Transcriptomic analysis of pathways regulated by toll-like receptor 4 in a murine model of chronic pulmonary inflammation and carcinogenesis
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Published on: July 7, 2017
Alison K Bauer1, Jennifer Fostel, Laura M Degraff
1Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI, USA. akbauer@msu.edu
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.
Insights
Toll-like receptor 4 (TLR4) plays a protective role in lung tumorigenesis by inhibiting pathways like EGFR signaling and inflammation. Understanding these TLR4-mediated pathways can help identify prognostic indicators for lung cancer susceptibility.
Area of Science:
- Immunology
- Genetics
- Oncology
Background:
- Therapeutic strategies for human pulmonary neoplasia are often ineffective due to disease heterogeneity.
- Toll-like receptor 4 (TLR4), an innate immunity gene, demonstrates protective effects against chronic pulmonary inflammation and tumorigenesis in mice, though the underlying mechanism remains unclear.
- This study aimed to identify TLR4-mediated gene expression pathways for prognostic indicators of lung tumorigenesis susceptibility in mice.
Purpose of the Study:
- To identify TLR4-mediated gene expression pathways involved in lung tumorigenesis.
- To elucidate the mechanism by which TLR4 protects against lung cancer.
- To explore potential prognostic indicators for lung cancer susceptibility.
Main Methods:
- Compared whole lung mRNA from Tlr4-mutant (BALB(Lps-d)) and normal (BALB/c) mice after butylated hydroxytoluene (BHT) treatment.
- Analyzed mRNA from micro-dissected tumors and adjacent tissues from both mouse strains after carcinogen exposure (3-methylcholanthrene) with or without BHT.
- Assessed bronchoalveolar lavage fluid for inflammatory cells and protein, and performed immunohistochemical staining for immune cells in tumor-bearing mice.
Main Results:
- In Tlr4-mutant mice, elevated expression of genes in the epithelial growth factor receptor (EGFR) signaling pathway (e.g., epiregulin, secreted phosphoprotein 1) was observed during tumor promotion and progression.
- Significantly higher inflammation, chemotactic, and immune response gene expression occurred in Tlr4-mutant mice compared to normal mice during progression.
- Normal BALB/c mice exhibited significantly reduced expression of immune and inflammatory genes in both tumor and non-tumor tissues.
Conclusions:
- TLR4 confers a protective effect against lung carcinogenesis by inhibiting multiple pathways, including EGFR signaling, inflammatory response, chemotaxis, and cell proliferation.
- Identified specific genes (e.g., Ereg, Cxcl5, Ccr1, Arg1, Pthlh) associated with TLR4-mediated inhibition of lung tumorigenesis.
- Future research will investigate the utility of these identified pathways as indicators of immune system deficiencies and tumorigenesis risk.
