呼吸道合胞病毒感染巨噬细胞活化Toll样受体3介导的抗病毒作用研究

目的探讨呼吸道合胞病毒(RSV)感染巨噬细胞(RAW264.7细胞)后,Toll样受体3(TLR3)的水平变化及其介导产生的I型干扰素的抗病毒作用。方法 RSV感染体外培养的小鼠巨噬细胞RAW264.7细胞,并给予TLR3特异性抗体处理,分别于感染的4、8、12、16和24h后收集各组细胞。以未感染病毒的细胞为对照组。用Trizol提取细胞总RNA,半定量RT-PCR法检测TLR3、干扰素α(IFN-α),干扰素β(IFN-β),RSVF蛋白的mRNA表达量变化。结果 (1)RSV感染RAW264.7细胞后,TLR3、IFN-α、IFN-β、RSVF蛋白的mRNA表达量均升高且有时间依赖性,TLR3mRNA24h表达量是基础表达量的6倍多,IFN-α,IFN-βmRNA24h表达量是基础表达量的4倍多,RSVF蛋白mRNA是基础表达量的近1.8倍。(2)预先给予TLR3抗体处理以抑制TLR3受体后,再行RSV感染,IFN-α和IFN-β的mRNA表达量虽有升高,但较感染组相比均有下降,mRNA表达在12h后显著降低,且IFN-β的mRNA表达量下调更明显。但RSVF基因的mRNA表达在12h后升高有统计学差异,24h升高有显著性差异。结论 RSV感染RAW264.7巨噬细胞后可上调TLR3表达,其活化细胞介导产生的I型干扰素起抗病毒作用,在一定程度上可抑制病毒的增殖水平。     

Respiratory syncytial virus causes increased bronchial epithelial permeability

BACKGROUND: Respiratory syncytial virus (RSV)-induced diseases are mediated through active cytokines released during infection. We hypothesized that RSV infection causes bronchial epithelial monolayer permeability in vitro via induction of vascular endothelial growth factor (VEGF). METHODS: Human bronchial epithelial cells were infected with RSV. In some cultures, VEGF antibody was included to block VEGF response; in other cultures, palivizumab was added to block RSV infection. Permeability was assessed in real-time using electric cell-substrate impedance sensing. VEGF release was assessed using enzyme-linked immunosorbent assay. Gap formation was assessed using live cell imaging. RESULTS: RSV-infected cells demonstrated a decrease in the resistance of the monolayer indicating an increase in permeability; this increase was blocked with VEGF-specific antibody, and palivizumab. Intercellular gap formation developed in RSV-infected epithelial monolayers. CONCLUSION: RSV increases permeability of the bronchial airway epithelial monolayer via VEGF induction.     

呼吸道合胞病毒感染对豚鼠咳嗽相关气道功能及其神经递质的影响

目的 观察呼吸道合胞病毒RSV(感染)对豚鼠咳嗽相关的气道功能及神经递质的影响,探讨病毒感染后咳嗽的发病机制.方法 雄性SPF级豚鼠60只,按数字随机法随机分成正常对照组、病毒感染组和哮喘组,病毒感染组按病毒感染后天数分为6、12、28和42 d四个组,每组10只.通过滴鼻方法接种RSV.Buxco肺功能仪测定咳嗽反射...     

