Whole transcriptome profiling of neutrophils (Gr1+) flow-sorted from the BM of PU.1 WT and PU.1 KO mice

Neutrophils are essential first line defense cells against invading pathogens, yet their inappropriate activation contributes to immunological diseases and can cause collateral tissue damage. However, if and how neutrophils cell-intrinsically titrate their inflammatory response remains unknown. Here, we conditionally deleted PU.1, a key myeloid transcription factor, from the neutrophils of mice undergoing fungal infection, and then performed comprehensive epigenomic profiling. We find that a major function of PU.1 is to restrain the neutrophils’ immune response by broadly suppressing genomic enhancer outputs via recruiting histone deacetylase activity, thereby limiting the immune-stimulatory AP1-transcription factor JUNB from entering chromatin. Thus, neutrophils rely on a direct PU.1 repressor function as rheostat of the inflammatory chromatin state, safeguarding their epigenome from undergoing uncontrolled activation prior to pathogenic stimulation. We performed whole transcriptome profiling of neutrophils (Gr1+) flow-sorted from the BM of PU.1 WT and PU.1 KO mice, and found nearly 1000 differentially expressed transcripts, 443 of which were decreased and 555 increased in PU.1-deleted neutrophils. Functional annotations of the differentially expressed transcripts revealed that differentiation-associated gene sets were not significantly changed in the neutrophils from PU.1∆Neu mice. In contrast, the top ranked gene ontology (GO) sets downregulated in PU.1∆Neu neutrophils represented immunological terms such as inflammatory response, NFκB activity and Toll-like receptor (TLR) signalling. Neutrophils were flow-sorted from PU.1 WT and PU.1 KO mice for RNA extraction and hybridization on Affymetrix microarrays.