Abstract
The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes. Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa. To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophore Mnemiopsis leidyi and the oyster Crassostrea gigas. Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage – possess immune-like cells capable of phagocytosis and ETosis. We demonstrate that both Mnemiopsis and Crassostrea immune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. We thus propose that ETosis is an evolutionarily conserved metazoan defense against pathogens.
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Introduction
Response to invading microbes is a primary physiological task for all metazoans. In vertebrates, specialized immune cell types such as macrophages and neutrophils detect the presence of microbes. These immune cells perform specific behaviors like phagocytosis or secretion of antimicrobial compounds to sequester and eliminate microbial invaders1,2. The release of extracellular DNA traps (ETs) is a relatively recently described immune cell behavior – a morphologically and molecularly distinct cell death process called ETosis – during which immune cells cast filamentous nets composed of nuclear chromatin material or mitochondrial DNA into the surrounding extracellular space, trapping and killing invading microbes3,4,5,6. Initially believed to be a behavior exclusive to vertebrate neutrophils, studies have highlighted ETosis as an anti-microbial response in non-vertebrate taxa7,8,9,10,11,12,13. Intriguingly, ETs have been reported as a mechanism of anti-microbial defense in social amoebas14 and some plant species15. Whether this cellular behavior has evolved independently in divergent lineages or represents a shared immune behavior in diverse eukaryotes is not clear.
In mammalian cells, ETosis and other immune responses are often initiated by pattern recognition receptors (PRRs) that bind to molecular motifs on microbes commonly referred to as pathogen-associated molecular patterns (PAMPs)16. PRR protein sequences and domain architectures can vary between taxa, as they evolve to detect host specific pathogens17,18,19. For example, mussels have a radiation of TLRs that may recognize a suite of specific pathogens20, while Drosophila uses Toll receptors to identify the protein Spätzle21. Despite these broad patterns of clade-specific immune receptor diversity, the signaling cascades and effectors downstream of PRRs have been shown to be well conserved22, including intracellular signaling pathways involving calcium, MAP kinases, and reactive oxygen species (ROS)23,24,25. In vertebrate leukocytes, exposure to cytokines, microbes, PAMPs, or pharmacological agents activate specific cellular pathways involved in ET formation, such as a response to ion imbalance (calcium, potassium) or reactive oxygen species (ROS) burst26,27. Although the detailed mechanisms of these pathways remain unknown in non-vertebrates6,8,26,28,29, genes involved in vertebrate immune cell signaling cascades have been identified in diverse metazoans, including non-bilaterians30,31,32,33,34.
Non-vertebrate ETosis has been best studied in molluscan model systems, particularly in bivalves like the model oyster Crassostrea gigas8,9,11,29,35,36. However, attempts to stimulate production of ETs in Crassostrea via exposure to some microbial signatures as well as ETosis-stimulating drugs has yielded variable results29. For example, robust ETotic response in oyster hemocytes was observed following challenge with Vibrio9; in contrast, induction of ETosis with PAMPs or other microbes has been either modest or not observed29. Without a consensus assessment of ETosis stimulation, the conservation of ET induction in non-vertebrates and whether ETosis represents a fundamental metazoan immune response remains unclear8,9,29.
Ctenophores, also known as “comb jellies,” are a clade of gelatinous planktonic marine animals that represent one of the earliest-branching metazoan lineages37,38,39,40. Ctenophores have two distinct germ layers (ectoderm and endomesoderm) separated by a jelly-like layer of collagenous mesoglea and lack a circulatory system. Understanding functional attributes of ctenophore physiological systems has provided fundamental insights into the conservation of animal cell types and signaling pathways, as well as revealing mechanisms for the emergence of evolutionary novelties32,41,42,43,44. Currently, the ctenophore immune system remains almost entirely undescribed32,45. Specific immune cell types have not been explicitly identified in ctenophores, though they possess motile amoebocyte-like cells that are abundant in the mesoglea and are capable of phagocytosis32,46. Whether ctenophores have additional cellular immune mechanisms has not been reported.
