Group A Streptococcus Modulates Host Inflammation by Manipulating Polymorphonuclear Leukocyte Cell Death Responses

Programmed cell death determines the fate of circulating polymorphonuclear leukocytes (PMNs) and those recruited to sites of infection. PMNs are rapidly and spontaneously apoptotic under physiological conditions, a phenotype also prominent during bacterial infection [1]. After phagocytosis, senescent PMNs initiate an apoptotic program that shuts down cellular processes and blunts inflammatory potential [2,3]. The initiation of phagocytosis-induced cell death triggers the expression of ‘eat-me' signals on these cells and marks them for phagocytosis by tissue-resident and recruited macrophages; this is called efferocytosis [4]. Macrophage efferocytosis of apoptotic PMNs reduces the accidental release of stored PMN granules, and thus prevents the potential for PMN-mediated collateral damage to the surrounding tissues [5]. However, numerous pathogens have been shown to be capable of prompting alternative leukocyte cell death mechanisms, including pyroptosis and oncosis [6,7]. Whilst the induction of PMN apoptosis and efferocytosis imparts a strong anti-inflammatory action on the surrounding tissues, pathogenic stimulation of pyroptosis and oncosis prompt pro-inflammatory cell phenotypes that lead to eventual cell death. Thus, dysregulation of native PMN apoptosis may stimulate gratuitous inflammatory responses via the uncontrolled release of granular contents and other pro-inflammatory cytosolic factors [8].

Group A Streptococcus (Streptococcus pyogenes; GAS) is the etiological agent of severe diseases with inflammatory involvement, including necrotizing fasciitis (NF) and septic shock [9,10]. Epidemiologically, GAS disease accounts for >663,000 cases of invasive infections globally [11]. Almost a third of NF cases lead to mortality in developed countries, a fraction that rises to 50% in cases with associated toxic shock [12]. Severe inflammation and destruction of focal tissues is a hallmark of NF infection. Furthermore, GAS sepsis is accompanied by the systemic release of numerous pro-inflammatory mediators, with the magnitude of inflammatory cytokine response strongly correlated to the severity of the disease [13]. GAS alteration of PMN cell death may therefore strongly influence clinical manifestations of inflammatory GAS disease. Here, we examine the potential of virulent and avirulent GAS strains to modulate PMN cell death responses as well as the associated effect of GAS-induced PMN cell death on inflammatory responses.

All work involving the use of human blood and blood products was conducted with the informed consent of the volunteers and was approved by the University of Wollongong Human Ethics Committee. All animal use and procedures were approved by the University of Wollongong Animal Ethics Committee.

The clinical GAS bacteremia isolate NS88.2 (emm98.1) encoding a nonfunctional control of virulence regulator (covRS) and the functional covRS derivative NS88.2rep strain have been previously described [14]. NS88.2 and NS88.2rep strains expressing enhanced green fluorescent protein (eGFP) were constructed via transformation with the pDC-eGFP vector [15]. GAS strains were routinely cultured at 37°C on solid horse-blood agar (Thermo Fisher Scientific, Waltham, Mass., USA) or Todd-Hewitt agar (BD Bioscience, Franklin Lakes, N.J., USA), in static cultures of yeast-supplemented (1% w/v) Todd-Hewitt broth (THY) or in static THY cultures supplemented with 2 µg/ml erythromycin for maintenance of the pDC-eGFP vector.

Human venous blood was taken into citrated polypropylene tubes, overlaid onto PolyMorphPrep (Axis-Shield, Oslo, Norway). PMNs were purified as per the manufacturer's instructions and maintained in Roswell-Park Memorial Institute (RPMI) medium containing 2% heat-inactivated plasma. The purity of the human PMN preparations via this method was estimated to be >98%, as exhibited by the characteristic forward- and side-scatter profiles and CD14 staining for contaminating monocytes of PMN samples (online suppl. fig. S1; for all online suppl. material, see www.karger.com/doi/10.1159/000430498).

