【作者】 张灯；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的A型土拉热弗朗西斯氏菌（Francisella tularensis）是一种革兰氏阴性专属细胞内细菌,也是导致兔热病（tularemia）的病原体,可以引起哺乳动物包括人类的全身性感染。如果不予以治疗,毒性A型土拉热弗朗西斯氏菌,尤其是气溶胶型的病原体,很容易导致人类的致死性感染。10个毒性A型细菌就可导致严重的疾病,而且偶然的或者有预谋的污染水和食物也是一种引发感染的替代方法。因此,A型土拉热弗朗西斯氏菌被看作是生物战争武器。尽管它的临床和生物安全性都很重要,但是对A型土拉热弗朗西斯氏菌感染,特别是通过呼吸道和消化道引起的感染的免疫发病机理和分子机制目前尚不清楚。淋巴毒素又称TNF-β,与TNF-α是两个密切相关的细胞因子,同属TNF家族。根据TNF来源的不同,人们将单核/巨噬细胞所产生的TNF称为TNF-α;把T细胞产生的TNF称为TNF-β,或称为淋巴毒素（LT）。膜结合的淋巴毒素以LT-α和LT-β亚单位结合形成的异源三聚体复合物形式分布在细胞膜上,与LT-β受体结合,在外周淋巴器官发生形成中起作用。分泌到细胞外的可溶性的LT-α通过TNF受体传导通路激活各种炎性细胞因子和趋化性细胞因子。LT与TNF及LT相关的可诱导配合体LIGHT形成一个完整的信号网络,这个信号网络对调节先天性和获得性免疫应答都有很重要的作用。在这点上,LT-α参与宿主防御许多细菌、病毒和寄生性病原体的作用已经被认同。本研究的目的一是为了阐明在不同途径（吸入和经口）及不同剂量感染的情况下,LT-α在宿主防御A型土拉热弗朗西斯氏菌中所起的作用;二是观察小鼠在无外周淋巴结的情况下被细菌感染后是否能够存活及清除病原菌,并观察缺乏淋巴结的小鼠是否能够阻止或延迟细菌到其他组织的播散;三是观察吸入感染A型土拉热弗朗西斯氏菌对胸腺和胸腺细胞的影响与其作用机制以及胸腺在兔热病发病过程中的作用;四是观察经口感染不同剂量A型土拉热弗朗西斯氏菌后免疫活性小鼠和免疫缺陷小鼠的易感性、感染过程的特征以及相关的宿主反应。方法1实验动物与感染方法C57BL/6小鼠,BALB/C小鼠,B6.129S2-Ltα^tm1Dch/J（LT-α-/-）小鼠,B6.129S7-Ifng^{tm1 Agt}（IFN-γ-/-）小鼠,B6;129S-Tnfrsfla^{tm1 Imx}Tnfrsflb^{tm1 Imx}（TNFR1R2-/-）小鼠以及胸腺切除小鼠。菌株:FSC033/SnMF,A型,原始分离于美国的一只松鼠体内,本实验室保种于-80℃冰箱中。吸入感染时,解冻A型土拉热弗朗西斯氏菌贮存株,用含20%（V/V）甘油的Mueller Hinton肉汤稀释以保持其感染性。气溶胶型的A型土拉热弗朗西斯氏菌由Lovelace喷雾器产生。操作定在40psi的压力下,小鼠暴露在这个气溶胶环境中7min,导致约10个细菌进入肺中。经口感染时,用PBS稀释A型土拉热弗朗西斯氏菌,用18-gauge填喂针给予每只小鼠0.5ml菌液,实际的细菌数用培养皿法确定。2 LT-α在抵抗土拉热弗朗西斯氏菌吸入感染过程中的作用LT-α-/-和对照组C57BL/6（LT-α+/+）小鼠吸入感染小剂量（吸入约10个细菌）A型土拉热弗朗西斯氏菌,监测它们的存活情况和死亡时间。为了研究感染的病原体的剂量大小是否对LT-α控制呼吸感染产生影响,LT-α-/-和C57BL/6小鼠吸入不同剂量（10,100,1000 cfu/只）A型土拉热弗朗西斯氏菌,同时监测它们的生存情况,记录死亡时间。为了研究LT-α在机体抵抗吸入感染小剂量A型土拉热弗朗西斯氏菌过程中的作用及相关的宿主反应,取实验组LT-α-/-小鼠和对照组C57BL/6小鼠吸入感染小剂量A型土拉热弗朗西斯氏菌,分别编号称重。在感染细菌后0d,2d和4d处死小鼠。通过肉眼观察,证实LT-α缺陷小鼠很明显地缺乏外周淋巴结。用精确度为0.1mg的天平称重,称量每只小鼠的体重和胸腺的重量,结果以胸腺重量指数表示,即胸腺重量（g）比小鼠体重（g）×100。为了进一步估计A型土拉热弗朗西斯氏菌感染对胸腺细胞的影响,我们将分离出的胸腺放在冷的PBS中,挤压胸腺通过75μm孔径的一次性细胞滤网中,再用大口吸管反复吹打,分离单个细胞,用血细胞计数池计数每只小鼠的胸腺细胞总数。同时收集支气管肺泡灌洗液,计数细胞总数并进行白细胞分类计数,用来确定在感染后2d和4d是否有大量炎性细胞流入肺中。收集血样和支气管肺泡灌洗液,用于测定血清和支气管肺泡灌洗液的21种细胞因子和趋化性细胞因子水平和血清中10种临床生化指标。将肺、脾、肝和胸腺取出,用于定量细菌学分析和组织病理学分析。3胸腺损伤与兔热病的相关性观察取进行胸腺切除术的C57BL/6小鼠作为试验组,另外一组C57BL/6小鼠作为对照组。小剂量吸入感染A型土拉热弗朗西斯氏菌,然后记录这两组小鼠的死亡率和半数死亡时间。为了探索TNF与吸入感染引起的胸腺损伤的相关性,再取一组C57BL/6小鼠作为对照组,取TNFR1R2-/-小鼠作为实验组,吸入感染小剂量A型土拉热弗朗西斯氏菌。在感染后4d,测定胸腺重量指数和每只小鼠的胸腺细胞总数。用RPMI 1640细胞培养液体外培养胸腺细胞,用A型土拉热弗朗西斯氏菌感染,在不同时间点观察细胞的存活情况,用这个体外实验来进一步观察A型土拉热弗朗西斯氏菌对胸腺细胞的影响在体内和体外实验中是否一致。4 LT-α在抵抗土拉热弗朗西斯氏菌经口感染过程中的作用取C57BL/6小鼠、BALB/C小鼠、LT-α-/-小鼠,经口给予不同数量的A型土拉热弗朗西斯氏菌（C57BL/6小鼠和BALB/C小鼠的感染剂量为10⁴～10⁸CFU,LT-α-/-小鼠的感染剂量为10⁴～10⁶CFU）,感染后观察14d,纪录生存情况和半数死亡时间,计算存活率。为了排除胃酸对细菌的影响,另外取C57BL/6小鼠和BALB/C小鼠在进行实验前禁食一夜,在经口感染前用3%的碳酸氢钠中和胃酸,经口给予不同数量的A型土拉热弗朗西斯氏菌（10⁴～10⁸CFU）,观察14d,纪录生存情况和半数死亡时间,计算存活率。为了探索免疫缺陷小鼠对经口感染A型土拉热弗朗西斯氏菌的易感性,取LT-α-/-、IFN-γ-/-和TNFR1R2-/-三种免疫缺陷小鼠及对照组C57BL/6小鼠,经口感染5×10⁵CFU的A型土拉热弗朗西斯氏菌。观察14d,记录它们的存活率及半数死亡时间。14d后处死依然存活的C57BL/6小鼠和LT-α-/-小鼠,取出肠系膜淋巴结（LT-α-/-小鼠除外）和脾,进行组织细菌定量分析。为了观察经口感染大剂量A型土拉热弗朗西斯氏菌能否增强免疫缺陷小鼠的易感性,另取上述试验组和对照组小鼠,经口感染5×10⁷CFU的菌量,观察小鼠的存活率及半数死亡时间。为了探索经口感染引起的兔热病的发病过程和免疫机制,经口给予C57BL/6小鼠和LT-α-/-小鼠5×10⁵ CFU,C57BL/6小鼠5×10⁷CFU A型土拉热弗朗西斯氏菌,分别在感染后0、2、4d和0、2、3d处死小鼠,采血分离血清进行细胞因子和趋化性细胞因子的分析;同时进行细菌定量分析,检测肺、肠系膜淋巴结（LT-α-/-小鼠除外）、脾中的细菌量以及进行肺和脾的组织病理学分析。