Here we demonstrate that the model ctenophore Mnemiopsis leidyi possesses immune-like cells capable of ETosis in response to diverse stimuli known to activate conserved signaling cascades in vertebrate immune cells. To rapidly and accurately quantify ETosis we developed an automated imaging pipeline to identify ETs. We applied this imaging pipeline to comparatively assess ET production in Mnemiopsis and the bivalve mollusk Crassostrea gigas, a model for invertebrate ETosis. Our data from both non-vertebrate species suggest that cells undergoing ETosis following a range of stimuli may represent evolutionarily conserved anti-microbial defense mechanisms in metazoans.
Results
Isolated ctenophore cells display immune-like behaviors
To investigate whether the model ctenophore Mnemiopsis leidyi possesses cell types capable of specialized immune functions, we mechanically disassociated whole Mnemiopsis and examined isolated cells47,48. We observed morphologically distinct motile cell types displaying prominent intracellular granules and/or vesicles, including amoebocyte-like cells with relatively short pseudopodia and stellate cells with long pseudopodia (Fig. 1A–C, Supp. Movie 1). Time-lapse microscopy shows that some stellate cells have a dynamic morphology and can rapidly vary the length and number of pseudopodia (Supp. Movie 2). These motile cells are highly active in primary cell cultures and their morphologies and scavenging behaviors are reminiscent of immune cell types, such as macrophage-like cells, that have been described in diverse metazoans1.
A major role for macrophage-like cells is host defense against microbes. We therefore assessed whether the motile cells present in Mnemiopsis exhibited fundamental immune cell behaviors in response to bacteria. Mnemiopsis cells were incubated with killed fluorescently labeled Escherichia coli and imaged by combined DIC and fluorescence microscopy. We found that Mnemiopsis cells exhibiting diverse morphologies internalized fluorescent E. coli32,49; these included motile stellate and amoeboid cells (Fig. 1D–F; Supp. Movie 3). Furthermore, timelapse DIC microscopy revealed that amoeboid cells actively maneuvered their pseudopodia to engulf nearby bacteria, supporting a potential role in sensing and removing microbes (Supp. Movie 4).
Following exposure to E. coli, a conspicuous subset of stellate cells changed their morphology dramatically by retracting their pseudopodial processes and undergoing nuclear rotation, followed by a rapid expulsion of cellular material (Fig. 1G; Supp. Movie 5). This behavior is remarkably similar to vertebrate monocyte behavior preceding extracellular DNA trap (ET) formation26,50. This led us to speculate that some ctenophore immune-like cells were producing ETs in response to the presence of microbes.
Ctenophore immune-like cells produce extracellular traps composed of chromatin
In vertebrates, ETs are released in response to recognition of microbial components, and protect against infection by immobilizing and killing invading microbes1. To test whether incubation with microbes promoted extrusion of DNA from Mnemiopsis cells, we stained isolated cells with Hoechst to label DNA. In isolated cells incubated in seawater media, we observed intact nuclei with concentrated Hoechst labeling (Fig. 2A). After treatment with heat-killed fluorescent E. coli we observed networks of Hoechst-stained DNA filaments cast in large areas around some individual Mnemiopsis cell bodies. Three-dimensional rendering of confocal z-stacks showed that Mnemiopsis cells extruding DNA had bacteria entangled in the DNA networks, consistent with ETs (Fig. 2B; Supp. Movie 651;).
In vertebrate immune cells, ETs can originate either from the cell nucleus or from mitochondria52,53. To assess the organellar origin of the extruded DNA, we stained Mnemiopsis cells with an antibody that recognizes an array of nuclear histone proteins (H1, H2A, H2B, H3, H4). Nuclei of non-ETotic Mnemiopsis cells were labeled with this histone antibody, which showed stereotypical patterns of concentrated DNA and histone labeling, confirming the specificity of this antibody in Mnemiopsis cells (Fig. 2C)54,55. Notably, in cells that have undergone ETosis, the extracellular ET DNA also stained with this pan-histone antibody, demonstrating that the extruded DNA filaments are composed of chromatin (Fig. 2C, arrows). These data show that some Mnemiopsis cells form extracellular net-like structures composed of chromatin that can ensnare bacteria6.
