) and WT littermates were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care–accredited animal facility at the Burns and Allen Research Institute at Cedars-Sinai Medical Center. Animals were housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee. Cebpe–/–
mice kept under the above mentioned conditions showed no signs of infection during life or at autopsy, with survivals equivalent to those of WT mice. Sex-matched mice used throughout our study were 6 to 10 weeks old.
Bacterial strains and growth conditions.
Unless otherwise indicated, WT S. aureus
), isolated from the skin of a child with atopic dermatitis, was used in the experiments. The MRSA clinical isolate COL (NRS100; NARSA) was also used.
S. aureus were propagated in Todd-Hewitt broth (THB; Difco) at 37°C, with shaking at 250 rpm, or on THB agar (THA).
Overnight bacterial culture was diluted until an optical density at 600 nm, corresponding to approximately 108 CFU/ml, was reached. Bacteria were harvested by centrifugation at 3,300 g for 10 minutes at 4°C and then washed twice with PBS (without Ca2+ and Mg2+; Mediatech). S. aureus strains were routinely cultured on Tryptic Soy sheep blood agar plates, and colonies with comparable hemolysis phenotypes were selected for each experiment.
Murine skin infection model.
was pelleted, washed twice, and resuspended in PBS mixed 1:1 with sterile Cytodex beads (GE Healthcare), following an established protocol for generating localized S. aureus
and S. pyogenes
s.c. infection (4
). One hundred microliters of 2 separate inocula, as specified (see the legend for Figure ), were administered by s.c. injection into the respective 2 flanks of each mouse. Injections were performed with careful visualization of the needle to assure that they were not intramuscular. Serial dilutions were prepared and plated to confirm the actual inocula used.
Determination of lesion size and tissue bacterial CFUs.
Baseline weights of mice were recorded prior to infection and daily thereafter until sacrifice. Lesions were measured with a caliper, daily throughout infection. Lesions were defined by darkened areas of dermonecrosis. Our method to measure lesion size has been previously reported (61
). Briefly, skin lesions were quantified by multiplying the length and width of the lesion. Irregularly shaped lesions were broken down into smaller symmetrical pieces, and each piece was measured by the same method.
Following euthanization of mice, infected skin lesion tissue was aseptically excised, thoroughly homogenized, and mixed in 1 ml PBS, as previously shown (61
). Ten-fold serial dilutions of the homogenates were plated on THA for CFU determination. The spleen and both kidneys were aseptically removed from each animal and processed in the same way. When required, the appropriate homogenized suspensions (skin lesions) were centrifuged at 15,000 g
for 10 minutes, and supernatants were stored at –80°C for subsequent analysis by ELISA.
Quantification of neutrophils and macrophages in infected skin lesions.
WT and Cebpe–/– mice (n = 8 per group) were infected by s.c. injection of S. aureus at the specified inoculum (see the legend for Figure ) and sacrificed at 24 hours p.i. Infected tissues (skin lesions) were then excised and fixed in 10% formalin (Medical Chemical Corporation) overnight. Paraffin embedding and H&E staining were performed by the Department of Pathology at Cedars-Sinai Medical Center. Image acquisition was performed with the Zeiss Axio Imager M1 microscope and the AxioVision 4.6 software (Zeiss). A minimum of 3 mice (2 lesions per mouse) from each genotype were randomly selected for enumeration of macrophages and neutrophils. Two blinded observers counted the cells from 10 nonoverlapping high-power fields (original magnification, ×400) within each lesion. The average count was obtained from each mouse in each group, and SEM was calculated from these averages.
Mouse CXCL1-specific and CXCL2-specific ELISAs were performed according to the manufacturer’s instructions (R&D Systems).
Isolation of murine bone marrow mononuclear cells and cultivation of BMDMs.
Bone marrow cells were harvested from WT or Cebpe–/– mice. Bone marrow was flushed out of isolated femurs and tibiae with RPMI 1640 medium and 10% heat-inactivated FBS using a 25-gauge needle. Cells were then incubated for 4 hours in a humidified atmosphere at 37°C and 5% CO2 to deplete adherent cells. Nonadherent bone marrow cells were cultured with 10 ng/ml murine M-CSF (Peprotech) in RPMI 1640 with 10% FBS for 7 days to induce BMDMs. Bone marrow mononuclear cells were isolated from nondepleted bone marrow cells using Lymphocyte Separation Medium (Mediatech).
