Impact of Staphylococcus aureus Small Colony Variants on Human Lung Epithelial Cells with Subsequent Influenza Virus Infection

Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world’s population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.


Introduction
The respiratory tract is a major portal for microorganisms, through which virus infections can cause non-symptomatic, mild, and self-limiting but also severe diseases, sometimes with fatal outcomes [1].
A growing body of evidence shows that the human respiratory tract contains a highly adapted microbiota including commensal and opportunistic pathogens. Among those, Staphylococcus aureus (S. aureus) is of special importance, forming quasi-dormant subpopulations characterized by increased fitness compared to other phenotypes [2]. Colonization of S. aureus could either be persistent or

Super-Infection Protocol
Human lung epithelial cells were seeded in either 6-well plates (0.5 × 10 6 ) or 12-well plates (0.2 × 10 6 ) in 2 mL or 1 mL culture medium 24 h before infection. For bacterial infection, the overnight culture was set to OD 600nm = 1 to determine the multiplicity of infection (MOI). Cells were washed with PBS and infected with S. aureus 3878 SCV in invasion media (DMEM INV : DMEM supplemented with 1% human serum albumin, 25

Transfection Protocol
For transfection of the 3× NFκB reporter plasmid construct as described elsewhere [22] (0.1 µg/µL) A549 cells were seeded in 12-well plates as described above. Cells were transfected with 0.1 µg/µL of the indicated plasmid for 4 h with Lipofectamine ® 2000 (Invitrogen, Carlsbad, CA, USA) corresponding to the manufacturer's protocol. Afterwards, cells were washed with PBS and further incubated in cell culture media up to 24 h. Afterwards transfected cells were infected up to 8 hpvi. Performance of luciferase assay was done as described elsewhere [23].

Intra-and Extracellular Bacterial Titer Measurements
Extracellular bacterial titers were determined by collecting the supernatant of infected cells including the washing with PBS. Cells were lysed via hypotonic shock with 2 mL ddH 2 O according to Tuchscherr et al. [7,8] (37 • C, 30 min) to determine intracellular bacterial titers, including adherent bacteria at the cells surface. Bacterial suspensions were centrifuged (4000 rpm, 4 • C, 10 min), pellets were resuspended in 1 mL PBS, and serial dilutions (1:10) were plated on BHI agar plates and incubated for 32 h at 37 • C.

Standard Plaque Assay
Infectious virus particles in the supernatant were titrated to determine viral titers. A standard plaque assay was performed as described earlier [24].
Bacterial RNA was isolated with the RNeasy Protect Bacteria Mini Kit (Qiagen, Hilden, Germany), and cDNA synthesis was performed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. qRT-PCR was performed using a Roche LightCycler 480 (Basel, Switzerland) and Brilliant SYBRGreen Mastermix (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. The primers to determine the gene expression of gyrB, aroE, arg, hla, sarA, and sigB were already described elsewhere [7].

RT 2 Profiler Array Analysis
For pathway focused gene expression analysis, we used RT 2 Profiler PCR Arrays (Qiagen, Hilden, Germany). RNA isolation, cDNA synthesis and procedure were performed according to the manufacturer's protocol and instructions. Analysis of data was accomplished by using the GeneGlobe Data Analysis Center recommended by Qiagen [26].

FACS Analysis
Determination of secreted proteins in the supernatant was performed with BioLegend's LEGENDplex™ (San Diego, CA, USA) according to the manufacturer's protocol. The human anti-viral and pro-inflammatory chemokine panels were used. Results were analyzed by BioLegend's cloud-based LEGENDplex™ Data Analysis Software. To analyze apoptotic or necrotic cells, infection was performed as described above until 44 hpvi. Cells were treated with tumor necrosis factor related apoptosis inducing ligand (TRAIL; Enzo Life Sciences, Farmingdale, NY, USA) (150 ng/mL) 4.5 h before harvested and used as a positive control for apoptosis. The supernatant was collected for this purpose, and cells were detached from the wells with trypsin-EDTA and recombined with the supernatant. Cell suspension was centrifuged at 1000× g at room temperature (RT) for 5 min, and cells were washed with PBS supplemented with 5% FCS. Afterwards, cells were stained with annexin V FITC (20 µL) (ImmunoTool, Friesoythe, Germany) and 1:2000 eBioscience™ Fixable Viability Dye eFluor™ 660 (Thermo Fisher Scientific, Karlsruhe, Germany) in 100 µL 1× annexin V staining buffer (10× annexin V staining buffer: 0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 (pH 7.5)) for 30 min at RT in the dark. Further 150 µL of staining buffer were added and the supernatant was removed after centrifugation. Cells were fixed with 500 µL PBS containing 4% formaldehyde and 1.25 mM CaCl 2 for 20 min at RT in the dark. Cells were finally resuspended in 150 µL staining buffer and stored at 4 • C until measurement with the FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany), followed by the analysis with FlowJo software (v.10; Flow Jo, Ashland, OR, USA). Three gates were set as the following: annexin V positive cells (early apoptotic cells) and live/dead marker positive cells (cells with a membrane rupture tending to necrosis).

