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. 2019 May 24;63(6):e02409-18.
doi: 10.1128/AAC.02409-18. Print 2019 Jun.

Antifungal Drugs Influence Neutrophil Effector Functions

Frederic Ries  1 , Astrid Alflen  1 , Pamela Aranda Lopez  1 , Hendrik Beckert  2 , Matthias Theobald  1 , Hansjörg Schild  3 , Daniel Teschner  4 , Markus Philipp Radsak  4
Affiliations

Antifungal Drugs Influence Neutrophil Effector Functions

Frederic Ries et al. Antimicrob Agents Chemother. .

Abstract

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) release was measured by enzyme-linked immunosorbent assay. POS led to enhanced activation, degranulation, and generation of ROS, whereas IL-8 release was reduced. In contrast, ISA-pretreated PMN showed decreased activation signaling, impaired degranulation, and lower generation of ROS. MFG caused enhanced expression of activation markers but impaired degranulation, phagocytosis, generation of ROS, and IL-8 release. CAS showed increased phagocytosis, whereas degranulation and generation of ROS were reduced. AMB led to activation of almost all effector functions besides impaired phagocytosis, whereas LAMB did not alter any effector functions. Independent from class, antifungal agents show variable influence on neutrophil effector functions in vitro Whether this is clinically relevant needs to be clarified.

Keywords: IL-8; antifungal agents; degranulation; neutrophil effector functions; oxidative burst; phagocytosis; polymorphonuclear leukocytes.

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Figures

FIG 1
FIG 1
Posaconazole stimulates neutrophil effector functions. Isolated PMN were pretreated with POS and were analyzed for CD62L shedding (n = 3) (A), CD11b expression (n = 3) (B), CD66b expression (n = 3) (C), ROS release (n = 3) (D), phagocytosis [n (medium, zymosan) = 4, n (LPS) = 3] (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
Isavuconazole impairs PMN generation of ROS. Isolated PMN were pretreated with ISA and were analyzed for CD62L shedding (n = 4) (A), CD11b expression (n = 4) (B), CD66b expression (n = 4) (C), ROS release (n = 3) (D), phagocytosis (n = 3) (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01.
FIG 3
FIG 3
Caspofungin shows variable modifications on PMN effector functions. Isolated PMN were pretreated with CAS and were analyzed for CD62L shedding (n = 4) (A), CD11b expression (n = 4) (B), CD66b expression (n = 4) (C), ROS release (n = 3) (D), phagocytosis (n = 4) (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01.
FIG 4
FIG 4
Micafungin influences PMN effector functions in different ways. Isolated PMN were pretreated with MFG and were analyzed for CD62L shedding (n = 3) (A), CD11b expression (n = 3) (B), CD66b expression (n = 3) (C), ROS release (n = 3) (D), phagocytosis (n = 3) (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
Conventional amphotericin B stimulates neutrophil effector functions. Isolated PMN were pretreated with AMB and were analyzed for CD62L shedding (n = 3) (A), CD11b expression (n = 3) (B), CD66b expression (n = 3) (C), ROS release (n = 3) (D), phagocytosis (n = 3) (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
FIG 6
Liposomal amphotericin B has no significant impact on PMN abilities. Isolated PMN were pretreated with LAMB and were analyzed for CD62L shedding [n (medium, zymosan) = 5, n (LPS) = 4] (A), CD11b expression [n (medium, zymosan) = 5, n (LPS) = 4] (B), CD66b expression [n (medium, zymosan) = 5, n (LPS) = 4] (C), ROS release (n = 4) (D), phagocytosis (n = 5) (E), and IL-8 release (n = 3) (F). Data are shown as means ± SEM. *, P < 0.05.
FIG 7
FIG 7
Gating strategy to analyze activation, degranulation, and phagocytosis by flow cytometry. (A to C) Viable leukocytes were identified by FSC and SSC and PI staining. (D) Subsequently, PMN were characterized as CD11b and CD66b double-positive cells. (D) Activation was quantified by mean fluorescence intensity (MFI) of CD11b-positive PMN and percentage of L-selectin (CD62L)-negative cells. (F to H) Degranulation was measured by quantifying CD66b expression. Phagocytic activity was quantified by identifying the percentage of PE-positive PMN.
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