Modulation of phagocytosis-induced cell death of human neutrophils by Neisseria gonorrhoeae

Abstract

Optimal innate immune response to infection includes eradication of potential pathogens, resolution of associated inflammation, and restitution of homeostasis. Phagocytosing human polymorphonuclear leukocytes (hPMN) undergo accelerated apoptosis, a process referred to as phagocytosis-induced cell death (PICD) and an early step in their clearance from inflammatory sites. Among human pathogens that modulate hPMN apoptosis, Neisseria gonorrhoeae delays PICD, which may contribute to the exuberant neutrophilic inflammation that characterizes gonorrhea. To elucidate the mechanisms underlying delayed PICD, we compared features of hPMN cell death that followed phagocytosis of N. gonorrhoeae FA1090 wild-type (GC) or serum-opsonized zymosan (OPZ), a prototypical stimulus of PICD. Phosphatidylserine externalization required NADPH oxidase activity after ingestion of GC or OPZ, and annexin V staining and DNA fragmentation were less after phagocytosis of GC compared to OPZ. Caspase 3/7 and caspase 9 activities after phagocytosis of GC were less than that seen after ingestion of OPZ, but caspase 8 activity was the same after ingestion of GC or OPZ. When hPMN sequentially ingested GC followed by OPZ, both caspase 3/7 and 9 activities were less than that seen after OPZ alone, and the inhibition was dose dependent for GC, suggesting that ingestion of GC actively inhibited PICD. Sequential phagocytosis did not block caspase 8 activity, mitochondrial depolarization, or annexin V/propidium iodide staining compared to responses of hPMN fed OPZ alone, despite inhibition of caspases 3/7 and 9. Taken together, these data suggest that active inhibition of the intrinsic pathway of apoptosis contributes to the delay in PICD after hPMN ingestion of N. gonorrhoeae.

Keywords: apoptosis; caspase 3; caspase 8; caspase 9; intrinsic pathway; mitochondrial depolarization.

Figure 1.. Delayed PICD of hPMN in suspension by GC

(A) PS exposure and plasma membrane integrity loss, markers of PICD, were quantitated with Annexin V-FITC and PI-PE staining, respectively. The increase in the percent of apoptotic hPMN (Annexin V⁺/PI⁻) after phagocytosis of GC (10:1 MOI) was significantly less than that after ingestion of OPZ (10:1 MOI), both at 2 and 6 hours, as was the percent of Annexin V⁺/PI⁺ hPMN. (B) DNA fragmentation, another marker of PICD, was decreased in hPMN fed GC (10:1 MOI) compared to that of OPZ (10:1 MOI) at 6 hours. Bars represent the average ± SEM from at least three independent experiments. p-values were determined using a two-way ANOVA and Turkey’s posttest (** p < 0.005; * p < 0.05).

Figure 2.. NADPH oxidase activity and hPMN cell death

(A) The NADPH oxidase activity of hPMN was assessed by monitoring luminol-enhanced chemiluminescence of stimulated cells. The soluble agonist PMA was used as a positive control for oxidase activity. Both OPZ (10:1 MOI) and GC triggered oxidant production and the response to GC was dose dependent. (B) At 2 hours after phagocytosis in DPI-treated hPMN, the percent of Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells in OPZ-fed hPMN were less than those fed OPZ without DPI treatment. Although DPI treatment did not significantly reduce Annexin or PI positivity of hPMN fed GC at 10:1 MOI, reduction in Annexin V⁺/PI⁺ cells was statistically significant at 50:1 MOI GC. Bars represent the average ± SEM from three independent experiments. p-values were determined using a two-way ANOVA and Turkey’s posttest (* p < 0.05). ¹ denotes statistical significance in Annexin V⁺/PI⁺ population, and not in Annexin V⁺/PI⁻.

Figure 3.. Caspase 3/7 activity in hPMN after phagocytosis

(A) At 6 hours after phagocytosis, the activity of caspase 3/7 in hPMN fed GC was decreased compared to that of hPMN fed OPZ as well as to hPMN in buffer alone. (B) hPMN sequentially fed GC followed by OPZ had a GC dose-dependent decrease in caspase 3/7 activity compared to that of hPMN fed OPZ alone. Bars represent the average ± SEM from three independent experiments. p-values were determined using a one-way ANOVA and Turkey’s posttest (* p < 0.05).

