Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils

Abstract

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

NETosis and Calcium influx in Dromedary Camel Neutrophils after in vitro Toll-like Receptor Stimulation

“…TLRs are membrane and intracellular pattern recognition receptors interacting with PAMPs (Newton and Dixit, 2012;Zindel and Kubes, 2020). Although few recent studies analyzed the impact of some TLR-agonists on some functions of neutrophils in the dromedary camel (Hussen et al, 2023a;Hussen et al, 2023b), many questions still exist regarding the modulatory effect of TLR stimulation on neutrophils phenotype and function. Especially the potential of bacterial and viral TLR-agonists to induce NETformation or Ca 2+ influx in camel neutrophils has not been investigated so far.…”

“…Camel neutrophils were separated as previously described by Hussen et al (2023a). Briefly, 5 mL camel blood was diluted with 5 mL phosphate buffered saline (PBS), and diluted blood was then layered (carefully without mixing them) on 5 mL of the lymphocyte separation medium Lymphoprep™ (Stemcell Technologies, Vancouver, Canada) in Corning® 15 mL centrifuge tubes.…”

Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel

“…Lymph node cells, blood MNC, or total leukocytes were labeled with monoclonal antibodies (mAbs) to camel leukocyte antigens and analyzed by flow cytometry ( 13 ). Separated cells (1 × 10 6 cells/well) were incubated in the wells of a 96-well plate with mAbs cross-reactive with the homologous camel molecules: CD45, CD44, BAQ44A, WC1, CD4, CD18, CD172a, CD14, CD163, and MHCII ( 15 , 16 ). After incubation (15 min; 4°C), cells were washed twice and were incubated with mouse secondary antibodies (IgM, IgG1, IgG2a; Invitrogen) labelled with different fluorochromes or with mouse isotype control antibodies (Becton Dickinson Biosciences).…”

Interaction of Specific Monoclonal Antibodies with Leukocyte Antigens in Camels

“…Monoclonal antibodies (mAbs) play a crucial role in the process of identifying immune cells and monitoring their distribution and mobilization in distinct tissues such as the lungs, the ovary, and the lymph node (Gunnes et al, 2003;Maecker et al, 2012;Rivers et al, 2022). In order to avoid the laborious procedure of generating species-specific mAbs, researchers frequently employ cross-reactive mAbs (Farady et al, 2009;Irani et al, 2016;Grandoni et al, 2020;Grandoni et al, 2023;Hussen et al, 2023a;Hussen et al, 2023b). Over the past few years, several studies have been performed to test the cross-reactivity of mAbs against leukocyte antigens sourced from humans or other veterinary animals with their corresponding camel antigens (Hussen et al, 2017;Hussen and Schuberth, 2020;.…”

“…The list of mAbs and the target cluster of differentiation antigen molecules have been recently reviewed (Hussen and Schuberth, 2020). The identification of such mAbs has been employed in several important studies on camel immune cells (Hussen et al, 2020a;Hussen et al, 2020b;Hussen et al, 2021;Hussen et al, 2023a;Hussen et al, 2023b).…”