5统计学处理各组中所有的数据都以平均数士标准差的形式表示,定量变量参数检验用t检验或单因素或双因素方差分析,然后再做各组间的两两比较。分类变量用Fisher精确检验和卡方检验。检验水平为α=0.05。结果1小鼠吸入感染后的存活时间年龄和性别匹配的22只实验组LT-α-/-和14只对照组C57BL/6（LT-α+/+）小鼠吸入感染小剂量气溶胶型A型土拉热弗朗西斯氏菌后,除了2只LT-α-/-小鼠和1只LT-α+/+小鼠外,所有的小鼠都在4-7d时死亡,死亡时间中位数为5d（LT-α-/-小鼠4-6d,LT-α+/+小鼠5-7d,P>0.05）。LT-α-/-和LT-α+/+小鼠吸入感染不同剂量（10,100,1000 cfu/只）后,虽然感染细菌数不同,但不论试验组还是对照组,感染后小鼠的死亡时间都是5d,试验组和对照组间的差异无统计学意义。2小鼠吸入感染后胸腺和胸腺细胞的变化LT-α+/+小鼠和LT-α-/-小鼠吸入感染小剂量A型土拉热弗朗西斯氏菌,两组小鼠体重在感染后2d几乎都没有变化,胸腺体积轻微降低。胸腺体重指数轻微降低。LT-α+/+小鼠胸腺细胞的总数也降低了约35%,但与0d组相比及两种小鼠间相比,差异无统计学意义。在感染后4d两种小鼠体重都出现中等程度的降低,胸腺体积显著降低。一般在死前24h左右,胸腺出现萎缩。C57BL/6小鼠胸腺体重指数严重降低,胸腺细胞的总数也降低了约85%,两者与0d组的差异均有统计学意义。LT-α-/-小鼠在感染后4d胸腺体重指数降低,胸腺细胞的总数也降低了约30%。与C57BL/6小鼠相比,两者的差异无统计学意义,与0d组的差异有统计学意义。TNFR1R2-/-小鼠体重和胸腺体积在感染后4d后均未出现变化。其胸腺重量指数和胸腺细胞数量与C57BL/6小鼠组的差异有统计学意义。3小鼠吸入感染后不同器官内的细菌定量分析在吸入感染小剂量A型土拉热弗朗西斯氏菌后2d,LT-α+/+小鼠和LT-α-/-小鼠都在组织中发现大量的细菌。但在作为原发感染灶的肺内,两组小鼠的细菌数差异无统计学意义。在LT-α-/-小鼠的脾内,细菌数却比LT-α+/+小鼠脾内细菌数低大约1到1.5对数倍（P<0.05）。在感染后4d,LT-α-/-小鼠的肺内又比LT-α+/+小鼠肺内高出10倍的细菌数（P<0.05）。在LT-α-/-小鼠的脾内细菌数也高,但与LT-α+/+小鼠相比,其差异无统计学意义。在小鼠的胸腺组织内,感染后4d发现大量A型土拉热弗朗西斯氏菌,而且LT-α-/-小鼠的胸腺组织内的细菌数比LT-α+/+小鼠的高出10倍,两组间的差异有统计学意义（P<0.05）。细菌数的差异经过3次完全独立的实验始终存在,重复性很好。4小鼠吸入感染后的病理、生化及细胞因子的变化在吸入感染小剂量A型土拉热弗朗西斯氏菌后2d,两种小鼠的肝和脾组织都显示中等程度的炎性渗出。同时出现轻微的、局部的支气管炎。在感染后4d,出现严重的坏死性肝炎和脾的淋巴滤泡破坏及支气管炎。但是吸入感染小剂量A型土拉热弗朗西斯氏菌的两组小鼠在组织病理学的改变方面没有差异。LT-α-/-小鼠和LT-α+/+小鼠在血液临床生物化学指标方面（包括总蛋白、白蛋白、球蛋白、白/球比率、胆红素、天冬氨酸转氨酶、丙氨酸转氨酶、葡萄糖、尿素、肌酸酐）、支气管肺泡灌洗液的细胞总数、白细胞分类计数方面的差异均无统计学意义。吸入感染后2d和4d的LT-α-/-和LT-α+/+两组小鼠的支气管肺泡灌洗液和血清中21种细胞因子和趋化性细胞因子（包括GM-CSF、IFN-γ、IL-10、IL-12（p40）、IL-12（p70）、IL-13、IL-17、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、KC、MCP-1、MCP-1β、RANTES、TNF-α、VEGF）水平都仅有小的变化。其中吸入感染A型土拉热弗朗西斯氏菌后2d支气管肺泡灌洗液中出现MCP-1的大辐度增长和KC的中等程度增长。在感染后4d,支气管肺泡灌洗液中和血清中都出现IFN-γ、IL-6、KC和MCP-1的大辐度增长;但在实验组和对照组之间的差异无统计学意义,其中唯一的例外是在LT-α-/-小鼠的支气管肺泡灌洗液中的IL-6明显高于LT-α+/+小鼠支气管肺泡灌洗液中的IL-6。5胸腺损伤与兔热病的临床相关性进行胸腺切除术的C57BL/6小鼠和对照组C57BL/6小鼠在感染后4d开始出现死亡,在5d超过半数死亡,6d时全部死亡。平均死亡时间都是5d。两组死亡率和死亡时间的差异都无统计学意义。体外培养的胸腺细胞贴壁生长良好,显示A型土拉热弗朗西斯氏菌没有导致体外培养的胸腺细胞萎缩死亡。6小鼠经口感染A型土拉热弗朗西斯氏菌半数致死量与对胃酸的抵抗力经口感染C57BL/6小鼠和BALB/C小鼠（10⁴-10⁸CFU）,LT-α-/-小鼠（10⁴-10⁶CFU）A型土拉热弗朗西斯氏菌,半数致死量为10⁶CFU。LT-α-/-小鼠未显示出强的易感性。将实验小鼠中和胃酸后重复做相同实验,与未中和胃酸的小鼠的实验结果相同,未增强易感性。而且经过体外实验,发现A型土拉热弗朗西斯氏菌在体外对酸有相当强的抵抗力。7免疫低下小鼠对A型土拉热弗朗西斯氏菌的易感性免疫低下小鼠LT-α-/-、IFN-γ-/-和TNFR1R2-/-小鼠及对照C57BL/6小鼠经口感染5×10⁵的A型土拉热弗朗西斯氏菌后,免疫低下小鼠显示出与正常小鼠相同的易感性。感染后14d没有死亡的C57BL/6小鼠和LT-α-/-小鼠肠系膜淋巴结(LT-α^-/-小鼠除外)和脾组织中,都没有培养出A型土拉热弗朗西斯氏菌。当经口感染大剂量5×10⁷CFUA型土拉热弗朗西斯氏菌时,免疫低下小鼠比对照小鼠显示出更高的易感性。8小鼠吸入感染后不同器官内的细菌定量分析LT-α+/+小鼠在经口感染5×10⁵CFUA型土拉热弗朗西斯氏菌后2d,在肺中几乎没有发现细菌,仅一只小鼠脾中细菌数高达10⁵。在感染后4d,肺中细菌数还是很少,在脾中的细菌数增高。LT-α-/-小鼠在感染后2d,肺和脾中细菌数较LT-α+/+小鼠高,在感染后4d,肺中细菌数变化不大,但有一只高达10⁷,在脾中的细菌数增高较多。但两种小鼠间和不同感染天数间的差异都无统计学意义。说明在这个感染剂量,LT-α-/-小鼠没有表现出高的易感性。LT-α+/+小鼠在感染5×10⁷ CFU A型土拉热弗朗西斯氏菌后2d,肠系膜淋巴结中的细菌数增高,在3d时已经低到了检测不出来的程度。感染后2d肺组织内细菌量仍然很低,但有一只高达10⁶。在3d后发现很少量的增高。在感染后2d脾内细菌数大增,在感染后3d达高峰。9小鼠经口感染后的血清细胞因子和趋化性细胞因子的变化C57BL/6小鼠和LT-α-/-小鼠经口感染5×10⁵CFUA型土拉热弗朗西斯氏菌后2d,血清中IFN-γ,IL-6,TNF-α,IL-12,IP-10显著增高;KC,MIP-1α,IL-1β,MIG,MCP-1和IL-10水平中等程度增高。在感染后4d,IL-6和MCP-1增高更多,其余因子维持在2d的水平。IL-2,IL-4和IL-5这3种细胞因子在感染后始终无明显变化。除了在感染后2d,LT-α-/-小鼠KC水平显著低于LT-α+/+小鼠;在感染后4d,LT-α-/-小鼠的TNF-α和MCP-1水平显著高于LT-α+/+小鼠,其余两组间的细胞因子和趋化性细胞因子的水平差异无统计学意义。