Recording of Cytopathic Effect of Infected Cells
To record the CPE at different time points, cells were visualized with Canon (EOS 500D) by light microscopy (Axiovert 40C, ZEISS, Jena, Germany) with a 10× magnification.

Lactate Dehydrogenase (LDH) Assay
The lactate dehydrogenase assay (CellBiolabs, San Diego, CA, USA) was used to measure the cell cytotoxicity and was used according to the manufacturer's instructions. Cells were infected as described previously, and 90 µL of the supernatant was mixed with 10 µL of the LDH cytotoxicity reagent in a 96-well plate. This plate was incubated at 37 • C and 5% CO 2 for 30 min, and the OD 450nm was measured on a Spectromax M2 Instrument (Molecular Devices, Munich, Germany). Triton X-100 used according to the manufacturer's instructions served as a positive control.

Primary S. aureus 3878 SCV Infection Provokes a Cytopathic Effect in Presence of IV
Cell death mechanisms induced by S. aureus or IV alone are very well investigated and described [28][29][30][31][32][33]. With respect to IV and S. aureus super-infection, we recently were able to show a S. aureus-mediated switch from IV-induced apoptosis to necrosis [27]. It is known that IV infection paves the path for secondary bacterial infection, resulting in enhanced pathogen-load [15,34], cytokine expression [35,36], and cell death [27]. Since S. aureus often persist in humans without any harm, we aimed to investigate the effects of colonizing S. aureus SCVs on secondary IV super-infection.
In a first set of experiments, we focused on the cell morphology of A549 human lung epithelial cells in absence and presence of S. aureus 3878 SCV and IV. For this reason, A549 human lung epithelial cells were infected with S. aureus 3878 SCV , which is a well described SCV patient isolate [10,37], for 24 h followed by infection with IV strain A/Puerto Rico/8/34 (PR8-M; H1N1) for the indicated points in time. The morphology of single-and super-infected cells was analyzed by light microscopy in comparison to uninfected control (mock) (Figure 1). While the cell monolayer is still intact in un-, single-, and super-infected cells up to 32 hpvi (hours post-viral infection), first changes in the cell morphology were visible 48 hpvi in single virus-infected and super-infected cells. Pictures of virus-infected cells showed a less confluent cell monolayer compared to uninfected cells, and in super-infected samples a clear cytopathic effect was observed, indicated by cell monolayer disruption and floating cells. To be able to ascribe these findings to the SCV phenotype, we additionally specified the pathological difference between S. aureus wildtype phenotype and SCV phenotype (S. aureus 3878 WT and S. aureus 3878 SCV ) by infecting A549 human lung epithelial cells. Cell morphology was monitored by light-microscopy (Supplementary Figure S1A  These results point to an altered cell culture environment and/or cellular signaling upon super-infection with S. aureus 3878SCV and IV, which could be triggered by increased pathogen load or cell intrinsic signaling changes in presence of both pathogens.