Ctenophore ETosis is an immune response to microbial exposure
To assess whether the formation of ETs in Mnemiopsis is a response to microbial challenge, we measured ET production after incubation of Mnemiopsis cells with E. coli and two additional microbial particles: heat-killed gram-positive S. aureus, and the yeast cell wall extract zymosan. We found that all three microbial particles stimulated ET formation in Mnemiopsis cells when compared with untreated cells incubated with media alone (Fig. 2D). Numbers of ETotic cells were manually quantified based on release of Hoechst-positive material by fluorescent microscopy (Fig. 2E). Exposure to S. aureus and zymosan promoted considerably higher rates of ET formation (mean increases of 98.2% and 117.7% over control respectively) than E. coli (43.7%) (Fig. 2E). These data demonstrate that production of ETs represents a cellular response to the presence of microbes in Mnemiopsis.
Ctenophore ETs form filamentous networks that capture microbes
Scanning electron microscopy (SEM) is commonly used to confirm the structure of ETs, and is informative in distinguishing ETosis from DNA release secondary to other forms of cell death such as necrosis or apoptosis56. To examine the structure of the extruded DNA in Mnemiopsis cell cultures, we performed SEM on isolated cells that had been incubated with S. aureus or with seawater media alone. Images of untreated Mnemiopsis cells confirm the presence of diverse morphological cell types observed by DIC, including stellate cells (Fig. 3A; Supp. Fig. 1). Following incubation with S. aureus, we observed long filamentous networks surrounding some Mnemiopsis cells (Fig. 3B, C; Supp. Fig. 1), which are similar to ETs produced by vertebrate and crustacean immune cells3,8,57,58. We also observed a close association between individual S. aureus and the filamentous nets produced by Mnemiopsis cells, providing further evidence that these structures are able to capture microbes, a critical function of ETs (Fig. 3B, C insets)59. Together, these data identify that some Mnemiopsis cells produce bona fide extracellular DNA traps.
PMA induces ETosis in Mnemiopsis
We hypothesized that ETosis in ctenophores may be initiated by engaging evolutionarily conserved signaling pathways known to precede ETosis in other metazoans. For example, phorbol 12-myristate 13-acetate (PMA) is commonly used to induce ET production in vertebrate immune cells through activation of NADPH-mediated ROS26. This pathway has been proposed to induce ET formation in immune cells in annelids and crustaceans8,10, suggesting that it may be evolutionarily ancient and well conserved in metazoans. We treated Mnemiopsis cells with PMA for four hours, stained DNA with Hoechst, and visualized cells by fluorescence microscopy. We observed the extrusion of DNA after PMA treatment, which was similar to ETs produced following micobial challenge (Figs. 2D and 4A).
Importantly, exposure to PMA can also trigger non-ETotic cell death pathways60, raising the possibility that the extracellular DNA we observed was a result of cell rupture and release of DNA secondary to cell death. To distinguish between these possible scenarios, we used a combination of two DNA dyes: Hoechst, which is a membrane-permeable dye that labels DNA in both live cells and dead cells, and SytoxGreen, a non-permeable cell stain that selectively labels DNA in cells with compromised cell membranes, as observed in cells undergoing cell death (Fig. 4A). Both DNA dyes label ETs8,61, and ETotic cells have diffuse gradients of both Hoechst and Sytox staining (Fig. 4A, B). We then developed a semi-automated imaging analysis approach that allowed us to measure Hoescht and SytoxGreen labeling in individual cell nuclei and accurately quantify both ETosis and cell death simultaneously (Fig. 4B; Supp. Fig. 2). ET production has been successfully identified and quantified in neutrophils using a combination of machine learning and high-throughput imaging62,63, and we deployed similar techniques to assess ETosis in ctenophore cells. An approach similar to those implemented in flow cytometry analyses was used in which we plotted the intensity of SytoxGreen fluorescence against Hoechst signal for each individual cell (Fig. 4B, C). This allowed for the identification of three distinct populations in PMA-treated Mnemiopsis cells: Hoechsthigh/ SytoxGreenlow live cells that show no DNA release and intact cell membranes; Hoechstlow/ SytoxGreenlow cells which display diffuse extracellular Hoechst and SytoxGreen fluorescence, representing ETotic cells8,61; and SytoxGreenhigh cells with a range of Hoescht staining, which are dead or dying non-ETotic cells.
