宿主的遗传背景对呼吸道感染沙眼衣原体后Treg产生的影响

目的 探讨宿主的遗传背景对呼吸道感染沙眼衣原体后调节性T细胞(Treg)产生的影响.方法 对衣原体感染具有明显易感性差异的C57 BL/6(C57)和C3H/HeN(C3H)小鼠鼻腔吸入1×103 IFU沙眼衣原体小鼠肺炎菌株(Chlamydia muridarum,Cm),于感染后不同天数处死小鼠.利用细胞内细胞因子染色技术检测小鼠脾脏单个核细胞CD4+ CD25+T细胞、Foxp3+ CD4+ CD25+T细胞百分率,利用RT-PCR技术检测小鼠肺组织Treg细胞分泌的相关细胞因子IL-10和IL-2的mRNA表达水平,并比较Cm呼吸道感染不同时期C57和C3H小鼠Treg免疫应答水平的差异.结果 Cm感染在两组小鼠均诱导较高水平的CD4+ CD25+T细胞、Foxp3+ CD4+ CD25+T细胞产生及IL-10、IL-2mRNA表达.感染后第3天和第7天,高易感性的C3H小鼠脾脏CD4+ CD25+T细胞、Foxp3+ CD4+CD25+T细胞扩增水平,以及肺组织细胞因子IL-2 mRNA的表达水平均高于C57小鼠,感染后第14天,C3H小鼠IL-10 mRNA表达水平明显高于C57小鼠.结论 衣原体呼吸道感染在高易感性的C3H小鼠诱导高水平的Treg的增殖及Treg相关细胞因子IL-10、IL-2的表达,从而对衣原体特异的Th1免疫应答抑制作用增强,在小鼠衣原体呼吸道感染易感性差异中发挥重要作用.     

沙眼衣原体小鼠肺炎株呼吸道感染诱导巨噬细胞浸润并向M1极化

目的:探讨沙眼衣原体小鼠肺炎株( Chlamydia muridarum, Cm)呼吸道感染对巨噬细胞浸润及向M1/M2极化的影响。 方法:经鼻腔给予C57BL/6小鼠1×10 3包涵体形成单位(inclusion-forming units, IFU) Cm建立小鼠沙眼衣...     

树突状细胞在沙眼衣原体小鼠肺炎株呼吸道感染中免疫保护作用的研究

目的探讨树突状细胞(dendritic cells, DC)在沙眼衣原体小鼠肺炎株(Chlamydia muridarum, Cm)呼吸道感染中的作用以及对适应性免疫应答的影响。方法 C57BL/6小鼠经鼻腔吸入1×103包涵体形成单位(inclusion-forming units, IFU)Cm建立小鼠呼吸道感染模型。流式细胞术检测感染后0、3、7 d脾组织中总CD11c+MHCⅡ+DC比例和共刺激分子(CD40、CD80和CD86)表达情况;qPCR检测小鼠脾组织中DC细胞因子IL-12p40、IL-10和IL-6 mRNA的表达;磁珠分选出Cm感染后7 d的小鼠脾组织DC并过继转移给受体小鼠, 于感染后14 d利用细胞内细胞因子染色检测受体小鼠Th1应答水平。结果 Cm感染诱导小鼠脾组织DC大量浸润并且共刺激分子表达增加;脾组织DC细胞因子IL-12和IL-10 mRNA的表达在感染后3 d达到高峰;将Cm感染小鼠脾组织DC过继转移给受体小鼠, 受体小鼠感染Cm后疾病减轻, 肺组织衣原体繁殖降低, 且肺和脾组织中的Th1细胞比例显著升高。结论 DC在Cm感染后成熟、活化并促进T...     

