Differential Innate Immune Cell Signatures and Effects Regulated by Toll-like Receptor 4 During Murine Lung Tumor Promotion

Tumor promotion is an early and critical stage during lung adenocarcinoma (ADC). We previously demonstrated that Tlr4 mutant mice were more susceptible to butylated hydroxytoluene (BHT)-induced pulmonary inflammation and tumor promotion in comparison to Tlr4-sufficient mice. Our study objective was to elucidate the underlying differences in Tlr4 mutant mice in innate immune cell populations, their functional responses, and the influence of these cellular differences on ADC progenitor (type II) cells following BHT-treatment. BALB (Tlr4-sufficient) and C.C3-Tlr4(Lps-d)/J (BALB(Lpsd); Tlr4 mutant) mice were treated with BHT (promoter) followed by bronchoalveolar lavage (BAL) and flow cytometry processing on the lungs. ELISAs, Club cell enrichment, macrophage function, and RNA isolation were also performed. Bone marrow-derived macrophages (BMDM) co-cultured with a type II cell line were used for wound healing assays. Innate immune cells significantly increased in whole lung in BHT-treated BALB(Lpsd) mice compared to BALB mice. BHT-treated BALB(Lpsd) mice demonstrated enhanced macrophage functionality, increased epithelial wound closure via BMDMs, and increased Club cell number in BALB(Lpsd) mice, all compared to BALB BHT-treated mice. Cytokine/chemokine (Kc, Mcp1) and growth factor (Igf1) levels also significantly differed among the strains and within macrophages, gene expression, and cell surface markers collectively demonstrated a more plastic phenotype in BALB(Lpsd) mice. Therefore, these correlative studies suggest that distinct innate immune cell populations are associated with the differences observed in the Tlr4-mutant model. Future studies will investigate the macrophage origins and the utility of the pathways identified herein as indicators of immune system deficiencies and lung tumorigenesis.