The F4/80 antigen, encoded by the Adgre1 locus, has been widely-used as a monocyte-macrophage marker in mice, but its value as a macrophage marker in other species is unclear, and has even been questioned. ADGRE1 is a seven transmembrane G protein-coupled receptor with an extracellular domain containing repeated Epidermal Growth Factor (EGF)-like calcium binding domains. Using a new monoclonal antibody, we demonstrated that ADGRE1 is a myeloid differentiation marker in pigs, absent from progenitors in bone marrow, highly-expressed in mature granulocytes, monocytes, and tissue macrophages and induced by macrophage colony-stimulating factor (CSF1) treatment in vivo. Based upon these observations, we utilized RNA-Seq to assess the expression of ADGRE1 mRNA in bone marrow or monocyte-derived macrophages (MDM) and alveolar macrophages from 8 mammalian species including pig, human, rat, sheep, goat, cow, water buffalo, and horse. ADGRE1 mRNA was expressed by macrophages in each species, with inter-species variation both in expression level and response to lipopolysaccharide (LPS) stimulation. Analysis of the RNA-Seq data also revealed additional exons in several species compared to current Ensembl annotations. The ruminant species and horses appear to encode a complete duplication of the 7 EGF-like domains. In every species, Sashimi plots revealed evidence of exon skipping of the EGF-like domains, which are highly-variable between species and polymorphic in humans. Consistent with these expression patterns, key elements of the promoter and a putative enhancer are also conserved across all species. The rapid evolution of this molecule and related ADGRE family members suggests immune selection and a role in pathogen recognition.

Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow–derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction.