Chemokine receptors coordinately regulate macrophage dynamics and mammary gland development.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We have previously demonstrated that the atypical chemokine receptor ACKR2 contributes to the control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor that mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here, we reveal reciprocal regulation of branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition, oestrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets.

Analysis of lung stromal expression of the atypical chemokine receptor ACKR2 reveals unanticipated expression in murine blood endothelial cells.

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa-Fluor-labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa-Fluor-labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment.

Enhanced CCR2 expression by ACKR2-deficient NK cells increases tumoricidal cell therapy efficacy.

Chemokines regulate leukocyte navigation to inflamed sites and specific tissue locales and may therefore be useful for ensuring accurate homing of cell therapeutic products. We, and others, have shown that atypical chemokine receptor 2 (ACKR2), deficient mice (ACKR2-/-) are protected from metastasis development in cell line and spontaneous mouse models. We have shown that this relates to enhanced CCR2 expression on ACKR2-/- NK cells allowing them to home more effectively to CCR2 ligand expressing metastatic deposits. Here we demonstrate that the metastatic-suppression phenotype in ACKR2-/- mice is not a direct effect of the absence of ACKR2. Instead, enhanced NK cell CCR2 expression is caused by passenger-mutations that originate from creation of the ACKR2-/- mouse strain in 129 embryonic stem cells. We further demonstrate that simple selection of CCR2+ NK cells enriches for a population of cells with enhanced anti-metastatic capabilities. Given the widespread expression of CCR2 ligands by tumors, our study highlights CCR2 as a potentially important contributor to NK cell tumoricidal cell therapy.

Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice.

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally distinct fluorescent reporters mark expression of CCRs 1, 2, 3, and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.

Milk yield, milk composition, and milk metabolomics of dairy goats intramammary-challenged with lipopolysaccharide under heat stress conditions.

Heat stress and mastitis are major economic issues in dairy production. The objective was to test whether goat's mammary gland immune response to E. coli lipopolysaccharide (LPS) could be conditioned by heat stress (HS). Changes in milk composition and milk metabolomics were evaluated after the administration of LPS in mammary glands of dairy goats under thermal-neutral (TN; n = 4; 15 to 20 °C; 40 to 45% humidity) or HS (n = 4; 35 °C day, 28 °C night; 40% humidity) conditions. Milk metabolomics were evaluated using 1H nuclear magnetic resonance spectroscopy, and multivariate analyses were carried out. Heat stress reduced feed intake and milk yield by 28 and 21%, respectively. Mammary treatment with LPS resulted in febrile response that was detectable in TN goats, but was masked by elevated body temperature due to heat load in HS goats. Additionally, LPS increased milk protein and decreased milk lactose, with more marked changes in HS goats. The recruitment of somatic cells in milk after LPS treatment was delayed by HS. Milk metabolomics revealed that citrate increased by HS, whereas choline, phosphocholine, N-acetylcarbohydrates, lactate, and ß-hydroxybutyrate could be considered as putative markers of inflammation with different pattern according to the ambient temperature (i.e. TN vs. HS). In conclusion, changes in milk somatic cells and milk metabolomics indicated that heat stress affected the mammary immune response to simulated infection, which could make dairy animals more vulnerable to mastitis.