呼吸道合胞病毒感染巨噬细胞诱导炎性基因表达的部分机制研究

目的探讨呼吸道合胞病毒(RSV)感染巨噬细胞时前炎介质肿瘤坏死因子-α(TNF-α)和诱导型一氧化氮合酶(iNOS)的基因表达变化及其调控的相关机制,为研究RSV的致病机制及有效预防和治疗RSV疾病提供新的思路。方法以RSV感染RAW264.7巨噬细胞,并设立不同的感染时间点(1h、4h、8h、16h和24h),同时给予PDTC(核转录因子NF-κB的特异性抑制剂)处理。以紫外线灭活RSV(UV-RSV)来分析有传染性的病毒的感染变化。收集各组细胞,用Western blot法检测细胞核内活性NF-κBp65蛋白的表达,半定量RT-PCR法检测TNF-α和iNOS mRNA表达量。结果RSV感染4h后,细胞核内活性NF-κBp65蛋白、TNF-α和iNOS mRNA表达均明显升高,各指标的变化与正常对照相比,差异均有显著性,并且与RSV感染存在时间依赖关系。当加入PDTC抑制NF-κB的入核活化后,则可显著下调相应时间点的RSV感染升高的TNF-α和iNOS mRNA表达量,使其降低至基线水平。而UV-RSV感染后并不引起NF-κB蛋白、TNF-α和iNOS mRNA的表达量增加(P>0.05)。结论RSV感染巨噬细胞可诱导前炎基因TNF-α和iNOS的大量表达,其表达可能主要依赖NF-κB活化,并且与病毒复制有关。提示在RSV感染的巨噬细胞中,NF-κB活化对TNF-α和iNOS基因表达具有重要的正调控作用。     

Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and gob-5

Respiratory syncytial virus (RSV) is associated with wheezing and childhood asthma. We previously reported that RSV infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin (OVA)-sensitized mice. In addition, allergically sensitized RSV-infected (OVA/RSV) mice had more abundant airway epithelial mucus production compared with OVA mice 14 days after infection, whereas there was almost no mucus in mice that were only RSV infected. We hypothesized that this increased mucus was associated with mucosal expression of Muc5ac, a mucus gene expression in airways, and gob-5, a member of the Ca(2)(+)-activated chloride channel family. By histochemical analysis, we found that there was significantly increased staining for gob-5 and Muc5ac in the airways of OVA/RSV mice compared with either OVA mice or allergically sensitized mice that were challenged with inactivated RSV, and virtually no detectable staining in the RSV group. These findings were confirmed by Western blot analysis. The increased mucus expression in the OVA/RSV group was associated with increased lung levels of interleukin-17, a factor known to stimulate airway mucin gene expression. The impact of virus infection combined with allergic inflammation on mucus production may partially explain the more severe disease and airway hyperresponsiveness associated with RSV in the setting of atopy.     

己酮可可碱对呼吸道合胞病毒感染的人肺泡巨噬细胞TNF—α表达水平的影响

目的 观察己酮可可碱（PTX）对呼吸道合胞病毒9RSV）所诱导的人肺泡灌洗液巨噬细胞肿瘤坏死因子（TNF－α）含量及TNF-α mRNA表达水平的影响。方法 收集咳嗽患者正常镜像的支气管肺泡灌洗液巨噬细胞,随机分成三组：（1）对照组（NOR组）;（2）感染组（RSV组）加入10^6pfu的RSV;（3）己酮可可碱组（PTX组）RSV感染后加入PTX（1mg/ml）。每组于RSV感染20h后,用ELISA法测定培养细胞上清中TNF－α含量的变化,用逆转录聚合酶链反应（RT－PCR）测定各组巨噬细胞TNF-α mRNA表达水平。结果 感染组中TNF-α mRNA表达水平与对照组相比明显增多。PTX组TNF-α mRNA的表达和感染组相比明显降低。TNF－α的含量也有相应的变化：与对照组比较,感染组明显升高（P＜0.01);而PTX组与感染组比较明显下降（P＜0.01）。结论 PTX能抑制SV所诱导的人肺泡巨噬细胞TNF－α的基因表达,并减少TNF-α的产生。     

RSV-induced bronchial epithelial cell PD-L1 expression inhibits CD8+ T cell nonspecific antiviral activity

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8(+) T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8(+) T cells, we demonstrated that RSV-infected BECs increased CD8(+) T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8(+) T cells enhanced CD8(+) T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.     

