Project description: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 Protocol 1 - 3 biological replicates after chronic dosing in each mouse strain Protocol 2 - multiple replicates after MCA/BHT tumor progression model used

Project description:Background: Human lung adenocarcinoma (ADC), the predominant subtype of lung cancer, are at a disadvantage as the disease lacks available biomarkers and is not usually diagnosed until its later stages. We previously demonstrated that Tlr4-mutantmice were more susceptible to BHT-induced pulmonary inflammation (bronchoalveolar lavage macrophages and lymphocytes) and tumorigenesis in comparison to Tlr4-sufficient control mice. Differential transcriptional profiles were also noted in the whole lung tissue of the mice. The study objective was to elucidate the mechanisms underlying the protective effect of Tlr4 including differences in specific innate immune cell populations, their functional responses, and the influence of these cellular differences on the growth of ACD progenitor cell types. Methods: Following butylated hydroxytoluene (BHT) treatment (tumor promoter;4 weekly ip.injections), BALB (Tlr4-sufficient) and C.C3-Tlr4Lps-d/J (BALBLpsd; Tlr4 mutant) lungs were lavaged, processed for flow cytometry, and frozen for molecular analysis (ELISAs for IGF1, KC, Raybiotech Mouse Cytokine Array). BALF-derived macrophages were collected for phagocytosis and efferocytosis assays. BALF macrophage RNA was also isolated and utilized for Affymetrix gene arrays and real-time quantitative PCR. Enrichment of bronchiolar Clara cells, an ADC progenitor cell, was performed and cells enumerated. Femur bone marrow cells were differentiated into macrophages, and co-cultured with C10 cells, a type II cell line, for a wound healing assay. Results: Tlr4-deficiency resulted in significantly increased numbers of innate immune cells in whole lung tissue in response to BHT, including macrophages, PMNs, myeloid-derived suppressor cells (MDSC)s and dendritic cells (DCs), in comparison to Tlr4-sufficient mice (p<05). Macrophage functionality was also affected. BHT treated Tlr4-mutant mice demonstrated enhanced phagocytic and efferocytic abilities and wound healing in comparison to macrophages from Tlr4-sufficient and control treated mice (P<0.05). Additionally, Clara cell proliferation was significantly increased in Tlr4-mutant compared to Tlr4-sufficient mice in response to BHT. Pulmonary cytokine, chemokine and growth factor protein expression also significantly differed among the strains and within macrophages, gene expression and cell surface markers combined demonstrated a more plastic macrophage phenotype in the Tlr4-mutant mice. The transcriptome study identified immune pathways important in inflammation, such as phagosome. Conclusion: There are distinct pulmonary innate immune cell populations, with particular emphasis on macrophages, that are likely influencing the enhanced tumor promotion observed in Tlr4-mutant mice. Though not as striking, the pulmonary lymphoid populations may also be affecting the promotion environment, if only via their influence upon the innate immune cells. Future studies will investigate the origins of the macrophages and continued delineation of the lymphoid cell populations. Two strains of mice were used for these studies. The C.C3-Tlr4Lps-d/J (BALBLpsd) with a dominant negative Tlr4 on a BALB/c background and the BALB/cJ mice with a sufficient Tlr4. Total RNA was extracted from bronchoalveolar lavage fluid macrophages 3 days following a chronic BHT protocol. (oil controls vs BHT treated for both strains. BALB oil and BHT treatment n=3 mice per treatment group; BALBLpsd oil controls n=2 per treatment group; BALBLpsd BHT treated n = 3 per treatment group). Therefore a total of 13 arrays with one BHT exposure times and oil (vehicle) controls.

Project description:Background: Human lung adenocarcinoma (ADC), the predominant subtype of lung cancer, are at a disadvantage as the disease lacks available biomarkers and is not usually diagnosed until its later stages. We previously demonstrated that Tlr4-mutantmice were more susceptible to BHT-induced pulmonary inflammation (bronchoalveolar lavage macrophages and lymphocytes) and tumorigenesis in comparison to Tlr4-sufficient control mice. Differential transcriptional profiles were also noted in the whole lung tissue of the mice. The study objective was to elucidate the mechanisms underlying the protective effect of Tlr4 including differences in specific innate immune cell populations, their functional responses, and the influence of these cellular differences on the growth of ACD progenitor cell types. Methods: Following butylated hydroxytoluene (BHT) treatment (tumor promoter;4 weekly ip.injections), BALB (Tlr4-sufficient) and C.C3-Tlr4Lps-d/J (BALBLpsd; Tlr4 mutant) lungs were lavaged, processed for flow cytometry, and frozen for molecular analysis (ELISAs for IGF1, KC, Raybiotech Mouse Cytokine Array). BALF-derived macrophages were collected for phagocytosis and efferocytosis assays. BALF macrophage RNA was also isolated and utilized for Affymetrix gene arrays and real-time quantitative PCR. Enrichment of bronchiolar Clara cells, an ADC progenitor cell, was performed and cells enumerated. Femur bone marrow cells were differentiated into macrophages, and co-cultured with C10 cells, a type II cell line, for a wound healing assay. Results: Tlr4-deficiency resulted in significantly increased numbers of innate immune cells in whole lung tissue in response to BHT, including macrophages, PMNs, myeloid-derived suppressor cells (MDSC)s and dendritic cells (DCs), in comparison to Tlr4-sufficient mice (p<05). Macrophage functionality was also affected. BHT treated Tlr4-mutant mice demonstrated enhanced phagocytic and efferocytic abilities and wound healing in comparison to macrophages from Tlr4-sufficient and control treated mice (P<0.05). Additionally, Clara cell proliferation was significantly increased in Tlr4-mutant compared to Tlr4-sufficient mice in response to BHT. Pulmonary cytokine, chemokine and growth factor protein expression also significantly differed among the strains and within macrophages, gene expression and cell surface markers combined demonstrated a more plastic macrophage phenotype in the Tlr4-mutant mice. The transcriptome study identified immune pathways important in inflammation, such as phagosome. Conclusion: There are distinct pulmonary innate immune cell populations, with particular emphasis on macrophages, that are likely influencing the enhanced tumor promotion observed in Tlr4-mutant mice. Though not as striking, the pulmonary lymphoid populations may also be affecting the promotion environment, if only via their influence upon the innate immune cells. Future studies will investigate the origins of the macrophages and continued delineation of the lymphoid cell populations.

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

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