GAS cultures were routinely prepared for in vitro human PMN infection via growth to the mid-logarithmic phase, washing twice with sterile PBS and diluted to the required inoculum in RPMI containing 2% heat-inactivated human plasma. Purified human PMNs were seeded into either 96-well (for phagocytosis, cytotoxicity and the production of reactive oxygen species (ROS)) or 24-well plates (for microscopy, TUNEL and Western blot) and GAS was added to the appropriate multiplicity of infection (MOI, PMNs:GAS). PMN phagocytosis was synchronized via centrifugation at 380 g for 8 min at 4°C prior to incubation at 37°C in 5% CO₂. In some experiments, PMNs were treated with 5 µg/ml of cytochalasin D (Cayman Chemicals, Ann Arbor, Mich., USA) for 30 min at room temperature prior to infection.

To measure GAS uptake by PMNs, 5 × 10⁵ cells were infected with 5 × 10⁶ CFU of eGFP-expressing GAS and incubated for varying times (5, 40, 80 and 120 min) or for 40 min at varying MOIs (1:5, 1:10, 1:20 and 1:40). The association of GAS with PMNs was measured using an LSR II flow cytometer (BD Bioscience), calculated as the percentage of PMNs with eGFP fluorescence relative to uninfected PMNs. The relative quantity of GAS associated with PMNs was estimated by the mean fluorescence intensity (MFI) of eGFP-positive PMNs. Double immunofluorescence of infected PMNs was conducted by in vitro infection of 1 × 10⁶ PMNs with 1 × 10⁷ GAS on prepared coverslips, with subsequent incubation for 30 min. After incubation, cells were fixed with 4% paraformaldehyde and extracellular GAS was labeled with polyclonal rabbit anti-GAS antiserum and mouse anti-rabbit Alexa Fluor 488-conjugated IgG (Dako, Glostrup, Denmark). Samples were then permeabilized with 0.1% Triton-X for 5 min and extracellular and intracellular GAS were labeled with polyclonal rabbit anti-GAS antiserum and mouse anti-rabbit Alexa Fluor 568-conjugated IgG (Dako). Slides were mounted with Prolong Gold containing DAPI (Invitrogen, Carlsbad, Calif., USA) and then visualized using an Axio Observer inverted microscope (Carl Zeiss, Oberkochen, Germany). Microscope images were altered for brightness and contrast to highlight the infecting GAS cells.

Kinetic measurement of PMN ROS production was conducted essentially as previously [2]. Briefly, purified PMNs were loaded with 25 µM of dichlorofluorescein (DCF, Molecular Probes, Eugene, Oreg., USA) for 40 min in the dark at room temperature, prior to the infection of 5 × 10⁵ PMNs with 5 × 10⁶ GAS CFU. PMN ROS production was measured fluorometrically (λ_ex: 488 nm, λ_emm: 515 nm) using a POLARstar Omega fluorescent plate reader (BMG Labtech, Carlsbad, Calif., USA).

The depolarization of mitochondrial membranes of infected PMNs was estimated using an established technique [6]. Briefly, purified PMNs were loaded with 5 µM of 3,3′-dihexyloxacarbocyanine [DiOC₆(3)] iodide for 20 min at room temperature prior to 2 h of GAS infection. Perturbation of the Ψ_m (mitochondrial membrane potential) was indicated as a loss of DiOC₆(3) fluorescence compared to time 0 uninfected PMNs determined by means of a FACSCalibur flow cytometer (BD Bioscience).

Caspase-3 cleavage was detected via Western blotting of cell lysates from infected PMNs. In vitro infections of 2 × 10⁶ PMNs with 2 × 10⁷ GAS CFU were conducted for the time points indicated and cell lysates were run on SDS-PAGE gels prior to transfer to nitrocellulose membranes and probing using mouse anti-caspase-3 IgG (Biolegend, San Diego, Calif., USA), rabbit anti-actin IgG (Sigma-Aldrich, St. Louis, Mo., USA) and detection using HRP-conjugated IgGs (Bio-Rad, Berkely, Calif., USA).

Assessment of apoptosis induced nuclear DNA fragmentation was quantified by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) using the DEADend fluorometric TUNEL kit (Promega, Fitchburg, Wis., USA) as per the manufacturer's instructions and an LSR II flow cytometer (BD Bioscience).

