BRAF-V600E utilizes posttranscriptional mechanisms to amplify LPS-induced TNF alpha production in dendritic cells in a mouse model of Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is an inflammatory disease characterized by abnormal dendritic cells (DCs) with hyperactive ERK signaling, called "LCH cells." Since DCs rely on ERK signaling to produce inflammatory molecules in response to pathogenic cues, we hypothesized that hyperactive ERK enhances DCs inflammatory responses. We specifically investigated TLR4-induced TNF alpha production in LCH cells by utilizing the BRAF-V600E(fl/+):CD11c-Cre mouse model of LCH, which hyperactivates ERK in DCs. We measured LPS-induced TNF alpha production both in vivo and in vitro using splenic CD11c+ cells and bone marrow-derived DCs with or without pharmacologic BRAF(V600E) inhibition. We observed a reversible increase in secreted TNF alpha and a partially reversible increase in TNF alpha protein per cell, despite a decrease in TLR4 signaling and Tnfa transcripts compared with controls. We examined ERK-driven, posttranscriptional mechanisms that contribute to TNF alpha production and secretion using biochemical and cellular assays. We identified a reversible increase in TACE activation, the enzyme required for TNF alpha secretion, and most strikingly, an increase in protein translation, including TNF alpha. Defining the translatome through polysome-bound RNA sequencing revealed up-regulated translation of the LPS-response program. These data suggest hyperactive ERK signaling utilizes multiple posttranscriptional mechanisms to amplify inflammatory responses in DCs, advancing our understanding of LCH and basic DC biology.