Intracellular survival assay (gentamicin protection assay) in macrophages.
Macrophages were seeded at a density of 5 × 104 cells per well (100 εl) in 96-well tissue culture plates. Macrophages were infected with S. aureus at the specified MOI (see Figure G and Figure ). To promote infection, bacteria were spun down onto the macrophages at 500 g for 10 minutes at room temperature, before incubating the cells in a humidified atmosphere at 37°C and 5% CO2. After 30 minutes, macrophages were washed 3 times with prewarmed media to remove extracellular bacteria. Gentamicin (Invitrogen) was then added to each well at a final concentration of 400 εg/ml for 1.5 hours. At this time, the concentration of gentamicin in the media was reduced to 100 εg/ml for the remainder of the assay. At 24 hours p.i., cells were washed 3 times with PBS, then 100 εl of 0.02% Triton X-100 in water was added to each well and pipetted vigorously 10 times to promote macrophage lysis and release intracellular bacteria. Ten-fold serial dilutions of each cell lysate were immediately plated onto THA, and CFUs were enumerated following overnight incubation at 37°C. Data are representative of at least 2 independent experiments performed in triplicate.
For assays involving PMA-differentiated human U937 macrophages, 24 hours prior to infection, media was replaced without G418 and PMA for the remainder of the time. Zinc (100 μM Zn2SO4) was added to the respective pMTε and control groups 24 hours prior to infection and was present for the remainder of the assay.
Neutrophil depletion in vivo.
Depletion of neutrophils was carried out as described previously (62
). Briefly, mice were made neutropenic by i.p. administration of 150 εl rabbit anti-mouse PMN antibody (Cedarlane Labs) 24 hours prior (day –1) to s.c. infection with S. aureus
on day 0 and every 24 hours thereafter, until sacrifice on day 4. The manufacturer certified that the antibody was sterile and suitable for use in cytotoxic assays and in vivo depletion. Control groups received equal amounts of normal rabbit serum (Sigma-Aldrich; sterile-filtered, cell culture and endotoxin tested) by i.p. injection.
WT and Cebpe–/– mice receiving either anti-mouse PMN antibody or normal serum (control) were infected s.c. with S. aureus on day 0 (refer to Murine skin infection model). Skin lesion areas were measured daily, and on day 4 (sacrifice) the CFUs in skin lesions, spleens, and kidneys were determined.
To confirm depletion of neutrophils after antibody injection, WT mice (n = 3/group) were sacrificed at day 0 and day 4 of infection. Splenocytes were collected after sacrifice and homogenized, and cells were stained with PE–anti-Ly6G monoclonal antibody and PE.Cy5–anti-CD11b monoclonal antibody (eBiosciences). The population of neutrophils (Ly6G+, CD11b+) were determined using a CyAnTM flow cytometer (Beckman Coulter), and the data were analyzed by Summit (Dako) software. The total population of neutrophils of mice treated with antibody was highly reduced on day 0 (–72%) and day 4 (–96%; data not shown) compared with that of mice treated with normal serum.
Intracellular survival assay (gentamicin protection assay) in murine whole blood.
Bacteria were pelleted, washed twice, diluted to 1.5 × 107 CFU/ml in 50 εl PBS (without Ca2+ and Mg2+), and immediately mixed with 150 εl of freshly drawn murine (WT or Cebpe–/–; n = 8/group) peripheral whole blood in sterile heparinized 2-ml round-bottom Eppendorf tubes. Notably, blood from WT and Cebpe–/– mice contained approximately the same number of neutrophils per microliter (Supplemental Figure 4). Reactions were incubated at 37°C on a rotary shaker.