TLR2/TLR4在失血性休克引发的脾组织TIPE2和TLR2/TLR4信号通路分子表达上调中的作用北大核心CSCD

目的研究发现,肠淋巴液回流是导致失血性休克后免疫功能紊乱的重要因素。本研究应用TLR2^(-/-)和TLR4^(-/-)小鼠探讨失血性休克对脾组织TIPE2和TLR2/TLR4信号下游分子表达的影响。方法将不同(C57BL/6J、TLR2^(-/-)、TLR4^(-/-))来源雄性小鼠随机分为Sham组、Shock组、Shock+Drainage组,分别予以不同手术处理后,无菌获取各实验组小鼠的脾组织。应用RTPCR技术分别检测WT小鼠脾组织TIPE2、TLR2、TLR4、MyD88、TRIF、TRAF3、TRAF6的mRNA表达。应用Western blotting技术分别检测不同小鼠(C57BL/6J、TLR2^(-/-)、TLR4^(-/-))脾组织TIPE2、TLR2、TLR4、MyD88、TRIF、TRAF3、TRAF6的蛋白表达。结果与Sham组相比,失血性休克后脾脏组织TIPE2、TLR2/TLR4及其下游分子表达水平均上调,肠淋巴液引流显著降低了失血性休克导致的TIPE2、TLR2/TLR4及其下游分子的高表达。TLR2的缺失降低了Shock组脾组织TIPE2、TLR4、MyD88、TRAF6的蛋白表达。TLR4缺失降低了Shock组TIPE2、TLR2、MyD88、TRIF、TRAF6的蛋白表达。结论以TLR2、TLR4作为干预靶点,TIPE2可能通过负反馈机制调控TLR2/TLR4信号通路下游分子mRNA和蛋白的表达,有利于促进失血性休克后免疫平衡状态的恢复。     

IFN-γ在沙眼衣原体呼吸道感染中免疫防御机制的探讨

目的:检测与IFN-γ作用相关的酶吲哚胺2,3二氧化酶(IDO)、诱导性一氧化氮合成酶(iNOS)和NADPH氧化酶(ox)gp91在沙眼衣原体呼吸道感染中的表达及与机体防御的关系,探讨衣原体感染中IFN-γ免疫防御作用的机制.方法:用沙眼衣原体小鼠肺炎株(MoPn)通过鼻腔感染C57BL/6(H-2b)小鼠,用过氧化物酶连接的鼠抗衣原体脂多糖单抗染色HeLa 229细胞,检测衣原体在肺组织的生长;用RT-PCR检测衣原体感染后第7及14天小鼠肺组织IFN-γ、IDO、iNOS和gp91NADPH ox mRNA表达.结果:MoPn呼吸道感染后小鼠肺组织匀浆衣原体活性测定,于感染后第2天,HeLa 229细胞内可见有衣原体包涵体生长,IFU值增高,于感染后第7天IFU达最高水平,以后逐渐下降,至感染后21天基本恢复到基线水平.与未感染的对照组比较,Th1细胞因子IFN-γ于感染后第7天表达显著增高,感染后14天有所降低,但仍维持较高水平;同时衣原体感染可显著诱导与IFN-γ作用相关的三种酶IDO、iNOS和gp91 NADPH ox在小鼠肺组织的表达,感染后第7及14天,IDO,iNOS及gp91 NADPH ox的表达与对照组比较均有显著差异,其中IDO和gp91 NADPH ox于感染后第7天mRNA表达增高显著(P＜0.01),14天略有下降(P＜0.05).结论:衣原体呼吸道感染诱导Th1细胞因子IFN-γ mRNA高表达,参与宿主对衣原体的清除及机体免疫防御,此作用可能与其相应的酶IDO、iNOS和gp91 NADPH ox表达增高有关.     

TLR2-MyD88通路在B细胞交叉提呈肿瘤来源自噬小体抗原过程中作用的初步研究

为研究B细胞表面TLR2在B细胞交叉提呈肿瘤来源自噬小体(tumor derived-autophagosome,DR)抗原诱导效应T细胞再活化过程中的作用,DR经淋巴结注射方式免疫C57BL/6小鼠,收获免疫小鼠的淋巴结细胞作为效应T细胞;以负载DR的C57BL/6、TLR2~(-/-)和MyD88~(-/-)及TLR2抗体封闭的小鼠脾脏B细胞作为pAPC。DR分别与分离纯化的WT C57BL/6、TLR2~(-/-)和MyD88~(-/-)及TLR2抗体封闭后的小鼠B细胞共孵育,然后与免疫小鼠CD4~+T和CD8~+T细胞共孵育,流式细胞术及ELISA法检测IFN-γ的分泌水平。结果显示,与WT C57BL/6小鼠B细胞组相比,负载DR的TLR2~(-/-)B细胞和MyD88~(-/-)B细胞及TLR2抗体封闭的B细胞活化效应CD8~+T和CD4~+T细胞产生的IFN-γ水平显著降低。以上结果提示B细胞交叉提呈DR抗原、诱导效应T细胞活化依赖于TLR2/MyD88信号通路。     

