This information is current as

of October 20, 2016.

Phagocytosis of Staphylococcus aureus by

Human Neutrophils Prevents Macrophage
Efferocytosis and Induces Programmed
Necrosis
Mallary C. Greenlee-Wacker, Kevin M. Rigby, Scott D.
Kobayashi, Adeline R. Porter, Frank R. DeLeo and William
M. Nauseef

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J Immunol 2014; 192:4709-4717; Prepublished online 11

April 2014;
doi: 10.4049/jimmunol.1302692
http://www.jimmunol.org/content/192/10/4709

The Journal of Immunology

Phagocytosis of Staphylococcus aureus by Human Neutrophils

Prevents Macrophage Efferocytosis and Induces Programmed
Necrosis
Mallary C. Greenlee-Wacker,*, Kevin M. Rigby, Scott D. Kobayashi,x Adeline R. Porter,x
Frank R. DeLeo,x and William M. Nauseef*,

ommunity-associated methicillin-resistant Staphylococcus

aureus (CA-MRSA) pose a significant threat to human
health, and infection can manifest as a variety of diseases,
including pneumonia, necrotizing skin infections, endocarditis,
arthritis, and sepsis (reviewed in Ref. 1). Human polymorphonuclear leukocytes (PMN) or neutrophils are the first responders in

*Inflammation Program, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Veterans Administration Medical Center, Iowa City, IA 52240;

Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine,

University of Iowa, Veterans Administration Medical Center, Iowa City, IA 52240;

Molecular Medicine Program, University of Utah School of Medicine, Salt Lake

City, UT 84112; and xLaboratory of Human Bacterial Pathogenesis, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
Received for publication October 4, 2013. Accepted for publication March 14, 2014.
This work was supported by T32 Training Grant 2T32AI007260-26A1 from the
University of Iowa and the National Institutes of Health (to M.C.G.-W.), National
Institutes of Health Grants AI70958 and AI044642 (to W.M.N.), and the Intramural
Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to F.R.D.). The Nauseef laboratory was supported by
a merit review award and use of facilities at the Iowa City Department of Veterans
Affairs Medical Center, Iowa City, IA.
Address correspondence and reprint requests to Dr. William M. Nauseef, Inflammation Program and Department of Medicine, Roy J. and Lucille A. Carver College of
Medicine, University of Iowa, D160 MTF, 2501 Crosspark Road, Coralville, IA
52241. E-mail address: william-nauseef@uiowa.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: CA-MRSA, community-associated methicillin-resistant Staphylococcus aureus; CTFR, CellTrace Far Red DDAO-SE; HBSS++, HBSS
with divalent cation; HSA, human serum albumin; IL-1RA, IL-1 receptor antagonist;
MFI, mean fluorescence intensity; MOI, multiplicity of infection; Nec-1, necrostatin-1;
PI, propidium iodide; PMN, polymorphonuclear leukocyte; PMN-SA, PMN harboring viable CA-MRSA strain USA300; PS, phosphatidylserine; RIP-1, receptorinteracting protein 1; sGFP, superfolded GFP.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302692

most bacterial infections, including those due to S. aureus (reviewed in Ref. 2). In general, PMN readily phagocytose bacteria
and use a variety of agents stored in granules and PMN-generated
HOCl to kill ingested microbes (3, 4). However, in the case of
S. aureus, 1550% of the initial ingested inoculum survives within
the PMN phagosome (57), and PMN containing viable S. aureus
initially exhibit some features consistent with accelerated apoptosis but 6 h following phagocytosis abruptly undergo lysis (7).
How the presence of viable bacteria within PMN directs the fate
of the phagocyte and the extent to which the induced changes in
PMN contribute to disease pathogenesis are unknown. Generally,
macrophages ingest spent or apoptotic PMN in a process termed
efferocytosis, thereby clearing damaged or dead host cells and
microbes to restore tissue to an uninflamed state. The interaction
of human macrophages and PMN harboring viable S. aureus has
not been explored.
Reasoning that events early in the interaction between innate
immune cells and invading S. aureus likely influence the subsequent course of disease, we examined the roles played by PMN
harboring viable CA-MRSA strain USA300 (PMN-SA) and macrophages in controlling or contributing to infection. Using human
PMN and a pulsed fieldtype USA300 strain, we demonstrate that
PMN-SA initially exhibited signs of apoptosis, including surface
exposure of phosphatidylserine (PS) and mitochondrial membrane
depolarization, but failed to activate caspase-3, -8, -9, and -2.
PMN-SA increased expression of the dont eat me signal CD47,
resulting in decreased uptake by macrophages and a secondary
skewing of the cytokine profile. Cytoplasmic levels of proliferating cell nuclear Ag (PCNA) were sustained in PMN-SA and the
eventual lysis of PMN-SA was blocked by the receptor-interacting

