Neutrophils From Patients With Invasive Candidiasis Are Inhibited by Candida albicans Biofilms

Abstract

Invasive candidiasis frequently involves medical device placement. On the surfaces of these devices, Candida can form biofilms and proliferate in adherent layers of fungal cells surrounded by a protective extracellular matrix. Due in part to this extracellular matrix, biofilms resist host defenses and antifungal drugs. Previous work (using neutrophils from healthy donors) found that one mechanism employed to resist host defenses involves the inhibition of neutrophil extracellular traps (NET) formation. NETs contain nuclear DNA, as well as antimicrobial proteins that can ensnare pathogens too large or aggregated to be effectively killed by phagocytosis. Given that these neutrophil structures are anticipated to have activity against the large aggregates of C. albicans biofilms, understanding the role of this inhibition in patients could provide insight into new treatment strategies. However, prior work has not included patients. Here, we examine NET formation by neutrophils collected from patients with invasive candidiasis. When compared to neutrophils from healthy participants, we show that patient neutrophils exhibit a heightened background level of NET release and respond to a positive stimulus by producing 100% more NETs. However, despite these physiologic differences, patient neutrophil responses to C. albicans were similar to healthy neutrophils. For both groups, planktonic cells induce strong NET release and biofilms inhibit NET formation. These results show that a mechanism of immune evasion for fungal biofilms translates to the clinical setting.

Keywords: Candida; biofilm; invasive candidiasis; neutrophil; neutrophil extracellular trap; patients; reactive oxygen species.

Figure 1

Comparison of PMA-induced NET formation and ROS production by neutrophils from healthy participants and patients with invasive candidiasis. (A) Scanning electron microscopy of patient and healthy neutrophils in the absence and presence of the NET stimulus PMA for 4 h. Measurement bars represent 10 µm and 1 µm for images taken at 2000X and 10,000X, respectively. (B) Patient and healthy neutrophils were incubated with or without PMA for 4 h and NET release was estimated by free DNA measurement following staining with cell-impermeable Sytox Green. (C) Neutrophils were incubated with or without PMA for 4 h, fixed, and stained with anti-neutrophil elastase rabbit primary antibody and chicken anti-rabbit IgG, DyLight 594 conjugated secondary antibody. (D) To measure ROS, neutrophils were pre-incubated with free radical sensor CM-H2DCFDA prior to incubation with or without PMA for 4 h, and fluorescence was measured. Statistical significance was analyzed by two-way ANOVA with Sidak’s multiple comparisons test, *p < 0.05, NS, not significant, mean with 95% confidence interval shown.

Figure 2

Response to planktonic and biofilm C. albicans for neutrophils from healthy participants and patients with invasive candidiasis. (A) Scanning electron microscopy of patient and healthy neutrophils co-incubated with planktonic or biofilm C. albicans for 4 h. Measurement bars represent 10 µm and 1 µm for images taken at 2,000× and 10,000×, respectively. (B) Patient and healthy neutrophils were co-incubated with planktonic or biofilm C. albicans for 4 h, and NETs were estimated by measurement of free DNA using Sytox Green. (C) Patient and healthy neutrophils were incubated with C. albicans planktonic cells or biofilm for 4 h, fixed, and stained with anti-neutrophil elastase rabbit primary antibody and chicken anti-rabbit IgG, DyLight 594 conjugated secondary antibody. Following fixation, neutrophils were stained for neutrophil elastase. (D) To measure ROS, neutrophils were pre-incubated with free radical sensor CM-H2DCFDA prior to incubation with planktonic or biofilm C. albicans for 4 h, and fluorescence was measured. Statistical significance was analyzed by two-way ANOVA with Sidak’s multiple comparisons test, *p < 0.05, NS, not significant, mean with 95% confidence interval shown.

Figure 3

C. albicans biofilms inhibit PMA-induced activation of neutrophils collected from patients with invasive candidiasis. (A) Patient neutrophils were co-incubated with C. albicans biofilm in the absence or presence of PMA for 4 h and NET formation was estimated by measurement of free DNA using Sytox Green. (B) To measure ROS, neutrophils were pre-incubated with free radical sensor CM-H2DCFDA prior to incubation with C. albicans biofilm in the presence or absence of PMA for 4 h, and fluorescence was measured. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, NS, not significant, mean with 95% confidence interval shown.