Primary Infection with S. aureus 3878SCV Followed by IV Infection Had No Impact on Bacterial or Viral Titers
First, we analyzed whether the observed cytotoxicity of co-infected A549 human lung epithelial cells with S. aureus 3878SCV and IV was due to increased pathogen load. For this, we infected A549 cells with S. aureus 3878SCV for 24 h and super-infected with two different IV strains for the indicated points in time to determine the amount of plaque forming units (PFU) or colony forming units (CFU) of viruses or bacteria, respectively ( Figure 2).
In general, titers of IV and SCVs increased with time, but neither viral (  These results point to an altered cell culture environment and/or cellular signaling upon super-infection with S. aureus 3878 SCV and IV, which could be triggered by increased pathogen load or cell intrinsic signaling changes in presence of both pathogens.

Primary Infection with S. aureus 3878 SCV Followed by IV Infection Had No Impact on Bacterial or Viral Titers
First, we analyzed whether the observed cytotoxicity of co-infected A549 human lung epithelial cells with S. aureus 3878 SCV and IV was due to increased pathogen load. For this, we infected A549 cells with S. aureus 3878 SCV for 24 h and super-infected with two different IV strains for the indicated points in time to determine the amount of plaque forming units (PFU) or colony forming units (CFU) of viruses or bacteria, respectively ( Figure 2).
In general, titers of IV and SCVs increased with time, but neither viral ( Thus, these data indicate that the disruption of the cell monolayer upon super-infection is not induced by increased amounts of pathogens but by a different mechanism that is altered by the presence of both pathogens. Thus, these data indicate that the disruption of the cell monolayer upon super-infection is not induced by increased amounts of pathogens but by a different mechanism that is altered by the presence of both pathogens.

Pro-Inflammatory Gene Expression Is Highly Upregulated after Super-Infection of S. aureus 3878SCV and IV
Given the observation that super-infection of S. aureus 3878SCV and IV PR8-M induced a cytopathic effect (Figure 1), which was not caused by increased pathogen load (Figure 2), we aimed to elucidate if changes of cell intrinsic signaling and inflammatory gene expression might be responsible for this phenomenon. We analyzed the gene expression of 84 different genes, involved in different signaling cascades by use of a RT 2 profiler Array (Qiagen, Hilden, Germany) in a single experiment to gain a first insight in the complexity of cellular signaling (Figure 3). This enables a quick analysis of expression levels of different genes that are organized by their function to be able to limit the amount of genes, altering the cell intrinsic signaling. Here, we used the anti-viral immune response panel, including pattern recognition receptors (PRRs), cytokines, and chemokines