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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pose a significant threat to human health. Polymorphonuclear leukocytes (PMN) are the first responders during staphylococcal infection, but 1550% of the initial ingested
inoculum survives within the PMN phagosome and likely contributes directly or indirectly to disease pathogenesis. We hypothesize
that surviving intracellular CA-MRSA undermine effective phagocyte-mediated defense by causing a decrease in macrophage
uptake of PMN containing viable S. aureus and by promoting PMN lysis. In support of this hypothesis, PMN harboring viable CAMRSA strain USA300 (PMN-SA) upregulated the dont eat me signal CD47, remained bound to the surface, and were
inefficiently ingested by macrophages. In addition, coculture with PMN-SA altered the macrophage phenotype. Compared to
macrophages fed USA300 alone, macrophages challenged with PMN-SA produced more IL-8 and less IL-1 receptor antagonist,
TNF-a, activated caspase-1, and IL-1b. Although they exhibited some features of apoptosis within 3 h following ingestion of
S. aureus, including phosphatidylserine exposure and mitochondrial membrane depolarization, PMN-SA had sustained levels of
proliferating cell nuclear Ag expression, absence of caspase activation, and underwent lysis within 6 h following phagocytosis.
PMN lysis was dependent on receptor-interacting protein 1, suggesting that PMN-SA underwent programmed necrosis or
necroptosis. These data are the first demonstration, to our knowledge, that bacteria can promote sustained expression of proliferating cell nuclear Ag and that human PMN undergo necroptosis. Together, these findings demonstrate that S. aureus surviving
within PMN undermine the innate immune response and may provide insight into the pathogenesis of S. aureus disease. The
Journal of Immunology, 2014, 192: 47094717.

4710

S. AUREUS PROMOTES RIP-1DEPENDENT CELL DEATH OF NEUTROPHILS

protein 1 (RIP-1) kinase inhibitor necrostatin-1 (Nec-1). Taken

together, our findings demonstrate the complex manner in which
viable S. aureus undermine early phagocyte-mediated defenses
and promote persistent infection and inflammation.

Materials and Methods

Reagents, Abs, and cells

S. aureus culture
Assays were performed using the USA300 LAC wild-type strain as described previously (5). Nonpathogenic RN6390 were obtained from
Dr. Alex Horswill (Department of Microbiology, University of Iowa, Iowa
City, IA). To obtain a USA300 stain expressing superfolded GFP (sGFP),
a LAC-derivative strain that has been cured of the cryptic plasmid pUSA03
[AH1263 (8)] was transduced with plasmid pCM29 (9) and selected with
10 mg/ml chloramphenicol. The AH1263 strain was also used for the
Luminex and ELISA assays. All USA300 strains are referred throughout as
USA300 or SA. S. aureus strains were grown in tryptic soy broth overnight
at 37C with shaking at 180 rpm. Bacteria were then diluted (OD550 0.05)
in TBS containing 0.1% HSA and subcultured at 37C with shaking at
180 rpm until early-logarithmic phase (OD550 0.10.2). Bacteria were
then suspended in HBSS with divalent cations containing 20 mM HEPES
or RPMI 1640 medium containing 10 mM HEPES and opsonized with
human serum (10% pooled or 50% autologous serum) for use in the assay.

PMN isolation
PMN were isolated from normal healthy volunteers and purified from
venous blood (as described in Ref. 10). Written consent was obtained from
each volunteer in accordance with a protocol approved by the Institutional
Review Board for Human Subjects at the University of Iowa or the National Institute of Allergy and Infectious Diseases, National Institutes of
Health. Briefly, heparinized blood was collected and PMN were isolated
following sedimentation with 3% dextran, separation on Ficoll Hypaque
gradient, and hypotonic lysis of RBCs. PMN were then kept on ice in
HBSS without divalent cations or RPMI 1640 medium containing 10 mM
HEPES and counted. This technique resulted in 9399% PMN purity as
measured by staining with HEMA-3 and analysis by light microscopy or as
determined by flow cytometry.

PMN phagocytosis assay and flow cytometry

PMN phagocytosis assays were performed as described previously (9).
Briefly, bacteria opsonized with pooled human serum were mixed with
PMN at the desired multiplicity of infection (MOI) in a 5-ml round-bottom
polypropylene tube and tumbled for 10 min at 37C. Following 10 min

Macrophage phagocytosis assay

Phagocytosis assays were performed (as described in Ref. 13) with the
following modifications. Macrophages were obtained (as described in Ref.
14). After 68 d of culture in Teflon jars with RPMI 1640 containing
20% autologous serum, cells were plated overnight in RPMI 1640 containing 10% pooled human serum. Macrophages adhered to plastic, and nonadherent cells were washed away prior to the start of the assay. Apoptotic
PMN (hereafter referred to as Aged PMN) were generated by culturing
freshly isolated PMN in 10% FBS in DMEM for 1824 h. Aged and
freshly isolated PMN were labeled with CTFR at a 1:1000 dilution for
20 min at 37C, washed with HBSS++ containing 0.1% HSA, and resuspended at 1 3 107 cells/ml. Following CTFR labeling, freshly isolated
PMN were fed USA300-expressing sGFP for 10 min, spun at 500 3 g to
remove extracellular bacteria, and incubated for 60 min at 37C while
tumbling. PMN containing viable bacteria were defined as PMN-SA.
PMN, Aged PMN, and PMN-SA were then opsonized with 10% pooled
human serum, centrifuged at 500 3 g, and resuspended in phagocytosis
buffer (RPMI 1640 containing 5 mM CaCl2). Macrophages were cocultured with PMN, Aged PMN, or PMN-SA at a ratio of 1:20 (macrophage/
target) in four-well Lab Tek chamber slides. Phagocytosis was synchronized by centrifugation at 500 3 g for 5 min at 4C, and cells were incubated for indicated time points at 37C in 5% CO2. Nonadherent cells
were washed away with PBS, and the remaining cells were harvested by
trypsinization. Cells were washed three times with FACS buffer and analyzed using an Accuri flow cytometer (BD Biosciences). Each data set was
first gated on CD14+ macrophages. Percent internalized was calculated as
(CTFR+CD152 cells/total CD14+ gated cells 3 100), and percent bound
was calculated as (CTFR+CD15+ cells/total CD14+ gated cells 3 100).
Three populations of CD14+ macrophages fed PMN-SA were assessed for
the presence of GFP+ USA300: CTFR2 (no PMN associated), those with
bound PMN, and those with internalized PMN. USA300 association was
calculated as (GFP+CD14+/total CD14+ macrophages) for each population
of cells. For CD47 blocking experiments, the anti-CD47 B6H12.2 hybridoma cell line was obtained from American Type Culture Collection
and grown in IMDM containing 20% low IgG heat-inactivated FBS (Life
Technologies, Grand Island, NY). Hybridoma cultures were grown for 5 d
postconfluence, IgG was purified using Gamma-Blind Sepharose beads
according to the manufacturers instructions (GE Healthcare). F(ab)2
fragments were subsequently generated with pepsin digestion and purified
(as described in Ref. 15). Mouse IgG F(ab)2 isotype control was obtained
from Thermo Scientific. Cells were washed twice with FACS buffer
(HBSS++, 0.2% HSA, and 0.2% sodium azide) and incubated with Abs for
30 min on ice with gentle shaking every 10 min.