Figure 4.. Caspase 8 and 9 activities in hPMN after phagocytosis

(A) Caspase 8 activity in hPMN fed OPZ, GC or both (sequentially, GC followed by OPZ) was greater than that of hPMN in buffer alone. (B) In contrast, activity of caspase 9 in hPMN fed GC was lower than that of OPZ-laden hPMN or hPMN in buffer alone. Sequential ingestion of GC followed by OPZ triggered less caspase 9 activity in hPMN than did ingestion of OPZ alone. Bars represent the average ± SEM from three independent experiments. p-values were determined using a one-way ANOVA and Turkey’s posttest (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Figure 5.. Extracellular superoxide anion generation in hPMN after phagocytosis

Extracellular superoxide anion generated by hPMN was measured as SOD-inhibitable reduction of ferricytochrome C. GC (50:1 MOI) triggered little extracellular superoxide production by hPMN, and sequential phagocytosis of GC then OPZ did not decrease the amount of extracellular superoxide anion produced by hPMN. Bars represent the average ± SEM from three independent experiments.

Figure 6.. Effect of NADPH oxidase on caspase 3/7 activity in hPMN

hPMN in the absence or presence of DPI were stimulated with GC, OPZ or GC followed by OPZ, and caspase 3/7 activity was measured 6 hours after phagocytosis. (A) Inhibition of NADPH oxidase activity with DPI increased caspase 3/7 activity in hPMN independent of the stimulus and even in buffer alone, with the exception of hPMN fed GC at 50:1 MOI. (B) hPMN fed sequentially GC and OPZ had less caspase 3/7 activity than did hPMN fed OPZ alone. The inhibition was in a GC dose-dependent manner and the same in the absence or presence of DPI. Bars represent the average ± SEM from three independent experiments. p-values were determined using a two-way ANOVA and Sidak’s posttest (**** p < 0.0001; *** p < 0.001; ** p < 0.01).

Figure 7.. Mitochondrial depolarization in hPMN after phagocytosis

Mitochondrial depolarization was measured fluorometrically after staining with JC-1. At 1 hour after phagocytosis, mitochondrial depolarization in hPMN fed GC at 10:1 MOI was decreased compared to that of OPZ. Phagocytosis of GC at higher MOI led to increased mitochondrial depolarization in hPMN in a MOI-dependent manner and sequential phagocytosis of GC followed by OPZ did not decrease mitochondrial depolarization of hPMN compared to that in OPZ alone. Bars represent the average ± SEM from three independent experiments. p-values were determined using a one-way ANOVA and Turkey’s posttest (** p < 0.01; * p < 0.05).

Figure 8.. Effect of sequential phagocytosis of GC followed by OPZ on hPMN PICD

hPMN PICD 2 hours after phagocytosis was assessed as PS externalization and plasma membrane permeabilization, measured with Annexin V and PI, respectively. The percent of Annexin V⁺/PI⁻ hPMN in buffer alone was similar to that after phagocytosis of GC at 10:1 MOI, but was less than that of hPMN fed GC at 50:1 MOI (inset A) Sequential ingestion of GC, either at 10:1 or 50:1 MOI, followed by OPZ led to fewer Annexin V⁺/PI⁻ cells than when hPMN were fed GC at 50:1 alone but similar to after ingestion of OPZ alone (inset A). Similar to the previous data, hPMN fed OPZ had more late-stage PICD cells (Annexin V⁺/PI⁺) than did hPMN in buffer alone or hPMN fed GC at 10:1 MOI (inset B). The percent of Annexin V⁺/PI⁺ cells was greater after sequential ingestion of GC (50:1) and OPZ (10:1) than in hPMN fed OPZ or GC alone (inset B). hPMN fed GC at 10:1 MOI followed by OPZ had a percent of Annexin V⁺/PI⁺ hPMN similar to that after ingestion of OPZ alone but higher than hPMN that ingested GC alone (inset B). Bars represent the average ± SEM from at least three independent experiments. p-values were determined using a one-way ANOVA and Turkey’s posttest (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).