TLR2基因敲除下调高脂饮食诱导的肥胖小鼠脂肪组织炎症因子表达和细胞凋亡

目的研究Toll样受体2(TLR2)基因敲除对高脂饮食诱导的肥胖小鼠脂肪组织炎症因子表达和细胞凋亡的影响。方法雄性C57BL6小鼠和TLR2基因敲除小鼠分为:正常对照组、肥胖组、TLR2基因敲除组和TLR2基因敲除肥胖组,并给予普通饮食或高脂饮食喂养。16周后检测各组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白和游离脂肪酸水平。分光光度计法检测小鼠脂肪组织Caspase3活性,Western blot检测Bcl2、Bax、MyD88和p65NFκB蛋白相对表达量。荧光实时定量PCR检测小鼠脂肪组织IL-6和TNF-αmRNA相对表达量。结果与正常对照组相比,肥胖组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白水平降低和游离脂肪酸水平升高;脂肪组织Caspase3活性、Bax蛋白、MyD88蛋白、p65NFκB蛋白、IL-6 mRNA和TNF-αmRNA相对表达量升高,Bcl2蛋白相对表达量减少。与肥胖组相比,基因敲除肥胖组小鼠空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白水平降低和游离脂肪酸水平降低;脂肪组织Bcl2蛋白相对表达量增加,Caspase3活性、Bax蛋白、MyD88蛋白、p65NFκB蛋蛋白、IL-6 mRNA和TNF-αmRNA相对表达量减少。结论 TLR2基因敲除下调了高脂饮食诱导的肥胖小鼠脂肪组织炎症因子的表达和细胞凋亡。     

贞芪扶正胶囊介导TLR4/MyD88/NF-κB信号通路抑制慢性湿疹小鼠炎症反应

目的 探讨贞芪扶正胶囊对慢性湿疹小鼠炎症反应的抑制作用.方法 SPF级C57BL/6小鼠30只,按随机数字表法随机分为对照组(control组)、模型组(CAD组)与贞芪扶正胶囊组(ZQFZ组).观察各组小鼠双耳肿胀程度、双耳变应评分;HE染色观察病灶处皮肤病理学变化;ELISA法检测血清炎性因子TNF-α、IL-6及IL-1β水平;RT-PCR检测皮肤中TNF-α、IL-6及IL-1β mRNA表达;Western blot检测TLR4、MyD88及NF-κB蛋白表达;RT-PCR检测皮肤中TLR4、MyD88及NF-κB mRNA表达.结果 ZQFZ可以显著改善小鼠双耳肿胀程度,降低小鼠双耳变应评分(P＜0.05);减轻小鼠皮肤炎症浸润并降低血浆及皮肤中TNF-α、IL-6及IL-1β蛋白与mRNA表达(P＜0.05);同时ZQFZ还可显著下调小鼠皮肤组织中TLR4、MyD88、NF-κB蛋白与mRNA表达(P＜0.05).结论 贞芪扶正胶囊可以通过抑制炎性反应达到治疗慢性湿疹作用,其作用机制可能与其调控TLR4/MyD88/NF-κB信号通路有关.     

内源性IL-17诱导MIP-2与IL-6表达在衣原体肺炎中促进中性粒细胞循环

目的 探讨衣原体肺炎中白细胞介素-17(interleukin -17,IL-17)对中性粒细胞(polymorphonuclear leucocyte,PMN)循环的调节作用及机制.方法 用40 μl含1×103包涵体形成单位(inclusion-forming units,IFU)的衣原体鼠肺炎株(Chlamydia muridarum,Cm)呼吸道感染BALB/c小鼠,诱导鼠衣原体肺炎.用抗鼠IL-17单克隆抗体吸入中和内源性IL-17,以相应独特型抗体(IgG2α)作为对照.用RT-PCR检测小鼠肺组织及肺上皮细胞系巨噬细胞炎性蛋白-2 (macrophage inflammatory protein-2,MIP-2)和IL-6 mRNA的表达.取小鼠支气管肺泡灌洗液细胞染色计数PMN,感染肺组织进行病理染色.结果 衣原体肺炎中,内源性IL-17中和小鼠肺组织PMN浸润显著降低,支气管肺泡灌洗液PMN数量显著低于对照组.IL-17与TNF-α协同可上调肺上皮细胞MIP-2和IL-6 mRNA表达,且内源性IL-17中和小鼠肺组织MIP-2和IL-6表达显著降低.结论 衣原体肺炎中IL-17通过促进肺组织细胞分泌趋化性细胞因子MIP-2和前症性细胞因子IL-6,诱导PMN循环,参与宿主抗衣原体炎性应答.     

