Macrophage colony-stimulating factor (CSF1) controls monocyte production and maturation and the steady-state size of the liver in pigs.

Macrophage colony-stimulating factor (CSF1) is an essential growth and differentiation factor for cells of the macrophage lineage. To explore the role of CSF1 in steady-state control of monocyte production and differentiation and tissue repair, we previously developed a bioactive protein with a longer half-life in circulation by fusing pig CSF1 with the Fc region of pig IgG1a. CSF1-Fc administration to pigs expanded progenitor pools in the marrow and selectively increased monocyte numbers and their expression of the maturation marker CD163. There was a rapid increase in the size of the liver, and extensive proliferation of hepatocytes associated with increased macrophage infiltration. Despite the large influx of macrophages, there was no evidence of liver injury and no increase in circulating liver enzymes. Microarray expression profiling of livers identified increased expression of macrophage markers, i.e., cytokines such as TNF, IL1, and IL6 known to influence hepatocyte proliferation, alongside cell cycle genes. The analysis also revealed selective enrichment of genes associated with portal, as opposed to centrilobular regions, as seen in hepatic regeneration. Combined with earlier data from the mouse, this study supports the existence of a CSF1-dependent feedback loop, linking macrophages of the liver with bone marrow and blood monocytes, to mediate homeostatic control of the size of the liver. The results also provide evidence of safety and efficacy for possible clinical applications of CSF1-Fc.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Bovine NK subsets in the afferent lymph and lymph nodes have distinct expression of naïve and activation-associated cell surface expressed molecules, and are differentially stimulated by BCG vaccination.

Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.

A novel monoclonal antibody against porcine macrophage colony-stimulating factor (CSF1) detects expression on the cell surface of macrophages.

Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system through binding to the receptor CSF1R. The expression and function of CSF1 has been well-studied in rodents and humans, but knowledge is lacking in other veterinary species. The development of a novel mouse anti-porcine CSF1 monoclonal antibody (mAb) facilitates the characterisation of this growth factor in pigs. Cell surface expression of CSF1 was confirmed on differentiated macrophage populations derived from blood and bone marrow monocytes, and on lung resident macrophages, the first species for this to be confirmed. However, monocytes isolated from blood and bone marrow lacked CSF1 expression. This species-specific mAb delivers the opportunity to further understanding of porcine myeloid cell biology. This is not only vital for the role of pigs as a model for human health, but also as a veterinary species of significant economic and agricultural importance.

Characterisation of dendritic cell frequency and phenotype in bovine afferent lymph reveals kinetic changes in costimulatory molecule expression.

The bovine afferent lymphatic cannulation model allows collection of large volumes of afferent lymph and provides an opportunity to study lymphatic cells trafficking from the periphery directly ex-vivo. The technique requires surgical intervention, but influence of the procedure or time post-surgery on cells trafficking in the lymph has not been well documented. Here, we measured the volume of lymph and number of cells/mL collected daily over a two week time-course. Animal to animal variability was demonstrated but no consistent changes in lymph volume or cell density were observed in relation to time post-cannulation. Cell populations (dendritic cells, αß T-cells, γδ T-cells and NK cells) were analysed by flow cytometry at 1, 3 and 10 days post-cannulation (DPC) and a reduced percentage of γδ T-cells in afferent lymph was observed at 1 DPC. In addition, cell surface molecule expression by afferent lymphatic dendritic cells (ALDC) was assessed due to the key role of these cells in initiating an adaptive immune response. Co-stimulatory molecules CD80 and CD86 were upregulated by CD172a+ve ALDC early in the time-course, suggesting that the cannulation procedure and duration of experiment may impact the activation state of DCs in the naïve host. This should be considered when analysing the response of these cells to vaccines or pathogens.

Anatomical distribution of respiratory tract leukocyte cell subsets in neonatal calves.

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.

Immunohistochemical study of morphology and distribution of CD163+ve macrophages in the normal adult equine gastrointestinal tract.

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 14 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).

Functional Annotation of the Transcriptome of the Pig, Sus scrofa, Based Upon Network Analysis of an RNAseq Transcriptional Atlas.

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 meta-analytic 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.

Species-Specific Transcriptional Regulation of Genes Involved in Nitric Oxide Production and Arginine Metabolism in Macrophages.

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.

ADGRE1 (EMR1, F4/80) Is a Rapidly-Evolving Gene Expressed in Mammalian Monocyte-Macrophages.

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.