Figure 2.

MEM enhanced bacterial killing by neutrophils. (A) Intracellular and extracellular killing of E. coli E44 after treatment with MEM. Neutrophil killing of bacteria includes two parts: intracellular killing and extracellular killing. A intracellular killing inhibitor of Cytochalasin D was used to distinguish intracellular killing of PMNs and extracellular killing microbes; (B) NETs trapping promoted by MEM. Neutrophils were stimulated for NET formation and infected with E44 at an MOI of 50. Different concentration of MEM was incubated with the neutrophils. Viable bacteria were quantified by plating of serial dilutions. The percentage of CFU trapped by NETs was calculated according to the formula described in section Materials and Methods; (C) MEM could enhance the intracellular killing ability of PMNs. Different MOI (5, 50, 100) was used for infection with E44-GFP, and a concentration of 50 μM MEM was added and incubated with PMNs for 1 h. PMNs without bacteria infection was used as a control group. Then the cells were collected by centrifuge and fixed on slide. DAPI was used for cell staining of nucleus (blue fluorescence). The intracellular killing of bacteria (green fluorescence) by neutrophils was counted, and 100 of neutrophils was assessed for each sample; (D) Phagocytized particles in PMNs treated with E44 or/and MEM. Phagocytized particles were counted under a fluorescence microscopy. CON represents cell treated with E44, and MEM group represents cell treated with E44 and memantine. Each bar represents the average of three different experiments ± SD (n = 3). Scatter plots in the bar graphs represent the three biological replicates. ^#P < 0.05 (P value of the intracellular killing %), *P < 0.05, **P < 0.01, ***P < 0.001 (P value of the extracellular killing %).