Luminex assays and ELISA

For measuring cytokines by Luminex or ELISA the following modifications
were made to the protocol. PMN were not stained, and macrophages were
fed PMN-SA at a 1:5 and 1:15 ratio or USA300 at an MOI of 1:1 in a 24-well
cell-culture plate. After 1 h of phagocytosis, uningested cells or bacteria
were washed away, and RPMI 1640 was added to each well. After 6 or 12 h,
supernatants were collected, centrifuged at 20,000 3 g to remove cell
debris, and frozen. Human cytokine 10-plex, including GM-CSF, IL-1b,
IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-g, and TNF-a, was purchased
from Invitrogen. Supernatants were minimally diluted 1:10, and the assay
was performed according to the manufacturers protocol and analyzed

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All reagents were purchased from Fisher Scientific (Pittsburgh, PA) unless
otherwise indicated. Clinical grade dextran T500 (Pharmacosmos, Holbaek,
Denmark), Ficoll-Hypaque PLUS (GE Healthcare, Piscataway, NJ), sterile
endotoxin-free water, and 0.9% sterile endotoxin-free sodium chloride
(Baxter, Deerfield, IL) were used in neutrophil preparations. HEPES, HBSS
(with [HBSS++] and without divalent cations), and Dulbeccos PBS were
purchased from Mediatech (Manasssas, VA). A HEMA-3 staining kit was
obtained from Fisher Scientific, and 25% human serum albumin (HSA)
was purchased from Talecris Biotherapeutics (Raleigh, NC). Heparin was
purchased from APP Pharmaceuticals (Schaumburg, IL). Tryptic soy agar
and broth were obtained from BD Biosciences (San Jose, CA). Poly- Llysinecoated poly-prep slides, diphenyleneiodonium, and Nec-1 were purchased from Calbiochem/EMD Biosciences (La Jolla, CA) and SigmaAldrich (St. Louis, MO). RPMI 1640 was purchased from Lonza (Hopkinton,
MN). Hyclone FBS was purchased from Thermo Scientific (Waltham,
MA) and heat inactivated for 30 min at 56C. Annexin VFITC Apoptosis
Detection Kit and anti-human caspase-3 Ab were obtained from BioVision
(Mountain View, CA). JC-1 assay kit was purchased from Invitrogen
(Grand Island, NY). CellTrace Far Red DDAO-SE (CTFR; Invitrogen,
Grand Island, NY) was reconstituted to 2 mM in DMSO and stored at
220C. Anti-human CD15-PE and anti-human CD14-PE Cy5.5 (Beckman
Coulter, Fullerton, CA) were used for phagocytosis assays. Anti-human
CD47 and caspase inhibitor Q-VD-OPh were obtained from R&D Systems
(Minneapolis, MN), anti-human caspase-1 was obtained from Cell Signaling Technology (Danvers, MA), human Fc block was obtained from
eBioscience (San Diego, CA), anti-human PCNA (sc-56) was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA), mouse antib-actin
and mouse anti-Fas (human, activating) were purchased from Millipore
(Temecula, CA), and a Cytotoxicity Detection KitPLUS was purchased
from Roche (Indianapolis, IN).

of phagocytosis, cells were spun at 500 3 g for 5 min and remaining

extracellular bacteria were aspirated into waste. PMN containing bacteria
were then resuspended in HBSS with divalent cations containing 1% HSA.
At indicated time points, PMN were removed from the tube and assayed
for S. aureus viability or analyzed by flow cytometry. Bacterial viability
was routinely determined by PMN lysis using H2O brought to a pH of 11
with NaOH immediately before use, subsequent plating of serial dilutions
on tryptic soy agar plates overnight, and quantification of CFU (11).
Alternatively, loss of GFP fluorescence, which correlates with loss of
viability at late time points, was measured by flow cytometry using sGFPexpressing S. aureus (12). Mean fluorescence intensity (MFI) for each
sample was calculated as the geometric mean of the (GFP+ population) 3
(percent of cells gated). Percent viability and MFI were normalized to
values for diphenyleneiodonium-treated PMN. Under the same conditions,
PMN were assayed for viability by staining with Annexin VFITC and
propidium iodide (PI) or mitochondrial membrane depolarization by
staining with JC-1 according to manufacturers protocol. Expression of
CD47 was monitored following challenge as follows: 1 3 105 PMN were
incubated with Fc Block at room temperature for 15 min, stained for
CD47-PE at 4C for 30 min, washed twice with PBS, and analyzed by
flow cytometry.