To confirm that the image analysis pipeline accurately distinguishes non-ETotic cell death from ETosis, we induced death in Mnemiopsis cells by exposure to a moderate osmotic shock (dilute saline solution, 2X PBS). We measured a significant increase in non-ETotic dead cells compared with normal seawater media controls (Fig. 4C). Notably, dying cells were clearly separated from cells undergoing ETosis. Using this pipeline, we then quantified ETosis and cell death in cells treated with PMA. Confirming our initial observations by microscopy, PMA promoted significant induction of ETosis, with a mean increase of 57.2% over cells incubated with seawater media alone (Fig. 4C, D). We also observed a modest but statistically significant increase in non-ETotic cell death following PMA incubation (Fig. 4D).
We sought to establish that the automated image analysis pipeline could detect ETosis induced by microbial exposure. Because we observed clear ETs with SEM following S. aureus challenge, we then applied this automated imaging approach to the microbial stimulus. Samples that are incubated with microbes contain more background fluorescence due to Hoescht and Sytox labeling of S. aureus nuclear material; however, we observed a clear increase in ETosis following S. aureus challenge using automated imaging analysis, as well as an increase in non-ETotic cell death (Fig. 4C, D). Together, these data confirm that PMA induces ETosis in Mnemiopsis. Further, the semi-automated image analysis pipeline presented here can accurately detect and quantify invertebrate ETosis and distinguish ETosis from total cell death.
ETosis in Mnemiopsis is initiated by agonists that induce ion flux
In vertebrates, ET formation can be induced by membrane ion flux triggered by exposure to bacterial toxins that act as ionophores26,55,64. Because stimulation of ETosis via ion flux has limited reports in non-vertebrates27,29, we assessed whether well-characterized bacterially-derived ionophores nigericin (potassium ionophore) and calcimycin (A23187; calcium ionophore) could also elicit ETosis in Mnemiopsis cells. We incubated Mnemiopsis cells with the K+ ionophore nigericin and observed a mean increase of 367% over untreated cells (Fig. 5A, B). Incubation with the Ca2+ ionophore calcimycin resulted in a mean increase of 225% over untreated control cells (Fig. 5A, B). Furthermore, although incubation with either ionophore also significantly increased Mnemiopsis cell death, consistent with their effects in vertebrate cells65,66,67, our image analysis pipeline was able to clearly distinguish ETosis from other forms of cell death. These data therefore support evolutionarily conserved signalling pathways for ET formation in response to immune stimulation or cellular damage.
