ATAC-sequencing of BTBR and C57BL6/J BMDM with Repeated LPS Treatment

The ability of peripheral innate immune cells to control pro-inflammatory cytokines during periods of repeated or prolonged exposure to immune activating stimuli is critical for preventing tissue damage. In macrophages, repeated exposure to lipopolysaccharide (LPS) induces a refractory period of immune activation termed endotoxin tolerance. Macrophages from the BTBR mouse strain have a skewed pro-inflammatory phenotype and show a hyper-response to LPS challenge. To identify disrupted regulatory mechanisms governing control of cytokine expression in BTBR compared to C57BL/6J mice (C57), we assessed bone marrow derived macrophages (BMDM) treated with either media, one dose or two doses of LPS. Differences in chromatin accessibility using ATAC-sequencing were used to identify regions of differential chromatin accessibility both under baseline media conditions and in response to LPS. Bone marrow was extracted from postnatal day 28 (P28) male BTBR or C57 mice and differentiated in culture for seven days (DIV 7) to form bone marrow derived macrophages (BMDM). BMDM from each strain were re-seeded into six well plates and treated with either media, one dose of LPS for 24hrs (LPSx1) or given a 4 hour LPS pretreatment followed by a 24hour LPS treatment (LPSx2). All LPS treatments were at 100ng/ml of culture media. Following treatment, cells were collected for ATAC-sequencing. After quality control assessment, reads were aligned to the mouse genome (mm10) and filtered for nucleosome free regions (<147bp). HOMER was then used to call peaks for each biological replicate.