Activation of TLRs/NF-κB signal pathway and occurrence of different functional cytokines during invasive pulmonary aspergillosis

Objective To study the activation of TLRs/NF-κB signal pathway and production of different functional cytokines during invasive pulmonary aspergillosis( IPA) , in order to probe the pathogene-sis of IPA. Methods Mouse were randomly divided into normal, normal + inoculated with Aspergillus fumigatus( normal inoculation group), and immune suppression + inoculation with Aspergillus fumigatus (IPAmodel group) , the mouse were killed at different time points after inhaling Aspergillus fumigatus spores by nose. Removing the lung tissue in a sterile manner and making pathological section respectively, counting Aspergillus fumigatus colony, dynamiclly detecting the expression of TLR2, TLR4 mRNA, variation of NF-κB p65 protein, pro-inflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-10 levels in the lung tissue by RT-PCR and Western blot method during Aspergillus fumigatus infection in mouse. Results (1) When it's 72 h after inhaling Aspergillus fumigatus by nose, IPA model emerged severe lung tissue inflammation, and generated a large number of hyphae, meanwhile, burthen of Aspergillus fumigatus was higher than normal inoculation group at each time point. (2)Compared with the normal inoculation group, IPA group whose TLR2 mRNA was low expression at early stage of infection (24 h), and emerged high expression at late stage of infection (120 h, 144 h); and TLR4 mRNA has been at a state of low expression in the infection process; NF-κB p65 suddenly increased at early stage of infection(24 h) and then continued to decline. (3) After infected by Aspergillus fumigatus in normal mouse, proinflammatory cytokine TNF-α, IL-1β in lung exhibited high expression at the early stages of infection, and the highest expression levels appeared at 48 h or 72 h, then decreased and recovered to normal level. And the expression level of anti-inflammatory cytokine IL-10 rised at late stage of infection; The IP A mouse released a lot of anti-inflammatory cytokine IL-10 at early stage of infection, which significantly reduced at late stage, and released pro-inflammatory cyto-kines TNF-α, IL-1β at slow and low level. Conclusion The abnormal activation of TLRs/NF-β signaling pathway caused the loss of dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines, leading to the occurrence and development of IPA.     

Activation of TLRs/NF-κB signal pathway and occurrence of different functional cytokines during invasive pulmonary aspergillosis

Objective To study the activation of TLRs/NF-κB signal pathway and production of different functional cytokines during invasive pulmonary aspergillosis( IPA) , in order to probe the pathogene-sis of IPA. Methods Mouse were randomly divided into normal, normal + inoculated with Aspergillus fumigatus( normal inoculation group), and immune suppression + inoculation with Aspergillus fumigatus (IPAmodel group) , the mouse were killed at different time points after inhaling Aspergillus fumigatus spores by nose. Removing the lung tissue in a sterile manner and making pathological section respectively, counting Aspergillus fumigatus colony, dynamiclly detecting the expression of TLR2, TLR4 mRNA, variation of NF-κB p65 protein, pro-inflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-10 levels in the lung tissue by RT-PCR and Western blot method during Aspergillus fumigatus infection in mouse. Results (1) When it's 72 h after inhaling Aspergillus fumigatus by nose, IPA model emerged severe lung tissue inflammation, and generated a large number of hyphae, meanwhile, burthen of Aspergillus fumigatus was higher than normal inoculation group at each time point. (2)Compared with the normal inoculation group, IPA group whose TLR2 mRNA was low expression at early stage of infection (24 h), and emerged high expression at late stage of infection (120 h, 144 h); and TLR4 mRNA has been at a state of low expression in the infection process; NF-κB p65 suddenly increased at early stage of infection(24 h) and then continued to decline. (3) After infected by Aspergillus fumigatus in normal mouse, proinflammatory cytokine TNF-α, IL-1β in lung exhibited high expression at the early stages of infection, and the highest expression levels appeared at 48 h or 72 h, then decreased and recovered to normal level. And the expression level of anti-inflammatory cytokine IL-10 rised at late stage of infection; The IP A mouse released a lot of anti-inflammatory cytokine IL-10 at early stage of infection, which significantly reduced at late stage, and released pro-inflammatory cyto-kines TNF-α, IL-1β at slow and low level. Conclusion The abnormal activation of TLRs/NF-β signaling pathway caused the loss of dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines, leading to the occurrence and development of IPA.     