The Journal of Immunology

using Bio-Rad Bioplex (Bio-Rad). GM-CSF, IL-2, IL-4, IL-5, IL-10, and
IFN-g were below the level of detection at the 1:10 dilution and therefore
were not measured in the assay. Abs for IL-1b ELISA were purchased
from BD Biosciences, the IL-1 receptor antagonist (IL-1RA) DuoSet
ELISA was purchased from R&D Systems, and IL-8, TNF-a, and IL-6
ELISA were purchased from eBioscience.

Immunoblot analyses

Caspase activity assays

To measure PMN caspase activity following phagocytosis of serum opsonized S. aureus, synchronized phagocytosis assays were performed as
described previously using 96-well culture plates (7). The bacteria-to-PMN
ratio was 10:1 for these experiments, and assay plates were incubated at
37C with 5% CO2 in a humidified incubator for up to 6 h. At the desired
time point, assay plates were centrifuged to pellet cells, culture supernatant
was removed, and cells were stored at 280C. PMN caspase activity was
determined using ApoAlert Caspase Assay Plates (Clontech, Mountain
View, CA) according to the manufacturers instructions.

Cytotoxicity assays
For experiments using a caspase inhibitor, PMN were pretreated with or
without 40 mM Nec-1 and/or 10 mM Q-VD-OPh for 20 min prior to
challenge with USA300 at an MOI of 1:1. Alternatively, PMN were pretreated with 0, 10, 50, 100, 200, or 500 mM Nec-1, and USA300 was added
to assays at an MOI of 10:1 as indicated. Lactate dehydrogenase (LDH)
activity was measured from supernatants in duplicate wells 6 h following
phagocytosis as reported previously (7).

Statistical analyses
One-way ANOVA and Dunnett or Tukey posttests were used to calculate
statistical significance. Unless indicated otherwise, p values were obtained
from the posttests used to correct for multiple comparisons (*p , 0.05).

Results
PMN fed S. aureus differentially regulate eat me and dont
eat me signals
Despite efficient uptake by PMN, 1550% of the ingested inoculum of USA300 or RN6390 remained viable 180 min after phagocytosis, as judged by two independent measures of bacterial viability
(12) (Supplemental Fig. 1). Furthermore, PMN acquire many of
the morphologic signs of apoptosis early on, including exposure of
PS and nuclear condensation, when challenged with USA300 at an
MOI of 10:1 (7). PMN fed USA300 or RN6390 at an MOI of 1:1
exhibited greater Annexin V staining than did control cells, and
this Annexin V staining increased over time (Fig. 1A). Importantly,
the majority of Annexin V+ cells remained negative for PI during
this time (Fig. 1A), indicating that the SA-laden PMN remained
viable. Increasing the MOI to 5:1 resulted in a similar percentage
of Annexin V+ cells, but a greater percentage of cells positive for

FIGURE 1. PMN challenged with S. aureus upregulate dont eat me signal CD47. PMN were in buffer alone or fed USA300 or RN6390 at an MOI of
1:1 and monitored for 60180 min. Cells were stained for Annexin VFITC and PI to distinguish apoptotic and necrotic cells, respectively (A). Shown on
the left axis and connected by a solid line are Annexin V+ cells, and on the right axis and connected by a dashed line are Annexin V+PI+ cells. Symbols
represent the mean of at least five experiments 6 SEM (A). PMN were stained after 60 min with JC-1 dye to measure mitochondrial depolarization and
analyzed by flow cytometry. p values were determined using repeated measures one-way ANOVA and Dunnett posttest [(B) n = 5 6 SEM]. PMN were
isolated and either aged for 1824 h (Aged) or challenged with USA300 at an MOI of 1:1 for 60 min. Cells were stained for CD47 and analyzed by flow
cytometry. Shown is a representative of three experiments (C) and average MFI from the five individual experiments 6 SEM (D). p values were determined
using one-way ANOVA and Tukey posttest. *p , 0.05 versus PMN, #p , 0.05 versus Aged PMN.

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For caspase-1 detection, macrophages were lysed (50 mM Tris-HCl [pH 8],
5 mM EDTA, 150 mM NaCl, and 1% Triton X-100 containing protease
inhibitor mixture of 0.5 mM benzamidine, 0.5 mM PMSF, 0.1 mg/ml
aprotinin, 0.1 mg/ml phosphoarmidone, 0.1 mg/ml tosyllysine chloromethylketone hydrochloride, 0.1 mg/ml tosylphenylalanyl chloromethyl ketone,
0.1 mg/ml 4-amidinophenylmethanesulfonyl fluoride, 0.1 mg/ml E-64,
0.05 mg/ml leupeptin, and 0.01 mg/ml pepstatin) at indicated time points,
proteins were resolved in a gradient SDS-PAGE gel, and transferred to
polyvinylidene difluoride membrane for immunoblotting. For caspase-3,
PCNA, and b-actin detection, PMN were lysed as previously described
(16). For immunoblotting, Fas stimulation was used as a positive control
for apoptosis using 500 ng/ml anti-Fas Ab. The following dilutions were
used for immunoblots: 1:1,000 anticaspase-1, 1:200 anticaspase-3, 1:200
anti-PCNA, and 1:20,000 antib-actin. The pixel intensity of bands in
immunoblots was measured using a phosphoimager (Typhoon 9410 Variable Mode Imager; GE Healthcare).