Subcutaneous GAS challenge of C57BL/6J mice has been described previously [14]. Estimation of bacterial survival in vivo and GAS interaction with murine PMNs were conducted by the method of Ly et al. [16], C57BL/6J mice were anesthetized via isoflurane inhalation, and 2 × 10⁷ CFU of mid-logarithmic phase eGFP-expressing GAS were injected intradermally into the left and right flanks prior to incubation for 6 h. The mice were subsequently euthanized via CO₂ asphyxiation and the intradermal sites of injection were lavaged with two 1-ml quantities of sterile 0.7% saline. The lavage fluid was adjusted to a fixed volume and bacterial survival was estimated via serial dilution of the lavage fluid, plating out and enumeration of the recovered bacteria after overnight incubation. Survival was expressed as: recovered GAS CFU/CFU of inoculum*100%. Remaining lavage fluid was washed once with sterile PBS and labelled with rat anti-mouse Ly6-G phycoerythrin-conjugated IgG (clone: 1A8, Biolegend) and GAS association with murine PMNs was assessed as described for purified human PMNs. For animal studies involving histological staining, including by TUNEL, C57BL/6J mice were infected with 5 × 10⁷ CFU of mid-logarithmic phase, non-eGFP-expressing GAS and euthanized by CO₂ 24 h after infection. Infected murine dermal tissue was then harvested, formalin-fixed and paraffin-embedded prior to microtome sectioning and hematoxylin and eosin (HE) staining, using standard protocols. Identification of apoptotic cells was conducted using the DEADend fluorometric TUNEL kit (Promega) instructions for paraffin-embedded tissues. TUNEL-stained tissues were analyzed using a Leica TCS SP5 laser scanning confocal microscope (Leica, Wetzlar, Germany). For animal cytokine studies, C57BL/6J mice were subcutaneously infected with 1 × 10⁷ CFU for 48 h. Blood was harvested from the apex of the heart and serum was collected and stored at -20°C until use. Murine TNF-α, IL-6 and IL-1β levels were quantified in duplicate by ELISA as per the manufacturer's instructions (Biolegend).

GAS-induced PMN cytotoxicity was assessed essentially as previously [6]. Briefly, 2 × 10⁵ human PMNs were infected with 2 × 10⁶ GAS CFU and incubated for varying periods of time, 50-µl aliquots of cell supernatants were then sampled and the lactate D-dehydrogenase (LDH) concentration was measured using the CytoTox 96 kit (Promega) according to the manufacturer's instructions. Cytotoxicity was calculated as LDH release induced by either GAS strain over maximum LDH release (total LDH from lysed uninfected cells) corrected for the spontaneous release of LDH from uninfected cells. For some experiments, PMNs were infected in the presence of 100 mM of glycine.

For scanning electron microscopy, 5 × 10⁶ PMNs were infected with 5 × 10⁷ GAS CFU for 5 h. Samples were fixed with 5% formaldehyde and 2% glutaraldehyde in HEPES buffer, and kept at 4°C before dehydrating with a graded series of acetone, critical-point-dried with CO₂ and sputter-coated with gold-palladium. Samples were examined under a Zeiss Merlin microscope (Carl Zeiss) at an acceleration voltage of 5 kV using the Everhart-Thornley SE-detector and the Inlens SE-detector in a 25:75 ratio. For transmission electron microscopy, PMNs were infected and samples fixed as above, and treated with 1% aqueous osmium tetroxide for 1 h prior to dehydration with a graded series of acetone, treatment with 2% uranyl acetate in 70% acetone for overnight, further dehydration with acetone, and embedding in Spurr epoxide resin. Ultrathin sections were cut with a diamond knife, counterstained with uranyl acetate and observed under a Zeiss EM910 microscope at an acceleration voltage of 80 kV. Images were recorded digitally with a slow-scan CCD camera (ProScan, 1024 × 1024) with ITEM-Software (Olympus, Tokyo, Japan). Contrast and brightness of images were adjusted with Adobe Photoshop (Adobe Systems, San Jose, USA).