C57BL/6小鼠经口感染5×10⁷CFUA型土拉热弗朗西斯氏菌后,首先出现在肠系膜淋巴结上,然后迅速的转移到肝和脾。在那里细菌大量繁殖达到很高的细菌量,同时引起显著的嗜中性细胞的渗透聚集和严重的组织坏死。在感染后3d,在脾的红髓和白髓边缘区出现中性粒细胞和坏死淋巴细胞的大量聚集,淋巴滤泡结构破坏。肺中仅出现少量中性粒细胞聚集。受感染的C57BL/6小鼠血清中的细胞因子和趋化性细胞因子水平迅速提高;在感染后2d,IFN-γ,IL-1β,IL-6,TNF-α,MIP-1α,IP-10,MCP-1,MIG,,KC和IL-10等等出现升高趋势。在感染后3d,IL-6和MCP-1升高更多。结论1 LT-α对吸入感染小剂量A型土拉热弗朗西斯氏菌后,小鼠的死亡率和死亡时间不产生显著影响。吸入感染不同剂量该菌对小鼠死亡率和死亡时间无影响。2吸入感染该菌可导致小鼠严重的胸腺萎缩和胸腺细胞死亡,但胸腺切除的小鼠未显示出高的死亡率和死亡时间。该菌未导致体外培养的胸腺细胞萎缩死亡。TNF受体在与该菌感染相关的胸腺萎缩过程中起一定作用。3经口感染该菌的小鼠的半数致死量为10⁶CFU;中和胃酸后未增强小鼠的易感性,体外实验发现该菌对酸有较强的抵抗力。4经口感染低剂量该菌时,免疫低下小鼠与免疫正常小鼠易感性相同;大剂量感染时免疫低下小鼠显示出更高的易感性。5 IFN-γ在机体抵抗经口感染该菌的过程中起到了一个微妙的,双面的作用。6胃肠道与肺仅作为此菌的进口,而不是感染的重要部位。全身性的感染比肺部和胃肠道的感染更易导致小鼠死亡,毒性的关键在于在机体器官中的传播和快速的细菌复制。7 LT-α在A型土拉热弗朗西斯氏菌由肺到脾、胸腺的分布过程中及后来病原体在肺、脾和胸腺中的复制方面起到了一定的作用。同时发现缺乏淋巴结短期延迟细菌到其他组织的播散。更多还原

【Abstract】 Goals:This study examined the role of lymphotoxin（LT）-αin host defense against airborne infection and oral infection with Francisella tularensis.At the same time,we examined the susceptibility of mice to intragastric inoculation with virulent type A F. tularensis and characterized the course of infection and the associated host responses. And we examined the effect of virulent strains of F.tularensis infection on the thymus and thymocytes and the potential mechanisms involved.Francisella tularensis is a gram-negative facultative intracellular bacterium and the causative agent of tularemia, a systemic infection of many mammals including humans.Left untreated,the virulent type A subspecies of F.tularensis routinely caused lethal infection in people particularly after aerosol exposure to the pathogen;as few as 10 virulent type A bacilli can initiate severe disease.Incidental or deliberate contamination of drinking water and the food supply could be an alternative means to initiate infection with this pathogen.Consequently,F.tularensis is considered a Category A biological warfare agent.Despite its clinical and biosecurity importance,the molecular basis for the immunopathogenesis of F.tularensis infection,particularly when initiated through the respiratory and intragastric tract,remain largely unknown. Lymphotoxin-α（LTα） is a member of the tumor necrosis factor（TNF） superfamily of cytokines and has two distinct roles:as a membrane-bound heterotrimer in combination with LTβ,it binds the LTβreceptor and is critical in the development and maintenance of organized secondary lymphoid organs.In this regard,LTαhas been implicated in the host defense against several different bacterial,viral and parasitic pathogens.Given that LTαis important in the control of these intracellular bacterial pathogens,in the present study we sought to determine whether it also plays a role in host defense against low-dose aerosol infection and oral infection with a virulent type A strain ofF.tularensis.Methods:1 Mice and Inoculation C57BL/6,BALB/c,Transgenic mice B6.129S2-Ltα^{tm1 Dch}/J （LTα-/-）,B6.129S7-Ifng^{tml Agt}Nmg(IFN-γ^-/- and B6.129S-Tnfrsfla^{tm1 Imx} Tnfrsfib^{tm1 Imx} (TNFR1R2^-/-. Type A F.tularensis strain FSC033/snMF（strain FSC033） was originally isolated from a squirrel in