Transcriptomic analysis of TLR4 pathways in a murine model of chronic pulmonary inflammation and carcinogenesis. Mus musculus

Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study Overall design: Protocol 1 - 3 biological replicates after chronic dosing in each mouse strain Protocol 2 - multiple replicates after MCA/BHT tumor progression model used

Toll样受体4（Toll-like receptor 4, TLR4）作为一种先天免疫基因，参与多种肺部激动剂的应答过程，包括脂多糖（endotoxin, LPS; Poltorak等, 1998）、臭氧（O₃; Kleeberger等, 2001）、铜绿假单胞菌（Pseudomonas aeruginosa; Faure等, 2004）以及高氧（hyperoxia; Zhang等, 2005）。TLR4可部分介导LPS与O₃诱导的肺损伤应答，但同时，TLR4在铜绿假单胞菌感染中对肺损伤具有保护作用，还可抵御高氧引发的急性肺损伤。目前该保护作用背后的分子机制尚未明确。
此前已有研究证实，TLR4同样可抵御BHT诱导的慢性炎症与肿瘤促发过程（Bauer等, 2005）。C.C3H-Tlr4Lps-d（简称BALBLps-d）小鼠为同类系小鼠，其C3H/HeJ品系第4号染色体上携带Tlr4基因的10 cM区域被导入至BALB/c遗传背景中（Vogel等, 1994），该品系存在一处错义突变，导致TLR4功能失常。该Tlr4突变可能通过破坏TLR4与其他信号分子的直接接触位点，从而阻断其下游信号转导通路（Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.）。
在慢性BHT处理后，与BALB/c（即BALB；Tlr4功能正常）小鼠相比，BALBLps-d小鼠的支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）中肺泡巨噬细胞、淋巴细胞总数以及总蛋白含量均显著升高（Bauer等, 2005）。此外，在MCA/BHT肿瘤促发方案处理后，BALBLps-d小鼠的肿瘤多发率较BALB小鼠显著升高60%。尽管这是首个证实TLR4在慢性肺部炎症与肿瘤发生中具有保护作用的动物模型，但调控该保护性应答的下游机制仍未阐明。
本研究通过Affymetrix基因芯片分析，结合GeneSpring与Ingenuity通路分析，鉴定出了可能参与TLR4介导的保护性机制的已知与新型下游通路及其相互作用。据此我们提出假说：在肿瘤促发/慢性炎症阶段鉴定出的这些通路以及基因间的相互作用，可在一定程度上影响肿瘤发生过程中观察到的品系差异性应答。
关键词：时间进程（time course）、肿瘤研究（tumor study）
整体实验设计：方案1——每个小鼠品系经慢性给药后设置3个生物学重复；方案2——采用MCA/BHT肿瘤进展模型，设置多个重复样本。