Statistical analysis of differences between conditions for PMN phagocytosis, LDH release, bacterial survival in vivo and systemic cytokine assays were conducted using the unpaired Student t test. Differences between conditions for TUNEL, Ψ_m depolarization and osmoprotection assays were analyzed using one-way ANOVA with the Tukey multiple-comparison test. Differences in survival curves were analyzed using a log-rank (Mantel-Cox) test. Differences were deemed statistically significant at p < 0.05. All statistical analyses were conducted using Prism 5 (GraphPad Software Inc., La Jolla, Calif., USA).

The initial interactions of PMNs with GAS are mediated via phagocytosis of the infecting bacteria. Incubation of eGFP-expressing virulent GAS (NS88.2) and avirulent GAS (NS88.2rep) with PMNs revealed that phagocytosis occurred rapidly for both NS88.2 and NS88.2rep GAS (<5 min), with a higher percentage of PMNs tending to associate with NS88.2rep compared with NS88.2 (fig. 1a). The relative quantity of NS88.2rep associated with each PMN cell was significantly higher than NS88.2, an effect that was exaggerated at increasing MOIs (fig. 1b, p < 0.001 for all). NS88.2 was frequently visualized in the extracellular space during PMN infection, whereas NS88.2rep was more often localized within infected PMNs (fig. 1c, d). This difference in phagocytosis was not due to differences in growth kinetics, as both strains showed equal growth rates in THY media alone (online suppl. fig. S2).

The PMN respiratory burst against ingested pathogens plays a key role in the destruction of phagocytosed microbes and in the regulation of downstream cell death pathways. Kinetic measurement of PMN ROS production during phagocytosis of GAS revealed that avirulent NS88.2rep GAS stimulated higher ROS activity than the virulent NS88.2 strain (fig. 1e). ROS also play an important function in the regulation of the Ψ_m, which, in turn, reflects cellular viability, as uncoupling of this proton gradient plays a central role in multiple cell death pathways [17]. Incubation with NS88.2rep elicited a large, significant reduction in Ψ_m compared with uninfected PMNs and cells infected with NS88.2 (p < 0.001; fig. 1f). Infection of PMNs with NS88.2 also elicited a significant, albeit smaller reduction in Ψ_m compared to uninfected cells (p < 0.001; fig. 1f). Collectively, these results suggest that downstream PMN cell death responses, such as apoptosis, may be influenced by differential ROS generation and Ψ_m depolarization after the phagocytosis of GAS.

With many forms of cell death pathways, cells undergo characteristic alterations in external morphology that are indicative of cell death mechanisms [18]. Visualization of GAS-induced PMN cell death was conducted by scanning electron microscopy 5 h after infection. Uninfected PMNs exhibited typical neutrophil exterior morphology with few membrane irregularities (fig. 2a). Comparatively, NS88.2-infected PMNs exhibited slight cellular swelling but membrane morphology similar to the uninfected cells (fig. 2b). In contrast, NS88.2rep-infected PMNs exhibited both cellular shrinkage and extensive membrane blebbing, both features indicative of apoptotic cell death (fig. 2c).

Biochemical interrogation of GAS-induced cell death was conducted via fluorescent measurement of nuclear DNA fragmentation (TUNEL staining) and caspase-3 cleavage. Infection of human PMNs with the avirulent NS88.2rep strain induced significantly higher TUNEL staining at 4 h (p < 0.01) and 6 h (p < 0.001) after infection compared to both NS88.2-infected and uninfected PMNs (fig. 2d), whereas NS88.2 infection induced comparable TUNEL staining to uninfected cells (p > 0.05). An increased amount of the active caspase-3 protein was present in NS88.2rep- infected cells relative to uninfected and NS88.2-infected cells 5 h after infection (fig. 2e), further indicating an apoptotic cellular response in the PMNs exposed to NS88.2rep. To determine whether PMN apoptotic responses were phagocytosis-dependent, PMNs were preincubated with the actin polymerization inhibitor cytochalasin D prior to infection to prevent GAS uptake. No significant difference in TUNEL staining was noted for PMNs treated with cytochalasin D relative to untreated PMNs for either the avirulent NS88.2rep or virulent NS88.2 strains (fig. 2f).