Georgia（United States）.For oral inoculation, thawed F.tularensis stocks were diluted in phosphate-buffered saline,and 0.5 ml of the inoculum was given to each mouse by using an 18-gauge gavage needle.For aerosol exposure,aerosols of F.tularensis strains were generated with a Lovelace nebulizer operating at a pressure of 40 psi.Mice were exposed to these aerosols for 7 min using a customized commercial nose-only exposure apparatus resulting in the implantation of 10-20 organisms into the lungs.2 The role of LT-αin host defense against aerosol infection with Francisella tularensis Groups of age matched female 22 LTα-/- and 14 LTα+/+ mice were challenged by low-dose aerosol（inhaled dose of～10 organisms） with virulent type A F.tularensis and their survival and median time to death were monitored.To examine the possible effect of inoculum size on the need for LTαexpression to control respiratory infection with type A F.tularensis,groups of LTα-/- and LTα+/+ mice were intranasally challenged with 10,100,and 1,000 cfu type A F.tularensis and their survival monitored.Groups of age matched LTα-/- and LTα+/+ mice were inoculated by low-dose aerosol with type A F.tularensis,and sacrificed at 0,2 and 4 days post inoculation （dpi）.The phenotype.of the LTα-/- mice was confirmed by visual inspection at necropsy to confirm the absence of peripheral lymph nodes.We determined total and differential leukocyte counts in the BAL fluid to identify the inflammatory cell influx into the lungs on dpi 2 and 4,and the changes in the mean body weights and relative weights of the thymus over the course of infection were assessed.The thymus were removed and weighed in a balance with accuracy to 0.1mg.The organ index was calculated as organ weight（in grams）/body weight（in grams）<100.Blood samples and bronchoalveolar lavage（BAL） fluid were collected for the determination of serum and BAL fluid cytokine levels and for serum clinical chemistry.In some experiments, the lungs,spleens and thymus were removed,homogenized and used for quantitative bacteriology or fixed immediately by immersion in 10%neutral buffered formalin for histopathology.Single thymocyte suspensions were prepared by pressing individual thymus in cold PBS through a 75-μm pore sized Falcon disposable cell strainer.The total thymocyte number in each mouse was determined using a hemocytometer.To determine the potential role of TNF in the development of type A F. tularensis infection-associated thymus atrophy,TNFR1R2-/-mice were challenged by aerosol with low-dose type A F.tularensis.3 Clinical relevance of thymus damage and tularemia in mice We compared the clinical outcome of thymectomized C57BL/6 mice and control C57BL/6 mice following low-dose aerosol infection with type A F.tularensis,monitor the mortality and the median time to death among these two mouse strains.We isolated the thymocytes from the thymus of untreated mice and culture in RPMI-1640 Culture medium with 10%FBS in 24 wells plates.Add fresh culture medium with the corresponding numbers of F.tularensis and negative control.Incubate and observe the