小鼠侵袭性肺曲霉病发病过程中TLRs/NF-κB信号通路的激活

目的:研究小鼠侵袭性肺曲霉病(IPA)发生过程中TLRs/NF-κB信号通路的激活,探讨IPA的发病机理。方法:小鼠随机分为正常组(N)、正常接菌组(N+Af)和IPA模型组(IPA),经鼻吸入烟曲霉(Af)孢子后在不同时相点处死小鼠,无菌取肺组织行病理切片、Af菌落计数,RT-PCR法检测TLR2和TLR4 mRNA、蛋白印迹法检测NF-κBp65、IL-1β的表达。结果:(1)鼻吸入Af后72小时时,IPA组肺组织出现严重炎症反应,并有大量的菌丝生成,同时各时相点的菌负荷均高于N+Af组;(2)与N+Af组比较,IPA组TLR2 mRNA后期异常高表达,TLR4 mRNA持续低表达,NF-κBp65早期骤然升高后又持续下降,IL-1β一直呈低表达状态。结论:TLRs的异常激活导致宿主下游信号的低反应性,宿主不能清除Af促使了IPA的发生发展。     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

锌指蛋白A20对Toll样受体信号途径的负反馈调控及其对机体的保护作用

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses at the early stage of virus invasion, promoting the maturation and differentiation of immune cells, regulating immune respon-ses,and triggering inflammatory responses. Lipopolysaccharide (LPS) activates the TLR4 signaling pathways which result in the activation of NFκB and JNK/SAPK and the expression of large amounts of inflammatory fac-tors,leading to systemic inflammatory response and multiple organ failure. A20, a NFκB-dependant cytosolic protein, via a negative feedback loop blocks NFκB activation and participates in the regulation of inflammatory responses and the inhibition of apptosis;therefore, it provides protective effect on the body.     

荧光定量PCR检测尖锐湿疣皮损Toll样受体的表达水平

目的 检测尖锐湿疣皮损中Toll样受体(TLRs)的mRNA表达水平,探讨TLRs在人乳头瘤病毒(HPV)感染免疫中的可能作用.方法 提取40例尖锐湿疣患者皮损和35例HPV阴性的慢性宫颈炎患者宫颈刮落细胞的总RNA,逆转录为cDNA后用实时荧光定量PCR法检测TLR1～10的mRNA表达水平,并检测40例尖锐湿疣的HPV分型.结果 40例尖锐湿疣患者以低危型HPV6和11型为主(77.5%和55%).55%尖锐湿疣患者感染两种以上HPV亚型,其中35%合并高危型HPV感染.尖锐湿疣组TLR3、7、8的mRNA表达水平高于其他TLRs,TLR9的mRNA表达水平低于其他TLRs(P均<0.05).尖锐湿疣皮损TLR1～10的mRNA表达水平在HPV6型和HPV11型感染间以及在低危型与合并高危型HPV感染间差异无统计学意义(P均>0.05).尖锐湿疣组TLR1～3、TLR5～8和TLR10的mRNA表达水平均高于HPV阴性的慢性宫颈炎组(P均<0.05),其中TLR2、7、8的mRNA表达水平升高更显著(P均<0.01).而TLR4、TLR9的mRNA表达水平与对照组差异无统计学意义(P>0.05).结论 尖锐湿疣皮损中部分TLRs(3、7、8)表达水平较高,但TLR9表达水平较低.相对于HPV阴性的慢性宫颈炎,尖锐湿疣皮损中TLR1~3、TLR5～8和TLR10的mRNA表达上调.不同HPV型别感染的尖锐湿疣皮损中TLRs表达谱差异无统计学意义.推测尖锐湿疣TLRs表达谱的改变可能与HPV感染有一定关系,是否与HPV感染的免疫逃逸机制及持续感染有关尚有待进一步明确.

Abstract：

Objective To investigate the expression of Toll-like receptors(TLRs) in condyloma acuminatum(CA) lesions and their possible roles in the pathogenesis of CA. Methods The expressions of TLR1-10 mRNA level in the lesions of CA and in the cervix scrape cells from the patients with human papillomavirus(HPV) negative chronic cervicitis were detected by real-time quantitative fluorescent PCR. HPV typing was detected by HPV GenoArray test kit. Results Low-risk HPV type 6 and type 11 were the most prevalent types in the forty CA cases with positive rate of 77.5% and 55% respectively. 55% CA patients were found infected with more than two types of HPV. 35% CA patients were concurrently infected with high-risk HPV. The expressions of TLR3, 7, 8 mRNA were higher than other TLRs and the expression of TLR9 mRNA was lower than others in the lesions of CA. No significant differences of the TLR1-10 mRNA levels were found between HPV6 and HPV11 positive CA lesions, so did it between low-risk and high-risk HPV concurrent infected CA lesions. The expressions of TLR1-3, TLR5-8, TLR10 mRNA, especially TLR2, TLR7 and TLR8 in the lesions of CA were significantly higher than that in cervix scrape cells of HPV negative chronic cervicitis. There were no significant differences of TLR4 and TLR9 mRNA levels between the two groups. Conclusion There were higher expressions of some TLRs (3, 7, 8) and lower expression of TLR9 in the lesions of CA. Compared with HPV negative chronic cervicitis, the expressions of TLR1-3, TLR5-8, TLR10 mRNA in the lesions of CA were up-regulated. The expression profile of TLRs in different type of HPV infected CA lesions had no significant differences. Our results suggested that the expression profile of TLRs in CA may be associated with the HPV infection. Whether it was associated with the immune escape mechanism and persistent infection of HPV need further demonstration.     