Pro-Inflammatory Gene Expression Is Highly Upregulated after Super-Infection of S. aureus 3878 SCV and IV
Given the observation that super-infection of S. aureus 3878 SCV and IV PR8-M induced a cytopathic effect (Figure 1), which was not caused by increased pathogen load (Figure 2), we aimed to elucidate if changes of cell intrinsic signaling and inflammatory gene expression might be responsible for this phenomenon. We analyzed the gene expression of 84 different genes, involved in different signaling cascades by use of a RT 2 profiler Array (Qiagen, Hilden, Germany) in a single experiment to gain a first insight in the complexity of cellular signaling (Figure 3). This enables a quick analysis of expression levels of different genes that are organized by their function to be able to limit the amount of genes, altering the cell intrinsic signaling. Here, we used the anti-viral immune response panel, including pattern recognition receptors (PRRs), cytokines, and chemokines involved in pathogen recognition and immune responses. The bioinformatic analysis is based on conventional ct-values and was performed with the recommended GeneGlobe online software [26]. A clustergram was generated to visually illustrate all up-and downregulated genes that were analyzed ( Figure 3A). To further interpret the results of the RT 2 profiler Array, we did an in silico clustering of the upregulated genes of the array that were highly upregulated (difference of an n-fold of 2) in S. aureus 3878 SCV and PR8-M super-infected cells compared to single-infected cells (APOBEC3G, CASP1, CASP10, CCL3, CCL5, CD40, CD80, CTSS,  CXCL10, CXCL11, CYLD, IL1B, IL6, CXCL8, MEFV, TLR3, TNF, B2M) (see Supplementary Table S1), with respect to their linkage to specific signaling pathways ( Figure 3B) by using the Kyoto Encyclopedia of Genes and Genomes mapper (KEGG mapper). KEGG mapper is a database resource of collected information about pathways and the involved genes representing a pool of molecular interactions, reactions, and their relation to each other [38][39][40]. Down-regulated genes were excluded, as the gene expressions were negligible (Supplementary Table S1). Besides gene clusters connected to expected PRR pathways including TLR-(11 genes involved, out of the 18 highly upregulated genes comparing co-and single-infected cells identified (11/18), NLR-(7/18), TNFR-(6/18), RLR-(5/18), and NFκB-(5/18) signaling pathways ( Figure 3B), we identified gene clusters belonging to two cell death mechanisms, necroptosis (5/18) and apoptosis (3/18). Furthermore, we identified genes involved in the IL-17 (5/18) and c-type lectin (5/18) signaling pathways. To further classify the activated genes leading to the observed cytopathic effect on human lung epithelial cells, we searched for a specific induction pattern in which super-infected cells led to upregulated genes. We, therefore, compared all upregulated genes of single-infected to super-infected samples in a Venn diagram ( Figure 3C,D). We identified 11 genes that were induced in all three infection-scenarios compared to uninfected cells and 9 genes that were upregulated in super-infected cells only. We also compared the upregulated genes for super-infection with IV Panama. Here, all infection scenarios shared the induction of 12 genes, where 7 genes were exclusively induced by the super-infection of S. aureus 3878 SCV and IV Panama. The upregulated genes of the Venn diagram are listed in Table S2A,B. With respect to the mRNA expression levels shown in Supplementary Table S1 and the cytopathic effect observed in super-infected cells (Figure 1), an induction of pro-inflammatory immune response can be concluded, which was further visualized by graphs, exhibiting the gene expression of the highly upregulated genes ( Figure 3E).
To confirm an increased pro-inflammatory status of the human lung epithelial cells upon super-infection, we analyzed the mRNA expression of different representative pro-inflammatory cytokines and chemokines (IL-6, IL-8, TNFα, IL-1β, and IFN-γ) in detail ( Figure 4A-E). Furthermore, we analyzed the mRNA expression of molecules that are involved in the induction of the type-I-IFN signaling (RIG-I, IFN-β, MxA, and OAS1) ( Figure 4F-I), since it was described that IV-induced type-I-IFN signaling had an impact on bacterial infections [41].
In S. aureus 3878 SCV colonized cells subsequently infected with IV PR8-M the mRNA expression of IL-6, IL-8, TNFα, and IL-1β 32 hpvi was induced if compared to uninfected cells or single-infected cells ( Figure 4A-D) and IFN-γ showed the same tendency ( Figure 4E). Single-infection of S. aureus 3878 SCV or IV PR8-M resulted in no significant induction of the mRNA expression 8 hpvi, 24 hpvi, or 32 hpvi, except for IL-8, which was significantly induced 8 hpvi in bacteria single-infected cells ( Figure 4B). Nevertheless, this induction was abolished over time. Genes, encoding key proteins involved in the recognition, and induction of type-I-IFN signaling were upregulated in IV PR8-M infected cells 8 hpvi (IFN-β by tendency) (Figure 4G     To analyze whether the induction of mRNA synthesis of pro-inflammatory genes could also be detected on protein level and to get further insights into the cell intrinsic innate immune status of super-infected A549 cells, the protein expression of exemplary cytokines and chemokines was monitored by FACS analysis (Figure 5A-H). Remarkably, FACS analysis verified the increased pro-inflammatory response of A549 human lung epithelial cells for the secretion of representative factors. In super-infected cells, protein levels of IL-6, RANTES (CCL5), IP-10, and I-TAC were significantly induced compared to uninfected or single-infected cells with either S. aureus 3878 SCV or IV PR8-M ( Figure 5A  These results emphasize the enhanced cell intrinsic pro-inflammatory status of the super-infected cells. We could confirm these results by the use of IV strain Panama, verifying a viral strain independent effect (Supplementary Figure S3A-H). Based on these results and due to the fact that the induction of pro-inflammatory cytokines and chemokines is mainly driven by NFκB activation, we hypothesized an induction of the pro-inflammatory response via specific PRRs, resulting in the activation of the NFκB-signaling cascade [42]. To confirm the induction of NFκB-signaling we transfected A549 cells with an artificial NFκB promoter-dependent luciferase reporter plasmid prior to super-infection with S. aureus 3878 SCV and subsequent IV PR8-M infection. An increase of NFκB activation was observed in super-infected cells compared to uninfected and IV PR8-M-infected cells, while S. aureus 3878 SCV infection only resulted in an increase of NFκB activation by trend ( Figure 5I). This induction pattern was also confirmed in cells super-infected with IV Panama (Supplementary Figure S3I).
The induction of pro-inflammatory responses via NFκB in epithelial cells after pathogen exposure can be triggered by both pathogens through different factors and their corresponding receptors [43][44][45], which were further analyzed. To exclude specific pathogen-mediated interference with cellular factors due to differences in protein expression, such as virulence factors or surface proteins, viral RNA (vRNA) and bacterial lipoteichonic acid (LTA, Invivogen, San Diego, CA, USA) were used as pathogen specific molecular stimuli. In human lung epithelial cells vRNA is mainly recognized by RIG-I, leading to a strong induction of the type-I-IFN-signaling cascade [46], while LTA is mainly recognized by TLR-2 [47]. We investigated mRNA expression of IL-6, IL-8, and TNFα after stimulating A549 cells with vRNA and LTA ( Figure 5J-L). The results matched our findings obtained from super-infected cells, since significant enhancement of mRNA expression of IL-6, IL-8, and TNFα was observed in presence of both stimuli. Artificial effects caused by RNA transfection could be excluded due to equal cytokine mRNA expression induced by cellular RNA (cRNA) and cRNA + LTA stimulated cells. Stimulation with vRNA tended to induce the expression of IL-6, IL-8, and TNFα, which, however, was not significant compared to unstimulated cells.
Overall, these data suggest an induction of pro-inflammatory gene expression responses through the detection of bacterial and viral components via the pathogen-associated molecular pattern receptors (PAMP) RIG-I and TLR-2, followed by the induction of NFκB. To exclude bacterial strain-specific effects, another SCV strain (S. aureus 814 SCV ) was used to determine pathogen loads and pro-inflammatory gene expression in IV super-infection (Supplementary Figure S4). While neither viral titers nor intraand extracellular bacterial load were increased in presence of both pathogens, pro-inflammatory cytokine expression was enhanced, verifying the former observations.