After 15 minutes of infection, gentamicin (Invitrogen; working stock, 5 mg/ml in PBS) was added to each 200 εl blood reaction at a final concentration of 400 εg/ml to kill extracellular bacteria. At 75 minutes p.i., the concentration of gentamicin in the blood was reduced to approximately 130 εg/ml for the remainder of the assay by adding 240 εl PBS to each blood reaction.
Forty-microliter (35 and 60 minutes p.i.) or 120-εl (180 minutes p.i.) aliquots were taken from each blood reaction, diluted into 500 εl PBS (in sterile 0.6-ml Eppendorf tubes), and centrifuged at 15,000 g for 3 minutes at room temperature. After carefully removing the supernatant, pure water (200 εl) was added and pipetted vigorously 10 times to promote host cell lysis and release intracellular bacteria. Each 200-εl cell lysate was immediately plated onto THA using the spread-plate technique (30-ml agar plates to further dilute out any remaining gentamicin), and CFUs were enumerated following overnight incubation at 37°C.
Development of U937-pMTε cells.
As reported previously by our group (64
), pMTε was constructed by inserting a full length of CEBPE
cDNA at the XhoI and HindIII sites of the pMTCB6+ vector (pMT; gift from F.J. Rauscher III, The Wistar Institute, Philadelphia, Pennsylvania, USA). We used the human promonocytic U937 cell line (ATCC) stably transfected with pEGFP plasmid (Clontech Laboratories) and either zinc-inducible pMTε or control vector pMT. Cells were maintained at between 2 × 105
cells/ml and 1 × 106
cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated FBS (Gemini Bio-Products), 2 mM l
-glutamine, and G418 (neomycin, 900 μg/ml; Omega Scientific) for selection. Multiple polyclonal cultures (>98% GFP positive) were screened for zinc-inducible C/EBPε protein overexpression by Western blot analysis.
PMA-differentiated human U937 myeloid cells.
U937 cells alone or carrying either pMTε or vector control were seeded at an appropriate density in tissue culture plates. The cells were subsequently induced to differentiate by addition of 10 ng/ml PMA (Sigma-Aldrich) for 24 hours.
Participants in our study included healthy humans with a negative history of infection, no antibiotic treatments, and no immune-boosting supplements in the 4 weeks prior to donating blood. Peripheral blood was collected from individuals in a fasting condition. Blood was routinely collected in the presence of either heparin or K2 EDTA and was used immediately for downstream applications.
NAM (C6H6N2O; Fw 122.13), tested for cell culture and insect cell culture, was purchased from Sigma-Aldrich. On each occasion prior to use, NAM was prepared fresh in sterile endotoxin-free PBS (without Ca2+ and Mg2+) and sterilized through a nonpyrogenic 0.22-εm low-protein-binding filter (PALL Life Sciences). Each specific lot of NAM used in our study was confirmed to be endotoxin-free (pyrogen-free) using the end-point chromogenic Limulus amebocyte lysate endochrome method (refer to Supplemental Methods and Supplemental Figure 7 for more detail).
Assessing the effect of NAM on the growth and viability of S. aureus.
We assessed whether NAM, at the concentrations used in our study, adversely affect the growth and viability of S. aureus. PBS was chosen as an inert nongrowth medium, and THB was chosen as a suitable growth medium for S. aureus. Seventy-five microliters of NAM (1 mM and 10 mM final concentrations; in either THB or PBS) or THB or PBS alone (respective controls) were placed in sterile 2-ml round-bottom Eppendorf tubes and then inoculated with S. aureus (~1 × 104 CFU/ml in 25 εl of PBS or THB) and immediately briefly vortexed. Triplicate reactions were incubated at 37°C for 1, 3, and 6 hours on a rotary shaker, at which time 10-fold serial dilutions were plated on THA for enumeration of CFUs.
In a different assay, S. aureus (~1 × 108 CFU/ml in THB) was incubated either with or without 50 mM NAM. Triplicate 1-ml reactions were incubated at 37°C on a rotary shaker, and at various time points (6, 12, and 24 hours), 10-fold serial dilutions were plated on THA for enumeration of CFUs. These assays were performed on 3 independent occasions.
Murine and human whole blood assays.
