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.

This study is based on extensive studies in the mouse of the role of CSF1 in monocyte-macrophage production and differentiation and the function of macrophages in the control of hepatocyte proliferation. We use a novel form of CSF1, an Fc fusion protein, to demonstrate that the findings in mice can be extended to large animals. We discuss the possible role for CSF1 in homeostatic control of the size of the liver.

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.

The domestic pig (Sus scrofa) is both an economically important livestock species and a model for biomedical research. Two highly contiguous pig reference genomes have recently been released. To support functional annotation of the pig genomes and comparative analysis with large human transcriptomic data sets, we aimed to create a pig gene expression atlas. To achieve this objective, we extended a previous approach developed for the chicken. We downloaded RNAseq data sets from public repositories, down-sampled to a common depth, and quantified expression against a reference transcriptome using the mRNA quantitation tool, Kallisto. We then used the network analysis tool Graphia to identify clusters of transcripts that were coexpressed across the merged data set. Consistent with the principle of guilt-by-association, we identified coexpression clusters that were highly tissue or cell-type restricted and contained transcription factors that have previously been implicated in lineage determination. Other clusters were enriched for transcripts associated with biological processes, such as the cell cycle and oxidative phosphorylation. The same approach was used to identify coexpression clusters within RNAseq data from multiple individual liver and brain samples, highlighting cell type, process, and region-specific gene expression. Evidence of conserved expression can add confidence to assignment of orthology between pig and human genes. Many transcripts currently identified as novel genes with ENSSSCG or LOC IDs were found to be coexpressed with annotated neighbouring transcripts in the same orientation, indicating they may be products of the same transcriptional unit. The metaanalytic approach to utilising public RNAseq data is extendable to include new data sets and new species and provides a framework to support the Functional Annotation of Animals Genomes (FAANG) initiative.

Intestinal macrophages are the largest group of mononuclear phagocytes in the body and play a role in intestinal innate immunity, neuroimmune interactions and maintaining intestinal homeostasis. Conversely, they also are implicated in numerous pathologies of the gastrointestinal tract, such as postoperative ileus and inflammatory bowel disease. As a result, macrophages could be potential therapeutic targets. To date, there are limited studies on the morphology and distribution of macrophages in the equine gastrointestinal tract (GIT). The aim of this study was to identify the location and abundance of resident macrophages in the equine GIT using CD163 as an immunohistochemical marker. Tissue samples were obtained post-mortem from sites along the gastrointestinal tracts of 10 horses free from gastrointestinal disease; sample sites extended from the stomach to the small colon. CD163 +ve cells were present in all regions of the equine GIT from stomach to small colon. CD163 +ve cells were also identified in all tissue layers of the intestinal wall; namely, mucosa, submucosa, muscularis externa (ME), myenteric plexus and serosa. Consistent with a proposed function in regulation of intestinal motility, CD163 +ve cells were regularly distributed within the ME, with accumulations closely associated with the myenteric plexus and effector cells such as neurons and the interstitial cells of Cajal (ICC).

Neonatal calves are highly susceptible to a number of diseases including those that infect via the mucosal surfaces of the respiratory and gastrointestinal tracts. In order to determine appropriate vaccine design and delivery systems, or to identify suitable immunostimulatory methods to combat these infections, a detailed understanding of the immune cell populations present at clinically relevant sites is key. Few studies have assessed the immune cell composition of the neonatal calf lung and comparisons with circulating immune cells in the blood are lacking. We describe immune cell populations present in the peripheral blood, bronchoalveolar lavage (BAL) fluid and lung tissue of young disease-free calves. Flow cytometric analysis revealed significant differences in cell subset distribution between the peripheral blood and respiratory tract, and between compartments within the respiratory tract. Notably, whereas WC1 + γδ TCR + T lymphocytes dominate the peripheral blood, both the BAL fluid and lung tissue contained a high proportion of myeloid cells which expressed CD14 and CD172a (SIRPα). Very low numbers of tissue myeloid cells expressed MHC Class II in comparison to circulating myeloid cells in the blood. Respiratory tract tissues had low frequencies of CD4+ and CD8 + T lymphocytes, which were significantly lower than in the blood. Differences in the proportion of NKp46+ natural killer cells were also observed between tissue compartments. In order to target vaccines or immunostimulatory therapeutics appropriately, these differences in immune cell populations in tissue compartments should be taken into consideration.

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