4711

4712

S. AUREUS PROMOTES RIP-1DEPENDENT CELL DEATH OF NEUTROPHILS

Macrophages readily bind but inefficiently ingest

S. aureusladen PMN
Typically, macrophages rapidly bind and ingest apoptotic cells.
However, we reasoned that the atypical phenotype of PMN-SA
might prompt an interaction with macrophages that differed from
that normally observed between macrophages and apoptotic PMN.
To explore the fate of PMN-SA with respect to efferocytosis, we
developed a flow cytometrybased assay to assess both binding
and internalization of PMN by macrophages. As expected, PMN
that underwent spontaneous apoptosis after being cultured for
24 h (Aged PMN) transiently bound to and were readily engulfed
by macrophages in a time-dependent fashion (Fig. 2A, 2B,
Supplemental Fig. 2A). Freshly isolated PMN bound to macrophages to a similar degree but were not internalized; internalization of Aged PMN was 4.8-fold greater than that of freshly isolated
PMN after 30 and 60 min of phagocytosis (*p , 0.05; Fig. 2B). The
interactions between PMN-SA and macrophages were strikingly
different from those of macrophages with either fresh or Aged
PMN, whereas there was increased binding of PMN-SA to macrophages, internalization was significantly decreased in comparison with uptake of Aged PMN (*p , 0.05).
To determine if S. aureus used PMN as a vehicle to gain access
to macrophages, we examined the distribution of viable sGFPUSA300 associated with macrophages fed PMN-SA. In general,
USA300 could associate with macrophages in one of three different ways: as PMN-SA bound to the surface of the macrophage,

FIGURE 2. Interactions between PMN containing viable S. aureus and

human macrophages. Aged or freshly isolated PMN were stained with
CTFR, and freshly isolated PMN were either left in buffer or fed sGFPexpressing USA300 at an MOI of 1:1 for 60 min (PMN-SA). Cell suspensions (Aged PMN, fresh PMN, and PMN-SA) were then fed to human
monocyte-derived macrophages and monitored for 15, 30, and 60 min.
Cells were stained for CD14 and CD15 and analyzed by flow cytometry.
Percent surface-bound (A), percent internalized (B), and percent of SA
associated with CD14+ macrophages and CTFR+ PMN (C) were calculated
as described in Materials and Methods. Symbols represent the mean of
four experiments 6 SEM. The p values were determined using repeatedmeasures one-way ANOVA and Tukey posttest for each time point. *p ,
0.05 PMN-SA versus PMN and Aged PMN.

as PMN-SA ingested by macrophages, or as free S. aureus released from lysed PMN and ingested by macrophages. Overall, the
majority of CD14+ macrophages (50.2 6 17.9% at 60 min; n = 4)
associated with viable S. aureus that appeared to be distributed
in one of these three different populations (Fig. 2C). Most sGFPUSA300 were found within PMN bound extracellularly to macrophages, whereas 69% were in PMN that had been ingested by
macrophages, and only 2 to 3% were free within macrophages.
These data suggest that macrophages did not effectively ingest
PMN-SA and that USA300 did not efficiently exploit PMN as
a Trojan horse to gain entry into macrophages. The flow cytometry
results indicating that most PMN-SA were bound to the surface of
macrophages was confirmed by confocal microscopy (Supplemental
Fig. 2B).
To determine if soluble cues from PMN-SA inhibited the capacity of macrophages to mediate efferocytosis, we compared ingestion of Aged PMN by macrophages in the presence or absence

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both Annexin V and PI (data not shown), suggesting that the PMN
plasma membrane was more readily compromised at an increased
bacterial MOI.
Accompanying the increased expression of PS on the surface
of cells, PMN fed USA300 or RN6390 exhibited greater loss of
mitochondrial membrane potential compared with that in unstimulated PMN, as measured by the fluorescent membrane potential
sensor JC-1. After 10 min of treatment with the mitochondrial membrane depolarizer carbonyl cyanide 3-chlorophenylhydrazone, 97
99% of cells lost red/green fluorescence associated with JC-1
accumulation in intact, polarized mitochondria (data not shown).
In like fashion, PMN mitochondrial membrane depolarization
occurred 60 min following phagocytosis of USA300 or RN6390,
with the greatest loss occurring in PMN fed USA300 (Fig. 1B).
Increased mitochondrial membrane depolarization in PMN fed
USA300 was confirmed using DIOC6 to assess mitochondrial
integrity (data not shown). Taken together, these data demonstrate
that intact PMN containing a population of viable USA300 exhibited some features typical of apoptosis, results consistent with
previous studies (7).
In general, increased expression of PS by apoptotic cells is
accompanied by a loss of dont eat me signals such as CD47 (17,
18). To determine if ingestion of USA300 altered expression of
dont eat me signals on PMN-SA, we compared the expression
of CD47 on PMN that were freshly isolated, aged, or fed opsonized
USA300. Surprisingly, although PMN-SA exhibited enhanced PS
exposure, PMN-SA challenged at an MOI of 1:1 expressed 1.3
7.2- and 1.31.7-fold higher levels of CD47 compared with Aged
PMN and PMN left alone in buffer, respectively (Fig. 1C, 1D).
There was 31% less CD47 on the surface of apoptotic PMN
compared with that on fresh PMN, but the decrease was not statistically significant (Fig. 1D). These data suggest that PMN-SA
exhibited an altered phenotype that was distinct from that typical
of apoptotic cells. Furthermore, the increased expression of CD47
on PMN-SA may influence their subsequent interactions with
macrophages, as loss of CD47 from the surface of apoptotic cells
inhibits SIRPa signaling and promotes efferocytosis (17, 18).