Microbes, PMA, and calcium ionophore stimulates ETosis in Crassostrea gigas hemocytes
Our observations that ET formation in Mnemiopsis can be triggered by similar signaling pathways to those involved in mouse and human immune cell ETosis raise the possibility that ETosis may represent an evolutionarily ancient response to infection or cell damage activated by intracellular signaling pathways that are widely conserved throughout animals68,69,70. However, stimulation of ETosis has not been widely assessed outside of vertebrates27,29, nor compared between phyla. To compare stimulation of Mnemiopsis cells with another non-vertebrate metazoan in which ETosis has been established, we extended our analysis to the bivalve mollusc, Crassostrea gigas, a marine non-vertebrate bilaterian with a circulatory system possessing specialized immune cells (hemocytes). Some hemocyte cell types have been shown to display immune behaviors such as phagocytosis71. We incubated isolated hemocytes with fluorescent E. coli for 4 h to assess whether C. gigas cells were responding to the presence of bacteria by phagocytosing the E. coli (Fig. 6A, B) or undergoing ETosis (Fig. 6B, inset). When observing hemocytes by confocal microscopy, we found that some hemocytes are highly phagocytic, with individual cells ingesting multiple bacteria (Fig. 6A). Fluorescent imaging also identified networks of extracellular DNA surrounding some hemocytes after exposure to microbial particles, similar to previous reports of bivalve ET formation9 (Fig. 6B, insets).
While ETs have been reported in multiple bivalve species, successful induction of ETosis in hemocytes following incubation with either microbial stimuli or pharmaceuticals has varied8,9,29. To directly compare ET production in oyster hemocytes with observations in Mnemiopsis, we assessed ETosis in C. gigas hemocytes using the same panel of microbes. We first confirmed that Crassostrea immune cells undergo ETosis in response to microbial stimulation using our automated imaging analysis pipeline (Fig. 6C, D). Hemocytes were incubated with heat-killed E. coli, S. aureus, or zymosan, and then assessed using the automated image analysis pipeline for quantification of ETosis. We found that ETosis increased after exposure to all three microbes, compared to hemocytes incubated in vitro with seawater media alone (Fig. 6C, D). Crassostrea hemocytes undergo ETosis in response to bacterial challenge with both gram-negative E. coli and gram-positive S. aureus with mean increases of 52.3% and 41.5% over untreated hemocytes, respectively (Fig. 6D). The fungal PAMP zymosan also elicited a significant ETotic response in oyster hemocytes, with a mean increase of 112% over seawater media controls. Interestingly, S. aureus stimulation did not stimulate significant increases in non-ETotic cell death in hemocytes (Fig. 6D), in contrast to what we observed in Mnemiopsis cells (Fig. 2D, E). Hemocyte total non-ETotic cell death increased significantly with either E. coli or zymosan exposure.
We also measured ETosis in Crassostrea hemocytes after four-hour incubations with PMA, nigericin, calcimycin, or seawater media controls (Fig. 6E, F). Exposure to PMA induced significant ET production in isolated Crassostrea hemocytes, with a relative mean increase of 95.7% (Fig. 6F). A large proportion of Crassostrea hemocytes also produced ETs after exposure to calcimycin, with a mean increase of 209% over controls (Fig. 6F). These results are similar to the robust response reported in prior studies of bivalve hemocytes to Ca2+ ionophore stimulation29. In contrast to Mnemiopsis cells, incubation of hemocytes with the K+ ionophore nigericin showed no significant induction of ETosis (compare Figs. 2D, E and 6F). Notably, non-ETotic cell death does not increase in C. gigas hemocytes following drug incubation. Unexpectedly, both PMA and calcimycin treatment resulted in a relatively minor, but statistically significant, decrease in non-ETotic cell death.
Discussion
Identifying core molecular mechanisms of immune cells and their evolutionary origins is critical for understanding the evolution of metazoan immune cell function and behavior. Here we report that ctenophores, which diverged very early from the metazoan stem lineage, have immune cells capable of ETosis, a cellular response to microbes first described in mammalinan neutrophils. We also show that cells isolated from the model ctenophore Mnemiopsis leidyi undergo ETosis following exposure to diverse microbial components, and stimulation of intracellular signaling cascades known to precede ETosis in vertebrates. For example, we find that incubation with the potassium ionophore nigericin stimulates ETosis in Mnemiopsis cells, as has been reported for mammalian neutrophils, suggesting that K+ efflux may be an evolutionarily conserved trigger for ETosis. Based on our results, we propose that ETosis represents a integral mechanism of cellular immune defense in metazoans (Fig. 7).