cells at different time points.4 The role of LT-αin host defense against oral infection with Francisella tularensis To determine the relative susceptibility of mice to oral infection with type A F.tularensis,groups of LTα-/-mice were gavaged with 10⁴ to 10⁶ CFU and matched LTα+/+ mice were gavaged with 10⁴ to 10⁸ CFU of type A F.tularensis in some separate experiments.In some experiments,mice were fasted overnight and administered 0.2 ml of 3%sodium bicarbonate to neutralize gastric acidity 10 min prior to oral inoculation.Survival of the inoculated mice was monitored for 14 days.Groups of immunocompromised mice(TNFR1R2^-/-,IFN-γ^-/-,LTα-/-) as well as wild-type（WT） C57BL/6,were inoculated by gavage with 5×10⁵ CFU,and their survival rates and median times were monitored.To examine the effect of high inoculum,groups of immunocompromised mice(TNFR1R2^-/-,IFN-γ^-/-,LTα-/-) as well as WT control mice,were gavaged with 5×10⁷ CFU(～50 LD₅₀) of type A F. tularensis,and their survival rate were compared.In order to determined the bacterial burdens and examined the associated pathology in different organs of C57BL/6 mice and LTα-/- mice,C57BL/6 mice and LTα-/- mice were gavaged with 5×10⁵ CFU of type A F.tularensis and C57BL/6 mice were gavaged with 5×10⁷ CFU over the course of infection.The mice killed at dpi 0,2 and 3.Mesenteric lymph nodes（except LTα-/- mice）,spleens,and lungs were aseptically removed and homogenized in aerosol-proof homogenizers,and the number of viable bacteria in the respective organs was determined by quantitative bacteriology.The sera were collected.The serum levels of cytokines and chemokines were determined by using the mouse multiplex cytokine detection system 2 and the mouse chemokine 5-Plex on a Luminex 100 IS system.Additional groups of mice were sacrificed and the lung and spleen was removed from each animal;fixed immediately in 10%neutral buffered formalin;and processed by standard paraffin-embedding methods to do the histopathology assay.5 Statistical analyses Data are presented as means±the standard deviation for each group.Differences in quantitative measurements were assessed by one-way or two-way analysis of variance,followed by post hoc multiple comparison tests. Differences in nonparametric data were analyzed by the Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney U test.The Fisher exact test and the chi-square test were used for comparison of categorical variables.Differences were considered significant when the P value was<0.05. 1 Survival rate after aerosol infection A total of 22 LTα-/- and 14 LTα+/+ mice were challenged by low-dose aerosol with virulent type A F.tularensis and their survival monitored.With the exception of two LTα-/- mice and one LTα+/+ mouse,all mice succumbed to infection between day 4 and 7 with a median time to death of 5 days（range 4-6 days for LTα-/- mice and 5-7 days for LTα+/+ mice,p>0.05 by Kaplan-Meier survival analysis）,indicating that LTα-/- mice are no more susceptible to low-dose aerosol challenge with this strain of the pathogen than control LTα+/+ mice.To examine