TLR4、MyD88、NF-κBmRNA在实验性自身免疫性肌炎小鼠淋巴结中的表达及意义

目的:研究TLR4、MyD88、NF-κB mRNA在实验性自身免疫性肌炎小鼠淋巴结中的表达情况,探讨TLR4在肌炎发病中的作用。方法:将30只雌性BALB/c小鼠随机分为5组(每组6只):第1组为正常对照组,其它4组分别为肌炎模型1周处理组、2周处理组、3周处理组和4周处理组,后4组均应用肌球蛋白诱导实验性自身免疫性肌炎模型(EAM)。采用实时荧光定量PCR方法检测各组小鼠淋巴结TLR4、MyD88、NF-κB mRNA表达水平。结果:(1)与正常小鼠相比,肌炎各组小鼠淋巴结中TLR4、MyD88、NF-κB mRNA表达均有统计学差异,表现为不同程度升高(P<0.01),第3组升高最为显著(P<0.01),第4组较第5组升高(P<0.01);(2)各组淋巴结中TLR4 mRNA表达水平与MyD88 mRNA、NF-κBmRNA表达水平均呈正相关(r=0.906,r=0.967,P<0.01),MyD88 mRNA表达水平与NF-κB mRNA表达水平呈正相关(r=0.919,P<0.01)。结论:TLR4在自身免疫性肌炎发生发展过程中发挥重要作用,且以MyD88依赖型信号途径为主,并通过激活NF-κB促进炎性因子释放。     

MyD88 and TLR2, but not TLR4, are required for host defense against Cryptococcus neoformans

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.     

Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria

Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.     

Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata

We have analyzed the importance of proteases for the induction of allergic responses against the mold Alternaria alternata. Responses induced in vivo with untreated or heat treated (protease inactivated) extracts were compared in BALB/c, C57BL/6, TLR4 KO, and MyD88 KO mice. In BALB/c mice, both extracts induced similar lung inflammation, upregulation of inflammatory mediators, Th2 cytokines, and Alternaria‐specific antibodies. However heat inactivation abrogated polyclonal IgE production. Similar results were obtained in C57BL/6 albeit lung expression of some Th2 mediators was decreased in mice stimulated with the heat‐treated extract. Treatment of the extract with protease inhibitors did not affect the induction of the allergic response either, except again for the polyclonal IgE response. Th2 responses and lung inflammation were readily induced in TLR4 knockout mice. In contrast, lung inflammation, Th2 responses, cytokine productions, and antibody synthesis were strongly suppressed in MyD88‐deficient mice. Early lung IL‐33 and IL‐1‐α expression were also suppressed. In conclusion, albeit some heat labile proteases are required for the stimulation of the polyclonal IgE secretion, fungal proteases, and TLR4 signaling are not required while MyD88 is essential for triggering the systemic immune response and for the development of lung allergic inflammation in response to Alternaria extracts.     

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p<0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p<0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p<0.05) and 2.1-fold (p<0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A-I transfer. In vitro, supplementation of HDL or apo A-I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.     

The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol

Taxol can mimic bacterial lipopolysaccharide (LPS) by activating mouse macrophages in a cell cycle-independent, LPS antagonist-inhibitable manner. Macrophages from C3H/HeJ mice, which have a spontaneous mutation in Toll-like receptor 4 (TLR4), are hyporesponsive to both LPS and Taxol, suggesting that LPS and Taxol may share a signaling pathway involving TLR4. To determine whether TLR4 and its interacting adaptor molecule MyD88 are necessary for Taxol's LPS mimetic actions, we examined Taxol responses of primary macrophages from genetically defective mice lacking either TLR4 (C57BL/10ScNCr) or MyD88 (MyD88 knockout). When stimulated with Taxol, macrophages from wild-type mice responded robustly by secreting both TNF and NO, while macrophages from either TLR4-deficient C57BL/10ScNCr mice or MyD88 knockout mice produced only minimal amounts of TNF and NO. Taxol-induced NF-kappa B-driven luciferase activity was reduced after transfection of RAW 264.7 macrophages with a dominant negative version of mouse MyD88. Taxol-induced microtubule-associated protein kinase (MAPK) activation and NF-kappa B nuclear translocation were absent from TLR4-null macrophages, but were preserved in MyD88 knockout macrophages with a slight delay in kinetics. Neither Taxol-induced NF-kappa B activation, nor I kappa B degradation was affected by the presence of phosphatidylinositol 3-kinase inhibitors. These results suggest that Taxol and LPS not only share a TLR4/MyD88-dependent pathway in generating inflammatory mediators, but also share a TLR4-dependent/MyD88-independent pathway leading to activation of MAPK and NF-kappa B.     

