NETs are rapidly released from the nucleus

To confirm authentic in vivo NETs (histones + granular proteins + DNA^9,20), a second assay was developed using specific fluorescently conjugated antibodies to visualize NET constituents (Method 2). Significant release of histones and neutrophil elastase occurred within minutes in response to Gram-positive human pathogens (S. aureus and S. pyogenes) (Fig. 1b,c). Negligible NETs occurred during sterile inflammation. Histone stained areas expanded in large sheets (Fig. 1c, Supplementary Fig. 2a and Supplementary Video 2) identical to extracellular DNA. Injection of dead, washed bacteria resulted in NET release (Fig. 1b), demonstrating innate recognition is sufficient independent of active toxin release.

Mitochondrial DNA NETs exist^21,22, however in vivo mitochondrial imaging showed unaltered intracellular mitochondria during infection (Supplementary Fig. 2a,b). This does not exclude mitochondrial DNA, however the nucleus seems to be the major source of NETs in our model of infection.

Using a third technique, in vivo PMN nuclei were pre-stained with a cell-permeable DNA specific dye (SYTO 60, Method 3) allowing nuclear imaging throughout NETosis. Without infection, SYTO 60 is restricted to normal multilobar distinct nuclei however, abnormal PMN with indistinct diffuse nuclear remnants were observed during infection (Fig. 1d). The ratio of extracellular to intracellular DNA was quantified to demonstrate the increase in NETs during infection (Fig. 1d).

NET formation is tightly regulated and requires both Tlr2 and C3

NETs may cause bystander injury, therefore we hypothesized that release is tightly regulated. Two critical systems involved in host defense against S. aureus are Tlr2 and complement²³. NETs were absent in Myd88^−/− animals demonstrating Toll dependence (Supplementary Fig. 3a). Furthermore, both Tlr2^−/− and C3^−/− animals were incapable of releasing histones (Fig. 1e) or nuclear DNA (Fig. 1f) despite recruiting normal numbers of PMN (Supplementary Fig. 4a). Normal mouse serum restored NETosis in C3^−/− animals indicating the importance of opsonization (Fig. 1f). As Tlr2^−/− mice have intact complement and the C3^−/− mice have intact Tlr2, it is clear that neither of these molecules is sufficient to cause NETs in isolation. Collectively these data suggest a multi-tiered regulation for which Tlr2 and complement are essential but not individually sufficient.

Tlr2 stimulation did not induce NETs (Supplementary Fig. 3c). In preliminary experiments, the C3a receptor played a greater role in NET production compared to the C5a receptor (Supplementary Fig. 3b), however, recombinant C3a alone or in addition to PAM-3-CYS (Tlr2 stimulant) had no effect on NETosis (Supplementary Fig. 3c), revealing that purified molecules could not mimic a pro-bono Gram-positive infection.

Viable NET-forming PMN Remain Functional in vivo

PMN did not take-up SYTOX (indicating death) during NETosis. We pre-labeled the neutrophil nucleus in vivo (Method 3) to understand if NETosing neutrophils continue to function. We observed a stark contrast between normal and NETosing PMN using SDCIM and 3D reconstruction (Fig. 2a). To visualize the innermost structures, we have adjusted the levels of 3D transparency. Non-NETosing PMN have intact, fully intracellular nuclei (left panel). The NETosing PMN has released a NET that surrounds the entire outer membrane (middle panel)(Fig. 2a). Here the NET is made transparent in order to see the underlying PMN. Remarkably, the NETosing PMN remains viable and continues to chemotax towards a GFP-bacterium (Supplementary Video 3). Further 3D images of PMN releasing intracellular DNA are shown (Supplementary Fig. 5a).

