Phenotypic and Functional Heterogeneity of Bovine Blood Monocytes

Citation: Hussen J, D uvel A, Sandra O, Smith D, Sheldon IM, et al. ( Phenotypic and Functional Heterogeneity of Bovine Blood Monocytes Jamal Hussen 0 1 Anna Du vel 0 1 Olivier Sandra 0 1 David Smith 0 1 Iain Martin Sheldon 0 1 Peter Zieger 0 1 Hans- Joachim Schuberth 0 1 Gourapura J. Renukaradhya, The Ohio State University, United States of America 0 1 Immunology Unit, University of Veterinary Medicine , Hannover, Germany , 2 UMR1198 Biologie du De veloppement et Reproduction, Institut National de la Recherche Agronomique, Jouy-en-Josas, France, 3 Ecole Nationale Ve te rinaire d'Alfort, Maisons-Alfort, France, 4 Institute of Infection , Immunity and Inflammation , College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow , United Kingdom , 5 Institute of Life Science, School of Medicine, Swansea University , Swansea , United Kingdom , 6 Zoetis, Paris , France 1 Phagocytosis Assay Heat killed staphylococcus aureus (S. aureus) (Pansorbin , Calbio- chem, Merck, Nottingham , UK) or E. coli (Institute of Microbi- ology, University of Veterinary Medicine , Hannover , Germany) Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14+ CD162, intermediate monocytes (intM) CD14+ CD16+ and nonclassical monocytes (ncM) CD14+ CD16+ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1b after LPS stimulation. Based on IL-1b secretion after LPS/ ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNc increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes. - Funding: Funded by Bundesministerium fu r Bildung und Forschung, www.bmbf.de, Grant: FKZ 0315861, Project EMIDA-Era-Net: iPUD- integrierter systemischer Ansatz zur Verhinderung uteriner Erkrankungen beim Rind. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: PZ (Zoetis, formerly Pfizer Animal Health) is part of a European Union-funded project were a company is involved in the translation of basic science into a product. The partners of this European Union project, however, are funded by their governments (Germany) and not by the company (Zoetis). Regarding the submitted data there are absolutely no competing interests with the company. The participation of PZ (Zoetis) does not alter the authors adherence to all the PLOS One policies. Monocytes are immune cells linking innate and adaptive immunity and play an essential role as cells of the first line of defense against pathogens [1]. Monocytes originate in the bone marrow and are released into the peripheral blood, where they circulate for several days before entering tissues to replenish tissue macrophage or dendritic cell populations [2]. Recent studies have reported a consecutive migration of murine monocyte subsets: In response to tissue damage or infection, nonclassical monocytes (ncM) rapidly migrate out of the circulation and invade the damaged site. This early efflux of ncM is transient and rapidly followed by the recruitment of neutrophils and subsequently by classical monocytes (cM) [3]. Monocytes are equipped with a large array of receptors that recognize various pathogens and mediate phagocytosis, and stimulated monocytes can produce large amounts of reactive oxygen species (ROS), chemokines and cytokines (e.g. chemokine (C-X-C motif) ligand 8 (CXCL8), CXCL1, interleukin (IL)-1b and tumor necrosis factor (TNF)-a) which are involved in the defense against pathogens [4,5]. Studies in human and mice have shown that blood monocytes are a heterogeneous population and that monocyte subsets exert different functions. [6]. Two subpopulations of human monocytes (CD14++CD162 and CD14+CD16+ monocytes) were initially defined based on the differential expression of the lipopolysaccharide (LPS) receptor CD14 and the FccIIIR CD16 [7]. Subsequent studies identified considerable heterogeneity within the CD16positive monocyte population and allowed for discrimination between CD14++CD16+ and CD14+CD16++ subsets [8]. Based on the new nomenclature, human monocytes are subdivided into three subsets on the basis of surface CD14 and CD16 expression. The major population of human monocytes (90%) with high CD14 but no CD16 expression (CD14++ CD162) are now termed cM, whereas the minor population (10%) of human monocytes contain the intermediate monocytes (intM) subset, with low CD16 and high CD14 expression (CD14++ CD16+), and the ncM subset, with high CD16 and low CD14 expression (CD14+ CD16++) [9]. In the murine system classical monocytes (cM) are defined as Ly6C++CD43+, intermediate monocytes (intM) as Ly6C++CD43++ and nonclassical monocytes (ncM) as Ly6C+CD43++ [10]. Human cM have been shown to act as professional phagocytes, to generate ROS and to secrete cytokines in response to bacterial stimulation [9]. Recently, human intM were considered as the monocytes subpopulation with the highest inflammatory potential, being the main producers of ROS [11] and inflammatory cytokines [12]. Human and mouse ncM have been characterized as the monocyte subset which patrol and crawl along the endothelium in a leukocyte function-associated antigen (LFA)-1 dependent manner [12]. NcM have further been shown to sense the presence of viruses and nucleic acids via toll-like receptor (TLR)-7 and TLR-8 [12]. Some studies proposed that monocytes leave the bone marrow as classical cells, which can either directly invade inflamed tissues and differentiate into macrophages and/or dendritic cells, or they can differentiate into intermediate (CD14++CD16+) monocytes in the circulation [9,13,14]. Studies on bovine monocytes were so far restricted to the CD14positive mononuclear cells (MNC) population [15,16,17,18]. Therefore, this study aimed at the definition of bovine blood monocyte subsets and the characterization of their phenotypical and functional heterogeneity. Materials and Methods Ethics Statement This study was approved by the Niedersachsisches Landesamt fu r Verbraucherschutz und Lebensmittelsicherheit (AZ 33.942502-05-09A/598). All procedures involving animals were carried out in accordance wi (...truncated)