Staphylococcus aureus-induced corneal inflammation is dependent on Toll-like receptor 2 and myeloid differentiation factor 88

Toll-like receptors (TLRs) expressed by the corneal epithelium represent a first line of host defense to microbial keratitis. The current study examined the role of TLR2, TLR4, and TLR9 and the common adaptor molecule myeloid differentiation factor 88 (MyD88) in a Staphylococcus aureus model of corneal inflammation. The corneal epithelia of C57BL/6, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice were abraded using a trephine and epithelial brush and were exposed to heat- or UV-inactivated S. aureus clinical strain 8325-4 and other clinical isolates. Corneal thickness and haze were measured by in vivo confocal microscopy, neutrophil recruitment to the corneal stroma was quantified by immunohistochemistry, and cytokine production was measured by enzyme-linked immunosorbent assay. The exposure of corneal epithelium to S. aureus induced neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in control C57BL/6 mice but not in TLR2(-/-) or MyD88(-/-) mice. The responses of TLR4(-/-) and TLR9(-/-) mice were similar to those of C57BL/6 mice. S. aureus-induced cytokine production by corneal epithelial cells and neutrophils was also significantly reduced in TLR2(-/-) mice compared with that in C57BL/6 mice. These findings indicate that S. aureus-induced corneal inflammation is mediated by TLR2 and MyD88 in resident epithelial cells and infiltrating neutrophils.     

小檗碱和育亨宾对内毒素血症小鼠脾脏TLR4信号通路中相关基因表达的影响

目的: 观察小檗碱和α₂肾上腺素能受体拮抗剂育亨宾对内毒素血症小鼠脾脏Toll样受体4(TLR4)信号通路84种基因表达的影响,并初步探讨其作用机制。方法: 雄性BALB/c小鼠随机分为对照组、脂多糖(LPS)组、小檗碱+LPS组、小檗碱+育亨宾+LPS组、育亨宾+LPS组、小檗碱组、小檗碱+育亨宾组和育亨宾组。分别用蒸馏水、小檗碱(50 mg/kg)、小檗碱+育亨宾(50 mg/kg+2 mg/kg) 和育亨宾(2 mg/kg)灌胃,每天1次,连续3 d,第3 d灌胃1 h后,腹腔注射LPS(20 mg/kg)或生理盐水。腹腔注射1 h后,用RT² Profiler^TM PCR Array分析技术检测小鼠脾脏TLR4信号通路84种基因mRNA的表达;用Western blotting分析小鼠脾脏TLR4信号通路的抑制分子细胞因子信号抑制物(SOCS)1、SOCS3和白细胞介素-1受体相关激酶(IRAK)-M蛋白的表达。结果: LPS可上调小鼠脾脏TLR4信号转导通路中相关炎症因子的mRNA表达,包括CXCL10、TNF-α、IL-1α、IL-1β、IL-6、IFN-γ和IFN-β。小檗碱能显著下调下调髓样分化因子(MyD88)依赖信号通路下游TNF-α、IL-1α、IL-1β和IL-6 mRNA的表达,也能MyD88非依赖信号通路下游基因IFN-β和CXCL10 mRNA的表达(P<0.05)。育亨宾能显著下调内毒素血症小鼠脾脏IL-1α、IL-1β 和IFN-β mRNA的表达(P<0.05),但对TNF-α、IL-6和CXCL10 mRNA表达的下调作用与LPS组相比没有显著差异(P>0.05)。小檗碱与育亨宾合剂能显著下调内毒素血症小鼠脾脏IFN-β和CXCL10 mRNA的表达,但不能显著下调内毒素血症小鼠脾脏IL-1α、IL-1β、TNF-α 和IL-6 mRNA的表达。LPS攻击后1 h,小檗碱和(或)育亨宾均不能增强内毒素血症小鼠脾脏SOCS1、SOCS3和IRAK-M蛋白的表达。结论: 小檗碱和育亨宾均能抑制LPS诱导的MyD88依赖和非依赖信号通路下游部分基因的表达,这种抑制作用的机制与SOCS1、SOCS3和IRAK-M蛋白无关。     

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.     

Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune disease and an animal model of multiple sclerosis. To investigate how upstream TLR signals influence autoimmune T cell responses, we studied the role of individual TLR and MyD88, the common TLR adaptor molecule, in the initiation of innate and adaptive immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in CFA. MyD88(-/-) mice were completely EAE resistant. Purified splenic myeloid DC (mDC) from MyD88(-/-) mice expressed much less IL-6 and IL-23, and serum and T cell IL-17 were absent. TLR4(-/-) and TLR9(-/-) mice surprisingly exhibited more severe EAE symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were higher in TLR4(-/-) mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6 expression by splenocytes was higher in TLR9(-/-) mice. Our data suggest that MyD88 mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization, which in turn drives IL-17-producing encephalitogenic Th17 cell activation. Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOG-induced EAE.