Regulation of mite allergen-pulsed murine dendritic cells by respiratory syncytial virus

Dendritic cells (DCs) are the only antigen-presenting cells that determine T-cell differentiation and play an important role in both allergy and viral infection. Respiratory syncytial virus (RSV) can infect DCs and affect their functions. The aim of this study was to determine the interaction between RSV infection and Dermatophagoides farinae allergen (D. farinae) sensitization on the development of allergy at the DC level. Murine bone marrow-derived DCs were prepared and treated as: control; D. farinae-pulsed DCs (D. farinae-DCs); ultraviolet-inactivated RSV challenged; RSV-infected, D. farinae-pulsed plus ultraviolet-inactivated RSV-challenged; and D. farinae-pulsed plus RSV-infected. In in vitro experiments, we compared the expression of costimulatory molecules and cytokine production between the six groups of DCs. Another group of naive mice were then intranasally inoculated with these DCs, after which intranasal challenge with D. farinae was performed to develop allergic airway inflammation in vivo. In comparison with D. farinae-DCs, D. farinae-pulsed plus RSV-infected DCs showed helper T cell (Th) 1-favored expression of costimulatory molecules and cytokine production. Allergic airway inflammation induced by intranasal instillation of D. farinae-DCs was abrogated when infected with RSV, which was associated with a concomitant suppression of Th2 response in the lung. Our results indicated that RSV suppresses D. farinae-DCs to induce Th2 response both in vitro and in vivo through regulation of expression of surface markers and production of immunoregulatory cytokines.     

Respiratory syncytial virus impairs T cell activation by preventing synapse assembly with dendritic cells

Respiratory syncytial virus (RSV) infection is one of the leading causes of infant hospitalization and a major health and economic burden worldwide. Infection with this virus induces an exacerbated innate proinflammatory immune response characterized by abundant immune cell infiltration into the airways and lung tissue damage. RSV also impairs the induction of an adequate adaptive T cell immune response, which favors virus pathogenesis. Unfortunately, to date there are no efficient vaccines against this virus. Recent in vitro and in vivo studies suggest that RSV infection can prevent T cell activation, a phenomenon attributed in part to cytokines and chemokines secreted by RSV-infected cells. Efficient immunity against viruses is promoted by dendritic cells (DCs), professional antigen-presenting cells, that prime antigen-specific helper and cytotoxic T cells. Therefore, it would be to the advantage of RSV to impair DC function and prevent the induction of T cell immunity. Here, we show that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells. Inhibition of T cell activation by RSV was observed independently of the type of TCR ligand on the DC surface and applied to cognate-, allo-, and superantigen stimulation. As a result of exposure to RSV-infected DCs, T cells became unresponsive to subsequent TCR engagement. RSV-mediated impairment in T cell activation required DC-T cell contact and involved inhibition of immunological synapse assembly among these cells. Our data suggest that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance.     

Respiratory syncytial virus alters electrophysiologic properties in cotton rat airway epithelium

The effect of respiratory syncytial virus (RSV) infection on the electrophysiologic properties of the airway epithelium was studied in tracheas obtained from cotton rats, after in vivo exposure to the virus. RSV infection was documented by tissue culture infectivity and immunofluorescent antibody techniques. Light microscopic studies of the tracheas 72 hours after exposure to RSV revealed normal epithelial morphology. RSV infection produced a 28% decrease in short-circuit current (Isc) and an almost 100% increase in tissue resistance. The decrease in Isc was partially attributed to a decrease in Cl secretion, while the increase in tissue resistance was associated with significant restriction to sodium and chloride movement through the paracellular pathway in RSV-infected tracheas. We conclude from these studies that RSV infection in the cotton rat trachea produces significant changes in active and passive ion flows. Alterations in ion transport across the airway epithelium may result in changes in secondary water transport across the airways and may contribute to the pathophysiology of RSV bronchiolitis and other locally invasive mucosal viral infections in children.     

