Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

Abstract

The dromedary camel (Camelus dromedarius) is well-adapted to the desert environment with the ability to tolerate increased internal body temperatures rising daily to 41-42°C during extreme hot. This study was undertaken to assess whether in vitro incubation of camel blood at 41°C, simulating conditions of heat stress, differently alters cell vitality, phenotype, and function of leukocytes, compared to incubation at 37°C (normothermia). Using flow cytometry, the cell vitality (necrosis and apoptosis), the expression of several cell markers and adhesion molecules, and the antimicrobial functions of camel leukocytes were analyzed in vitro. The fraction of apoptotic cells within the granulocytes, lymphocytes, and monocytes increased significantly after incubation of camel whole blood at 41°C for 4 h. The higher increase in apoptotic granulocytes and monocytes compared to lymphocytes suggests higher resistance of camel lymphocytes to heat stress. Functionally, incubation of camel blood at 41°C for 4 h enhanced the phagocytosis and ROS production activities of camel neutrophils and monocytes toward S. aureus. Monocytes from camel blood incubated at 41°C for 4 h significantly decreased their expression level of MHC class II molecules with no change in the abundance of CD163, resulting in a CD163^high MHC-II^low M2-like macrophage phenotype. In addition, heat stress treatment showed an inhibitory effect on the LPS-induced changes in camel monocytes phenotype. Furthermore, in vitro incubation of camel blood at 41°C reduced the expression of the cell adhesion molecules CD18 and CD11a on neutrophils and monocytes. Collectively, the present study identified some heat-stress-induced phenotypic and functional alterations in camel blood leukocytes, providing a paradigm for comparative immunology in the large animals. The clinical relevance of the observed changes in camel leukocytes for the adaptation of the camel immune response to heat stress conditions needs further in vitro and in vivo studies.

Keywords: ROS; camel (Camelus dromedarius); heat stress; immunity; leukocytes; phagocytosis.

Figure 1

Gating strategy for flow cytometric analysis of cell necrosis. Separated blood leukocytes were loaded with the DNA-sensitive dye propidium iodide (PI) and labeled cells were analyzed by flow cytometry. Cell duplicates were excluded using a FSC-H against FSC-A dot plot. After gating on single cells, camel granulocytes (G), lymphocytes (L), and monocytes (M) were identified based on their FSC and SSC properties. Viable (PI-negative cells) and necrotic (PI-permeable cells) were identified according to their staining with PI in a SSC-A against PI (FL-3).

Figure 2

Flow cytometric analysis of cell apoptosis. Separated blood leukocytes were loaded with the mitochondrial membrane potential (MMP) probe JC-1. After setting gates on camel granulocytes, lymphocytes, and monocytes based on their FSC and SSC characteristics and the exclusion of cell duplicates, the fractions of normal viable cells (orange JC-1 aggregates detected in FL-2) and apoptotic cells (green JC-1 monomers detected in FL-1) were calculated for blood incubated at 37 and 41°C for 1, 2, and 4 h.

Figure 3

The impact of heat stress on leukocyte cell apoptosis. Whole camel blood (A) or separated camel leukocytes (B) were incubated at 37 or 41°C for different times. The cells were loaded with the mitochondrial membrane potential (MMP) probe JC-1 and were analyzed by flow cytometry. The percentages of apoptotic cells were calculated for gated camel granulocytes, lymphocytes, and monocytes and presented as mean ± SEM. Different lower case letters indicate a significant difference between the groups as analyzed using one-way ANOVA (p < 0.05). For separated leukocytes, the student t-test was used for comparison between the means of cells incubated at 37 and 41°C (* indicates p < 0.05).

Figure 4

The impact of heat stress on bacterial phagocytosis by camel neutrophils and monocytes. (A) Separated camel leukocytes were incubated with heat-killed FITC-labeled S. aureus bacteria and their phagocytosis activity was analyzed by flow cytometry. After gating on neutrophils and monocytes, the percentage of FITC-positive, phagocytosis-positive cells and the phagocytosis capacity (indicating the number of bacteria phagocytosed by each cell as measured by MFI of phagocytosis-positive cells) were determined for each cell type. (B) Data were presented as means ± SEM for blood incubated at 37 and 41°C for 1, 2, and 4 h. Different lower case letters indicate a significant difference between the groups as analyzed using one-way ANOVA (p < 0.05).

Figure 5

The impact of heat stress on S. aureus-induced ROS-response of camel neutrophils and monocytes. (A) Flow cytometric analysis of ROS production by camel neutrophils and monocytes. Separated camel leukocytes were stimulated with heat-killed S. aureus bacteria in the presence of the ROS-sensitive dye dihydrorohdamin-123 (DHR-123) and the reactive oxygen-dependent generation of rhodamine-123 was analyzed by flow cytometry. After gating on neutrophils and monocytes, the mean fluorescence intensity (MFI) of rhodamine-123 was presented in a count/FL-1 histogram. (B) MFI values of rhodamine-123 were presented for granulocytes and monocytes (mean ± SEM) from whole blood incubated at 37 or 41°C for 1, 2, and 4 h. Different lower case letters indicate a significant difference between the groups, as analyzed using one-way ANOVA (p < 0.05). (C) MFI values of rhodamine-123 were presented for granulocytes and monocytes (mean ± SEM) from separated leukocytes incubated at 37 or 41°C for 4 h. For separated leukocytes, the student t-test was used for comparison between the means of cells incubated at 37 and 41°C (* indicates p < 0.05).

Figure 6

Modulatory effects of heat stress on the phenotype of camel monocytes. (A) Gating strategy for the flow cytometric analysis of expression of CD163 and MHC class II molecules on camel monocytes. Separated camel leukocytes were labeled with monoclonal antibodies to CD14, CD163, and MHC class II molecules and analyzed by flow cytometry. Monocytes were identified as CD14-positive cells within mononuclear cells after excluding cell duplets using SSC-A/SSC-H dot plot. The expression levels of CD163 and MHC class II molecules were presented as histograms. (B) The MFI values of CD163 and MHC class II molecules were calculated and presented (mean ± SEM) for blood incubated at 37 and 41°C for 1, 2, and 4 h. Different lower case letters indicate significant difference between the groups as analyzed using one-way ANOVA (p < 0.05). (C) Impact of heat stress on LPS-induced change in monocyte phenotype. Leukocytes were separated from camel blood incubated at 37 or 41°C with or without LPS stimulation. The MFI values of CD163 and MHC class II molecules were calculated and presented (mean ± SEM). Different lower case letters indicate a significant difference between the groups, as analyzed using one-way ANOVA (p < 0.05).

Figure 7

The effect of heat stress on adhesion molecules expression on camel neutrophils and monocytes. Leukocytes separated from heat-stressed whole blood (A) or heat-stressed leukocytes (B) were labeled with monoclonal antibodies to CD18 and CD11a and labeled cells were analyzed by flow cytometry. After setting gates on neutrophils and monocytes, the expression levels of CD18 and CD11a were calculated as MFI values and presented (mean ± SEM) for blood incubated at 37 and 41°C for 1, 2, and 4 h. Different lower case letters indicate a significant difference between the groups, as analyzed using one-way ANOVA (p < 0.05). For separated leukocytes, the student t-test was used for comparison between the means of cells incubated at 37 and 41°C (* indicates p < 0.05).