The Journal of Immunology

of supernatant conditioned by PMN-SA. Conditioned supernatants
failed to alter uptake or binding of Aged PMN (data not shown).
Therefore, soluble factors did not significantly contribute to enhanced binding or decreased engulfment of PMN-SA. The ability
of PMN-SA to resist uptake by macrophages could also likely reflect augmented expression of CD47 and possibly other "dont eat
me" signals by PMN harboring viable S. aureus. Our results indicate that blocking CD47 with F(ab)2 prepared from B6H12.2
(an Ab known to bind CD47) enhanced the engulfment of viable
cells, as previously reported by Gardai et al. (17). When compared
with ingestion of Aged PMN by macrophages, macrophages ingested 26% more viable PMN opsonized with anti-CD47 F(ab)2
than viable PMN opsonized with the isotype control (Supplemental
Fig. 2C). Using the same blocking Ab fragments, we were unable
to demonstrate that blocking CD47 significantly enhanced internalization of PMN-SA (data not shown), suggesting that CD47
modulation alone cannot account for altered efferocytosis of
PMN-SA.

Macrophages secrete cytokines when they ingest microorganisms

or apoptotic PMN (19, 20). To determine if PMN-SA were likewise stimulatory, we compared the cytokine profiles of macrophages
fed PMN-SA or USA300 alone (Fig. 3). We compared cytokine
production by macrophages after 6 h of exposure to PMN-SA with
that of macrophages cultured in the absence of agonists or fed
USA300 alone. Macrophages stimulated with USA300 produced
more IL-1RA, IL-6, and TNF-a than unstimulated macrophages.
There was a trend for enhanced IL-8 production from macrophages fed USA300 versus unstimulated macrophages, but this
difference was not statistically significant. Macrophages fed PMN-SA
at a ratio of five PMN-SA per macrophage produced significantly
less IL-1RA than those fed USA300 at a 1:1 ratio, and the level
of IL-1RA did not exceed basal levels produced by unstimulated
macrophages. Although IL-6, IL-8, and TNF-a were detected
above basal levels, production was relatively depressed (versus
USA300 alone); however, these decreases were not statistically

significant. Increasing the ratio of PMN-SA per macrophage to

15:1 resulted in levels of IL-6 and TNF-a equivalent to those
elicited by USA300 alone, triggered significantly more IL-8 compared with that from macrophages fed USA300, but caused no
change in IL-1RA release. Similar relative profiles were observed
after 12 h of stimulation (data not shown). Thus, the cytokine
profile of macrophages fed PMN-SA differed significantly from
that of macrophages fed USA300 alone.
Under identical experimental conditions, macrophage IL-10 was
measured 12 h after these phagocytes were fed USA300 alone,
PMN-SA, or Aged PMN (Supplemental Fig. 3). Macrophages fed
USA300 alone or PMN-SA at a ratio of five PMN per macrophage
produced more IL-10 than unstimulated macrophages. Increasing
the ratio of PMN-SA per macrophage to 15:1 resulted in significantly more IL-10 than unstimulated macrophages. In contrast, the
level of IL-10 produced from macrophages fed Aged PMN did not
exceed basal levels (Supplemental Fig. 3). Importantly, macrophages were capable of producing elevated levels of IL-10 in response to Aged PMN when the macrophages were cocultured with
Aged PMN in the presence of serum as previously reported (19
and data not shown). Overall, macrophages fed PMN-SA had
enhanced IL-10 production to compared with those exposed to
USA300 alone.
Inflammasome activation by PMN-SA versus S. aureus alone
Given that inflammasomes contribute to innate host response to
various microbial agents (21), including NLRP3 inflammasome activation by S. aureus in murine macrophages (22), we compared
IL-1b production and caspase-1 activation by macrophages stimulated with USA300 or PMN-SA. Macrophages fed USA300 alone
produced significantly more IL-1b than did unstimulated macrophages (Fig. 4A, 4B). Macrophages fed PMN-SA produced 22.6fold less IL-1b than did those fed USA300 alone when assayed
by Luminex (Fig. 4A). Cytokine analysis by ELISA resulted in
a similar trend, as macrophages that were fed PMN-SA compared
with those fed USA300 alone produced 6.6-fold less IL-1b. As a
positive control for NLRP3 inflammasome activation of macrophages, we measured IL-1b production in response to LPS and

FIGURE 3. Macrophage cytokine production is skewed by PMN-SA. Human monocyte-derived macrophages were left alone (Mac), fed USA300 (SA) at
an MOI of 1:1, or fed PMN-SA at a ratio of five PMN-SA per macrophage or 15 PMN-SA per macrophage. Following a 6 h incubation, supernatants were
analyzed by Luminex or ELISA. Bars represent the mean of three individual experiments 6 SEM. Statistical analyses were performed using one-way
ANOVA and Tukey posttest. *p , 0.05 versus macrophages, #p , 0.05 versus macrophages + SA.

