Transcription profiling by array of CD40L-knockout and wild-type mice with or without exposure to Pneumocystis murina for different duration

Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. To better understand the immune mechanisms contributing to clearance of infection, microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knock-out (CD40L-KO) mice over time following exposure to Pneumocystis. Immuncompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the upregulation of 349 primarily immune-response associated genes. Temporal changes in the expression of these genes suggested that there was an early (week 2) primarily innate response, that waned without controlling infection; this were followed by primarily adaptive immune responses that peaked at week 5 and successfully cleared the infection. In conjunction with the latter, there was an increased expression of B cell associated (immunoglobulin) genes at week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no upregulation of immune-response associated genes at days 35 to 75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust biphasic response to infection by Pneumocystis; CD40 ligand is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia. Keywords: Time course response Pneumocystis murina infection wild type versus CD40L-KO mice In experiment 1, C57BL/6 and CD40L-KO mice were co-housed in 2 cages; one cage was exposed to a P. murina-infected seeder, while the second was unexposed. Mice (3 per group) were sacrificed at day 32. Because the initial study demonstrated very little change in gene expression in CD40L-KO mice, only C57BL/6 mice were used for experiments 2 and 3. In Experiment 2, three cages (10 animals/cage) were set up: 2 cages were exposed to P. murina, and one was unexposed. Animals (5 per cage) were sacrificed at days 34 and 41. In Experiment 3, which focused on gene expression early after exposure to P. murina, five cages (9-10 animals/cage) were set up, of which 3 were exposed and 2 were unexposed. Animals (1-3 per cage) were sacrificed at days 7, 14 and 21 of exposure. Experiment 4 was designed to verify the initial results in CD40L-KO mice, and included 5 unexposed controls and 4 mice exposed for 35 days.肺孢子虫（Pneumocystis）是免疫受损宿主的病原体，但也可感染健康宿主，感染可在健康宿主中被快速控制并清除。为更好地明确参与感染清除的免疫机制，本研究采用微阵列（microarray）方法，检测了C57BL/6与CD40配体敲除（CD40L-KO）小鼠暴露于肺孢子虫后，肺部随时间变化的差异基因表达情况。 免疫健全的C57BL/6小鼠可高效控制并清除感染，其感染应答强烈，表现为349个主要与免疫应答相关的基因上调。这些基因的表达时序变化显示，早期（第2周）以固有免疫应答为主，该应答在未成功控制感染的情况下逐渐减弱；随后以适应性免疫应答为主，该应答在第5周达到峰值，并成功清除感染。伴随适应性免疫应答的激活，第6周时B细胞相关（免疫球蛋白）基因的表达升高，该表达持续至第11周。 与之形成对比的是，极易罹患重症肺孢子虫肺炎的CD40L-KO小鼠，在第35至75天几乎未出现免疫应答相关基因的上调。免疫组化染色（immunohistochemical staining）证实了上述观察结果：C57BL/6小鼠肺部的CD4+、CD68+及CD19+细胞数量增多，而CD40L-KO小鼠无此变化。 综上，健康宿主对肺孢子虫感染表现出强烈的双相应答；CD40配体是高效控制感染、阻止进行性肺炎发生的适应性免疫应答的关键上游调节因子。 关键词：时序应答 鼠肺孢子虫（Pneumocystis murina）感染 野生型与CD40L-KO小鼠 实验1中，C57BL/6与CD40L-KO小鼠共饲养于2个笼盒：1个笼盒接触感染了鼠肺孢子虫的播种小鼠，另一个为未接触对照组。每组3只小鼠在第32天处死。由于初始研究显示CD40L-KO小鼠的基因表达变化极微，因此实验2和3仅使用C57BL/6小鼠。 实验2中共设置3个笼盒（每笼10只动物）：2个笼盒接触鼠肺孢子虫，1个为未接触对照组。分别在第34天和第41天处死每组5只动物。 实验3聚焦于鼠肺孢子虫暴露后的早期基因表达，共设置5个笼盒（每笼9~10只动物），其中3个接触病原体，2个为未接触对照组。分别在暴露第7、14和21天处死每组1~3只动物。 实验4旨在验证CD40L-KO小鼠的初始实验结果，共设置5个未接触对照组及4只暴露35天的感染小鼠。