This well-described phagocytic survival assay has been previously reported (4
). Bacteria were pelleted, washed twice, diluted to the specified inoculum (see the legends for Figures , , and ) in 25 εl PBS (without Ca2+
), and immediately mixed with 75 εl of freshly drawn human or murine peripheral whole blood in sterile heparinized 2-ml round-bottom Eppendorf tubes. Reactions (performed in minimum in triplicate) were incubated at 37°C for 1 to 3 hours on a rotary shaker, at which time 10-fold serial dilutions were plated on THA for enumeration of surviving CFUs.
When required, freshly drawn human or murine peripheral blood was pretreated with NAM (1 mM or 10 mM) or PBS (without Ca2+ and Mg2+) prior to inoculation with bacteria. Pretreatment was performed in sterile, nontreated, low evaporation tissue culture plates (Becton Dickinson) for 24 hours in a humidified atmosphere (95% humidity) at 37°C and 5% CO2, with gentle mixing on a nutator. On each occasion, blood was aseptically taken from mice via cardiac puncture using a 22-gauge needle to minimize lysis and maintain the integrity of the blood for the duration of the respective assay.
Murine systemic infection.
Mice were systemically infected by i.p. injection of ~1 × 107 CFU/ml to 2 × 107 CFU/ml S. aureus for 48 hours. Following euthanasia, the spleen and both kidneys were removed for CFU determination.
CBC of human and mouse blood specimens.
CBCs were used to measure the viability of myeloid lineages in human and mouse blood. Blood specimens were collected in BD Microtainer tubes with K2 EDTA (Becton Dickinson). Specimens were analyzed for hematology testing within 3 hours of collection and were stored in racks or continuously mixed at room temperature during this time.
Human blood specimens were routinely analyzed by the Department of Pathology at Cedars-Sinai Medical Center. CBC with automated differential was performed using a Beckman Coulter LH 1500 Series Hematology Automation System.
Murine blood specimens were routinely analyzed by the Department of Comparative Medicine at Cedars-Sinai Medical Center. CBC with automated differential was performed using a Hemavet 950 FS Veterinary Hematology System (Drew Scientific Inc.). Automated functionality included wbc, rbc, Hb, HCT, MCV, MCH, MCHC, platelets, and 5-part wbc differential, including neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
Isolation of human neutrophils.
Polymorphprep (Axis-Shield) was used for the isolation of neutrophils from human peripheral blood according to the manufacturer’s instructions.
Human neutrophils were maintained ex vivo in RPMI 1640 supplemented with 10% heat-inactivated FBS and 2 mM l-glutamine in a humidified atmosphere at 37°C and 5% CO2. When indicated, immediately following isolation, human neutrophils were treated ex vivo with either NAM (1 mM) or PBS for 20 to 24 hours and subsequently used for Western blot or specific functional assays.
For some experiments, freshly drawn human peripheral blood was pretreated with either NAM (1 or 10 mM) or PBS (without Ca2+ and Mg2+), prior to isolating the neutrophils using Polymorphprep. Pretreatment of the human blood was performed in sterile, nontreated, low evaporation tissue culture plates (Becton Dickinson) for 20 to 24 hours in a humidified atmosphere (95% humidity) at 37°C and 5% CO2, with gentle mixing on a nutator.
Trypan blue exclusion, according to standard staining protocol, was used to measure the viability (over time) of neutrophils in culture or neutrophils isolated directly from blood.
Isolation of human monocytes.
Human peripheral blood was collected in the presence of heparin and used immediately. Blood was layered over Lymphocyte Separation media (Fisher Scientific) according to manufacturer’s instructions and centrifuged at 1,000 g for 25 minutes at room temperature without the break engaged. The cell layer was isolated and washed 3 times with RPMI 1640. Monocytes were isolated using Human Monocyte Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, the Monocyte Isolation Kit II is an indirect magnetic labeling system for the isolation of untouched monocytes from human peripheral blood mononuclear cells. Human monocytes were isolated by depletion of nonmonocytes (negative selection). Nonmonocytes were indirectly magnetically labeled with a cocktail of biotin-conjugated monoclonal antibodies, as primary labeling reagent, and anti-biotin monoclonal antibodies conjugated to MicroBeads, as secondary labeling reagent.
