Human neutrophils dump Candida glabrata after intracellular killing

Accepted Manuscript Video article Human Neutrophils dump Candida glabrata after intracellular killing Fabian Essig, Kerstin Hünniger, Stefanie Dietrich, Marc Thilo Figge, Oliver Kurzai PII: DOI: Reference:

S1087-1845(15)30032-3 http://dx.doi.org/10.1016/j.fgb.2015.09.008 YFGBI 2899

To appear in:

Fungal Genetics and Biology

Received Date: Revised Date: Accepted Date:

3 July 2015 27 August 2015 15 September 2015

Please cite this article as: Essig, F., Hünniger, K., Dietrich, S., Figge, M.T., Kurzai, O., Human Neutrophils dump Candida glabrata after intracellular killing, Fungal Genetics and Biology (2015), doi: http://dx.doi.org/10.1016/ j.fgb.2015.09.008

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VIDEO ARTICLE - RESEARCH

Title: Human Neutrophils dump Candida glabrata after intracellular killing

Authors: Fabian Essig1, 2, Kerstin Hünniger1, Stefanie Dietrich3,4, Marc Thilo Figge2,3,4, Oliver Kurzai1,2,5*

Affiliations: 1

Septomics Research Center, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology – Hans-Knoell-Institute, Jena, Germany

2

Center for Sepsis Control and Care, University Hospital Jena, Jena, Germany

3

Applied Systems Biology, Leibniz Institute for Natural Product Research and Infection Biology – Hans-KnoellInstitute, Jena, Germany

4 5

Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Jena, Germany German National Reference Center for Invasive Fungal Infections, Hans-Knoell-Institute, Jena, Germany,

*Correspondent Footnote: Oliver Kurzai Septomics Research Center Friedrich Schiller University Jena and Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knoell-Institute Albert-Einstein-Str. 10 07745 Jena [email protected] 1

Abstract Interaction between fungal pathogens and human phagocytes can lead to remarkably variable outcomes, ranging from intracellular killing to prolonged survival and replication of the pathogen in the host cell. Using live cell imaging we observed primary human neutrophils that release phagocytosed Candida glabrata yeast cells after intracellular killing. This process, for which we propose the name “dumping”, adds a new outcome to phagocyte – fungus interaction which may be of potential immunological importance as it allows professional antigen presenting cells to take up and process neutrophil-inactivated pathogens that in their viable state are able to evade intracellular degradation in these cells.

Keywords: Candida glabrata, Neutrophils, Phagocytosis

Highlights:  Human neutrophils can dump Candida glabrata after intracellular killing  Providing killed fungal cells to monocytes may avoid lysosome maturation block and intracellular replication and allow induction of T cell responses 2

Introduction Among Candida spp., Candida glabrata is the second most common pathogen after Candida albicans. Both species are phylogenetically distinct and use different strategies to evade from or adapt to attacks of the human immune system. C. albicans mainly relies on morphological plasticity, resulting in lysis of host cells and fungal escape (Kumamoto et al., 2005, Kurzai et al., 2005; Martin et al., 2013; Vylkova et al., 2014; Duggan et al., 2015). In contrast, the monomorphic yeast C. glabrata is capable of intracellular replication in monocytic cells (Kaur et al., 2007, Seider et al., 2011). Live cell imaging, especially in combination with automated image analysis, has become an important tool for studying host-pathogen interaction (Brandes et al., 2014, Medyukhina et al., 2015). We have used live cell imaging to show that C. albicans and C. glabrata are differentially recognized by human polymorphonuclear neutrophils (PMN). PMN are less effective in taking up C. glabrata and enhance recruitment und uptake of C. glabrata by monocytes (Duggan, Essig et al., 2015). Apart from undergoing intracellular killing or replicating inside the host cell, fungi have been shown to be actively expulsed from macrophages with both fungus and host cell remaining viable (Alvarez et al., 2006; Ma et al., 2006). For Cryptococcus neoformans, this process, termed vomocytosis (Chayakulkeeree et al. 2011), can occur in a significant proportion of macrophage interactions in vitro and has also been observed in vivo (Nicola et al., 2011). A similar process in which even filamentous forms are released after phagocytosis has been described for C. albicans (Bain et al., 2012). However, in contrast to vomocytosis of C. neoformans, expulsion of C. albicans filaments occurs only in rare instances in vitro (<1% of interactions according to Bain et al., 2012) and has not yet been observed in vivo. Here, we report that PMN can also release fungal cells, in this case C. glabrata, after phagocytosis and intracellular killing. This process adds a new outcome to fungus – immune cell interaction, which is of potential relevance for initiation of adaptive immunity. To describe the release of intracellularly killed microorganisms by PMN we propose the name “dumping”.

