Assay Development for Phagocytosis Activity Evaluation

: The efficiency of phagocytic activity is a significant organism’s indicator which decreases with the aging of the immune system. The medications are able to influence phagocytosis, having a blocking or activating effect, and therefore are important modulators of immune function. We are developing an ex vivo assay indispensable for medication screening in human and Macaca fascicularis whole blood. For assay verification several published control drugs were successfully used. This assay assists to find medications for enhancing the immune response in the elderly and to provide a deep comprehension of fundamental process of immune system aging.


Introduction
The increased risk of developing age-related diseases in the elderly is associated with an imbalance and deficiency of the immune response, therefore, specific treatment and vaccination approaches should be developed.The efficiency of phagocytic activity is a significant organism's indicator which decreases with the aging of the immune system.The medications are able to influence phagocytosis, having a blocking or activating effect, are important modulators of immune function.We are developing an ex vivo assay indispensable for medication screening in human and Macaca fascicularis whole blood.M.fascicularis is the well-recognized preclinical model having human-like immune responses.For assay verification several published control drugs [1] were successfully used for blocking and acceleration of phagocytosis.Experiments evaluating human and animal phagocytosis indices at different ages will provide statistical standards to be used for medication preclinical and clinical trials.This assay assists to find medications for enhancing the immune response and antigen presentation in the elderly.Moreover, screening of medications, having impact to phagocytosis and further researches of their mechanisms assist to a deep comprehension of fundamental process of immune system aging, and provide a perspective for immune response correction.

Materials and Methods
The object for phagocytosis was E. coli stained in house with FITC (Lumiprobe) for 3 h, 37 °C.Phagocytosis was carried out in whole blood without adhesion [2] under near physiological conditions (T = 37 °C, 5% CO2).After 1 h incubation with E. coli, cells were treated with Protein transport inhibitor, containing monensin (BD GolgiStop), according to the instruction, or placed on ice, or treated with combination of lidocaine (0.1-3 mg/mL)-epinephrine (0.34-10.2 μM) [1] for 5 min.Then the erythrocytes were lysed with BD Pharm Lyse according to the instruction.Besides, cells were pre-treated with these agents for 5 min before adding bacteria.Sample analysis was performed on a BD LSRFortessa flow cytometer.To correctly identify leucocytes, CD45 APC-eFluor780 (HI30, eBiosciencе; 1:100) was used.

Results and Discussion
Pre-treated with different agents blood cells were incubated with E. coli as described; after that percentage of phagocytosed сеlls (PC; activated monocytes or granulocytes, as the most prominent phagocytosed populations) was evaluated (Figure 1).In comparison to non-treated cells (PC ~ 33%), treated cells' phagocytic activity plummeted (PC 4.6-20.8%),and lidocaine-epinephrine treatment showed dose-dependent decrease, effectively blocked phagocytosis in the same manner as commercial GolgiStop in optimal concentration.This trend was linear for both granulocytes and monocytes (R 2 = 0.972 and R 2 = 0.997, respectively, Figure 2).Moreover, treatment after 1 h incubation with E. coli, was indistinguishable from control sample without inhibitors of phagocytosis (Figure 3).Despite there was no difference, it can be used in modifications of phagocytic dynamic test where both engulfed and adhered bacteria are detected in defined timelapse.In case of phagocytosis was stopped on ice, percentage of PC was lower in comparison with treated and non-treated samples, and it can be supposed that cells undergo degranulation and cell death on ice.Further analysis confirmed that degranulation and death of phagocytosed cells on ice occurs, and that additionally validates usage of chemical agents to block phagocytosis.

Conclusions
Lidocaine-epinephrine mixture inhibits phagocytosis and, if necessary, can be used as a stop-reagent as a cheaper and effective analogue to commercial reagents and ice.Phagocytic activity evaluation by FITC-conjugated bacteria and developed protocol in whole blood suggested to use as an essential part for medication screening.Validation of perspective medications is recommended for elderly people whose phagocytosis is impaired.Moreover, phagocytosis testing in whole blood under near physiological conditions offers useful and feasible approach for a personalized medicine.

Figure 2 .
Figure 2. Linear dependence of activated cells percentage on lidocaine-epinephrine concentrations.

Figure 2 .
Figure 2. Linear dependence of activated cells percentage on lidocaine-epinephrine concentrations.