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Figure 2

PMN are viable and functional during nuclear breakdown and chromatin decondensation. PMN and nuclei were evaluated in vivo following live S. aureus (GFP-USA300). (a) A normal PMN that has captured a live bacterium (green) is shown by 2D imaging (top left) and contrasted by a NET-forming PMN (top middle) that is chemotaxing towards a GFP-bacterium (green arrow) while releasing DNA. Beneath each 2D spinning disk image is a panel of confocal 3D reconstruction using various degrees of transparency to distinguish the DNA in relation to the PMN outer membrane. The NETosing PMN in this image (middle panels) can be observed crawling toward the live bacteria in Supplementary Video 3. Four-color spinning disk confocal and 3D reconstruction reveals a PMN (yellow, Alexa 750 conjugated GR-1 specific antibody) with a diffuse nucleus (cell permeable SYTO 60, blue) releasing an extracellular NET (cell impermeable SYTOX Orange, red) while retaining live S. aureus (GFP-bacteria, green). (b) Three nuclear phenotypes were visualized; normal, diffuse, or absent. Top panels show extracellular membrane and nuclear staining, while the lower panels show the nucleus alone. (c) 3D-reconstruction reveals the morphology of each nuclei group. The 3D image rendering results in an artificial white reflection on the cell surface that does not represent an authentic fluorescent stain or nuclei. (d) Phagocytosis of live GFP-staphylococcus by neutrophils was quantified in wildtype, Tlr2^−/− or C3^−/− mice. The ability for neutrophils with decondensed or absent nuclei to phagocytose was quantified in (e) wildtype, Tlr2^−/− or C3^−/− animals. (n = 3 experiments per group). (** = P < 0.01 and *** = P < 0.001 for phagocytosis versus no phagocytosis)

To directly visualize PMN releasing intracellular DNA that becomes a NET we developed a 4-color SDCIM strategy combining the cell permeable DNA dye SYTO 60 and the cell impermeable dye SYTOX Orange. Live PMN were visualized that contained diffuse nuclear staining with concurrent release of NETs. 3D imaging confirmed that the PMN with extracellular DNA (red) contained a diffuse abnormal nucleus (right panel)(Fig 2a). PMN that crawled while releasing NETs were quantified for velocity, meandering index and crawling track (Supplementary Fig. 5b–d and Video 4). Therefore NETosis is not limited to immobile and incapacitated cells.

Live NETosing PMN develop decondensed nuclei and become anuclear

Neutrophil nuclear swelling and changes in morphology predict chromatin decondensation and nuclear envelope breakdown^{6,14,19,24,25}. We observed three different nuclear morphologies of emigrated PMN exposed to S. aureus. Normal nuclei were distinct demarcated multilobar rings with uniform DNA-staining (Fig. 2b, Supplementary Fig. 6a and Video 5). The normal nucleus was malleable, but retained its structure during crawling. Diffuse nuclei lacked structure and had a heterogeneous, lower intensity staining pattern consistent with breakdown of the nuclear envelope, chromatin decondensation and intracellular chromatin dispersion (Fig. 2b, Supplementary Fig. 6b and Supplementary Video 5). Anuclear PMN had absent nucleic acid staining (Fig. 2b, Supplementary Fig. 6b and Supplementary Video 5). These observations were not due to photobleaching as all three nuclear phenotypes were observed in the same fields of view, over the same duration and laser intensity. To confirm structural nuclear differences, we used confocal z-stacks and 3D reconstruction (Fig. 2c). Completely anuclear PMN by 2D imaging were completely devoid of a nucleus on 3D reconstruction and no longer contained any stainable nucleic acids suggesting these cells had completed the NETosing process (Fig. 2c).

GFP-histone transgenic mice (Tg(HIST1H2BB/EGFP)1Pa) confirmed in vivo anuclear PMN (Supplementary Fig. 7a). The PMN on the left has a normal nucleus, whereas the PMN on the right has no detectable nucleus. PMN with normal and diffuse nuclei continued to crawl within the tissues. Flow cytometry of untreated animals reveal all PMN are uniformly and brightly positive for a nucleus prior to infection (Supplementary Fig. 7b). Bone marrow chimera experiments (GFP-expression restricted to hematogenous cells) support a subpopulation of PMN within infected tissue capable of surviving and functioning without a nucleus (Supplementary Fig. 7c).

