the cytoplasm to the nucleus and the stabilization of p65/RelA expression. Furthermore, Western blot analysis indicated that the inactivation of NF-κB was due to the failure of IκBα degradation. The data demonstrated that CSFV could be replicated in DCs and CSFV infection could modulate the secretion of crucial co-stimulatory molecules and cytokines which down-regulated maturation of DCs, without activating NF-κB in DCs. Thus, the results suggested a possible mechanism for CSFV evasion of innate host defenses, providing the basis for understanding molecular pathways in CSFV pathogenesis.     

Expression kinetics of chicken β2-microglobulin and Class I MHC in vitro and in vivo during Marek’s disease viral infections

Marek’s disease virus (MDV) is a highly cell-associated herpesvirus that causes a disease in chickens characterized by tumor formation and immunosuppression. The changes of major histocompatibility complex (MHC) expression in different MDV-infected cells are not completely understood. In this study, we investigated the expression of the Class I MHC and β2-microglobulin (β2m) genes in response to MDV infection at different time points by real-time PCR. In both in vitro and in vivo, the expression levels of Class I MHC and β2m genes were upregulated during early MDV infections in comparison to control cells; We also found that the expression of Class I MHC gene was downregulated in BudR (5-bromo-2′-deoxyuridine)-treated MSB1 cells at 48 h and MDV-infected chicken embryo fibroblast cells (CEF) at 120 and 168 h post infection (hpi); Furthermore, compared to control groups, Class I MHC and β2m expression levels were downregulated in peripheral blood lymphocytes (PBLC) from MDV-infected chickens at 14 and 28 days post infection (dpi); Interestingly, both Class I MHC and β2m gene expression levels increased again in PBLC from MDV RB1B-infected chickens at 35 dpi, in which MDV was in the latent or transformed infection stages. In addition, Class I MHC expression was clearly decreased in MDV-infected CEF at 120 hpi although β2m expression was significantly increased. These changes in Class I MHC and β2m gene expression might provide more insights into host-virus interaction.     

The activation of the IFNβ induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable

Background

It has been recognized that the expression of type I interferon (IFNα/β) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection.

Objective

Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNβ) and downstream signaling events.

Methods

In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNβ protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events.

Results

These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNβ protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNβ signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNβ production.

Conclusion

The results of this study demonstrate that the induction of IFNβ and signaling in PAMs after PRRSV infection is variable.

     

Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.     

Cytokine modulation of the interaction between bluetongue virus and endothelial cells in vitro.

An in vitro model was developed to examine the interaction between endothelial cells and the host inflammatory response in bluetongue virus (BTV) infections. Whole cell enzyme-linked immunosorbent assays, a tritiated thymidine uptake assay, and a colorimetric assay of mitochondrial function were used to assess how four cytokines (interleukin-1, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) affect endothelial cell metabolism and susceptibility to BTV infection. Concurrent alterations in major histocompatibility complex (MHC) antigen expression were also examined. BTV infection suppressed target cell mitochondrial function and DNA synthesis and enhanced MHC class I expression. Interferon-gamma and tumor necrosis factor alpha suppressed viral antigen expression and were synergistic early in the infection. Interferon gamma enhanced MHC class I and induced MHC class II antigen expression in both BTV infected and uninfected endothelial cells. The other cytokines had minimal effect on endothelial cell surface antigen expression, although interleukin-1 (IL-1) did inhibit cell growth. Infected endothelial cell cultures produced interferon at 20 hours and 40 hours after infection. Electron microscopic analysis confirmed previous findings in other cell lines regarding BTV morphogenesis in endothelial cells, the putative target cell population in vivo.     

