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

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

Immunomodulatory Effects of Bacterial Toll-like Receptor Ligands on the Phenotype and Function of Milk Immune Cells in Dromedary Camel.

(1) Toll-like receptors (TLR) are a family of pattern recognition receptors that sense distinct molecular patterns of microbial origin. Although the immune cell composition of camel milk has been recently described, host-pathogen interaction studies in the camel mammary gland are still scarce. The present study aimed to use a whole milk stimulation assay for investigating the modulatory effect of selected Toll-like receptor (TLR) ligands on the phenotype and function of milk immune cells. (2) Methods-camel milk samples (n = 7) were stimulated in vitro with the TLR4 ligand LPS or the TLR2/1 ligand Pam3CSK4, and separated milk cells were evaluated for stimulation-induced shape change, the expression of cell surface markers, phagocytosis, apoptosis, ROS production, and NETosis. Stimulation with PMA was used as a control stimulation. (3) Results-all stimulants induced shape change in milk cells, change in the expression of several cell markers, and increased cell apoptosis and NETosis. In addition, stimulation with Pam3CSK4 and PMA was associated with enhanced ROS production, while only PMA stimulation resulted in enhanced bacterial phagocytosis by milk immune cells. (4) Conclusions-our data indicates selective modulating effects of the TLR ligands LPS and Pam3CSK4 on camel milk phagocytes. These results may have implications for the use of synthetic TLR agonists as immunomodulatory adjuvants of the immune response to intra-mammary vaccines against mastitis pathogens.

Flow Cytometric Analysis of Leukocyte Populations in the Lung Tissue of Dromedary Camels.

Respiratory tract infections are among the most common infections in dromedary camels, with a high impact on animal health, production, and welfare. Tissue-specific distribution of immune cells is one of the important factors that influence the nature and outcome of the immune response to pathogens. Several protocols have recently been described for the flow cytometric analysis of immune cells in the lung tissue of several species. However, no such protocol currently exists for dromedary camels. The aim of the present study was, therefore, to establish a flow cytometric protocol for the identification of immune cell populations in the camel lung tissue and the evaluation of some of their phenotypic and functional properties. Combined staining of camel lung leukocytes with monoclonal antibodies to the pan-leukocyte marker CD45 and the myeloid cell marker CD172a allowed the identification of myeloid cells (CD45+CD172a+) and lymphoid cells (CD45+CD172a-) in the lung of healthy camels. The cell adhesion molecules CD11a and CD18 were found in a higher abundance on myeloid cells compared to lymphoid cells. Based on their differential expression of the LPS receptor CD14, macrophages (CD172a+CD14high cells) were identified as the most abundant immune cell population in the camel lung tissue. In contrast to their dominance in camel peripheral blood, granulocytes (CD172a+CD14low) presented only a minor population in the lung tissue. The higher frequency of γδ T cells in the lung tissue than in peripheral blood suggests a role for these cells in the pulmonary immune system. Flow cytometric analysis of bacterial phagocytosis and ROS production upon bacterial stimulation revealed high antimicrobial activity of camel lung phagocytes, which was comparable with the antimicrobial activity of blood granulocytes. Comparative analysis of immune cell distribution between the cranial and caudal lobes of the camel lung revealed a higher frequency of granulocytes and a lower frequency of macrophages in the cranial compared to the caudal lung lobe. In addition, the higher frequency of cells expressing the M2 macrophage marker CD163 in the caudal lung tissue, with a slightly higher fraction of MHCII-positive cells (M1 phenotype) in the cranial lung tissue, may suggest the distribution of different macrophage subtypes in the different lobes of the camel lung. Such differences between lung lobes could influence the effectiveness of the immune response to infection or vaccination with respiratory pathogens. Collectively, the present study identified some similarities and differences between camels and other farm animals regarding the distribution of the main immune cell populations in their lungs. Further studies are required for comprehensive immunophenotyping of the cellular pulmonary immune system in camels.

The Impact of the Animal Housing System on Immune Cell Composition and Function in the Blood of Dromedary Camels.

BACKGROUND: The dromedary camel (Camelus dromedarius) is an important livestock animal of desert and semi-desert ecosystems. In recent years, several elements of the camel immune system have been characterized. Stress and excitement induced by animal housing represent the most important environmental factors with potential modulatory effects on the immune system. The present study evaluated the impacts of a restricted-housing system on some phenotypic and functional properties of blood leukocytes in dromedary camels. METHODS: Immunofluorescence and flow cytometry were used to comparatively analyze samples collected from camels during a free-ranging time and samples collected from the same camels during movement-restricted housing. RESULTS: In comparison to blood samples collected from the camels during the free-ranging time, samples from movement-restricted camels showed elevated serum myeloperoxidase activity, a significant shape-change in their neutrophils, and higher reactive oxygen species content in their monocytes and neutrophils, indicating increased cellular oxidative stress under movement-restricted housing. The leukogram pattern of the camels under restricted housing was characterized by leukocytosis with increased numbers of neutrophils, eosinophils, lymphocytes, and monocytes, resembling an excitement leukogram pattern. Within the lymphocyte population, only the helper T cells and B cells were expanded in animals under restricted housing. The upregulation of CD163 together with the downregulation of MHC-II on monocytes from excited camels indicate a modulatory potential of animal excitement to polarize monocytes toward an anti-inflammatory phenotype. Functional analysis of bacterial phagocytosis indicates an impaired antibacterial function of phagocytes in excited camels. The downregulation of several cell adhesion molecules on leukocytes from excited camels suggests a role for impaired cell adhesion and tissue migration and leukocyte retention in blood in the observed leukocytosis in animals under excitement. CONCLUSIONS: The present study identified significant changes in blood immune cell composition, phenotype, and function in dromedary camels under restricted-housing conditions. The observed changes in leukocyte composition suggest the development of an excitement leukogram pattern in camels under movement-restricted housing. To evaluate the clinical relevance of the observed changes in immune cell phenotype and function for the immune competence of camels under restricted housing, further studies are required.