the possible effect of inoculum size on the need for LTαexpression to control respiratory infection with type A F.tularensis,groups of LTα-/- and LTα+/+ mice were intranasally challenged with 10,100,and 1,000 cfu type A F.tularensis and their survival monitored.This study revealed that the LD₁₀₀ and LD₁₀₀₀ of type A F. tularensis for LTα-/- and LTα+/+ mice were comparable in that all mice died with a median time of 5 days.These results indicate that LTαdoes not appear to play a significant role in determining the clinical outcome of respiratory infection with various doses of type A F.tularensis in mice.2 Low-dose aerosol infection of mice induces severe thymus atrophy and thymocytes depletion After the Low dose aerosol infection,the thymus size of LTα+/+ mice decreased slightly at dpi 2 and drastically by dpi 4（24-48 h prior to expected death）,concurrent with the decrease in the thymus size,the total number of thymocytes in the infected mice dropped by 85%at dpi 4.The Thymus weight index of LTα-/- mice decreased slightly at dpi 4 and the total number of thymocytes in the infected mice dropped by 30%.There are significant difference between two mice strain at dpi4 in Thymus weight index and the total number of thymocytes.Compared to low-dose type A F.tularensis infected conventional B6 mice at dpi 4, TNFR1R2-/-mice did not show any thymus changes.3 Bacterial burdens in different tissues after aerosol infection We examined whether LTαcontributes to the control of F.tularensis replication and systemic dissemination by comparing the bacterial burdens in the lungs and spleens of LTα-/-and LTα+/+ mice at dpi 2 and 4 following aerosol challenge.There was no.difference in the bacterial burdens in the lungs,the primary site of infection,between LTα-/- and LTα+/+ mice at dpi 2.However,the bacterial burdens in the spleens of LTα-/- mice were about 1 to 1.5 log lower than those in LTα+/+ mice at this time point.By dpi 4, LTα-/- mice had approximately 10-fold more bacteria in their lungs than did LTα+/+ mice,and the bacterial burdens in the spleens of LTα-/- mice were also higher, although not statistically significant,than those in LTα+/+ mice.We have cultured large numbers of F.tularensis consistently from the thymuses of our infected LTα+/+ mice and LTα-/- mice at dpi 4 and LTα-/- mice had approximately 10-fold more bacteria in their lungs than did LTα+/+ mice.The subtle differences in bacterial burdens were consistently observed in three independent experiments.4 Pathology,clinical chemistry,Cytokine and chemokine responses after aerosol infection Histopathologically,both LTα-/- and LTα+/+ mice showed moderate inflammatory infiltrations in the livers and spleens and mild,focal bronchopneumonia at dpi 2,and by dpi 4 moderately severe necrotic hepatitis,lymphoid follicle destruction in the spleen,and bronchopneumonia.However,as would be expected from the quantitative bacteriology and survival data,no overt differences in tissue histopathology or blood clinical chemistry were observed between LTα-/- and LTα+/+ mice following aerosol exposure to