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Results and Discussion Interaction of C. glabrata with human PMN was analysed in time-lapse live cell microscopy as previously described (Duggan, Essig et al., 2015). In accordance with our previous data, the movies clearly show that touching of C. glabrata cells frequently does not result in phagocytosis. In some cases repeated physical contact without initiation of phagocytosis can occur between a yeast cell and a PMN. Upon further analysis of the live cell imaging data, we noticed that in almost all experiments PMN could be detected that expulsed killed yeast cells. Video 1 shows an example of a PMN taking up a viable C. glabrata yeast cell (at time point 0 sec). After intracellularly carrying it for some time, the yeast cell is killed as indicated by turning positive for red PI staining in the video (at time point 6 sec). Immediately afterwards, the process of expulsion is started and after a short dragging, the killed yeast cell is released completely (at time point 7 sec).

Video 1. Dumping of an intracellularly killed C. glabrata cell by a human PMN (snapshot at 7 sec)

We propose the phrase “dumping” to describe the expulsion of killed yeasts/microorganisms by intact human immune cells and to discriminate this process from host cell lysis (lytic escape) and the release of viable pathogens by intact host cells (vomocytosis). To systematically analyze the occurrence of dumping, we evaluated 6 live cell imaging sequences each covering 2 h of co-incubation between human PMN and C. glabrata yeasts at ratios of either 1:1 or 1:5 with cells of different donors. In total, 152 phagocytosis events could be observed in these videos (homogenously distributed over all videos: 25.33 ± 21.16 per video), of which 78 (51%) resulted in killing of the yeast cell within the observation period. 4

Killing frequency in the different videos ranged from 20-64% (average: 45 ± 16% of phagocytosis events). Dumping was observed in 12 cases (8% of all phagocytosis events). Therefore, up to 15% of all intracellular killing events resulted in subsequent dumping. Dumping of killed C. glabrata cells was independent of the individual PMN donor and could be observed for 5 of 6 analyzed videos. In the remaining case, our live cell imaging video sequence covered only 9 phagocytosis events, of which 4 resulted in intracellular killing throughout the observation period. Therefore it seems likely that we failed to observe dumping due to the low number of host-fungal interactions and not due to the absence of dumping in the interaction of this donor’s PMN with C. glabrata. Importantly, dumping of phagocytosed C. glabrata occurs for killed yeast cells. Video 2 shows a PMN that takes up two C. glabrata cells (at time point 15 sec.). After intracellular killing of one C. glabrata, only the killed yeast cell but not the viable yeast cell is dumped (at time point 22 sec). The other yeast cell is killed later. Similar events of selective dumping of dead cells with viable fungi remaining inside the same PMN have been observed 5 times in 3 independent videos.

Video 2. Selective dumping of the killed C. glabrata cell by human PMN (snapshot at 22 sec)

In all imaging video sequences, a release of viable C. glabrata cells (vomocytosis) was only observed in two phagocytosis events (within the same movie). Thus, for C. glabrata dumping is more frequent than vomocytosis. The release of inactivated C. glabrata cells adds a new option to the repertoire of human immune cells getting into contact with fungal pathogens 5

(Table 1). Importantly, dumping of inactivated C. glabrata by PMN may provide other immune cells like monocytes, macrophages and dendritic cells with the opportunity to take up dead fungal cells and present the relevant antigens for induction of adaptive responses. This may be relevant for induction of adaptive immunity as macrophage phagosomes containing viable C. glabrata have been shown to acquire early and late endosomal markers, but fail to undergo further maturation and recruit the lysosomal marker cathepsin D (Seider et al., 2011). In contrast, inactivated C. glabrata undergo the complete maturation process, allowing digestion of the yeast and efficient antigen presentation (Seider et al., 2011). Therefore dumping of dead C. glabrata cells may actively promote the induction of protective T-cell responses in addition to contributing to innate pathogen elimination. Finally, it seems likely that dumping can also occur for other pathogens, including bacteria and parasites and could form an effective way for PMN to promote the activation of subsequent immune responses.

