Synergistic interaction between the macrophage polarizing signals IL-4 and IFNγ is controlled by the STAT6/STAT1/IRF1 axis.

IFNγ and IL-4, the canonical Th1 and Th2 cytokines, typically induce opposing macrophage polarization. Beyond their frequent coexistence and mutual antagonism in pathological settings, a synergistic crosstalk may also exist and remains to be defined. Through integrated transcriptomic, epigenomic, and CRISPR-based analyses, we identify that co-exposure to IL-4 and IFNγ results in an intermediate macrophage polarization state in murine macrophages, associated with a cohort of synergistically activated genes. We show that this synergistic activation is mediated by co-binding of STAT6 and STAT1 at regulatory regions marked with pronounced H3K27Ac accumulation and is dependent on the BRD4 cofactor. Deletion of either STAT factor or disruption of their binding motifs abolishes the synergistic transcriptional response. Our data support a model in which chromatin openness induced by one cytokine facilitates binding of the opposing cytokine-activated STAT transcription factor (TF), contributing to synergistic gene activation. We further show that IRF1, an IFNγ-induced TF whose expression persists in the presence of IL-4, is indispensable for a substantial subset of this program. Importantly, single-cell RNA sequencing of the PyMT murine breast cancer model reveals an in vivo tumor-associated macrophage subset showing STAT1 and STAT6 activation and is enriched for the synergistic gene signature.