To understand if NETosis affects other host-defense functions we quantified phagocytosis of live bacteria. In wild type animals approximately 50% of PMN phagocytosed bacteria (Fig. 2d). Although NETosis and phagocytosis were closely linked, phagocytosis alone was not sufficient to induce NETosis as Tlr2^−/− PMN took up bacteria without nuclear changes (Fig. 2e). C3^−/− mice could not phagocytose and did not have altered nuclei, or make NETS, however this impairment was reversed with normal serum demonstrating the importance of opsonization (Fig. 2e).

Unusual cellular phenotypes of NETosing PMN

Unique patterns of crawling and cell morphologies were observed during NETosis. Normal PMN with multilobar nuclei crawl with a single lead pseudopod (Fig. 3a). In contrast PMN with diffuse nuclei demonstrate hyperpolarization and multiple pseudopods (Fig. 3a). To test if the loss of the nucleus explains the bizarre crawling phenotype, we studied a well-defined induced anuclear human PMN cytoplast^26–29 (Fig. 3a). Indeed cytoplasts exhibit crawling behaviors similar to NETosing PMN suggesting that the intact nucleus provides the structural fulcrum for normal crawling similar to other mobile cells^17,18. Crawling was further analyzed in Tlr2^−/− and C3^−/− PMN. As anticipated Tlr2^−/− and C3^−/− PMN crawled with a normal single lead pseudopod and maintained morphology similar to non-NETosing PMN with normal nuclei (Fig. 3a).

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Figure 3

NET-forming PMN display a novel crawling phenotype in vivo related to nuclear structure. (a) 2D images of in vivo PMN with a normal nuclei, NET-forming cells with diffuse nuclei and an in vitro generated anuclear human PMN (cytoplast). Contouring analysis of each cell is shown beneath the image. The PMN outer membrane is traced every 30 s (PMN in the middle image with white arrow is traced). Typical crawling phenotypes are shown for Tlr2^−/− and C3^−/− animals. (b) Cell polarities in relation to nuclear morphology were quantified. (c) The relationship of pseudopod formation of crawling PMN compared to their nuclear architecture or between wild type, Tlr2^−/− and C3^−/− mice. (d) PMN velocity and meandering index during live staphylococcus infection. (e) Cellular tracking during live staphylococcus infection (*** = P < 0.001, ** = P < 0.01, * = P < 0.05 compared to sterile inflammation and ### = P < 0.001, # = P < 0.05 compared to wild type).

Polarization is normal for crawling cells and in vitro these cells become 1.5 to 2× longer than their width, the limitation of this value being the width of the nucleus. However in infected tissue PMN that had diffuse nuclei became hyperpolarized (Fig. 3b and Supplementary Fig. 4b). Both the NETosing PMN and the cytoplasts generated multiple pseudopods simultaneously, often at obtuse angles (90–180 °), a phenotype never observed in sterile inflammation. During sterile inflammation, the majority of PMN crawled with one dominant pseudopod, which rarely split to yield 2 pseudopods at an acute angle (< 90 °). Infection increased pseudopod formation, which was more common in cells with altered nuclei (Fig. 3c). Tlr2^−/− and C3^−/− PMN did not form multiple pseudopods. Despite having abnormal nuclei, hyperpolarization and multiple pseudopods, wild type PMN crawled with normal velocity and moved with purpose (i.e. a high meandering index)(Fig. 3d,e). We tracked individual PMN and found that NETosing PMN with diffuse nuclei crawled with high velocity and meandering index, while fully anuclear PMN crawled slowly (Supplementary Fig. 8a–c). Together these data suggest that structural nuclear breakdown is related to aberrant, but functional chemotaxis, similar to anuclear human PMN cytoplasts. By contrast, despite normal nuclei, Tlr2^−/− and C3^−/− had reduced crawling velocities and moved without purpose in random directions (Fig. 3d,e).

NETs are critical to contain an acute invasive infection in vivo

We followed staphylococcus skin infections over time using minimally invasive non-surgical luminescent imaging. Currently no specific inhibitor of NETosis exists, so exogenous DNase treatment was used to breakdown NETs. Animals treated with or without DNase had comparable S. aureus levels at 1 h, but DNase treatment decreased luminescence within 4 h (Fig. 4a–b). DNase did not alter bacterial growth in culture (data not shown). Disruption of NETs resulted in significant increase in bacteremia at 4 h and a decrease of skin CFUs at 24 h, indicating that bacteria can rapidly escape the portal of entry in the absence of NET trapping (Fig. 4c,d).