S. aureus 3878 SCV Provoke Enhanced Necrotic Cell Death in Presence of IV Infection
The observed disruption of the cell monolayer ( Figure 1) could be induced by a variety of mechanisms. Besides the involvement of pro-inflammatory cytokines in the innate immune response, these factors are also involved in the induction of cell death mechanisms, like apoptosis and necrosis. As the results shown in Figure 3 indicate, an upregulation of pro-inflammatory cytokines, the cell death mechanisms might be triggered by TLRs or cell death receptors through PAMPs or cytokines, like TNFα, among others [48][49][50]. Therefore, we further investigated the induction of apoptosis and necrosis, correlating to the cell death mechanisms identified in the RT 2 Profiler Array analysis ( Figure 3B).
As the results of the LDH assay led to the hypothesis of an induced necrotic cell death mechanism, we performed FACS analysis to determine the number of early apoptotic cells by detecting phosphatidylserine which switches to the cells' surface in early apoptotic cells and can be labeled with annexin V. Cells with a membrane rupture tending to necrosis were detected by using a viability marker comparable to 7-aminoactinomycin D and propidium iodide staining. Therefore, we performed the infection up to 44 hpvi to be able to still distinguish between early apoptosis and necrotic-like cells and stained the cells accordingly. The amount of necrotic cells significantly increased comparing un-or single-infected with super-infected cells, probably indicating necrosis ( Figure 6A). Furthermore, the amount of apoptotic cells was significantly higher in IV-infected cells compared to un-, bacteria-, or super-infected cells 44 hpvi ( Figure 6B).

