Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood

Abstract

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++CD16–/low), intermediate (intM; CD14++/+CD16+), and non‐classical (ncM; CD14–/lowCD16++) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no‐centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no‐wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++/CD16+. These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In‐house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In‐house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome‐conjugated antibodies

Hyperthermia-induced changes in leukocyte survival and phagocytosis: a comparative study in bovine and buffalo leukocytes

“…However, a role of the differential expression of other phagocytosis receptors (43) such as scavenger receptors, C-type lectin receptors, and toll-like receptors in buffalo and bovine phagocytes could be the focus of further studies. In addition, given that classical monocytes (cM) are the main phagocyting monocyte subset in both bovine and buffalo, the enhanced phagocytosis activity of bubaline monocytes could be linked to the specific phenotype of bubaline cM being positive for the activation marker CD16 + (CD14 ++ CD16 + ), which is in contrast to the CD16-phenotype (CD14 ++ CD16 − ) of bovine cM (24). Several previous in and in vivo studies have reported reduced phagocytosis in bovine neutrophils under heat stress conditions (11,44,45).…”

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1

“…In water buffalo, blood monocytes were recently classified into three different cell subsets based on the cell surface abundance of the CD14 LPS receptor and the CD16 FC receptor: CD14 +/++ CD16 −/+ classical monocytes (cMs), CD14 +/low CD16 +/++ intermediate monocytes (intMs), and CD14 -/low CD16 ++ non-classical (ncM) monocytes [ 18 ]. Although the dominant fraction of cMs (74–86% of total monocytes) with a minor population of intMs (5–8% of total monocytes) are in line with their distribution in bovine [ 33 ] and human blood [ 34 ], the frequency of bubaline ncMs (8–18% of total monocytes) seems to be higher than their bovine and human counterparts [ 33 , 34 ].…”

“…The cM, intM, and ncM subsets were detected using multicolor flow-cytometric panels: Pacific Blue anti-CD172 a (clone CC149; Biorad Laboratories, Hercules, CA, USA), BV510 anti-CD18 (clone 6.7; BD Pharmingen, Franklin Lakes, NJ, USA); FITC anti-CD16 (clone KD1; Bio-Rad Laboratories, CA, USA); and AF647 anti-CD14 (clone MM61 A, Washington State University, Pullman, WA, USA). Clones CC149 and MM61 a were conjugated in house as reported by Grandoni et al, 2023 [ 18 ]. All labeled samples were immediately acquired using a CytoFLEX flow cytometer, and the data were analyzed using the CytExpert software (Beckman Coulter, Brea, CA, USA).…”

“…Similarly, the heterogeneity of buffalo blood monocytes was recently reported with some similarities to and differences from bovine monocyte subsets. Using monoclonal antibodies to the signal regulatory protein (SIRP)-alpha protein (also known as CD172a), the CD14 LPS receptor, and the low-affinity CD16 FC receptor, bubaline classical (cM), intermediate (intM), and non-classical (ncM) monocyte subsets were characterized as CD14 +/++ /CD16 −/+ , CD14 +/low /CD16 +/++ , and CD14 -/low /CD16 ++ , respectively [ 18 ].…”