呼吸道合胞病毒诱导一氧化氮产生的机制

目的探讨呼吸道合胞病毒(RSV)感染时一氧化氮(NO)的水平变化及其产生的调控机制,为临床治疗RSV疾病提供有益的思路。方法以RSV感染人肺上皮细胞A549细胞,并设立不同的感染时间(4h、8h、16h和24h),同时分别给予PDTC(核转录因子NF-κB的特异性抑制剂)或AG(诱导型一氧化氮合酶i NOS的特异性抑制剂)处理。收集各组细胞和细胞培养上清,用Western blot法检测细胞核内活性NF-κBp65蛋白的表达,半定量RT-PCR和免疫细胞化学法检测i NOS mR-NA和蛋白表达,硝酸还原酶法检测细胞培养上清中NO含量。结果RSV感染4h后,核内活性NF-κBp65蛋白、i NOS mR-NA和蛋白、细胞培养上清中NO含量均升高,各指标的变化与正常对照相比,差异均有显著性,并且与RSV感染存在时间依赖关系。加入PDTC后可明显抑制NF-κB活化,同时下调i NOS mRNA和蛋白表达。加入AG后,则明显降低细胞培养上清中NO含量。结论RSV感染可诱导产生大量的NO,其主要受i NOS的调节。NF-κB活化对i NOS基因表达具有重要的正调控作用。提示RSV诱导NO生成可能通过活化NF-κB所致。     

呼吸道合胞病毒感染后气道上皮细胞Toll样受体4的表达及其功能

目的 观察呼吸道合胞病毒(RSV)感染后气道上皮细胞Toll样受体4(TLR4)表达变化及其信号通路的功能,探讨RSV诱导气道炎症的机制.方法 体外培养人气管上皮细胞株9HTEo,以RSV感染复数为10感染上皮细胞,用半定量逆转录PCR(RT-PCR)检测TLRl～10 mRNA表达;用定量PCR检测TLR4 mRNA表达的动态变化;用流式细胞术检测TLR4蛋白表达及与细胞凋亡的关系;感染后再用TLR4激动剂脂多糖(LPS)刺激细胞,用酶联免疫法(ELISA)检测上清液中白细胞介素-8(IL-8)含量以观察病毒诱导表达的膜TLR4蛋白功能.RT-PCR和流式细胞术实验分为正常组和RSV感染组,用GraphPad 4.0统计软件进行配对t检验;实时定量PCR实验分为正常组、RSV感染组和紫外线灭活RSV感染组,采用Kruskal-Wallis检验分析;ELISA实验分为正常组、RSV感染组、单独LPS刺激组和RSV-LPS共刺激组,采用One-way ANOVA检验分析.结果 (1)RSV感染组TLR2～10 mRNA表达均上调(t值为3.49～14.47,P均＜0.05),以TLR2和TLR6变化最为显著;定量PCR结果提示:感染组TLR4 mRNA 3 h后开始增高(Kruskal-Wallis检测值=8.82,P＜0.05,n=6),紫外灭活RSV组TLR4 mRNA表达无明显变化;(2)流式细胞术结果表明:RSV感染组膜上TLR4表达比正常组增高(平均荧光强度:1.27±0.48,0.97±0.25,t:2.39,P＞0.05,n=10),感染组胞内TLR4表达比正常组降低(平均荧光强度:3.08±1.38,3.36±1.31;t=2.92,P＞0.05,n=10),感染组膜TLR4阳性细胞中(93.32±1.7)%为膜黏连蛋白-5阳性细胞;(3)RSV-LPS共刺激组培养上清液中IL-8含量明显高于单纯LPS刺激组(F=59.29,P＜0.01,n=3).结论 RSV感染后,上皮细胞TLR4mRNA和蛋白表达水平上调,与TLR4信号通路相关的IL-8分泌增加;TLR4与上皮细胞凋亡的关系提示TLR4及其信号通路可能参与了RSV诱导的上皮细胞急、慢性炎症反应.     

Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV‐infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV‐mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.     