type A F.tularensis.We determined total and differential leukocyte counts in the BAL fluid to identify the inflammatory cell influx into the lungs on dpi 2 and 4.There is no significant difference in either the total cell number or the composition of cell populations（macrophage,neutrophil,and lymphocyte） in the lavage fluids of LTα+/+ and LTα-/- mice with the exception of a small but not significant increase in lymphocytes in LTα-/- mice on both dpi 2 and 4.To assess whether LTαdeficiency alters F.tularensis-induced cytokine responses following aerosol challenge with the pathogen,levels of a panel of 21 cytokines and chemokines,including IFN-γ,IL-6, KC and MCP-1,in the BAL and the sera of LTα-/- and LTα+/+ mice killed at dpi 2 and 4 were measured.Overall,there was little change in the levels of the majority of assayed cytokines in either the BAL or the sera at dpi 2 or 4 in either mouse strain. However,F.tularensis infection resulted in a substantial increase of MCP-1 and a moderate increase of KC in BAL fluid at dpi 2 and a substantial increase of IFN-γ, IL-6,KC and MCP-1 in both BAL and sera at dpi 4,but again no differences were observed between the two mouse strains with the exception of IL-6,which was significantly higher in BAL fluid of LTα-/- mice than that of LTα+/+ mice.5 Clinical relevance of thymus damage and tularemia in mice We compared the clinical outcome of thymectomized C57BL/6 mice and control C57BL/6 mice following low-dose aerosol infection with type A F.tularensis.There was no significant difference in the mortality or the median time to death among these two mouse strains.In vitro exposure of cultures of thymocytes,isolated from untreated mice,to F.tularensis failed to induce thymocyte death.6 LD₅₀ to oral inoculation and resistant to acid stress To determine the relative susceptibility of mice to oral infection with type A F.tularensis and the effect on the LT-a,groups of 8- to-10-week-old female C57BL/6 mice and matched LTα-/- mice were gavaged with various numbers of type A F.tularensis（10⁴ to 10⁸ CFU and10⁴ to 10⁶）.Survival of the inoculated mice was monitored for 14 days.Oral gavage of mice with 10⁶ CFU of virulent type A F.tularensis resulted in slightly more than 50% mortality by day 5 after inoculation.All C57BL/6 and LTα-/- mice except one of each group that received the lowest inoculum（10⁴ CFU） survived the infection,whereas all mice that received the highest inoculum（10⁸ CFU） died by dpi 5.A similar susceptibility was also observed in BALB/c mice.These results suggest that the 50% lethal dose(LD₅₀) for oral type A F.tularensis infection in mice is about 10⁶ CFU.We find that neutralization of gastric acidity with 3%sodium bicarbonate does not increase the susceptibility of mice to the infection.Type A F.tularensis is relatively resistant to acid stress in vitro in that there was no substantial reduction in the bacterial viability of the pathogen at different pH.7 Susceptibility of immunocompromised mice to oral inoculation Groups of immunocompromised mice(TNFR1R2^-/-,IFN-γ^-/-,LTα-/-) as well as