Since the first description of neutrophil extracellular traps1, multiple pathways involved in the generation of mammalian ETs have been identified26. However, the intracellular actors necessary for ET formation in non-vertebrate taxa are not well understood. For example, gene homologs for proteins known to be essential for ETosis in mammalian neutrophils, such as peptidylarginine deiminase 4 (PAD4), neutrophil elastase, myeloperoxidase (MPO) and pannexin-1 are absent outside of vertebrates. There is strong evidence for the presence of multiple ETosis pathways in non-vertebrates that can be activated via stimulation with microbial signatures, parasites, PMA, A23187, and UV light8,9,10,11,12,29. Potassium ion efflux, for example, is classically associated with cellular damage, including during bacterial infection72. Calcium flux has been implicated in ET formation in vertebrates either as a component of immune receptor-mediated signaling or as an activator of downstream effectors that drive DNA condensation preceding ETosis73. In addition to intracellular ion flux, components of vertebrate ETosis pathways, such as ERK, p38, or Akt, are foundational components of metazoan intracellular signaling68,69,70.
Our ability to make further direct comparisons of the molecular mechanisms of ETosis between vertebrates and Mnemiopsis is currently hampered by a lack of tractable forward genetic approaches and is therefore largely restricted to pharmacological perturbation. However, our data supports considerable apparent conservation of ET formation; firstly, bacterial and fungal components (E. coli, intact S. aureus and fungal particles) significantly stimulated ETosis in Mnemiopsis and Crassostrea hemocytes in our study. These results are in agreement with work in other non-vertebrates and supports the concept that ET formation is a fundamental component of metazoan anti-microbial defense8,9,10,11,12,29. We also demonstrate that ETosis in Mnemiopsis and Crassostrea is stimulated by PMA, which in mammalian cells stimulates protein kinase C and Ca2+ flux, and also by the Ca2+ ionophore A23187. These findings align with observations in vertebrate neutrophils26 supporting a role for Ca2+ transients in metazoan ETosis. Finally, the K+ ionophore nigericin promoted robust ETosis in Mnemiopsis, indicating additional conservation of K+ sensing and signaling pathways. Interestingly, Crassostrea hemocytes did not produce ETs in response to nigericin, raising the possibility that the nigericin-stimulated pathway is lost in this mollusc species. Signaling pathway inhibitors optimized for marine invertebrate cells will be useful for future studies of molecular actors involved in immune responses.
Within other non-bilaterian clades, ETosis has been reported in the sea anemone Actinia equina following stimulation with PMA8, but there are no reports assessing whether placozoans or poriferans have cells capable of ETosis (Supplemental Table 1). ETosis has now been reported in two non-bilaterian clades – Ctenophora and Cnidaria – as well as in ecdysozoans, spiralians, and deuterostomes; we thus propose that the most parsimonious hypothesis supports ETosis as a conserved immune behavior in metazoans. An alternative convergence hypothesis, invoking several independent emergences of this immune defense, would require a minuimum of two distinct origins within metazoans alone. Weighing past reports of ETosis across Metazoa as well as our findings of an additional occurrence of non-bilaterian ETosis, we do not feel that current data supports multiple origins for metazoan ET production. Futher, convergent acquisition of ETosis in ctenophores cannot be robustly assessed at present due to lack of data from Porifera and Placozoa. While the evolutionary history of Ctenophora has been a subject of recent debate37,38,39,40,74,75, ctenophores represent the earliest-diverging metazoan group in which ETosis has been reported.
In contrast to vertebrate immune cell nuclear morphologies that correspond with discrete immune cell functions (e.g., lobed nuclei in neutrophils and eosinophils76), no obvious differences in nuclear morphologies have been reported in either bivalve hemocytes or ctenophore immune-like cells. We did observe that Mnemiopsis cells that underwent ETosis in vitro after exposure to E. coli did not appear to have phagocytosed bacteria in large amounts (Supp. Movie 2). Intriguingly, we also observed other motile cells that had phagocytosed large amounts of bacteria without undergoing ETosis (Supp. Fig. 2, Supp. Movie 3; Supp. Movie 4). Our functional characterization of ETosis-competent Mnemiopsis cells suggests that future studies should assess whether ETosis-competent and highly phagocytic non-ETotic cells represent discrete immune cell types in ctenophores.