Parthenolide attenuates LPS-induced activation of NF-κB in a time-dependent manner in rat myocardium

Parthenolide(PTN),a selective nuclear factor kappa B(NF-κB)inhibitor,has been used extensively to inhibit NF-κB activation.The duration of the inhibitory effect of PTN on NF-κB in vivo remains unclear.This study was to determine whether a lipopolysaccharide(LPS)challenge 6,12 and 24 h after the administration of PTN could activate NF-κB.Rats were devided into five groups.The rats in the PTN,PTN+LPS and DMSO groups were injected intraperitoneally with PTN or DMSO.After 6,12 or 24 h,LPS was administered in LPS and PTN+LPS groups.The expressions of NF-κB p50,IκBα and p-IκBα were inhibited in both PTN and PTN+LPS group at end of 6 and 12 h and no effects at 24 h.In summary,myocardial NF-κB expression occurs 1 h after the administration of LPS.PTN blocks this effect given at 6 h and no inhibitory effect 24 h after administration in vivo.     

TLR2和TLR4单克隆抗体对DSS诱导的小鼠结肠炎肠黏膜细胞因子及肠道菌群的影响

Objective To evaluate the effect of TLR2McAb and TLR4McAb on intestinal flora of DSS-induced colitis in mice. Methods Fifty healthy male BALB/c mice (SPF level), were randomly assigned into five groups: the control group( group A), the UC model group( group B), TLR2McAb intervention group( group C), TLR4McAb intervention group( group D) and TLR2McAb + TLR4McAb intervention group(group E). Clinical symptoms were evaluated by the disease activity index(DAI), while tissue sam ples were evaluated by histological scoring(HS). The quantities of mRNA for IFN-γ, IL-4 and IL-17 were determined by real-time PCR. Meanwhile, fecal samples were obtained directly from the cecum for microbiological studies. Results After the treatment with TLR2McAb and TLR4McAb, DAI and HS were decreased significantly. Compared with group A, inflammatory cytokines such as IFN-γ, IL-4 and IL-17 in group B were higher. Compared with group B, expression of these three cytokines in group C to E was all markedly decreased. Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp,while the UC model group showed a conspicuous increase of Escherichia coli and decreases of Lactobacillus spp and Bifidobacterium spp. After treatment with TLR2McAb or/and TLR4McAb, Lactobacillus spp and Bifidobacterium spp increased to the normal level. But counts of E. Coli in the three intervention groups were not changed. Conclusion TLR2McAb and TLR4McAb suppressed the development of DSS-induced colitis and increase cecum counts of Lactobacilli and Bifidobacteria.     

TLR2和TLR4单克隆抗体对DSS诱导的小鼠结肠炎肠黏膜细胞因子及肠道菌群的影响

Objective To evaluate the effect of TLR2McAb and TLR4McAb on intestinal flora of DSS-induced colitis in mice. Methods Fifty healthy male BALB/c mice (SPF level), were randomly assigned into five groups: the control group( group A), the UC model group( group B), TLR2McAb intervention group( group C), TLR4McAb intervention group( group D) and TLR2McAb + TLR4McAb intervention group(group E). Clinical symptoms were evaluated by the disease activity index(DAI), while tissue sam ples were evaluated by histological scoring(HS). The quantities of mRNA for IFN-γ, IL-4 and IL-17 were determined by real-time PCR. Meanwhile, fecal samples were obtained directly from the cecum for microbiological studies. Results After the treatment with TLR2McAb and TLR4McAb, DAI and HS were decreased significantly. Compared with group A, inflammatory cytokines such as IFN-γ, IL-4 and IL-17 in group B were higher. Compared with group B, expression of these three cytokines in group C to E was all markedly decreased. Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp,while the UC model group showed a conspicuous increase of Escherichia coli and decreases of Lactobacillus spp and Bifidobacterium spp. After treatment with TLR2McAb or/and TLR4McAb, Lactobacillus spp and Bifidobacterium spp increased to the normal level. But counts of E. Coli in the three intervention groups were not changed. Conclusion TLR2McAb and TLR4McAb suppressed the development of DSS-induced colitis and increase cecum counts of Lactobacilli and Bifidobacteria.