wild-type（WT） C57BL/6,were inoculated by gavage with 5×10⁵ CFU,and their survival rates and median times were monitored.Immunocompromised mice displayed similar susceptibilities to oral inoculation with this dose of type AF tularensis.To examine the effect of high inoculum,those mice were gavaged with 5×10⁷ CFU(～50 LD₅₀) of type A F.tularensis,and their survival rate were compared.In contrast to WT mice, which showed an MTM of 5 days,mice with various immunodeficiencies had a shortened MTM（3 to 4 days）.8 Bacterial burdens in different tissues after oral infection When inoculated by gavage with 5×10⁵ CFU,small numbers of F.tularensis were cultured from the MLN,spleen,and lung of C57BL/6 mice at dpi 2.F.tularensis was not cultured from the lung at dpi 2.The bacterial numbers continued to increase significantly in the spleen at dpi 4.The bacterial numbers in lung and spleen of LTαmice are higher than C57BL/6 mice at dpi2.The bacterial numbers continued to increase significantly in the spleen at dpi 4.But there is no significant difference between those two strain of mice and between the dpi.When inoculated by gavage with 5×10⁷ CFU,the number of bacteria in the MLN peaked at dpi 2,and then fell to undetectable levels in most of the mice by dpi 3.F.tularensis,was present in one mouse at lung at dpi 2,and was only detected occasionally in small numbers at dpi 3.The bacterial numbers continued to increase significantly in the spleen between dpi 2 and 3.9 Cytokine and chemokine responses after oral infection In the C57BL/6 mice and LTα-/- mice,the serum levels of IFN-γ,IL-6,TNF-α,IL-12,IP-10,MIG,and MCP-1 were significantly increased;the KC,MIP-1α,IL-1β,and IL-10 levels were moderately increased at dpi 2;and most of these cytokines or chemokines maintained similar levels at dpi 3 except IL-6 and MCP-1,which increased further.On the other hand,there was little or no change in the serum IL-2,IL-4,IL-5.But To most of the cytokines or chemokines,there is no significant difference between those two mice strains.Conclusions1.) Type A,F.tularensis cause severe reduction in thymus weight and destruction of thymocytes and depletion of thymocytes.2.) The 50%lethal dose(LD₅₀) for oral type A F.tularensis infection in mice is about 10⁶ CFU.Neutralization of gastric acidity does not increase the susceptibility of mice to the infection.3.) Immunodeficiencies display similar susceptibilities to oral inoculation with 5× 10⁵ CFU and increase susceptibility to high dose of type A F.tularensis.4.) The gastrointestinal tract and lungs,acts primarily as a portal of entry for F. tularensis rather than as the critical site of infection.It is the disseminated rather than the pulmonary and gastrointestinal infection that kills the host.5.) LTαdoes play a subtle role in the multiplication/dissemination of F.tularensis.6.) The lack of draining lymph nodes may simply cause a delay in antigen presentation leading to a delayed or otherwise impaired antibacterial host response.7.) IFN-γmay play a divergent role in the pathogenesis of oral infection with virulent type A F.tularensis.更多还原