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Figure 4

NETs are essential to limit acute S. aureus dissemination. (a) Photon imaging of live luminescent S. aureus (Xen8.1, 1 × 10⁸ CFU per 100 μl saline injection) within the mouse skin at 1 h and 4 h. (b) Live luminescent S. aureus were quantified within the skin of mice pretreated with either DNase (1,000U i.p.) or saline (i.p.) at 1 h, 4 h and 8 h post-infection (6 mice). (c) Bacterial dissemination from the skin to the blood at 4 h post-infection (Xen8.1) (6 mice). (d) CFUs grown from a 3.5 mm skin biopsy of the injection site at 24 h (6 mice). (* = P < 0.05).

We examined dissemination in mutant mice that lacked NETosis. Wild type, Tlr2^−/− and C3^−/− animals received intraperitoneal injections of live S. aureus and blood CFUs were determined at 5 h. Compared to wild type mice, both Tlr2^−/− and C3^−/− mice had impaired ability to contain S. aureus dissemination (Supplementary Fig. 4c). Notably C3^−/− which were deficient in both phagocytosis and NETosis had more severe bacteremia than did Tlr2^−/− which could phagocytose but not NETose.

Green fluorescence protein (GFP) reporter vector construction and GFP labelling of S. aureus (MRSA, USA300)

The GFP reporter vector was constructed by gene fusion of the promoterless GFP⁺ with a truncated S. aureus protein. Briefly, the GFP⁺ (a GFP variant with increased fluorescence) coding sequence with an in-frame polylinker was cleaved with KpnI/SpeI from the plasmid pMUTIN-GFP^{+ 43} and subcloned into an E. coli-S. aureus shuttle vector pBT2⁴⁴ at the restriction site created by XbaI/KpnI. The recombinant plasmid contained the promoterless gfp⁺ reporter gene, designated pBT2-gfp⁺, was replicated in E. coli and verified with PCR, sequencing and restriction analysis. The genomic DNA of a CA-MRSA USA300 local clinical strain (USA300-2406) was digested with KpnI/EcoRI. The resultant KpnI-EcoRI fragments were inserted into pBT2-gfp⁺ plasmid at KpnI/EcoRI restriction site and transformed into E. coli DH5α. The clone with the brightest green fluorescence was selected and the plasmid was purified. The gene sequencing revealed that a 1.8kb KpnI-EcoRI fragment containing a truncated SAUSA300_1226 gene which encodes a homoserine dehydrogenase⁴⁵ was fused with the promoterless GFP⁺ in the subsequent plasmid, designated pBT2-gfp⁺ fused. This plasmid was further transformed into the laboratory chemical mutant strain S. aureus RN4220 by electroporation using tryptic soy agar (TSA) plates containing chloramphenicol. The pBT2-gfp⁺fused plasmids purified from RN4220 were then used for labelling of the clinical CA-MRSA (USA300) S. aureus isolate by electroporation and under chloramphenicol selection⁴⁶.

Animals

C57BL/6J, Complement-3 deficient (C3^−/−) and transgenic GFP fusion to histone H2B (Histone-GFP) (B6.Cg-Tg (HIST1H2BB/eGFP) 1Pa/J) mice were purchased from The Jackson Laboratory. Myd88^−/− and Tlr2^−/− mice were provided by Prof. Shizuo Akira (Osaka University, Japan). Dr. T.Graf (Barcelona, Spain) provided the Tg(LysMeGFP). Mice (20–35 g, 6–10 weeks old) were maintained in a pathogen-free environment. The mice had access to food and water ad libitum. All procedures performed were approved by the University of Calgary Animal Care Committee and were in accordance with the Canadian Guidelines for Animal Research.

