Release of chromatin extracellular traps by phagocytes of Atlantic salmon, Salmo salar (Linnaeus, 1758)

Abstract

Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5 μg/ml calcium ionophore (CaI) for 1 h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3 h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (p < 0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5 μg/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases.

Keywords: ETosis; Macrophage; NETosis; Neutrophil extracellular traps; Polymorphonucleocyte.

Graphical abstract

Fig. 1

Neutrophil-like cells isolated from Atlantic salmon head kidney spontaneously released structures resembling extracellular traps (ETs). The neutrophil-enriched cell fraction collected at the interface of the 1.060 and 1.072 g/ml Percoll layers was non-adherent in culture and characterised by cytology. a. Representative cytological spin slide of the isolated cells stained with Rapi-Diff II. Cells presented a characteristic eosinophilic polymorphonuclear morphology, with the nucleus divided into several lobes, and granulocytic cytoplasm; scale bar, 50 μm. b. Magnification of an optical field showing mononuclear cells infiltrating the cytology sample, with arrows indicating cells with mononuclear morphology; scale bar, 50 μm. c. Bar chart showing percentages of the different myeloid leukocyte subsets in the isolated cell population (mean ± SEM, n = 6 fish). Polymorphonuclear cells (PMNs; i.e., neutrophils) were the predominant subset (>70%) in the isolated population, followed by monocyte/macrophages (Mn/Mɸ). d. Correlation between the mass of each fish and yield of PMNs (mean percentage in each cell preparation) obtained from individual fish (r_s = 0.4824, p (two-tailed) = 0.0052, n = 32). e. Fluorescence microscopy image of neutrophil-enriched cell fraction stained with 5 μM Sytox Green, showing the spontaneous release of ETs in vitro after incubation (1 h, 15°C); scale bar, 100 μm. f. Association between the mass of each fish and the spontaneous release of ETs as measured by fluorescence after incubation for 1 h at 15°C (log₁₀ values used due to heteroscedasticity and non-linear decay shape to curve); r = −0.3814, p (two-tailed) = 0.0452, n = 28. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Nucleic acid structure of the extracellular trap (ET)-like structures confirmed by enzymatic digestion with DNase I. a−b. Fluorescence microscopy images of neutrophil-enriched cell suspensions from Atlantic salmon cultured in vitro and stained with 5 μM Sytox Green. a. After settling (30 min, 15 °C), control cells were incubated with RPMI-1640 culture medium for 30 min; scale bar, 100 μm. b. The nucleic acid nature of the structures was confirmed by degradation with medium containing 200 U/ml DNase I for 30 min; scale bar, 100 μm. c. Bar chart of fluorescence (mean ± SEM) of neutrophil-enriched cell suspensions incubated with culture medium lacking or supplemented with 200 U/ml DNase I for 30 min and stained with 5 μM Sytox Green; ∗ indicates a significant difference from the untreated control (t = 3.86046, p = 0.0048, n = 5). d. Bar chart showing the percentage of ETotic cells (mean ± SEM) in the neutrophil-enriched cell suspension following incubation with culture medium lacking or supplemented with 200 U/ml DNase I for 30 min; ∗ indicates a significant difference from the untreated control (t = 3.8871, p = 0.0177, n = 3; percentage data were arcsine transformed before statistical testing). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effects of previously characterised stimulants on extracellular traps (ETs) released from neutrophil-enriched cell suspensions from Atlantic salmon. a. Bar chart showing fluorescence (mean ± SEM) of neutrophil-enriched cell suspensions after incubation (1 h, 15 °C) with various proposed inducers and inhibitors of ETosis and stained with 5 μM Sytox Green showing cultures exposed to calcium ionophore (CaI) and LPS had significantly greater fluorescence compared to the untreated control (indicated by ∗; CaI: t = −5.3387, p = 0.0007, n = 5; lipopolysaccharide [LPS]: t = −3.9831, p = 0.0040, n = 5), which was not the case for phorbol 12-myristate 13-acetate (PMA) and diphenyleneiodonium chloride (DPI) (PMA: t = −1.7787, p = 0.1132, n = 5; DPI: t = −1.8394, p = 0.1031, n = 5), whilst exposure to polyinosinic–polycytidylic acid sodium salt (Poly I:C) yielded a significant reduction in fluorescence (t = 4.2372, p = 0.0028, n = 5). b−f. Fluorescence microscopy images of neutrophil-enriched cell suspensions incubated with different compounds (1 h, 15 °C); scale bars, 100 μm. b. Untreated controls. c. Incubation with 5 μg/ml CaI. d. Incubation with 10 nM PMA. e. Incubation with 50 μg/ml LPS. f. Incubation with 10 μM DPI. Note that the Poly I:C treatment image resembled closely the untreated controls (not shown). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Confirmation of decoration of the extracellular trap (ET)-like structures with characteristic protein signatures by immunostaining. Fluorescence microscopy images of neutrophil-enriched cell suspensions from Atlantic salmon cultured in vitro with 5 μg/ml CaI (a–f) or 10 nM PMA (g–h) for 1 h at 15 °C. a–c. Immunocytochemical detection of neutrophil elastase in ETotic neutrophils; scale bars, 50 μm. a. Localisation of neutrophil elastase by rabbit to human neutrophil elastase and stained with conjugated Alexa Fluor 488 (green). b. DNA stained blue with 4′,6-diamidino-2-phenylindole (DAPI). c. Merge of a–b. d–f. Immunocytochemical detection of histone H2A in different ETotic neutrophils; scale bars = 100 μm. d. Localisation of histone H2A by mouse to human histone H2A and stained with conjugated Alexa Fluor 488. e. DNA stained with DAPI. f. Merge of d–e. g–h. Immunocytochemical detection of histone H1 with DNA in an extended extracellular strand interlinking two cells; scale bars, 100 μm. g. Localisation of H1 with DNA by mouse to human histone H1/DNA and stained with conjugated Alexa Fluor 488. h. DNA stained with DAPI. i. Merge of g–h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Aeromonas salmonicida induced extracellular trap release in neutrophil-enriched cell suspensions from Atlantic salmon. After settling (30 min, 15 °C), neutrophil-enriched cell suspensions were incubated with A. salmonicida 30411 or Hooke at a multiplicity of infection of ca. 100 colony-forming units for 2 h at 22 °C. a. Bar chart showing fluorescence (mean ± SEM) of neutrophil-enriched cell suspensions incubated with bacteria and stained with 5 μM Sytox Green showing cultures exposed to A. salmonicida Hooke and 30411 had significantly greater fluorescence compared to the untreated control (indicated by ∗; Hooke: t = 7.1644, p = 0.0000, n = 6; 30411: t = 8.8521, p = 0.0000, n = 6). b−d. Fluorescence microscopy images of neutrophil-enriched cell suspensions after staining with 5 μM Sytox Green; scale bars, 100 μm. d. Untreated control neutrophil-enriched cell suspensions contained few ETs. c. Extracellular chromatin was extruded by neutrophils after incubation with A. salmonicida Hooke. d. Extracellular chromatin was extruded by neutrophils after incubation with A. salmonicida 30411. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Interaction between bacteria and extracellular traps (ETs). Neutrophil-enriched cell suspensions from Atlantic salmon were incubated with 5 μg/ml calcium ionophore (1 h, 15 °C) to induce ET release and then Aeromonas salmonicida Hooke was added at a multiplicity of infection of ca. 100 colony-forming units (CFU). a. Bar chart showing CFU (mean ± SEM) recovered from the wells containing ETs (No DNase I) or that had been digested away with 200 U/ml DNase I for 30 min (With DNase I) showing there was no significant change in CFU/ml for either treatment at 3 h when accounting for multiple comparisons (No DNase I: t = 2.6311, p = 0.0301, n = 5; With DNase I: t = −0.2414, p = 0.8153, n = 5). b–d. Neutrophil-enriched cell suspensions were incubated with A. salmonicida Hooke for 2 h and then their interaction was visualised by fluorescence microscopy; scale bars, 100 μm. b. DNA was stained with 5 μM Sytox Green. c. A. salmonicida Hooke was stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI). d. Merge of b–c. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Effects of previously characterised stimulants on extracellular traps (ETs) released from macrophage-enriched cell suspensions from Atlantic salmon.a. Light microscopy image of cytospin slide of isolated mononuclear cells stained with Rapi-Diff II; scale bar, 20 μm. b. Bar chart showing the percentage of monocyte/macrophages (Mn/Mɸ) in the isolated cell population (mean ± SEM, n = 9). c. Bar chart showing fluorescence (mean ± SEM) of macrophage-enriched cell suspensions after incubation (1 h, 15 °C) with various proposed inducers and inhibitors of ETosis and stained with 5 μM Sytox Green showing cultures exposed to calcium ionophore (CaI) had significantly greater fluorescence compared to the untreated control (indicated by ∗; CaI: t = −7.0314, p = 0.0001, n = 5), which was not the case for the other compounds (phorbol 12-myristate 13-acetate [PMA]: t = −0.1033, p = 0.9202, n = 5; lipopolysaccharide [LPS]: t = −0.1841, p = 0.8585, n = 5; diphenyleneiodonium chloride [DPI]: t = 0.0819, p = 0.9367, n = 5). d–h. Fluorescence microscopy images of macrophage-enriched cell suspensions incubated with different compounds (1 h, 15 °C); scale bars, 100 μm. d. Untreated controls spontaneously released a low abundance of ETs. e. Incubation with 5 μg/ml CaI. f. Incubation with 10 nM PMA. g. Incubation with 50 μg/ml LPS. h. Incubation with 10 μM DPI. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)