Human monocytes were maintained ex vivo in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, and 50 ng/ml recombinant human M-CSF (PeproTech) in a humidified atmosphere at 37°C and 5% CO2. Human monocytes were treated ex vivo with either NAM (1 mM) or PBS for approximately 20 hours and subsequently used for Western blot or functional assays.
Isolation of mouse neutrophils.
Isolation of neutrophils from murine whole blood was performed according to a protocol established by Cedarlane Labs. Blood was aseptically collected from mice via cardiac puncture using a 22-gauge needle to minimize lysis and maintain the integrity of the blood cells. Blood was collected in the presence of heparin and used immediately. rbc were pelleted by centrifugation at 1400 g for 10 minutes at room temperature, without the brake engaged. The buffy coat (wbc layer) and the top layer of the rbc was harvested with a Pasteur pipette and transferred to a fresh tube. PBS (without Ca2+ and Mg2+) was added to wash and fully suspend the cells, and the mixture was centrifuged (600 g, 10 minutes, room temperature) to re-pellet the cells. rbc were then lysed using standard protocol. wbc were subsequently resuspended in 4 ml of a serum-free medium (PBS without Ca2+ and Mg2+). 4 ml diluted wbc were then carefully layered over 3 ml Lympholyte-Mammal (Cedarlane Labs) at room temperature in a 15-ml centrifuge tube. The gradient was centrifuged at 800 g for 25 minutes at room temperature, without the brake engaged. The resulting interfacial band consisted of lymphocytes, and the pellet consisted of PMNs. Once the PMNs were obtained, further washing was carried out to remove any rbc membranes (rbc ghosts). The majority of the granulocytes were neutrophils (>70%).
Murine neutrophils were maintained ex vivo in RPMI 1640 supplemented with 10% FBS and 2 mM l-glutamine and incubated in a humidified atmosphere at 37°C and 5% CO2. Murine neutrophils were treated ex vivo with either NAM (1 mM) or PBS for approximately 20 hours and subsequently used for Western blot.
Intracellular survival assay (gentamicin protection assay) in human neutrophils.
S. aureus were opsonized with 10% prewarmed pooled human plasma and incubated for 20 minutes at 37°C under slow rotation. The preopsonized bacteria were pelleted, washed twice, diluted to the specified inoculum (see the legend for Figure ) in 50 εl RPMI, and immediately mixed with 150 εl human neutrophils (6 × 105 total neutrophils) in sterile 2-ml round-bottom Eppendorf tubes. Before being used in this assay, the neutrophils had been pretreated ex vivo with either NAM (1 mM) or PBS for 20 hours. Based on CBC data from at least 11 healthy human donors, a mean of 4,000 neutrophils per microliter of human blood was assumed when determining an appropriate MOI to use in this assay. Reactions were incubated at 37°C on a rotary shaker.
After 20 minutes of infection, gentamicin (Invitrogen; working stock, 5 mg/ml in RPMI) was added to each 200 εl neutrophil reaction at a final concentration of 400 εg/ml to kill extracellular bacteria. At 80 minutes p.i., the concentration of gentamicin in the reaction was reduced to approximately 130 εg/ml for the remainder of the assay by adding 240 εl RPMI to each neutrophil reaction.
Forty-microliter (55 and 80 minutes p.i.) or 120-εl (200 minutes p.i.) aliquots were taken from each neutrophil reaction, diluted into 500 εl RPMI (in a sterile 0.6-ml Eppendorf tube), and centrifuged at 15,000 g for 3 minutes at room temperature. After carefully removing the supernatant, pure water (200 εl) was added and pipetted vigorously 10 times to promote host cell lysis and release intracellular bacteria. The entire 200-εl cell lysate was immediately plated onto THA using the spread-plate technique (30 ml agar plates to further dilute out any remaining gentamicin), and CFUs were enumerated following overnight incubation at 37°C. A similar assay was performed using human monocytes.