Orchestrated necrotic leukocyte mechanisms (also referred to as oncosis) have been previously shown to result in proinflammatory phenotypes, and they precede cell death following infection by other pathogens [19,20]. To investigate the potential necrotic consequences of GAS infection of PMNs, cell membrane integrity (LDH release) was assayed (fig. 3a). Infection of PMNs with NS88.2rep resulted in minimal LDH release and maintenance of plasma membrane impermeability, in contrast to NS88.2-infected PMNs which provoked significantly higher LDH release (p < 0.05). Higher rates of LDH release in response to infection by NS88.2 but not NS88.2rep could be significantly reduced through the addition of the osmoprotectant glycine (fig. 3b). Substantiating this biochemical data, transmission electron microscopy revealed a large degree of vacuolization and evidence of cell membrane disintegration in NS88.2-infected PMNs in comparison to NS88.2rep- infected and uninfected cells 5 h after infection (fig. 3c-e). Both extensive vacuolization and loss of cell membrane integrity have been previously associated with necrotic cell death processes, leading to proinflammatory cell responses [21]. Although proinflammatory PMN cell death responses are well-characterized in vitro, the in vivo role of GAS-induced necrotic PMN cell death and necrotic PMN-mediated inflammatory responses is not so well-defined.

Necrotic PMN cell death results in eventual cell lysis and, as such, the release of damage-associated molecular pattern molecules (DAMPs) from these injured and dying cells [8]. GAS-induced dysregulation from a more physiological apoptotic PMN response may contribute to the destructive tissue pathologies noted in murine infection models, and in clinical manifestations of severe GAS disease [22]. To characterize the dynamics of PMNs recruited to virulent GAS infection in vivo, C57BL/6J mice were intradermally injected with eGFP-expressing NS88.2 and NS88.2rep, and the infection site was lavaged 6 h after infection. Murine neutrophils (Ly6-G⁺ cells) showed an increased uptake of NS88.2rep compared with NS88.2 (fig. 4a, p < 0.001), corroborating previous data described above using human PMNs. Reduced phagocytic uptake of NS88.2 by murine neutrophils is likely to contribute to the significantly enhanced survival of this strain within the dermis of infected mice at 6 h (fig. 4b, p < 0.01) and to NS88.2 virulence during 10-day infection, relative to NS88.2rep (fig. 4c, p < 0.001).

Histopathological assessment of HE-stained infected murine dermal tissues was undertaken 24 h after infection. Saline injection did not engender any adverse histopathologies (fig. 5a-c). Infection with virulent NS88.2 elicited suppurative inflammation manifested by robust PMN infiltration (fig. 5d, e, filled arrowheads). Infiltrating PMNs exhibited a high degree of pyknotic (fig. 5f, filled arrowheads) and karyorrhexic (fig. 5f, unfilled arrowheads) nuclei morphologies. Intradermal infection with avirulent NS88.2rep also elicited PMN infiltration (fig. 5g). Infiltrates to the site of NS88.2rep infection were primarily PMNs (fig. 5h, filled arrowheads), and displayed frequent pyknotic cell morphology (fig. 5i, filled arrowheads) with the appearance of numerous apoptotic body structures (fig. 5i, unfilled arrowheads). Systemic levels of several classic proinflammatory cytokines were also assessed 48 h after GAS infection (fig. 6a-c). The circulating levels of TNF-α and IL-6 were significantly higher in mice infected with NS88.2 than with NS88.2rep (fig. 6a, b, p < 0.05). Circulating levels of IL-1β were also increased in NS88.2-infected mice, though this difference was not statistically significant (fig. 6c, p > 0.05).

Biochemical analysis of murine infiltrates associated with GAS infection was assessed via TUNEL to confirm apoptotic nuclear DNA fragmentation (fig. 7a-i). Saline injection was not associated with significant TUNEL staining (fig. 7a-c). Cells associated with NS88.2 infection were infrequently TUNEL-positive, indicating a paucity of apoptotic cell nuclei (fig. 7d-f). Infiltrating cells within and surrounding the bolus of NS88.2rep infection exhibited stronger and more frequent positive TUNEL staining (fig. 7g-i). Thus, immunohistological evidence supports the in vitro data described above, and demonstrates that PMNs recruited to cutaneous, virulent GAS infection display a reduced apoptotic phenotype.