The failure of interleukin-10-deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen-sensitized/challenged mice

Interleukin-10-deficient mice develop a robust pulmonary inflammatory response but no airway hyperresponsiveness (AHR) to inhaled methacholine (MCh) following allergen sensitization and challenge. In the present study, we investigated the effect of respiratory syncytial virus (RSV) infection on AHR and pulmonary inflammation in allergic IL-10-/- mice. Unlike littermate control mice, RSV-infected or ovalbumin (OVA)-sensitized/challenged IL-10-/- mice failed to develop significant AHR. In contrast, sensitized/challenged IL-10-/- mice infected with RSV did develop AHR accompanied by increased eosinophil numbers, both in bronchoalveolar lavage (BAL) and pulmonary tissue, and mucin production in airway epithelium. The cytokine profile in OVA-sensitized/challenged IL-10-/- mice was skewed toward a Th1 response but after RSV infection, this response was more of a Th2 type, with increased IL-5 levels in the BAL. Studies with an RSV mutant that lacks the G and SH genes showed equal enhancement of the AHR response as the parental wild-type strain, indicating that G protein is not essential to this response. These data suggest that RSV infection can overcome the failure of development of AHR in allergic IL-10-/- mice.     

The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium

Respiratory synctial virus (RSV) infection of undifferentiated airway epithelial cells has been shown to induce the production of chemokines. The purpose of this study was to investigate the vectorial release of interleukin (IL-8) and Released on Activation, Normal T-cell Expressed and Secreted (RANTES) by polarized, well-differentiated respiratory epithelial cells after RSV infection. Human bronchial epithelial cultures were differentiated under air-liquid interface conditions and infected with RSV by the apical or basolateral route. RSV infection was specific to the apical surface. Supernatants were collected at 6 and 48 hours after RSV inoculation, and IL-8 and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Both IL-8 and RANTES were significantly released by 48 hours following inoculation with RSV. The secretion of each chemokine was greatest after apical inoculation, and secretion was polarized to the basolateral supernatant. Immunohistochemical staining confirmed that RSV infection was specific to ciliated cells, and immunohistochemical staining for chemokines was localized to RSV-infected ciliated cells. The authors conclude that, in differentiated human airway epithelium in vitro, RSV-induced increases in IL-8 and RANTES release are predominantly in the basolateral direction. In epithelial layers, virus-containing cells are the predominant source of the increased chemokine release. The authors speculate that similar processes in vivo influence recruitment of leukocytes to sites of RSV infection.     

Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice

BACKGROUND: Respiratory syncytial virus (RSV) infection is associated with acute morbidity (e.g., pneumonia and airway obstruction [AO]) and long-term complications (e.g., airway hyperresponsiveness [AHR]). We present a comprehensive evaluation of the acute and chronic phases of RSV respiratory tract infection, using a mouse model. METHODS: BALB/c mice were inoculated with RSV and monitored for 154 days. RSV loads and cytokines were measured in bronchoalveolar lavage (BAL) samples. Pneumonia severity was assessed using a standard histopathologic score, and pulmonary function was determined by plethysmography. RESULTS: RSV-infected mice exhibited viral replication that peaked on day 4-5 and became undetectable by day 7. These mice developed acute pneumonia (peak days, 4-5) and chronic pulmonary inflammatory infiltrates that lasted up to 154 days after inoculation. BAL concentrations of tumor necrosis factor- alpha, interleukin (IL)-6, interferon- gamma, IL-4, IL-10, KC (an IL-8 homologue), MIG (CXCL9), RANTES, macrophage inflammatory protein-1 alpha, and eotaxin were significantly higher in RSV-infected mice than in control mice. RSV-infected mice developed acute AO during the first week of infection that persisted for 42 days. RSV-infected mice also showed significant AHR in response to methacholine up to 154 days. CONCLUSION: This model provides a means to investigate the immunopathogenesis of RSV infection and its association with reactive airway disease.