The ChIP Assay Kit (Upstate Biotechnology) was used, and chromatin was prepared for immunoprecipitation as instructed by the manufacturer. The sonicated chromatin was immunoprecipitated with either 5 εg anti-acetylated histone H3 antibody or normal rabbit IgG antibody as negative control (Upstate Biotechnology). Immunoprecipitated DNA was subsequently analyzed by PCR using primers specific for the CEBPE
promoter region spanning the genomic region –797 to –518 base pairs upstream of the transcriptional start site (position: 21578530; Promoter ID 12948) that was localized by our group (10
); input chromatin was analyzed for β-actin mRNA as a positive control. The optimal reaction conditions for PCR were determined for each primer pair. Primers were denatured at 95°C for 1 minute and annealed at 60°C for 1 minute, followed by elongation at 72°C for 1 minute; each product was amplified 35 cycles. PCR products were analyzed by 2.5% agarose/ethidium bromide gel electrophoresis. Densitometry of all agarose gels was performed using Quantity One software 4.6.3 (Bio-Rad) to quantify fold changes. The primers used for ChIP analysis were as follows: human CEBPE
, 5′-GCTTTGGCCAAGCCCAGGGA-3′ (forward), 5′-TGCTGGGCTCCACCTACCCC-3′ (reverse); human ACTB
, 5′-CTCCTCGGGAGCCACACGCA-3′ (forward), 5′-TAGGGGAGCTGGCTGGGTGG-3′ (reverse); murine Cebpe
, 5′-TGAGGCTGCAGCTTGCCTGG-3′ (forward), 5′-ACCAAGCTACCCCTGGCCCT-3′ (reverse); murine Actb
, 5′-ACCTGTTACTTTGGGAGTGGCAAGC-3′ (forward), 5′-GTCGTCCCAGTTGGTAACAATGCC-3′ (reverse).
Western blot and immunoprecipitation.
Whole-cell extracts were produced by lysing cells (107) with 100 εl denaturing RIPA buffer (50 mM Tris HCl, pH 8; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) added with a protease inhibitor cocktail (Roche Molecular Biochemicals) on the day of extraction. Extracts were stored at –80°C until use.
For Western blot, protein lysates were boiled in Laemmli sample buffer (Bio-Rad), resolved on 4% to 15% gradient SDS-PAGE gels, and transferred to nitrocellulose membranes (Sigma-Aldrich). Immunoblots were probed with anti-C/EBPε antibody (Santa Cruz Biotechnology Inc.), anti-LTF antibody (Abcam), anti-CAMP antibody (Abcam), anti-gp91phox antibody (NOX2; Santa Cruz Biotechnology Inc.), and anti–lipocalin-2 antibody (NGAL; R&D Systems) and developed using the enhanced chemiluminescence kit (Pierce). ACTB (Sigma-Aldrich) was used as a control. Western blot data are representative of 1 out of 3 independently performed experiments. Densitometry of all blots was performed using Quantity One software 4.6.3 (Bio-Rad) to quantify fold changes.
We used an anti–acetyl-lysine antibody (ab21623; Abcam) for immunoprecipitation according to the manufacturer’s protocol. The input of the protein lysates was used as a loading control.
We used 2-tailed unpaired Student’s t test to compare 2 independent groups when using ex vivo data; nonparametric Mann-Whitney U test was applied for the independent comparison of the murine in vivo CFU data. One-way ANOVA was used for the comparison of more than 2 independent groups, and 2-way ANOVA, in combination with Bonferroni as post-hoc test, was used to compare murine body weight or lesion size data sets obtained over time. Paired Student’s t test was used for the comparison of human blood samples treated with either NAM or PBS. We deemed a P value below 0.05 as significant. GraphPad Prism was used for analyses.
All animal experiments were approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee and performed using accepted veterinary standards. Peripheral blood was obtained from healthy adult donors by the General Clinical Research Center at Cedars-Sinai Medical Center. All subjects provided written informed consent. Experimentation using human blood were approved by the Cedars-Sinai Medical Center Institutional Review Board and Office of Research Compliance. Experimental protocols were approved by the Cedars-Sinai Medical Center Biosafety Committee.