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
Toll양수체2화4재소서폐염련구균중이급성감염중적작용
Toll-like receptor 2 and Toll-like receptor 4 participates in mediation of acute otitis media and mortality in pneumococcal infections in mice
  • 万方数据
    中华耳鼻咽喉头颈外科杂志 중화이비인후두경외과잡지
    CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
    2011年 12期 1009-1018 ,共10页

    李胜利%张敏燕%李白芽%郑庆印%朱宏亮

    리성리%장민연%리백아%정경인%주굉량

    Toll样受体2%Toll样受体4%链球菌,肺炎%中耳炎,化脓性%细胞因子类%小鼠 Toll樣受體2%Toll樣受體4%鏈毬菌,肺炎%中耳炎,化膿性%細胞因子類%小鼠
    Toll양수체2%Toll양수체4%련구균,폐염%중이염,화농성%세포인자류%소서
    Toll-like receptor 2%Toll-like receptor 4%Streptococcus pneumoniae%Otitis media,suppurative%Cytokines%Mice
    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.
    목적 탐토Toll양수체2(Toll-like receptor 2,TLR2)화Toll양수체4(Toll-like receptor 4,TLR4)재소서폐염련구균중이감염중적작용.방법 야생형(wild type,WT)C57BL/6J소서、TLR2결함(TLR2-/-)화TLR4결함(TLR4-/-)소서분별통과중이고막접충폐염련구균현액[1×106균락형성단위(colony forming unit,CFU)].재세균접충전、접충후제3천화제7천분별진행은성뇌간반응(ABR)화고실성도항측시,혈액급중이적액중세균적도측정,섭골병리절편형태학관찰,반전록취합매련반응(RT-PCR)검측불동기인형소서염성세포인자IκB격매β(IκB kinase β,IKKβ)、핵인자κB(NF-κB)、종류배사인자α(TNF-α)、백세포개소-1β(interleukin-1β,IL-1β)、거서세포염성단백1α(MIP-1α)、점단백/점액소5AC화5B(MUC5AC화MUC5B)mRNA적표체.결과 중이접충폐염련구균3d후,TLR2-/-소서사망솔위58.8％(40/68),TLR4-/-소서사망솔위35.6％(21/59),이WT소서적사망솔부위17.3％(9/52).접충7d후,TUR2-/-소서사망솔위82.4％(54/68),TLR4-/-소서사망솔위54.2％(32/59),이WT소서적사망솔부위23％(12/52),삼조지간차이구유통계학의의(P＜0.01).ABR측시결과현시,접충후존활3d급7d적TLR2-/-화TLR4-/-소서ABR역치고우WT소서,삼조지간적차이구유통계학의의(P＜0.01).섭골병리발현,TLR2-/-화TLR4-/-소서접충폐염련구균후제3천중이출현적액화조직파배,접충후제7천감염겁위엄중.TLR2-/-서출현엄중적이와염성세포침윤,내외모세포파배、개막종대화혈관문퇴변,이급라선신경절세포적손상；TLR4-/-서가견이와내외모세포화라선신경절세포적손상；이WT서적이와미견염성세포침윤화조직파배,내외모세포기본정상.재접충세균3d화7d적TLR2-/-서중이점막미발현비대세포,단재TLR4-/-화WT서중이점막발현교다적비대세포병유탈과립현상.접충폐염련구균후3d화7d적TLR2-/-화TLR4-/-소서혈액중세균적도균고우WT소서,차이구유통계학의의(P치균＜0.05).접충폐염련구균3d후,TLR2-/-소서은포조직중NF-κB、TNFα、IL-1β、MIP-1α、MUC5AC、MUC5B등인자적mRNA표체수평저우TLR4-/-화WT소서,차이구유통계학의의(P치균＜0.05).결론 TLR2-/-서재폐염련구균격야후산생상대저수평적치염성세포인자,중이청제세균능력하강,인발농독혈증,도치고사망솔.TLR2화TLR4시2충중요적소서중이염성반응적수체화신호전도분자.
    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.
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