OP0179 Brafv600e promotes myeloid skewing in multisystemic langerhans cell histiocytosis humanized mouse model

Abstract

BackgroundMultisystemic Langerhans cell histiocytosis (mLCH) is an aggressive disease characterized by the accumulation of mononuclear phagocytes with immunohistochemical features of dendritic cell (histiocytes)1. Histiocytes infiltrate mostly the skin, bone marrow (BM), lung and spleen, and produce high levels of proinflammatory cyto/chemokines, leading to organ dysfunction2,3. In patients, around 10% of cells in lesions carries an oncogenic mutation in the MAPK pathway, mostly BRAFV600E (70% of cases). BRAFV600E can be detected also in monocytes and BM progenitors (HSPC) from these patients, whereas only a fraction of them carries a mutation in B cells and none in T cells4.ObjectivesTo study the role of BRAFV600E in the pathogenesis of mLCH, we set up a humanized mouse model of mLCH based on the transplantation into immunodeficient mice (NSG) of human HSPC expressing BRAFV600E.MethodsWe isolated HSPC from human cord blood and transduced them at two different levels (50% and 20%) with lentiviral vectors that ubiquitously express BRAFV600E, BRAFWT or GFP.ResultsAll BRAFV600E mice manifested severe weight loss within 7 weeks with a median of 3 and 5 weeks for 50% and 20% transduction groups, respectively. Mice showed dysplastic bone marrow (BM) with infiltration of histiocytes; lesions were present also in lungs, kidneys, CNS, spleen and liver. Immunophenotype of infiltrating histiocytes closely resembles mLCH, staining positive for CD14, CD68, S-100 and langerin. None of the control mice lost weight nor displayed organ alteration. Flow cytometry analyses of BM showed 5- to 7-fold reduction in engraftment of human cells in BRAFV600E vs GFP groups (p<0,001). Percentage of myeloid cells increased by 3- to 4-fold in BRAFV600E vs GFP groups (p<0,001). On the contrary, percentage of B cells was reduced by 3- to 6,5-fold in BRAFV600E vs GFP groups (p<0,001). Moreover, there was no difference in the percentage of GFP-positive cells between myeloid cells, whole BM and in vitro sample, suggesting a non-cell autonomous mechanism underlying this myeloid phenotype.ConclusionsIn summary, we generated for the first time a human xenogeneic transplantation mouse model of mLCH, showing that BRAFV600E in human HSPC promotes myeloid skewing rather than proliferation.References Wilejto et al. Curr Opin Rheumatol 2012. DOI: 10.1097/BOR.0b013e32834db53e.Donadieu et al. Histiocytic Disorders of Children and Adults 2005.Kannourakis et al. Br J Cancer 1994, Sep;23:S37–40.Berres et al. J Exp Med 2014, DOI: 10.1084/jem.20130977. Disclosure of InterestNone declared

OP0178 Fibroblast priming is common to many sites, and psoriatic skin fibroblasts may acquire inflammatory memory

“…In patients, around 10% of cells in lesions carries an oncogenic mutation in the MAPK pathway, mostly BRAF V600E (70% of cases). BRAF V600E can be detected also in monocytes and BM progenitors (HSPC) from these patients, whereas only a fraction of them carries a mutation in B cells and none in T cells 4 . Objectives: To study the role of BRAF V600E in the pathogenesis of mLCH, we set up a humanized mouse model of mLCH based on the transplantation into immunodeficient mice (NSG) of human HSPC expressing BRAF V600E .…”

“…Gilis 1,2 , J. Staal 3,4 , R. Beyaert 4,5 , D. Elewaut 1,6 . 1 Department of Internal Medicine, Ghent University; 2 Molecular Immunology and Inflammation Unit, VIB Inflammation Research Center; 3 Department of Biomedical Molecular Biology, Ghent University; 4 …”

“…Objectives: In this report we describe the in vitro and in vivo characterization of the small molecule GLPG1972, an inhibitor of ADAMTS-5. GLPG1972 anticatabolic activity was evaluated in murine and human cartilage explants and DMOAD activity was investigated in the destabilization of the medial meniscus (DMM) mouse model 4 .…”

OP0180 Pharmacological characterization of the ADAMTS-5 inhibitor GLPG1972: an oral anti-catabolic agent for the treatment of osteoarthritis

“…Objectives: In this report we describe the in vitro and in vivo characterization of the small molecule GLPG1972, an inhibitor of ADAMTS-5. GLPG1972 anticatabolic activity was evaluated in murine and human cartilage explants and DMOAD activity was investigated in the destabilization of the medial meniscus (DMM) mouse model 4 . Methods: The ADAMTS-5 biochemical assay is based on the cleavage of a fluorescent substrate by recombinant ADAMTS-5.…”

“…Human articular cartilage explants were stimulated with IL-1β for 12 or 19 days and the NITEGE epitope quantified using the AGNx1 assay. Unilateral OA was induced in C57BL6 mice by DMM 4 . Mice were treated with vehicle or GLPG1972 at 30, 60 or 120 mg/kg, b.i.d.…”