It remains unclear whether non-vertebrate immune cells are capable of vital (mitochondrial) ETosis, which involves production of ETs derived from mitochondrial DNA while maintaining cell viability13. Ctenophores have not previously had specific immune cell antimicrobial behaviors described beyond phagocytosis32,46. We demonstrate that Mnemiopsis leidyi possesses cells functionally competent for ETosis in response to a range of microbial challenges. Our data demonstrates that both Mnemiopsis leidyi and Crassostrea gigas immune cells can be stimulated to produce ETs with distinct pharmaceutical agents that, in vertebrate immune cells, differentially induce specific intracellular signaling pathways to activate ET production. The production of extracellular traps in Mnemiopsis suggests this specific immune cell antimicrobial defense behavior was likely present early in metazoan evolution, prior to the diversification of extant metazoan lineages.
Methods
Animal maintenance
This research complies with all relevant animal experimentation regulations. Laboratory cultures of Mnemiopsis leidyi were maintained as previously described44. Adult Mnemiopsis cells were isolated from whole animals following established protocols32,47,48,77. In brief, whole small adult Mnemiopsis (approximately 3 cm in length) were homogenized to a single-cell preparation using a loose-fitting dounce homogenizer. Cells were mixed with 0.22 µm filtered seawater (FSW) + 1% penicillin/streptomycin and filtered through a 70 µm sterile nylon mesh before plating. Crassostrea gigas were maintained under flowing seawater at approximately 13 °C and hemolymph was extracted from the adductor muscle with a syringe. Isolated cells from both taxa were maintained in vitro under sterile conditions in 0.22 µm filtered seawater (FSW) + 1% penicillin/streptomycin.
Scanning electron microscopy (SEM)
Mnemiopsis cells were seeded onto coverslips treated with poly-L-lysine. Cells were incubated with or without heat-killed E. coli for 4 h to induce ET formation. Cells were fixed in ½ strength Karnovsky’s fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3) overnight at 4 °C. Samples were then rinsed with 0.1 M cacodylate buffer and treated with 1% osmium tetroxide for 1 h. The samples were then dehydrated through a graded series of alcohols and critical point dried (Autosamdri, Tousimis Corp, Rockville, MD). Samples were mounted on stubs, sputter coated with gold/palladium (Denton Desk IV, Denton Vacuum, Moorestown, NJ) and imaged on a JSM 6610 LV scanning electron microscope at 5 kV (JEOL, Tokyo, Japan).
Stimulation of ETosis and imaging
For stimulation and quantification of ETosis, cells were isolated from 24 individual animals, plated in 96 well plates, and exposed to pHrodo-E. coli, Staphylococcus aureus, zymosan particles (Sigma Aldrich), 25μM nigericin (Thermo Fisher Scientific), 1 mg/mL PMA (Sigma Aldrich), or 4μM A23187 (Sigma). Each experimental condition for each animal was performed in triplicate. Live cell staining was performed following (2) and live imaging was performed using a JuLI Stage (NanoEntek). For immunofluorescence, ctenophore cells were prepared following established protocols47,48,78 and labeled with mouse anti-histone H11–4 (EMD Millipore, cat. MAB3422) at a dilution of 1:200 and goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific, cat. A28175) at a dilution of 1:500. This specific H11–4 histone antibody was selected because it recognizes histones H1, H2A/B, H3, and H4 proteins across eukaryotic species. Cells were imaged using ×60 objective and ×100 oil objective, on a Nikon Ti (Eclipse) inverted microscope with Ultraview Spinning Disc (CSU-X1) confocal scanner (Perkin Elmer). Images were captured with an Orca-ER Camera using Volocity (Quorum technologies). Post-acquisition analysis such as contrast adjustment, deconvolution through iterative restoration and colocalization were performed using Volocity software.
