Chromatin immunoprecipitation sequencing (ChIP-Seq) for Bhlhe40 from naïve and IL-4c-stimulated large peritoneal macrophages (LPMs)

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state, and can expand during type 2 immunity. Whether shared mechanisms regulate macrophage proliferation in homeostasis and disease is unclear. We found that the transcription factor Bhlhe40 was cell-intrinsically required in large peritoneal macrophages (LPMs) for self-renewal and maintenance, but was not required in other resident macrophages. Bhlhe40 was selectively necessary in LPMs for proliferation, but not polarization, in response to IL-4. During a helminth infection, Bhlhe40 was required for normal LPM cell cycling. Bhlhe40 repressed the expression of Maf and Mafb and directly promoted expression of cell cycle-related transcripts to enable proliferation of LPMs. Genomic sites bound by Bhlhe40 in LPMs included some co-bound by the macrophage lineage-determining factor PU.1 and others uniquely bound by Bhlhe40, including Maf and cell cycle-related loci. Our findings demonstrate a tissue-specific control mechanism regulating resident macrophage proliferation in homeostasis and type 2 immunity. LPMs were sorted from pooled naïve and pooled IL-4c-treated C57BL/6 mice. Chromatin was crosslinked and immunoprecipitation (Novus Biologicals, anti-Dec1, catalog NB100-1800, Lot C1) was performed for Bhlhe40-bound genomic regions, which were sequenced along with input DNA controls from the same samples. DNA from 1 naïve LPM ChIP and 1 IL-4c-stimulated ChIP were sequenced, as well as input DNA. Cells were pooled from multiple mice for each immunoprecipitation.