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PMN-SA alter macrophage proinflammatory cytokine

production

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S. AUREUS PROMOTES RIP-1DEPENDENT CELL DEATH OF NEUTROPHILS

silica (Fig. 4B). The decreased IL-1b production reflected depressed NLRP3 engagement, as caspase-1 activation was less than
when macrophages were fed PMN-SA versus USA300 alone
(Fig. 4C). Taken together, these data indicate that PMN-SA elicited altered cytokine profiles in macrophages and failed to activate the NLRP3 inflammasome.
PMN-SA exhibit limited features of accelerated apoptosis
Because macrophages did not scavenge PMN-SA, we reasoned
that PMN lysis would be the predominant fate for PMN following ingesting of CA-MRSA. In fact, we and others (7, 9) had

FIGURE 5. Analysis of PCNA and caspase-3 in

human PMN following phagocytosis. Lysates from PMN
that were in buffer alone, challenged with USA300 at an
MOI of 1:1 (+SA), or treated with anti-Fas Ab (+aFas)
as indicated were analyzed by immunoblot for PCNA,
caspase-3, and actin. Shown is a representative of three
experiments (A). To quantify band intensity, immunoblots were scanned and analyzed using a phosphorimager. Data were plotted as the signal above background versus time 0 for the indicated bands 6 SEM
[(B) n = 4].

demonstrated that PMN undergo lysis .180 min after phagocytosis of USA300, even at MOI as low as 1:1. Lysis requires viable
USA300 and is partially dependent on staphylococcal toxin production (7, 9, 23). Furthermore, PMN lysis late after ingestion of
USA300 is inhibited by pretreatment of PMN with inhibitors of
transcription or protein synthesis (7 and M.C. Greenlee-Wacker
and W.M. Nauseef, unpublished observations), demonstrating that
PMN actively engage endogenous responses that culminate in cell
disruption.
As demonstrated earlier (Fig. 1), PMN-SA initially exhibited
increased surface expression of PS and mitochondrial membrane

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FIGURE 4. Inflammasome activation is dampened by PMN-SA. Human monocyte-derived macrophages (Mac) were treated with buffer or primed with
LPS for 2 h prior to treatment buffer alone, silica, USA300, or PMN-SA. Supernatants were analyzed for IL-1b production by Luminex for following a
6 or 12 h incubation (A) or by ELISA following a 6 h incubation (B). Caspase-1 cleavage was analyzed by immunoblotting following a 6 h incubation (C).
Bars represent the mean of five experiments 6 SEM (B) or three experiments (C). For (A), p values were determined using one-way ANOVA and Tukey
posttest. *p , 0.05 versus macrophages, ^p , 0.05 versus macrophages + SA. For (B), an outlier experiment was identified using Prism software
(GraphPad) and excluded from the analysis. p values were then determined using repeated-measures one-way ANOVA. *p , 0.05 versus macrophages +
LPS, #p , 0.05 versus macrophages + LPS + Silica, ^p , 0.05 versus macrophages + SA.

The Journal of Immunology

S. aureus causes rapid destruction of PMN in an

RIP-1dependent manner
Because previous studies and our current observations indicate
that PMN-SA did not undergo apoptotic cell death but rather
eventually lysed (7), we tested the hypothesis that necroptosis was
the cell death program that dictated the fate of PMN-SA. Necroptosis is defined as RIP-1dependent cell death that can be
blocked by Nec-1 (26, 27). At an MOI of 10:1, 50 mM of Nec-1
inhibited lysis of PMN-SA by 21.1%, and there was a 53.7%
reduction in PMN lysis with 500 mM of Nec-1 (PMN lysis was
62.2 6 4.0% in the absence of Nec-1 [0 mM] versus 28.8 6 3.2%
for assays containing 500 mM Nec-1) (Fig. 7). At an MOI of 1:1,
caspase inhibition in combination with 40 mM of Nec-1 was sufficient to completely inhibit lysis of PMN-SA (Supplemental Fig.
4). These data suggest that the higher inoculum of SA was sufficient
to induce necroptosis of PMN-SA, whereas a lower bacterial burden
required additional caspase inhibition to favor the necroptotic
pathway. Importantly, concentrations of Nec-1 used in these assays
did not affect S. aureus survival, and Nec-1 alone did not interfere
with macrophage lysis (data not shown). In addition, Nec-1 did
not affect neutrophil function, including phagocytosis, reactive
oxygen species production in response to PMA, or CD11b upregulation in response to USA300 or fMLF (data not shown).
Combined, these data clearly demonstrate that USA300 induced
RIP-1dependent cell death.

FIGURE 6. Caspase activity in human PMN following phagocytosis.