Bacteria Strains

Three S. aureus strains were used in this study. S. aureus (Xen8.1 and Xen29), known pathogenic strains that have been used in soft tissue, biofilm and sepsis models, were purchased from Caliper Life Sciences. The GFP-transgenic CA-MRSA USA300-2406 was developed as previously described above. Staphylococcus Xen29 and Xen8.1 were grown in LB broth (200 μg ml⁻¹ kanamycin) and CA-MRSA was grown in brain heart infusion BHI (chloramphenicol 20 μg ml⁻¹) at 37 °C overnight. Bacteria were subcultured in fresh LB or BHI and incubated for 2 h in a 37 °C shaker to obtain bacteria in mid-log phase growth prior to injection. Streptococcus pyogenes was obtained from ATCC (ATCC19615). S. pyogenes was grown on a sheep blood plate overnight at 37 °C, and then a single colony from the blood agar plate was inoculated into 25 ml fresh BHI broth for 18 h at 37 °C.

Surgical procedures

Mice were anaesthetized (10 mg kg⁻¹ xylazine hydrochloride and 200 mg kg⁻¹ ketamine hydrochloride) and body temperature was maintained using a rectal probe and heating pad. The mice were pretreated with intradermal MIP-2 (0.2 μg) diluted in sterile normal saline or MIP-2 tissue superfusion (5 nM). The right jugular vein was cannulated to administer additional anesthetic and fluorescent dyes. The microcirculation of the dorsal skin was prepared for microscopy as previously described⁴⁷. Briefly, after shaving the mouse’s back, a midline dorsal incision was made extending from the tail region up to the level of the occiput. The skin was separated from the underlying tissue, remaining attached laterally to ensure the blood supply remained intact. The area of skin was then extended over a viewing pedestal and secured along the edges using 4.0 sutures. The loose connective tissue lying on top of the dermal microvasculature was carefully removed by dissection under an operating microscope. The exposed dermal microvasculature was immersed in isotonic saline and covered with a coverslip held in place with vacuum grease. Alexa Fluor 488, Alexa Fluor 649, PE or FITC conjugated GR-1 specific antibody (10 μl per mouse i.v.) or the neutrophil specific conjugated antibody to Ly6G (clone 1A8, Biolegend) was used to visualize neutrophils.

Three different methods were used to visualize NETs in vivo. Method 1 used the membrane impermeable SYTOX Orange dye (diluted 1:1,000 with sterile saline, 100 μl per mouse i.v.). MIP-2 superfusion (5 nM) was initiated 30 min prior to S. aureus administration (Xen29, DNase positive, 1 × 10⁸ CFU in 100 μl sterile saline i.d.). Method 2 used Xenon labeled histone specific antibody (M-20) or neutrophil elastase specific antibody (M-18) (20 μg per mouse i.v.). The Xenon labeling kit was modified from manufacturer’s instructions for in vivo experiments. Preliminary experiments were performed using various concentrations of component A and component B of the Xenon kit. Optimal visualization was found with 7.5 μl component A and 10 μl of antibody incubated at room temperature for 5 min, without the use of component B. Control IgG (20 μg per mouse i.v.) was labeled using the same Xenon kit and modified procedure. MIP-2 (0.2 μg, i.d.) was administered 60 min prior to S. aureus (Xen8.1, DNase negative, 1 × 10⁸ CFU in 100 μl of sterile saline, i.d.). For method 3 mice received an i.p injection of the cell-permeable nucleic acid dye SYTO 60 (5 μl of stock 5 mM diluted in 200 μl PBS, i.p.) 16 h prior to intravital microscopy or i.v. administration during surgery. MIP-2 (0.2 μg, i.d.) was administered 60 min prior to the clinical CA-MRSA transgenic-GFP bacteria (USA300-2406, 1 × 10⁸ CFU in 100 μl of sterile saline, i.d.).

Following baseline visualization, all bacteria were directly administered into the field of view using a tuberculin needle (1 × 10⁸ CFU in 100 μl of sterile saline, i.d.). Three to six separate areas were visualized in each experiment in order to minimize selection bias. For killed bacteria experiments (Xen8.1) bacteria was heated for 30 min at 65 °C and washed twice with sterile saline (n = 3).