原头蚴外泌体对树突状细胞活化和细胞因子分泌的影响

为分析囊型包虫原头蚴(PSC)分泌的外泌体(Exosome)对树突状细胞(DCs)活化和分泌细胞因子的影响,本研究采用高速离心法结合外泌体提取试剂盒分离原头蚴分泌的外泌体(PE),收集的PE沉淀中加入去离子水低渗裂解离心,提取超滤后的蛋白浓缩液即为原头蚴外泌体超滤裂解物(PEL)。利用免疫磁珠从健康ICR小鼠脾脏中分选DCs,分别加入PE、LPS、PEL、PBS进行刺激培养,24 h后收集培养细胞及培养上清,利用流式细胞术检测DCs表面活化分子的表达水平及细胞因子分泌情况。结果显示:4组DCs表面表达CD40、CD80、CD86、MHCⅡ的比例相比,PEL组结果明显高于PE组,这两组较PBS组均显著升高(p<0.05),但3组均低于LPS组。PE、PEL组DCs培养上清中IL-6、IL-10、IL-12、TNF-α均显著高于PBS组,且PEL组IL-12、TNF-α高于PE组,IL-6、IL-10水平低于PE组。以上结果初步证实PE能刺激DCs活化和释放细胞因子,低渗裂解超滤提纯外泌体蛋白并去除miRNA成分的PEL能够更有效激活DCs活化和促进细胞因子释放,表明外泌体蛋白可诱导抗感染免疫应答,exosomal miRNA成分可能参与虫体免疫逃逸。本研究为后续进一步探索包虫感染及抗感染免疫应答机理提供实验依据。     

Interaction between innate immunity and porcine reproductive and respiratory syndrome virus

Innate immunity provides frontline antiviral protection and bridges adaptive immunity against virus infections. However, viruses can evade innate immune surveillance potentially causing chronic infections that may lead to pandemic diseases. Porcine reproductive and respiratory syndrome virus (PRRSV) is an example of an animal virus that has developed diverse mechanisms to evade porcine antiviral immune responses. Two decades after its discovery, PRRSV is still one of the most globally devastating viruses threatening the swine industry. In this review, we discuss the molecular and cellular composition of the mammalian innate antiviral immune system with emphasis on the porcine system. In particular, we focus on the interaction between PRRSV and porcine innate immunity at cellular and molecular levels. Strategies for targeting innate immune components and other host metabolic factors to induce ideal anti-PRRSV protection are also discussed.     

N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro

Nitric oxide (NO) was proposed to be an important molecule against some microorganisms. In this study, we investigated the inhibitory effect of NO on the infection by porcine reproductive and respiratory syndrome virus (PRRSV) in vitro and the role of NO in the defense against PRRSV. Our results indicated that exogenous NO did not inhibit PRRSV infection. Unexpectedly, N-acetylpenicillamine (NAP), a commonly used compound as negative control for NO-producing reagents, inhibited PRRSV replication. Thus, the inhibition effect of NAP on PRRSV replication was further explored. We found that the maximal inhibition effect of NAP on PRRSV replication was achieved upon treatment 1 h after virus infection and the virus yield was reduced by approximately 50 fold in the presence of 400 μM NAP. An obvious inhibitory effect on viral RNA and protein synthesis was also observed. However, the inhibitory effect was only achieved at early phase of virus infection. The normal virus yield could be restored upon the removal of NAP treatment. The inhibitory effect might be caused by sulfhydryl-reducing capacity and metal chelating properties of NAP. These studies suggested that (i) NO production or NO synthase (NOS) expression profiling may not be a reliable index for the immune response to PRRSV; (ii) NAP could inhibit the replication of PRRSV.     

Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates

ABSTRACT: The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers.     

Acute phase response in porcine reproductive and respiratory syndrome virus infection

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.     

Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

To investigate cytokine alterations in pigs infected in-utero with porcine reproductive and respiratory syndrome virus (PRRSV), constitutive mRNA expression by peripheral blood mononuclear cells (PBMCs) was measured. PBMC from in-utero PRRSV-infected pigs displayed significantly increased IL-6, IL-10, and IFN-gamma mRNA expression at 0 and 14 days of age compared with age-matched control pigs. There were no significant differences in IL-2, IL-4, and IL-12 mRNA expression between in-utero PRRSV-infected and control pigs. However, the IL-10/IL-12 ratio was significantly increased in in-utero PRRSV-infected pigs at 0 and 14 days of age, suggesting the imbalance of IL-10 and IL-12 mRNA production. The abnormal mRNA expression of cytokines in in-utero PRRSV-infected pigs occurred concurrently with a significant decrease in the CD4(+)/CD8(+) T-cell ratio in peripheral blood. PRRSV was not isolated from the sera of pigs at 9 weeks of age that had been viremic at 0 and 14 days old. Delayed type hypersensitivity (DTH) responses to Tuberculin and analysis of cytokine mRNA expression by PBMC showed that cell-mediated immune response and cytokine message profiles in pigs infected in-utero with PRRSV had returned to levels similar to those of control pigs by 9 weeks of age. We conclude that in-utero infection with PRRSV results in significant alteration of cytokine mRNA expression that may cause transient immunomodulation. However, at 10 weeks of age the pigs' immune responses seemed to recover. This may help to understand the immunopathogenesis of in-utero PRRSV infection and the increased susceptibility to secondary bacterial pathogens in neonatal piglets.     