Microorganisms 2020, 8, x FOR PEER REVIEW
14 of 20 ( Figure 6A). Furthermore, the amount of apoptotic cells was significantly higher in IV-infected cells compared to un-, bacteria-, or super-infected cells 44 hpvi ( Figure 6B).  As cell death mechanisms like necrosis can be further defined in specific mechanisms and to compare our findings to previously described inductions of cell death mechanisms upon infection with SCV [51] or in co-infection scenarios [27], we performed western blot analysis to be able to differentiate between necroptosis and apoptosis.
Necroptosis is an inflammatory programmed form of necrosis, which was already described in a recent publication reporting its induction by S. aureus SCV in single-infected human primary keratinocytes [51]. Necroptosis is induced via a receptor-interacting protein (RIP) kinase-mediated activation, resulting in the phosphorylation and oligomerization of mixed lineage kinase domain like pseudokinase (MLKL) and pore formation, leading to the release of inflammatory cytokines. To distinguish necroptosis from apoptosis induction, we monitored the induction of phosphorylated MLKL and PARP cleavage, which are indications for both cell death mechanisms [52]. We infected A549 human lung epithelial cells with S. aureus 3878 SCV for 24 h, followed by IV infection with PR8-M for 32 h (Figure 6C-E).
In super-infected cells, induction of pMLKL 32 hpvi was observed in comparison to uninfected, bacteria-, or virus single-infected cells, respectively. PARP cleavage was more likely to be induced in IV PR8-M-infected cells, and was slightly decreased in super-infected samples 32 hpvi ( Figure 6C). However, the densitometrical analysis of three independent experiments could only confirm a trend of activated MLKL due to induced phosphorylation in super-infected cells ( Figure 6D), whereas an induction of apoptosis upon IV infection could be verified ( Figure 6E). Additionally, pyroptosis as another form of regulated necrotic cell death mechanism, which is activated via the induction of the inflammasome resulting in the cleavage of gasdermin D, could not be detected by cleaved gasdermin D (Supplementary Figure S5). The original blots are shown in the Supplementary Figure S6.
Thus, our results indicate a necrotic cell death induction, most likely induced by increased pro-inflammatory gene expression response after super-infection with S. aureus 3878 SCV , followed by secondary IV infection with PR8-M and Panama.