Spinning-Disk Confocal Intravital Microscopy (SDCIM)

Spinning-disk confocal intravital microscopy was performed using an Olympus BX51 (Olympus) upright microscope equipped with a 20×/0.95 XLUM Plan Fl water immersion objective. The microscope was equipped with a confocal light path (WaveFx, Quorum) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation). Laser excitation at 488, 561, 649 and 730 nm (Cobalt), was used in rapid succession and fluorescence in green, red and blue channels was visualized with the appropriate long pass filters (Semrock). Exposure time for all wavelengths was constant at 1 s. Sensitivity settings were maintained at the same level for all experiments. A 512×512 pixels back-thinned EMCCD camera (C9100-13, Hamamatsu) was used for fluorescence detection. Volocity Acquisition software (Improvision Inc.) was used to drive the confocal microscope. Images captured using the spinning disk were processed and analyzed in Volocity 4.20. NET area was quantified using the Volocity software.

Bone Marrow Chimera Animals

Recipient C57BL/6 mice were irradiated with 2 doses of 5 Gy (Gammacell 40 ¹³⁷Cs γ-irradiation source; Nordion International). An interval of 3 h was allowed to pass between the first and second irradiations. Transgenic histone-GFP bone marrow was isolated from mice euthanized by spinal cord displacement and 8 × 10⁶ donor bone marrow cells were injected into the tail vein of recipient irradiated mice. The mice were kept in microisolator cages for 8 weeks to allow full humoral reconstitution. This protocol previously confirmed that 99% of leukocytes in Thy1.1 and Thy1.2 congenic recipient mice were from donor bone marrow⁴⁸.

Neutrophil Behavior

Individual neutrophils were manually tracked to determine velocity, distance, displacement, pseudopod formation and cell contouring. Cells that remained within the field of view for 5 min were examined. Volocity software was used to calculate velocity, distance and displacement.

Bacterial Dissemination

S. aureus (Xen8.1) were grown to mid-log phase, washed and suspended in saline. Mice were injected i.p. with 1 × 10⁸ CFUs of the bacterial preparation in a volume of 200 uL. At 5–6 h after inoculation, mice were anaesthetized and whole blood was collected. Serial dilutions were cultured on LB agar plates overnight at 37 °C. To directly assess the effect of NETs on bacterial dissemination mice received a dorsal subcutaneous live mid-log phase inoculation of staphylococcus (Xen8.1, 1 × 10⁸ CFU in 100 μl injection of saline). 1 h post bacterial administration mice received an injection of either DNase (1,000 U) or saline into the infected skin. Dissemination was quantified by obtaining blood via a cardiac puncture in anaesthetized animals at 4 h post-bacterial inoculation, diluting it 1:1 with PBS and plating it on LB kanamycin for 24 h.

In vivo Bacterial Monitoring

An IVIS Imaging System 200 (Caliper Life Sciences) was used to quantify luminescent live staphylococcus (Xen8.1) over time. Live mid-log phase Staphylococcus aureus (Xen8.1, 1 × 10⁸ CFU in 100 μl injection of saline) was administered subcutaneously into shaved mice. Animals were anesthetized with 1.5% isoflurane while in the imaging chamber. Photons were measured during a 30 s exposure. Total photon emissions from uniform area of each mouse were quantified by using the Living Image software (Caliper Life Sciences). Mice were imaged at 1 h, 4 h and 8 h post-bacterial inoculation. At 24 h, the mice were euthanized and a skin biopsy (3.5 mm punch) was obtained from within the injection site. The tissue was homogenized and plated on LB agar plates with kanamycin and grown overnight to quantify CFU.

Human PMN Isolation

Whole blood was collected into acid citrate dextrose (1:5 blood, v/v). Erythrocytes were removed using dextran sedimentation followed by two rounds of hypotonic lysis. Neutrophils were isolated from the resulting cell suspension using Ficoll-Histopaque density centrifugation. PMN were stained ex vivo using PE-conjugated CD16 specific antibody and SYTO 60 (1:1,000 dilution) for 15 min prior to directed intradermal injection into the mouse skin prep. Ethical approval for obtaining healthy human volunteer blood was provided by The University of Calgary research ethics committee and each human subject provided informed consent.

