Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.

Ross S Osgood

doi:10.4172/2155-9929.1000160

Abstract

TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALBLps-d) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-d over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-d BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALBLps-d BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening.

Highlights

Lung cancer is a largely preventable disease in the U.S and abroad [1]
We demonstrated an increase in total Bronchoalveolar Lavage Fluid (BALF) protein content and macrophage infiltration in all mice exposed to Butylated Hydroxytoluene (BHT) (Figure 1A and 1B), similar to previous studies [3], for comparison to the analysis of gap junction activity in the experiments that followed (Figures 3 and 4)
Total BALF protein significantly increased in both strains in response to BHT compared to oil controls (p≥0.02), the BALBLps-d mice had significant elevations above that observed in the BALB BHTtreated mice (Figure 1A)

Summary

Introduction

Lung cancer is a largely preventable disease in the U.S and abroad [1]. Lung tumor formation is often a 2-stage process, starting with initiation, a direct mutagenic event, followed by promotion, a nongenotoxic event leading to the clonal expansion of initiated cells and tumor development. A common 2-stage model in the lung uses 3-Methylcholanthrene (3-MCA) followed by sequential dosing of Butylated Hydroxytoluene (BHT) in mice [2]. In more recent and past studies, the inflammatory characteristics of Bronchoalveolar Lavage Fluid (BALF) from treated mice have been used to describe changes in the local microenvironment that may be predictive of tumorigenesis [3,4,5,6].We previously demonstrated that functional Toll-Like Receptor 4 (TLR4), an innate immune receptor, is protective against inflammation and primary tumor formation in the mouse lung in vivo using the MCA/BHT 2-stage promotion model [3,7]. TLR4 both exacerbates and protects from inflammation and injury in pulmonary models. TLR4 exacerbates lipopolysaccharide (LPS)-induced lung injury [8]. TLR4 confers protection in other organ systems, such as human gastric carcinomas [12]

Methods

Results

Conclusion