Induction of PMN apoptosis is critical for the resolution of inflammation in a variety of noninfectious and infectious scenarios, including wound-healing, meningitis and pneumonia [23]. This process is essential for preventing excessive inflammatory reactions, whereby aging cells and cells recruited to sites of infection may be disposed of safely without the sustained stimulation of immune responses. In animal models of meningitis, the persistence of PMNs lacking crucial apoptotic factors is strongly associated with adverse clinical outcomes [24]. Our study indicates that interaction with avirulent GAS elicits a programmed PMN cell death mechanism that is apoptotic in nature. Avirulent GAS elicits robust PMN ROS production, triggering downstream mitochondrial membrane depolarization and activation of the primary apoptotic effector, caspase-3. Despite higher rates of phagocytosis of avirulent GAS by both murine and human PMNs, the inhibition of human PMN phagocytosis did not reduce TUNEL staining of human PMNs (fig. 2f). This suggests that avirulent GAS may induce human PMN apoptosis via secreted products, as has been described for the interaction of GAS cysteine protease SpeB with epithelial cells [25].

These data indicate that exposure to avirulent GAS elicits a regulated PMN cell death mechanism reflective of the caspase-dependent, intrinsic apoptotic pathway [26]. The induction of apoptotic PMN cell death by avirulent GAS could be crucial for preventing excessive inflammatory reactions, whereby PMNs exhausted from microbicidal responsibilities may be disposed of safely, without undue immune stimulation [23]. As apoptotic cell death does not induce further inflammatory responses, it is considered to be immunologically silent [27]. However, exposure to virulent GAS appears to elicit a distinct PMN cell death process that lacks typical apoptotic markers.

Rigorous biochemical identification of oncosis is problematic, given that few broadly applicable markers have been identified [18]. In this regard, we did not observe the rapid ATP depletion or ionic disturbances in response to virulent GAS infection, as has been noted in previous accounts of cellular oncosis (data not shown) [21,28]. Recent guidelines governing the molecular definitions of cell death mechanisms recommend the use of general, as opposed to specific, nomenclature regarding cell death programs [17]. As such, we report that virulent GAS induce a proinflammatory form of cell death in PMNs which lacks common apoptotic markers and shares many of those reported for regulated necrosis.

Necrotic leukocyte cell death responses have been previously shown to result in a proinflammatory phenotype, and they precede leukocyte cell death following infection by other pathogens [19,20]. However, there is still a dearth of literature concerning the mechanisms by which these processes proceed in PMNs, the relationship between infection-mediated PMN cell death responses and inflammation during disease. Here, we show that, in contrast to avirulent GAS, PMNs infected with virulent GAS undergo mitochondrial membrane depolarization, as shown for avirulent GAS, but this event precedes the early loss of plasma membrane integrity and vacuolization. In vivo data describing the pathophysiological relevance of leukocyte cell death has been lacking. We propose that the manner of PMN cell death by either apoptosis or more necrotic forms affects the magnitude and nature of subsequent host immune responses.

Multiple reports have described a pivotal role for pore-forming toxins in other bacteria in shaping host leukocyte responses, including the GAS cytotoxins SLO and SLS [29,30]. Virulent GAS bestowed with covRS mutations are highly encapsulated, and express higher levels of SLO, so PMN necrosis is likely due in part to cytolysin-expressing extracellular GAS. Induction of PMN apoptosis has been postulated as a viable strategy to reduce harmful pathologies during acute inflammation [23,31]. In support of this proposition, there are studies showing treatment of mice with cyclin-dependent kinase inhibitors that induce PMN apoptosis and improve the resolution of inflammation and clinical symptoms during experimental pneumococcal infection [32,33]. Cyclin-dependent kinase inhibitors show promise as novel anti-inflammatory therapeutics [34].

In summary, this work describes dynamic host PMN cell death responses to GAS infection. PMN cell death in response to avirulent GAS infection is associated with an apoptotic program whereas PMNs recruited to virulent GAS infection in the dermis lack apoptotic markers and bear hallmarks of proinflammatory cell death, which may amplify immune reactions during infection.

We thank I. Schleicher for help with electron microscopic analyses. This work was supported by the National Health and Medical Research Council of Australia (grant numbers 635218 and 1009369 to M.L.S.-S.).

The authors have no conflicts of interest to declare.