Human Abscesses

The infectious disease specialty consultation service of the Division of Infectious Diseases in the Dept. of Medicine, Alberta Health Services – Calgary and Area, identified patients who were clinically diagnosed with cellulitis and secondary abscesses due to Gram-positive bacteria requiring wound aspiration as part of routine medical care. All patients were fully reviewed by a physician with specialty training in infectious diseases. Specimens were sent for routine Gram-stain and culture. Remaining samples were used for electron microscopy, immunofluorescence and in vivo spinning disk confocal experiments with consent of each patient. The University of Calgary research ethics committee approved the use of discarded human clinical samples.

Transmission Electron Microscopy

Fresh clinical aspirates were fixed with 2.5% glutaraldehyde. The cells were spun into a pellet using a microcentrifuge. After washing three times, the pellets were postfixed in 1% osmium tetroxide in cacodylate buffer for 1 h, dehydrated through a graded series of acetone, and embedded in epoxy-based resin. Ultrathin sections were cut in a Reichert-Jung Ultracut E microtome using a diamond knife and stained with 4% aqueous uranyl acetate and Reynolds’ lead citrate. The sections were observed in a Hitachi H7650 transmission electron microscope at 80 kV, and images were taken with an AMT 16000 digital camera mounted on the microscope microscope¹⁴.

Immunofluorescence Microscopy

Slides (smears) were prepared from clinical aspirates and allowed to air dry. Slides were fixed in ice-cold acetone for 10 min and dried. Slides were then blocked for 2 h at room temperature in PBS containing 2% BSA followed by staining for 1 h with a cocktail of fluorescently-labeled Ab together with SYTOX Green. Slides were washed and coverslips mounted with ProLong Gold antifade reagent (Invitrogen). Slides were imaged using a spinning-disk confocal microscope consisting of an Olympus IX81 inverted microscope (Olympus) coupled to a confocal light path (WaveFx; Quorum Technologies) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation). Excitation by each of 491-, 561-, and 642-nm laser wavelengths (Cobolt) were sequentially controlled and merged into a single optic cable using an LMM5 laser merge module (Spectral Applied Research) and fluorescence was visualized through band pass emission filters (Semrock) driven by a Modular Automation Controller (Ludi Electronic Products, Ltd.) and detected with a 512×512 pixels back-thinned EMCCD camera (C9100-13, Hamamatsu). Volocity Acquisition software (V5.2.1) (Improvision Inc.) was used to drive the confocal microscope

The pBT2 was a gift from R. Brückner (University of Kaiserslautern, Germany). We thank D. Knight and C. Baddick for technical assistance and ongoing support. We acknowledge P. Colarusso and the support staff of the Snyder Institute Live Cell Imaging Facility for assisting in the image capturing and analysis (Canada Foundation for Innovation funded). We also thank the University of Calgary Flow Cytometry Facility and L. Kennedy for their assistance. We are grateful to P. Forsyth for use of the IVIS 200. We thank the physicians, nurses and support staff in the Division of Infectious Diseases in the Dept. of Medicine, Alberta Health Services-Calgary and Area for assistance in obtaining clinical samples. We thank E. Yung, C. Horn, K. Nelson and K. Headley from Calgary Lab Services for assistance with the electron microscopy experiments.

The Canadian Institute of Health Research (CIHR) provided the operating grants to support this work. P. Kubes is an Alberta Innovates – Health Solutions (AIHS) Scientist, Canada Research Chair, and the Snyder Chair in Critical Care Medicine. B. Petri received an AIHS postdoctoral fellowship. B. Yipp is a Clinical Scholar (Department of Critical Care Medicine, Calgary), an AIHS clinical fellow and a CIHR fellow. During a portion of this study he received salary support from the Rockefeller University by Grant Award Number UL1RR024143 from the National Center for Research Resources (NCRR), a component of the US National Institutes of Health (NIH) and NIH Roadmap for Medical Research. The contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.