Neutrophil Functional Heterogeneity: Identification of Competitive Phagocytosis

Abstract

Introduction: Phagocytosis by neutrophils is a key process in the innate immune response against invading microorganisms. Despite reported heterogeneity in other neutrophils functions, little is known regarding differences in phagocytosis by individual cells. Therefore, we tested the hypothesis that heterogeneity is present in the neutrophil compartment in its potency to phagocytize bacteria.

Methods: Phagocytosis assays were performed in suspension with isolated neutrophils and Staphylococcus aureus expressing different fluorescent proteins at MOIs between 1 and 10. Repetitive addition of bacteria with different fluorescent proteins and MOIs was used to compare the phagocytic capacity of S. aureus-green fluorescent protein (GFP)-positive and negative neutrophils and exclude randomness.

Results: The percentage and mean fluorescence intensity (MFI) of S. aureus-GFP-positive neutrophils increased with higher MOIs. The increase in MFI was due to phagocytosis of multiple bacteria per neutrophil as was confirmed by confocal imaging. Sequential phagocytosis of GFP- and mCherry-expressing S. aureus showed a non-random process, as S. aureus-GFP-positive neutrophils preferentially phagocytized S. aureus-mCherry.

Conclusion: All neutrophils were able to phagocytize S. aureus, but some were much more potent than others. Therefore, at physiologically relevant MOIs these potent phagocytizing neutrophils will outcompete the uptake of bacteria by less competent cells in a process we propose to name "competitive phagocytosis."

Keywords: PMN; Staphylococcus aureus; bacteria; granulocyte; innate immunity.

Figure 1

Active phagocytosis in only part of the neutrophils population. (A) Percentage of Staphylococcus aureus- green fluorescent protein (GFP)-positive neutrophils over time. The percentage of positive neutrophils is depicted when incubated with an MOI 1. A plateau is reached at 40 min. Data are presented as mean ± SEM [n = 17 (5 min), 15 (10 min), 17 (20 min), 13 (30 min), 29 (40 min), 10 (50 min), 12 (60 min)]. (B) Percentage of S. aureus-GFP-positive neutrophils with increasing MOI. The percentage of positive neutrophils in conditions with increasing MOI is depicted. An increase in the percentage of S. aureus-GFP-positive neutrophils as well as concurrent increase in mean fluorescence intensity (MFI) is observed (****P < 0.0001). Data are presented as mean ± SEM [n = 8 (MOI 1), 5 (MOI 2), 6 (MOI 5), 5 (MOI 7), 8 (MOI 10)]. Data were analyzed with one-way ANOVA. (C) Confocal image showing a neutrophil (cell membrane is visualized in red by staining of CD16-V500) with multiple intracellular bacteria (green, GFP), and two empty neutrophils.

Figure 2

Repeated phagocytosis experiment. Representative experiment for two individual experiments. (A) Phagocytosis of Staphylococcus aureus- green fluorescent protein (GFP) by neutrophils (5 × 10⁶/ml) was initiated with a MOI of 1 for 40 min. The GFP-negative cells were sorted and the sorted cells underwent the same procedure as the cells depicted in panel (A). (B) Sorted GFP-negative neutrophils were mixed with S. aureus-GFP for 40 min and analyzed again. A comparable distribution of S. aureus-GFP is seen in the formerly GFP-negative population.

Figure 3

Analysis of randomness in the interaction between Staphylococcus aureus- green fluorescent protein (GFP) and neutrophils. The experiment was performed with a MOI of 1 S. aureus-GFP in the first round (A) and with -mCherry in the second round (B). A representative experiment is shown. Ratios in the mCherry positive (/+) and negative cells (/−) were determined in the GFP-positive (+/) and negative (−/) (C) and MOI 1 and 2 (D) cells. (C) A relative enrichment of +/+ and −/− neutrophils is seen compared to the +/− and −/+ cells, which is not to be expected in a random process (***P = 0.0005). Differently colored dots represents results from the same experiments, different experiments can be executed with different MOIs. (D) An increase in mean fluorescence intensity can be observed in the mCherry positive group of the MOI 2 condition compared to a MOI 1 (ns, P = 0.125). Data were analyzed with the Wilcoxon test.

Figure 4

Analysis to test the feed-forward hypothesis. Neutrophils were first mixed with Staphylococcus aureus -Cherry with an MOI of 1 and a clear distribution is seen into positive and negative cells (A). (B) The same experiment was performed in cells from the same donor preincubated with S. aureus-GFP at an MOI of 10. All neutrophils were characterized in the first round by 100% phagocytosis all neutrophils become GFP-positive[panel (B)]. Adding S. aureus-mCherry with a MOI of 1 to these GFP-positive neutrophils resulted the same distribution pattern that was observed before adding S. aureus-GFP [see panel (A)]. Depicted are representative samples for three independent experiments.