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Interferon gamma (IFN-γ) is the main cytokine driving organ dysfunction in Familial Hemophagocytic Lymphohistiocytosis (FHL). Blockade of IFN-γ pathway ameliorates FHL hepatitis, both in animal models and in humans with FHL. Hepatocytes are known to express IFN-γ receptor (IFN-γ-R). However, whether IFN-γ induced hepatitis in FHL is a lymphocyte or liver intrinsic response to the cytokine has yet to be elucidated. Using a IFNgR−/− bone marrow chimeric model, this study showed that non-hematopoietic IFN-γ response is critical for development of FHL hepatitis in LCMV-infected Prf1−/− mice. Lack of hepatic IFN-γ responsiveness results in reduced hepatitis as measured by hepatomegaly, alanine aminotransferase (ALT) levels and abrogated histologic endothelial inflammation. In addition, IFN-γ non-hematopoietic response was critical in activation of lymphocytes by soluble interleukin 2 receptor (sIL-2r) and recruitment of CD8+ effector T lymphocytes (CD8+ CD44hi CD62Llo) (Teff) and inflammatory monocytes. Lastly, non-hematopoietic IFN-γ response results in increased hepatic transcription of type 1 immune response and oxidative stress response pathways, while decreasing transcription of genes involved in extracellular matrix (ECM) production. In summary, these findings demonstrate that there is a hepatic transcriptional response to IFN-γ, likely critical in the pathogenesis of FHL hepatitis and hepatic specific responses could be a therapeutic target in this disorder.

Background: Langerhans Cell Histiocytosis (LCH) is an inflammatory disease driven by abnormal dendritic cells (DCs) with hyper-MAPK/ERK signaling, usually due to Braf-V600E mutation. Since DCs are poised with Toll like Receptors (TLRs), which utilize MAPK/ERK signaling to incite an immune response, we hypothesize that LCH cells have a hyper-TLR response. Methods: We use an animal model of LCH (CD11c-Cre:BRAFV600E-flox; which expresses Braf-V600E in DCs) to measure serum TNFa and tnfa transcripts in CD11c+ splenocytes after LPS injection. Cultured BMDCs were pre-treated with/without a V600E-inhibitor before LPS stimulation. We measured intracellular TNFa, secretion and reuptake of TNFa, tnfa transcripts, NFkB signaling, TNFa degradation, and TACE activity. Results: LCH mice have increased LPS-induced circulating TNFa compared to WT. LCH-BMDCs have reversible LPS-induced increase in TNFa secretion and intracellular accumulation, but decreased tnfa and tlr4 transcripts and NFkB signaling compared to WT. There is no difference in TACE activity, TNFa reuptake, or protein degradation. Conclusions: Hyper-MAPK/ERK signaling in DCs causes a hyper LPS-induced TNFa response, despite diminished LPS signaling. Increased intracellular TNFa indicates a cell intrinsic, post-transcriptional mechanism. Current experiments are analyzing translation of TNFa, which is implicated by MAPK/ERK signaling. These data indicate novel effects of Braf-V600E on LPS-induced TNFa production, and current studies are investigating whether this is a more global phenomenon.

Background The origin of inflammation in Langerhans Cell Histiocytosis (LCH), an inflammatory disease of dendritic cells (DCs) in children, remains elusive. Over 85% of patients harbor somatic, gain-of-function mutations in the Ras-ERK signaling pathway in DCs, which leads to hyper-pERK activity. We hypothesize that DCs with hyper-pERK signaling are intrinsically hyper-responsive to TLR stimuli, resulting in increased levels of cytokines that contribute to LCH etiology. Methods To determine the TLR response in hyper-pERK DCs, we generated bone marrow derived DCs (BMDCs) from wild-type and CD11c-Cre:BRAF-V600E flox mice (V600E-DCs). Cells were pre-treated with or without a V600E-inhibitor and stimulated with TLR ligands. Cytokine levels were measured by ELISA and qPCR. TLR levels were measured by qPCR. Results V600E-DCs secreted increased LPS-induced TNFa and IL-6 compared to wild type, which was reversible with acute inhibition of V600E. Intriguingly, this was despite V600E-DCs having decreased tnfa, il-6, and tlr4 transcripts. Osteoponton (OPN) secretion was irreversibly increased in V600E-DCs both at baseline and after stimulation with LPS, CpG, and Poly(I:C); however, its gene, spp1, was not increased. Conclusions DCs harboring the BRAF-V600E allele have a reversible increase in LPS induced inflammatory cytokine expression, suggesting that increased pERK directly caused a hyper-response to TLR4 stimulation. The irreversible increase in OPN secretion suggests a permanent defect in V600E-DCs independent of pERK levels at time of stimulation. These data taken together suggest that the increased pool of pERK in V600E-DCs contributes to a complex disruption of cytokine secretion that may play a role in LCH etiology.

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 TNFα production in LCH cells by utilizing the BRAF‐V600Efl/+:CD11c‐Cre mouse model of LCH, which hyperactivates ERK in DCs. We measured LPS‐induced TNFα production both in vivo and in vitro using splenic CD11c+ cells and bone marrow‐derived DCs with or without pharmacologic BRAFV600E inhibition. We observed a reversible increase in secreted TNFα and a partially reversible increase in TNFα protein per cell, despite a decrease in TLR4 signaling and Tnfa transcripts compared with controls. We examined ERK‐driven, posttranscriptional mechanisms that contribute to TNFα production and secretion using biochemical and cellular assays. We identified a reversible increase in TACE activation, the enzyme required for TNFα secretion, and most strikingly, an increase in protein translation, including TNFα. 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.