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Different Outcomes of Pathogen – Host Cell Interaction

Table 1

Candida albicans Killing

Candida glabrata

Cryptococcus neoformans

in PMN1:

in PMN1

in PMN1, M2:

inhibition of intracellular filamentation and killing

rapid intracellular killing

intracellular killing can occur

(Duggan, Essig, et al., 2015)

(Miller and Mitchell, 1991)

(Wozniok et al., 2008) 4

in DC

killing by both oxidative and non-oxidative mechanisms (Wozniak and Levitz, 2008; Hole et al., 2012) 2

Survival/ Replication

3

4

5

2

3

3

in M , M , DC , NK :

in M , M :

in M :

intracellular filamentation and host cell lysis

block of late phagosome maturation, intracellular replication by budding in intact host cell

efficient intracellular replication in acidified but potentially non-intact or not fully matured phagolysosomes of intact host cells, may result in cell division or lateral transfer of cryptococcal cells between M

(Kumamoto and Vinces, 2005; Kurzai et al., 2005, Voigt et al., 2014)

(Seider et al., 2011)

(reviewed in García-Rodas and Zaragoza, 2012)

Vomocytosis

in M3

in PMN:

in M3:

rarely vomocytosis like expulsion of filaments

very rare

frequently observed, also occurring in vivo

(this study)

(Bain et al., 2012)

(Alvarez et al., 2006; Ma et al., 2006; Chayakulkeeree et al. 2011; Nicola et al., 2011)

not yet observed / unstudied

Dumping

by PMN1

not yet observed / unstudied

(this study)

Table 1: Summary of current knowledge on different outcomes of the interaction between major pathogenic yeasts and human innate immune cells. Killing refers to more or less rapid inactivation of the fungus by intracellular effector mechanisms; Survival/Replication describes prolonged survival of a fungal pathogen inside a host cell, which may lead to replication inside the intact host cell or lysis of the host cell and release of the viable fungus; Vomocytosis describes release of a viable intracellular fungal cell without damage of the host cell (Chayakulkeeree et al. 2011); Dumping refers to release of intracellularly killed fungal cells as described in this study. neutrophil;

2

M = monocyte;

3

M= macrophage;

4

1

PMN = polymorphonuclear

DC = dendritic cell;

5

NK = natural killer

cell. Literature references refer to important exemplary descriptions but are not complete due to manuscript length restrictions. 7

Methods Ethics statement Human peripheral blood was collected from healthy volunteers after written informed consent. The study was conducted in accordance with the Declaration of Helsinki, all protocols were approved by the Ethics Committee of the University Hospital Jena (permit number: 273-12/09).

Isolation of primary human immune cells Venous blood of healthy volunteers was collected in EDTA monovettes. PMN were subsequently purified as described elsewhere (Wozniok et al., 2008). Cells were resuspended in RPMI1640 (Gibco) containing 5% heat inactivated (56°C for 60 min) human serum (type AB, Sigma-Aldrich).

Fungal cells and culture C. glabrata expressing GFP (Seider et al., 2011) were grown overnight in M199 medium (9,8 g/l M199, 35,7 g/l HEPES, 2,2 g/l NaHCO3), pH4 at 37°C to stationary phase in a shaking incubator. Cells were then reseeded in M199 medium, pH8 and cultured for one hour at 37°C prior to confrontation assays.

Time-lapse microscopy of confrontation assays Live cell imaging was performed as described in (Duggan, Essig, et al., 2015). Briefly, 2x105 and 1x106 C. glabrata expressing GFP, respectively, were seeded in a µGrid dish (MoBiTec GmbH) and confronted with 2x10 5 PMN (Candida:PMN = 1:1 or 5:1) in a total volume of 2 ml RPMI1640 containing 5% heat inactivated human serum. 2.5 ng/ml propidium iodide (PI, Sigma) was added. PI is excluded from viable cells and selectively stains dead cells, which lack an intact plasma membrane. Therefore, death of a fungal cell (or a PMN) can be identified in the video sequence by the respective cell/fungus turning red fluorescent.

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Confrontation assays were incubated in an environmental control chamber at 37°C and 5% CO2. Images were acquired every 10 seconds with an LSM 780 confocal microscope. Manual analysis of all video files was done by two independent persons and all nonconcurrent events were re-checked.

Acknowledgements We are grateful to all anonymous blood donors that contributed to our study. Cindy Reichmann contributed to the work with expert technical assistance. FE received a stipend from the Center for Sepsis Control and Care (CSCC, Jena) and this work was financially supported by the CSCC.

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Videos

Supplemental Video 1 Supplemental Video 2

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