Chemokine receptors ACKR2 and CCR1 coordinate macrophage dynamics and mammary gland development

Abstract

13Macrophages are key regulators of developmental processes. We previously demonstrated 14 that the scavenging atypical chemokine receptor, ACKR2 has profound effects on branching 15 morphogenesis, by regulating macrophage dynamics during pubertal mammary gland 16 development. ACKR2 is a non-signalling receptor, which mediates effects through 17 collaboration with inflammatory chemokine receptors. Here we reveal that the inflammatory 18 chemokine receptor CCR1, is the reciprocal receptor controlling branching morphogenesis in 19 the mammary gland. Delayed development, with decreased area of the ductal epithelial 20 network and CD206+ macrophages, was observed in the glands of pubertal CCR1-/-mice. 21This is an inverse phenotype to that observed in ACKR2-/-mice. Subcutaneous 22 administration of CCL7, a shared ligand between CCR1 and ACKR2, is sufficient to drive 23 increased numbers of CD206+ macrophages and branching morphogenesis. In addition, 24 estrogen, which is essential for ductal elongation during puberty, upregulates CCR1 25 expression on the surface of pubertal mammary gland macrophages. These data 26 demonstrate that ACKR2 and CCR1 co-ordinately regulate the rate of branching 27 2 morphogenesis in the pubertal mammary gland, whereby stromal ACKR2 modulates levels 28 of CCL7 to control the movement of macrophages expressing CCR1 to the ductal 29 epithelium. The age at which girls develop breasts is steadily decreasing, which increases 30 the risk of many diseases including breast cancer. This study presents a previously unknown 31 immunological mechanism underpinning the rate of development during puberty which could 32 reveal novel therapeutic targets. 33 35Of note, in contrast to ACKR2-/-mice, no difference was observed in the density of, 126 or distance between, branches, measured within a 5 x field of view (F.O.V.) between WT 127 and CCR1-/-mice at any of the time points investigated ( Supplementary Fig. 2). This reveals 128 that CCR1 does not regulate the density, but the spread of the network. CCR3-/-mice 129 display reduced distance between branches at 7 weeks, suggesting that ACKR2 and CCR3 130 may regulate the proximity of branches within mammary gland. Considerable data support 131 potential redundancy in roles for iCCRs in vivo. Therefore, to assess redundant and 132 combinatorial effects of multiple chemokine receptors, whole mounts were obtained from 133 iCCR-/-mice which have a compound deletion of the entire iCCR locus encoding CCR1, 134