Discussion
The first occurrence of persisting bacteria or SCVs was already described about 100 years ago [53]. Even though they are known for such a long time, not many studies were undertaken to elucidate their impact on cellular responses or their impact on additional infections with other pathogens. Our aim was to investigate the interaction of S. aureus SCVs with a subsequent IV infection in respect to epithelial cell responses, which built the first cellular barrier for pathogens in the lung. Here, we demonstrate that invasive S. aureus SCVs do have an impact on the cell intrinsic response in human lung epithelial cells, as indicated by highly secreted pro-inflammatory cytokines and chemokines ( Figure 5 and Supplementary Figure S3) and, furthermore, an induction of necrotic cell death of super-infected compared to single-infected cells ( Figure 6). This was somehow surprising, since the majority of SCVs are not described to significantly induce cell intrinsic responses, due to decreased secretion of virulence factors [54], a feature that would match their dormant status. In particular, not much is known about the impact of SCVs on lung tissue responses and nothing so far about their impact on a secondary IV infection. In this study, we were able to show a cytopathic effect accompanied by increased pro-inflammatory cytokine and chemokine release and necrotic cell death through colonizing S. aureus 3878 SCV and different IV strains, such as PR8-M and Panama.
Typically, super-infections with pathogenic S. aureus strains and IV led to increased pathogen loads accompanied with the induction of pro-inflammatory responses [13,35]. However, this could not be confirmed within the present study in the SCV and IV super-infection scenario. It was shown previously that super-infection with pathogenic S. aureus leads to the enhancement of viral titers due to the inhibition of STAT1 and STAT2 dimerization, resulting in decreased production of anti-viral factors [15]. This inhibitory effect could be excluded since mRNA expression of RIG-I, IFNβ, MxA, or OAS1 in super-infected cells compared to IV-infected cells were not altered ( Figure 4 and Supplementary Figure S2). Based on these results, we could exclude an effect of the anti-viral response and the involvement of an altered pathogen load. Nevertheless, we identified a clear induction of pro-inflammatory cytokines and chemokines in human lung epithelial cells. Besides the attraction of immune cells and the induction of an anti-pathogen status of the cell, pro-inflammatory cytokines induce a stress response leading to the induction of cell death mechanisms via TLRs or death receptors [55][56][57].
To proof the impact of two main cell death mechanisms we performed FACS analysis to monitor early apoptotic and necrotic-like cells. Correlating to the LDH assays, we could confirm an increase in necrosis during super-infection ( Figure 6A). Besides, the amount of apoptotic cells was decreased in super-compared to IV-infected cells. Further specifications of cell-death mechanisms by western blot analysis revealed the tendency for an increase of phosphorylated MLKL in super-infected cells, giving the hit of probably induced necroptosis. Concomitantly, IV-induced PARP cleavage was reduced in super-infected cells compared to IV PR8-M-infected cells by trend.
Interestingly, there are two different mechanisms described, how pathogenic S. aureus and S. aureus SCVs are able to induce necroptosis [27,51]. During the critical phase of S. aureus infection the virulence factor agr is induced [8], resulting in possible secretion of different toxins, which induces necroptosis [27,33]. In SCV-infected keratinocytes, necroptosis was driven by the activation of glycolysis [51]. S. aureus adopts its whole metabolism to persist within the host. The metabolic changes of S. aureus were already described elsewhere [58]. As the utilization of the tricarboxylic acid cycle for the host cell and the persisting bacteria is decreased, the glycolysis is stronger induced to generate adenosine triphosphate (ATP). As we observed a disruption of cell monolayer and a possible induction of phosphorylated MLKL upon super-infection, we linked our findings more to necroptosis (Figures 1 and 6). Even though S. aureus-induced necroptosis might be independent of TLR stimulation [59], our data indicate a synergistic effect of S. aureus 3878 SCV and IV inducing cell death, which can be related to TLR2-and RIG-I-mediated pro-inflammatory response induction. In addition, our data show that the superinfection could be imitated with the stimuli LTA and vRNA. This underlines that the initial induction of the pro-inflammatory response and the subsequent cell death must be different from that of pathogenic bacterial strains that induce cell death much more quickly. In case of SCV, this indicates a lower virulence probably due to the decreased secretion of virulence factors. Nonetheless, dormant SCVs can work synergistically and affect the virus-induced immune response. As we performed pure ligand experiments, inhibitory effects of molecules of this pro-inflammatory cell intrinsic response is supposed to trigger cellular stress in the form of reactive oxygen species [60].
So far, the impact of S. aureus SCVs with subsequent IV infection had not been investigated. Interestingly, we could give first insights in this super-infection scenario and unravel one extraordinary role of a SCV patients' isolate S. aureus 3878 SCV with subsequent IV infection. We observed an induction of pro-inflammatory cytokines and chemokines, which underlines the severity of the coincident occurrence of S. aureus SCVs and IV. These data point to a cross-interaction of necrotic cell death and pro-inflammatory cell intrinsic response, as the pathogens alone can induce an inflammatory response through PAMPs and secreted cell damage-associated molecular patterns (DAMPs). Upon necrotic cell death induction, further pro-inflammatory responses are induced via DAMP receptors [50], leading to an enhancement of pro-inflammatory cytokines and chemokines seen on transcriptional and translational level.
In summary, we were able to show that persistent S. aureus SCV and subsequent IV infection affects cell-internal immune response by inducing the release of pro-inflammatory cytokines and chemokines, resulting in cell death induction.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/12/1998/s1, Table S1: List of ct-values analyzed by GeneGlobe online software of the RT 2 Profiler Array plate. Table S2: (A,B) Listed gene names of the venn diagrams shown in Figure 3. Figure S1: Wildtype phenotype S. aureus 3878 is more virulent compared to S. aureus 3878 SCV , but S. aureus 3878 SCV induces LDH release upon super-infection with PR8-M. Figure S2: Pro-inflammatory cytokines and chemokines are enhanced after S. aureus 3878 SCV colonization and subsequent IV Panama infection. Figure S3: Secretion of the pro-inflammatory cytokines and chemokines are enhanced after S. aureus 3878 SCV colonization and subsequent IV Panama infection regulated by TLR2-and RIG-I-mediated NFκB promoter activation. Figure S4: Pathogen load and pro-inflammatory cytokines and chemokines are enhanced after super-infection with the SCV strain S. aureus 814 SCV . Figure S5: S. aureus 3878 SCV colonization and subsequent influenza virus infection has no effect on the induction of pyroptosis. Figure S6: Original western blots of Figure 6C