For quantification of ETosis and total cell death, cells were treated with Hoechst 33342 (Sigma Aldrich) and SytoxGreen (Invitrogen) for 20 min, before imaging at 20X on an automated imaging plate reader, Cytation 3 (Biotek, software Gen5 v4.2).
Automated image-based profiling
We analyzed approximately 28,000 total images using CellProfiler (v4.1.3). Image quality was assessed by calculating a focus score using two classes Otsu thresholding method, weighted variance on 20 × 20 pixel measurements. We calculated and applied an illumination correction for each fluorescent channel (SytoxGreen and Hoechst) using a background illumination function of 50 pixels block size, without smoothing. Each corrected image was then segmented using a global robust background method (0.05–50), with a smoothing scale of 1.3488 and a correction factor of 0.89. Clumped objects were identified and split by shape. For each segmented object we measured the number and intensity of pixels in each fluorescent channel. Each image and each segmented object, along with Metadata, were exported as csv files by experiment. R (v4.0.5) software with tidyverse (v1.3.1), dplyr (v1.0.7) and readr (v1.4.0) packages were then used to transform the datasets (https://github.com/carolinestefani/ETosis-and-death-automated-pipeline). Data from images and objects were merged, and measurements from individual images with a Focus Score <0.2 were removed from further analysis. This allowed us to identify and select only images that were in focus. Surface area, Hoechst intensity and SytoxGreen intensity per object (nucleus) and per individual animal were then imported into FlowJo (v10.8.0), and percentages of cells per delineated population (dead/dying cell, live cell, and ETotic cell) were calculated. Dying and ETotic cells were gated as indicated on the figures. Finally, percentages per individual animal surveyed were combined and tested for statistical significance using GraphPad Prism (v9.2.0). All statistical tests were performed using two-tailed unpaired student t-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Statistics and reproducability
No statistical method was used to predetermine sample size. No data were excluded from the analyses.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The imaging and statistical data generated in this study are provided in the Supplementary Information/Source Data file. Source data are provided with this paper.
Code availability
Detailed computational methods and associated code may be found at https://github.com/carolinestefani/ETosis-and-death-automated-pipeline
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Acknowledgements
The authors are grateful to Anna Bruchez and Rachel Prins for imaging support. This research was supported by the National Oceanographic and Atmospheric Administration, a National Research Council Postdoctoral Fellowship to LEV, and the National Science Foundation under Grant No. 2013692. The authors thank Steve Ziegler for technical and experimental support. The authors gratefully acknowledge the Fred Hutchinson Cancer Center Electron Microscopy Shared Resource (EMSR). The EMSR is supported in part by the Cancer Center Support Grant P30 CA015704-40.
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Conceptualization, C.S. and L.E.V.; Methodology, L.E.V., P.P.D., and C.S.; Formal Analysis, C.S. and L.E.V.; Investigation, L.E.V. and C.S.; Visualization, C.S., L.E.V., and W.E.B.; Writing – Original Draft, L.E.V. and C.S.; Writing – Review & Editing, C.S., A.L.H., W.E.B., F.W.G., and L.E.V.; Funding Acquisition, A.L.H., W.E.B., N.T.K., F.W.G., and L.E.V.; Resources, A.L.H., W.E.B., and F.W.G.; Supervision, A.L.H., W.E.B., and F.W.G.
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Vandepas, L.E., Stefani, C., Domeier, P.P. et al. Extracellular DNA traps in a ctenophore demonstrate immune cell behaviors in a non-bilaterian. Nat Commun 15, 2990 (2024). https://doi.org/10.1038/s41467-024-46807-6
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DOI: https://doi.org/10.1038/s41467-024-46807-6
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