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages. Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli. At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells. However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.     

布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

为探究Ⅳ型分泌系统在布鲁氏菌(Brucella)感染过程中的作用,深入了解布鲁氏菌Ⅳ型分泌系统在疫苗开发中的潜力,本研究以牛种布鲁氏菌A19疫苗株为研究对象,使用A19 VirB启动子缺失株感染小鼠树突状细胞(DCs),通过菌落计数(CFU)评估Ⅳ型分泌系统对布鲁氏菌黏附侵袭及胞内生存的影响,同时对感染的细胞进行RNA和总蛋白的提取,分别通过实时荧光定量PCR和Western blotting检测自噬基因Beclin-1的转录和表达情况;收集感染后的细胞上清液,利用ELISA检测炎症因子白细胞介素-6(IL-6)和IL-10的分泌水平。黏附侵袭结果显示,布鲁氏菌VirB启动子缺失株与亲本株A19的黏附侵袭水平无显著差异(P>0.05);胞内生存试验发现,感染的4 h,布鲁氏菌VirB启动子缺失株的胞内存活能力显著低于亲本株A19(P<0.05),感染后0、24和48 h极显著低于亲本株A19(P<0.01);实时荧光定量PCR和Western blotting结果显示,感染后4、8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生Beclin-1的水平极显著高于亲本株A19(P<0.01);ELISA结果显示,感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生IL-6的水平显著高于亲本株A19(P<0.05),而在感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞分泌IL-10的水平显著低于亲本株A19(P<0.05),感染24 h时极显著低于亲本株A19(P<0.01)。综上所述,当VirB启动子缺失后,布鲁氏菌对DCs的黏附侵袭能力并未明显改变,但显著降低了布鲁氏菌在DCs内的存活能力,提升了DCs的自噬水平,促进了DCs IL-6的分泌,抑制了IL-10的分泌。本研究初步探究了布鲁氏菌Ⅳ型分泌系统在感染DCs过程中的生物学作用,为后续布鲁氏菌疫苗改造研究奠定了理论基础。     

Persistent upregulation of MHC class II antigen expression on T-lymphocytes from cats experimentally infected with feline immunodeficiency virus.

A significant elevation in the percentage of CD4+ and CD8+ T-lymphocytes expressing major histocompatibility complex (MHC) Class II antigens was observed in the blood of cats shortly after they were experimentally infected with feline immunodeficiency virus (FIV). In addition to an increase in the relative proportion of T-lymphocytes expressing Class II antigens, there was an increase in the density of Class II antigens on the cell surface. These elevations were still evident at the completion of the 5 month study. A second group of cats that had been infected with FIV for almost 5 years, and with either normal or abnormally low levels of CD4+ T-lymphocytes, had similar elevations in MHC II expression, suggesting that such abnormalities are lifelong. Cats with chronic (2 year) feline leukemia virus (FeLV) infection or dual FIV/FeLV infections also showed similar alterations in MHC II expression on CD4+ and CD8+ T-lymphocytes, suggesting that these alterations were not FIV specific. Feline T-lymphocytes expressed more MHC II antigen and interleukin-2 (IL-2) receptor following stimulation in vitro with conconavalin A and IL-2, demonstrating that feline T-lymphocytes respond to activation signals in a manner similar to T-lymphocytes of other species. However, changes in MHC II expression on T-cells of FIV infected cats were not explainable by viral induced T-cell activation alone, because FIV infected cats with elevated MHC II expression did not have coincident elevations in IL-2 receptor expression.     

病毒免疫逃逸防卫机制

从各种各样的病毒免疫逃逸机制中归纳概括出病毒逃避宿主抗感染反应的三个大方面,包括病毒逃避体液免疫系统的识别、病毒抑制细胞免疫应答以及病毒干扰免疫效应功能,以期为研制新的病毒疫苗和抗病毒药物提供参考。在宿主和病毒的长期共同进化过程中,宿主发展了各种免疫机制以清除病毒,而病毒则进化出各种免疫逃逸机制来躲避宿主的免疫应答。     

Maturation of bovine dendritic cells by lipopeptides

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFalpha. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNgamma secretion. In contrast, exposure of MPhi to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFalpha secretion. These different changes in bovine DC and MPhi may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MPhi having a much less marked effect on immune responses.