Transcriptomic analysis of pathways regulated by toll-like receptor 4 in a murine model of chronic pulmonary inflammation and carcinogenesis.

BACKGROUND

Therapeutic strategies exist for human pulmonary neoplasia, however due to the heterogeneity of the disease, most are not very effective. The innate immunity gene, toll-like receptor 4 (TLR4), protects against chronic pulmonary inflammation and tumorigenesis in mice, but the mechanism is unclear. This study was designed to identify TLR4-mediated gene expression pathways that may be used as prognostic indicators of susceptibility to lung tumorigenesis in mice and provide insight into the mechanism.

METHODS

Whole lung mRNA was isolated from C.C3H-Tlr4(Lps-d) (BALB(Lps-d); Tlr4 mutant) and BALB/c (Tlr4 normal) mice following butylated hydroxytoluene (BHT)-treatment (four weekly ip. injections; 150-200 mg/kg/each; "promotion"). mRNA from micro-dissected tumors (adenomas) and adjacent uninvolved tissue from both strains were also compared 27 wks after a single carcinogen injection (3-methylcholanthrene (MCA), 10 microg/g; "control") or followed by BHT (6 weekly ip. injections; 125-200 mg/kg/each; "progression"). Bronchoalveolar lavage fluid was analyzed for inflammatory cell content and total protein determination, a marker of lung hyperpermeability; inflammation was also assessed using immunohistochemical staining for macrophages (F4/80) and lymphocytes (CD3) in mice bearing tumors (progression).

RESULTS

During promotion, the majority of genes identified in the BALB(Lps-d) compared to BALB/c mice (P < 0.05) were involved in epithelial growth factor receptor (EGFR) signaling (e.g. epiregulin (Ereg)), secreted phosphoprotein 1(Spp1)), which can lead to cell growth and eventual tumor development. Inflammation was significantly higher in BALB(Lps-d) compared to BALB/c mice during progression, similar to the observed response during tumor promotion in these strains. Increases in genes involved in signaling through the EGFR pathway (e.g. Ereg, Spp1) were also observed during progression in addition to continued inflammation, chemotactic, and immune response gene expression in the BALB(Lps-d) versus BALB/c mice (P < 0.05), which appears to provide more favorable conditions for cell growth and tumor development. In support of these findings, the BALB/c mice also had significantly reduced expression of many immune response and inflammatory genes in both the tumors and uninvolved tissue.

CONCLUSION

This transcriptomic study determined the protective effect of TLR4 in lung carcinogenesis inhibition of multiple pathways including EGFR (e.g. Ereg), inflammatory response genes (e.g. Cxcl5), chemotaxis (e.g. Ccr1) and other cell proliferation genes (e.g. Arg1, Pthlh). Future studies will determine the utility of these pathways as indicators of immune system deficiencies and tumorigenesis.