Toll样受体2和4在小鼠肺炎链球菌中耳急性感染中的作用

目的 探讨Toll样受体2(Toll-like receptor 2,TLR2)和Toll样受体4(Toll-like receptor 4,TLR4)在小鼠肺炎链球菌中耳感染中的作用.方法 野生型(wild type,WT)C57BL/6J小鼠、TLR2缺陷(TLR2-/-)和TLR4缺陷(TLR4-/-)小鼠分别通过中耳鼓膜接种肺炎链球菌悬液1×106菌落形成单位(colony forming unit,CFU)].在细菌接种前、接种后第3天和第7天分别进行听性脑干反应(ABR)和鼓室声导抗测试,血液及中耳积液中细菌滴度测定,颞骨病理切片形态学观察,反转录聚合酶链反应(RT-PCR)检测不同基因型小鼠炎性细胞因子IκB激酶β(IκB kinase β,IKKβ)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎性蛋白1α(MIP-1α)、黏蛋白/黏液素5AC和5B(MUC5AC和MUC5B)mRNA的表达.结果 中耳接种肺炎链球菌3d后,TLR2-/-小鼠死亡率为58.8％(40/68),TLR4-/-小鼠死亡率为35.6％(21/59),而WT小鼠的死亡率仅为17.3％(9/52).接种7d后,TUR2-/-小鼠死亡率为82.4％(54/68),TLR4-/-小鼠死亡率为54.2％(32/59),而WT小鼠的死亡率仅为23％(12/52),三组之间差异具有统计学意义(P＜0.01).ABR测试结果显示,接种后存活3d及7d的TLR2-/-和TLR4-/-小鼠ABR阈值高于WT小鼠,三组之间的差异具有统计学意义(P＜0.01).颞骨病理发现,TLR2-/-和TLR4-/-小鼠接种肺炎链球菌后第3天中耳出现积液和组织破坏,接种后第7天感染极为严重.TLR2-/-鼠出现严重的耳蜗炎性细胞浸润,内外毛细胞破坏、盖膜肿大和血管纹退变,以及螺旋神经节细胞的损伤;TLR4-/-鼠可见耳蜗内外毛细胞和螺旋神经节细胞的损伤;而WT鼠的耳蜗未见炎性细胞浸润和组织破坏,内外毛细胞基本正常.在接种细菌3d和7d的TLR2-/-鼠中耳黏膜未发现肥大细胞,但在TLR4-/-和WT鼠中耳黏膜发现较多的肥大细胞并有脱颗粒现象.接种肺炎链球菌后3d和7d的TLR2-/-和TLR4-/-小鼠血液中细菌滴度均高于WT小鼠,差异具有统计学意义(P值均＜0.05).接种肺炎链球菌3d后,TLR2-/-小鼠听泡组织中NF-κB、TNFα、IL-1β、MIP-1α、MUC5AC、MUC5B等因子的mRNA表达水平低于TLR4-/-和WT小鼠,差异具有统计学意义(P值均＜0.05).结论 TLR2-/-鼠在肺炎链球菌激惹后产生相对低水平的致炎性细胞因子,中耳清除细菌能力下降,引发脓毒血症,导致高死亡率.TLR2和TLR4是2种重要的小鼠中耳炎性反应的受体和信号转导分子.

Toll-like receptor 2 and Toll-like receptor 4 participates in mediation of acute otitis media and mortality in pneumococcal infections in mice

Objective To investigate the roles of Toll-like receptor 2(TLR2)and Toll-like receptor 4(TLR4)in host defense against Streptococcus pneumoniae infection in the middle ear.Methods Wild-type(WT)C57BL/6J,TLR2-deficien(TLR2-/-)and TLR4-deficien(TLR4-/-)mice were inoculated with Streptococcus pneumoniae(1 × 106CFU)through the tympanic membrane.All animals were tested the mouse ABR thresholds and tympanometry measurement before,and 1 day,3 days and 7 days following pneumococcal challenge.Blood bacterial titer were determined by plating 50 μl volumes of 10-fold diluted blood.Histological analysis of middle ear and inner ear were performed by fixation,decalcification,embedded section,and counterslained with hematoxylin/eosin and toluidine blue staining.Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes.Results Forty of 68 TLR2-/-mice and twenty-one of 59 TLR4-/-mice showed bactermia and died within 3 days after the pneumococcal challenge,however,only 9 of 52 WT mice died.The survive mice were shown have more severe heating loss in the TLR2-/-and TLR4-/-mice than in the WT mice,indicated by ABR thresholds,at 3 or 7 days postinoculation.The histological pathology was characterized by effusion and tissue damage in the middle ear,and in the TLR2-/-and TLR4-/-mice,the outcome of infection became more severe at 7 days.At both 3 and 7 days after challenge,the TLR2-/-mice had higher blood bacterial titers than WT mice(P ＜ 0.05).Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge,the TLR2-/-and TLR4-/-mice showed effusion and tissue damage in the middle ear,and the infection became more severe at 7 days postinoculation.TLR2-/-mice showed severe inflammatory cell infiltration in the cochlear,the organ of Corti showed the outer hair cells damage,the tectorial membrane swelling,degeneration of the stria vascularis,and severe loss of spiral ganglion cells; However,the WT mice was not found the cell infiltration and tissue damage in the cochlear,the organ of Corti shown normal of outer hair cells.Mast cells were not found in the middle ear mucosa of TLR2-/-mice,but in the TLR4-/-and WT mice,more mast cells were found in the middle ear mucosa of effusion ear by 3 and 7 days postchallenge.Moreover,by 3 days postchallenge,the mRNA accumulation levels of NF-κB,tumor necrosis factor alpha(TNFα),interleukin13,MIP-1α,MUC5AC and MUCSB were significantly lower in the ears of TLR2-/-mice than that in WT and TLR4-/-mice.Conclusions TLR2-/-mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge,thus hindering the clearance of bacteria from the middle ear and leading to sepsis and high mortality rate.This study indicated that TLR2 and TLR4 are important in the molecular pathogenesis and host response to otitis media.