Epithelial Ikkβ deletion modulates immune responses and the IFNγ/CXCL9 axis during early esophageal carcinogenesis

Abstract

Esophageal cancer is a major cause of cancer-related death, often preceded with chronic inflammation and injuries. The NF{kappa}B/IKK{beta} pathway plays a central role in inflammation, yet its role in early esophageal carcinogenesis remains unclear. This study investigated the role of epithelial IKK{beta} in early esophageal carcinogenesis. Mice were treated with the carcinogen 4-nitroquinoline-1-oxide (4-NQO) or a vehicle for one month to induce precancerous lesions. Esophagi were harvested and examined through histological, protein, flow cytometry, and RNA analyses. Histological analysis revealed that 4-NQO treatment led to increased inflammation, intraepithelial CD45+ immune cells, and elevated IKK{beta} phosphorylation levels. Mice with esophageal epithelial-specific Ikk{beta} deletion (4-NQO/Ikk{beta}EEC-KO) showed delayed progression to a precancerous state, with reduced immune cell recruitment compared to 4-NQO/controls. Immunophenotyping showed decreased recruitment of T cells, including CD4+, CD8+ and regulatory (Tregs) T cells, and increased recruitment of macrophages in 4-NQO/Ikk{beta}EEC-KO mice compared to 4-NQO/controls. RNA sequencing data identified 262 differentially expressed genes in 4-NQO/Ikk{beta}EEC-KO mice, implicating pathways related to inflammation and wound healing. Notably, the chemokine CXCL9, a T cell chemoattractant, was significantly upregulated in 4-NQO control mice, but not in 4-NQO/Ikk{beta}EEC-KO mice. Further analysis identified IFN{gamma} as an upstream regulator of Cxcl9 expression, and neutralization of IFN{gamma} reduced Cxcl9 expression levels in 4-NQO treated mice. Additionally, in vitro studies demonstrated that IFN{gamma} upregulates Cxcl9 in an NF-B dependent manner in esophageal keratinocytes. These findings suggest that epithelial IKK{beta} regulates the immune microenvironment in early esophageal carcinogenesis through the IFN{gamma}/CXCL9 axis and influencing T cell recruitment and inflammatory responses. SummaryIn a mouse model of early esophageal squamous cell carcinogenesis, loss of epithelial Ikk{beta} reduced inflammation and T cell recruitment, increased macrophage recruitment, inhibited IFN{gamma}/CXCL9 signaling, and delayed the transition to a precancerous state.