(AD) Human neutrophils were cultured with IgG and serum complementcoated latex beads (Beads; filled squares, 10 beads/PMN), USA300 (SA; red
circles, MOI of 10:1), or anti-FAS Ab (a-FAS; filled triangles) as indicated.
At each time point, samples were clarified by centrifugation, and caspase
activity in culture supernatants and stimulated PMNs was determined with
an ApoAlert Caspase Assay Plate kit (Clontech) according to the manufacturers instructions. Data for PMNs are the mean 6 SEM of two to three
experiments for all assays except the 2 h time point, in which case there is
a single data point. The level of caspase activity for FAS-stimulated PMNs at
6 h is set as 100% (positive control), and all other data are expressed relative
to the aFAS data points at 6 h. Statistical analyses were performed using
one-way ANOVA and Tukey posttest. *p , 0.05 for a-FAS versus SA, #p ,
0.05 for Beads versus SA, ^p , 0.05 for a-FAS versus Beads.

their potential to contribute to persistence or progression of infection during human disease. Based on these clinical observations
and experimental findings, we reasoned that the fate of PMN-SA

Discussion
Staphylococcal infections are generally characterized by resistance
to antimicrobial therapy, local persistence, distant metastases, and
tissue necrosis (28). Despite their important role in the innate
immune host response to invading microbes, PMN fail to kill and
degrade all ingested S. aureus that are trapped in phagosomes.
Furthermore, PMN-SA can transmit infection to naive mice or
rabbits in experimental models (29, 30), thereby demonstrating

FIGURE 7. Nec-1dependent inhibition of PMN lysis. Human PMN

were cultured with USA300 at an MOI of 10:1 in the presence of Nec-1 or
vehicle (0 mM Nec-1) or without bacteria (DMSO), and cell lysis was
determined by release of LDH. Data for PMNs are the mean 6 SEM of
four to eight experiments. Statistical analyses were performed using oneway ANOVA and Dunnett posttest. *p , 0.05 for PMNs + USA300 treated
with Nec-1 versus vehicle control (0 mM).

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depolarization, features characteristic of the initial stages of apoptosis. In contrast to apoptotic PMN, however, PMN-SA upregulated surface expression of CD47 and were not engulfed by
macrophages (Figs. 1, 2). These data suggest that PMN-SA may
derail the apoptotic cell death pathway. PCNA, a cell-cycle protein
expressed in PMN cytoplasm, promotes PMN survival by scavenging procaspases and thereby preventing their activation (24).
As decreased levels of cytoplasmic PCNA typically accompany
PMN apoptosis, we speculated that PMN-SA may increase PCNA
over time, sequester procaspases, and thereby contribute to prolonged survival. PCNA decreased and caspase-3 increased in
cultured PMN in a time-dependent fashion, results consistent with
the induction of apoptosis in normal PMN over time in culture
(Fig. 5A). In contrast, PMN-SA exhibited a sustained level of
PCNA compared with that of PMN in culture and cytoplasmic
PCNA was observed as late as 24 h following phagocytosis
(Fig. 5B). Furthermore, whereas anti-Fas Ab promoted caspase-3
activation in PMN, as previously demonstrated (25), PMN-SA
failed to activate caspase-3 with similar kinetics (Fig. 5). To determine if a higher MOI would induce caspase activation in
PMN, we challenged PMN with USA300 at an MOI of 10:1 and
assessed caspase activation (Fig. 6). Even at the higher MOI,
PMN fed USA300 failed to activate caspase-3 (Fig. 6A). In addition, PMN-SA failed to activate caspase-8, -9, and -2, whereas
opsonized beads or anti-FAS Ab prompted time-dependent increase in all caspases tested (Fig. 6BD). Taken together, the
sustained levels of cytoplasmic PCNA and the failure to activate
caspase-3 demonstrate a divergence of the phenotype of PMN-SA
from that typical for the apoptotic death pathway.

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isoforms exceed those of cellular FLICE/caspase 8 inhibitory protein long isoforms, direct cells to undergo programmed necrosis
(40). Although additional studies are required to investigate the
downstream signaling events in PMN-SA, it is noteworthy that
necroptosis of PMN-SA at low MOI required the addition of caspase inhibitors, whereas no supplemental inhibition was required
at the higher MOI (10:1).
From a clinical perspective, necroptosis of PMN-SA would be
anticipated to exacerbate staphylococcal disease, both locally and
systemically. Lysis of PMN-SA releases viable SA that could
propagate infection to distant sites, perhaps with organisms phenotypically more virulent by virtue of adaptations that allowed
them to survive attack in PMN phagosomes. In addition, necroptosis of PMN-SA releases PMN constituents that would serve as
danger-associated molecular pattern molecules that would amplify
local tissue damage.
In summary, we demonstrate that CA-MRSA undermines effective innate immune response of human phagocytes in several
ways. Diversion of PMN-SA from a typical apoptotic pathway
compromised macrophage efferocytosis and cytokine production,
promoted programmed necrosis, and culminated in escape of viable
bacteria. Collectively, these events may contribute to the persistent
nature of staphylococcal infection as well as its propensity for
causing local tissue necrosis. Elucidation of the staphylococcal
signals that trigger the PMN responses that drive the downstream
cellular changes dictating PMN-SA fate may provide important and
novel insights with therapeutic potential.

Acknowledgments
We thank Sarah Bloomberg, Michael Carlson (Beckman Coulter), Matthew
Long, Jamie Schwartz, and Dr. Mark White for excellent technical
assistance.

Disclosures
The authors have no financial conflicts of interest.

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