Abstract 3607: Toll-like receptor (TLR) 4 and innate immune cells are involved in the early stages of murine lung tumor promotion.

Chronic inflammatory lung diseases predispose to lung neoplasia. Activation of certain innate immune pathways may confer protection against lung adenocarcinoma (AC) development. Lack of the innate immunity toll-like receptor 4 (Tlr4) gene enhances susceptibility to lung inflammation via bacterial infections while its presence can mediate anti-cancer efficacy. We previously found that Tlr4 is protective in butlylated hydroxytoulene (BHT)-induced pulmonary chronic inflammation and turmorigenesis and using transcriptomics, analyzed the downstream mechanisms involved (Bauer et al., 2005, 2009). The objective of this study was to further characterize the early stage of tumor promotion by determining the phenotypes of the immune cells in the lungs and the interactions between the alveolar immune cells and pulmonary epithelial cells. C.C3H-Tlr4 Lps-d (BALB Lps-d ;Tlr4 mutant) and BALB/c (BALB;Tlr4 wild-type) mice were chronically injected weekly for a min. of 4 weeks with BHT or oil vehicle. Mice were then euthanized at several time points (1, 3, and 30 dy) following the last injection. Subsequently, lungs were half-lung bronchoalveolar lavaged (BAL) for BAL fluid (BALF), while the unlavaged lung was snap frozen. BALF was analyzed for cellular infiltrates by differentials and protein content (indicative of hyperpermeability). Staining and flow cytometric analysis was performed on the same lungs using markers to differentiate between lymphocytes, neutrophils and several pulmonary macrophage (MΦ) populations, including alveolar, interstitial, and activated macrophages. BHT-induced chronic pulmonary inflammation in the BALB Lps-d mice had significantly elevated levels of inflammatory cells and hyperpermeability during the early stages of promotion above that observed in the BALB mice. Results from the flow cytometric analysis suggest that there are increased numbers of MΦs and neutrophils in the lungs of BALB Lps-d mice compared to the BALB mice. In further phenotyping of the MΦs via flow cytometry, alternatively activated MΦs (M2; CD206 + ), were found in greater abundance than classically activated MΦs (M1, iNOS + ) in both strains with an increase in BALB Lps-d vs. BALB mice. These findings, along with several genes upregulated in the BALB Lps-d but not the BALB mice (Pparg, Arg1, Ccl17), suggest that the absence of TLR4 function may lead to an increase in the M2 phenotype, which has been found to assist tumorgenesis. The results demonstrate the importance of TLR4 in the immune response against BHT-induced lung inflammation and subsequently lung tumorgenesis. Future experiments will further investigate the interactions between pulmonary MΦs and pulmonary epithelial cells. Funded by ACS RSG-10-162-01-LIB (AKB). Citation Format: Carla-Maria Alexander, Thomas Hill, Ross Osgood, Alison K. Bauer. Toll-like receptor (TLR) 4 and innate immune cells are involved in the early stages of murine lung tumor promotion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3607. doi:10.1158/1538-7445.AM2013-3607