The rat mAb M1/70 and mouse mAb 44a, which recognize the mouse and human α_M (CD11b) integrin subunit of integrin Mac-1, respectively, were purified from conditioned media of hybridoma cells obtained from the American Type Culture Collection (ATCC, Manassas, VA) using protein A agarose. The mouse Alexa Fluor 488-conjugated anti-Ly6G directed against neutrophil-specific lymphocyte antigen 6, locus G (catalog #127625) was from BioLegend (San Diego, Ca). The secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog #A-10680), Alexa Fluor 633-conjugated goat anti-rabbit IgG (catalog #A-21071), and Alexa Fluor 633-conjugated goat anti-rat IgG (catalog #A-21094) were from Thermo Fisher Scientific. The polyclonal anti-PF4 antibody was raised in rabbits using recombinant PF4 as an antigen. Calcein-AM (catalog #C3100MP), NHS-Fluorescein (catalog #46410). Heparan sulfate sodium salt (catalog #7640) and 4% Brewer thioglycolate (TG) solution were from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 568-conjugated phalloidin (catalog #A12380) and the LIVE/DEAD BacLight Bacterial Viability kit (catalog #13152) were from Thermo Fisher Scientific.

To produce recombinant PF4 (rPF4), cDNA encoding human PF4 ORF was cloned into the pET-15b vector (Novagen, Madison, WI) and transformed into Origami B (DE3) competent cells (Novagen, Madison, WI). rPF4 was purified from soluble fractions of cell lysates by affinity chromatography using a 5-ml HiTrap heparin-agarose column (GE Healthcare). Endotoxin-free rPF4 was prepared as previously described (Lishko et al., 2018). rPF4 was iodinated with ¹²⁵I using IODO-GEN (Pierce, Rockford, IL).

C57BL/6 and Mac-1^-/- (B6.129S4-Itgam^tm1Myd/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All procedures were performed under the animal protocols approved by the Institutional Animal Care and Use Committee of Arizona State University. Animals were maintained under constant temperature (22°C) and humidity on a 12-h light/dark cycle in the Animal Facility of Arizona State University. Eight- to 12-week-old male and female mice were used in all experiments with age- and sex-matched mice selected for side-by-side comparison.

The antibiotic-susceptible strain of S. aureus subsp. aureus Rosenbach (catalog #25923) and methicillin-resistant S. aureus subsp. aureus Rosenbach strain (catalog #33591) were obtained from ATCC. Bacteria were grown overnight at 37°C on Luria-Bertani (LB) agar plates. A single colony was transferred to LB media and incubated with shaking in screwcap 15-ml plastic tubes containing 3 ml medium for 16 h at 37°C. The culture was diluted 100 times in fresh media and grown at 37°C until the mid-log phase. The bacteria were harvested by centrifugation at 4000xg for 20 min, washed with PBS and adjusted to desirable CFU/ml. Colony forming units (CFU) per milliliter values were determined by colony counts of serial dilutions on agar plates incubated overnight at 37°C. To produce encapsulated S. aureus, bacteria were grown under conditions that enhanced capsule production. Briefly, a single colony from an LB agar plate was grown in 10 ml LB media in a shaker incubator under aerobic conditions for 20 h at 37°C to reach the post-exponential growth phase and then harvested and adjusted to desirable CFU/ml.

Resident peritoneal macrophages were obtained from mice by lavage using cold PBS containing 5 mM EDTA as described (Podolnikova et al., 2016). Peritoneal lavage containing neutrophils and monocytes was isolated from mice 4 h and 3 days after intraperitoneal injection of 0.5 mL of a 4% TG solution. Inflammatory macrophages from a 3-day peritoneum were isolated using the EasySep Mouse selection kit (StemCell Technologies, Vancouver, BC, Canada) with mAb F4/80 conjugated to PE according to the manufacturer’s protocol. The IC-21 murine macrophage cell line and human monocytic U937 cells were obtained from ATCC and grown in RPMI containing 10% FBS and antibiotics. The HL-60 human leukemia cells (ATCC) were cultured in IMDM (Iscove’s Modified Dulbecco’s medium, Invitrogen) supplemented with 10% FBS and antibiotics. The cells were differentiated into granulocytes by culturing in the same medium containing 1.3% DMSO for 5 days (Hauert et al., 2002; Yakovlev et al., 2011). Human embryonic kidney (HEK) 293 cells stably expressing Mac-1 were previously described (Yakubenko et al., 2002).

96-well plates (Immulon 4HBX) were coated with different concentrations of heparan sulfate in PBS for 16 h at 4°C, washed with PBS, and different concentrations of rPF4 (0-40 μg/ml) were added to the wells for 3 h at 37°C. After washing, rabbit polyclonal anti-PF4 antibodies (5 μg/ml) were added for 2 h at 37°C. The microtiter plates were incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase, and the binding was detected by reaction with p-nitrophenyl phosphate, measuring the absorbance at 405 nm. Background binding to heparan sulfate was subtracted.

96-well plates (Immulon 4HBX) were coated with different concentrations of heparan sulfate in PBS for 16 h at 4°C and blocked with 1% polyvinylpyrrolidone (PVP) for 1 h at 37°C. The cells were labeled with 7.5 μM calcein-AM for 30 min at 37°C. The labeled cells were washed with Hanks’ balanced salt solution (HBSS) containing 0.1% BSA and resuspended in the same buffer at a 5 × 10⁵/ml concentration. Aliquots (100 μl) of labeled cells were added to each well and incubated for 30 min at 37°C. Nonadherent cells were removed with two washes of PBS, and fluorescence was measured using a fluorescence plate reader (Perceptive Biosystems, Framingham, MA). To test the effect of PF4 on cell adhesion, different concentrations of rPF4 were added to the wells pre-coated with 10 μg/ml heparan sulfate and incubated for 3 h at 37°C.

Phagocytosis assays with adherent resident and inflammatory mouse macrophages and the mouse IC-21 macrophages were performed using S. aureus bioparticles conjugated to a pH-sensitive pHrodo dye (Invitrogen, catalog #A10010). Bioparticles (100 μg/ml) were incubated with different concentrations of rPF4 (10-40 µg/ml) for 1 h at 37°C. Cells were resuspended in DMEM+10% FBS and cultured in Costar 48-well plates (2.5 x10⁵/well) for 3-5 h at 37°C. After the medium was aspirated, adherent cells were washed and incubated with 0.5 ml of S. aureus bioparticle suspensions supplemented with rPF4 for 1 h at 37°C. Cells were washed thrice with 1 ml of PBS, and photographs of five fields for each well were taken using an EVOS FL Auto microscope (Thermo Fisher Scientific, Waltham, MA). Phagocytosed bioparticles were counted using ImageJ software.

Solution-phase phagocytosis assays with pHrodo-conjugated S. aureus bioparticles were performed with IC-21 cells and HL-60 cells. S. aureus bioparticles (10 µg) were incubated with different concentrations of rPF4 for 1 h at 37°C. Aliquots (100 μl) of IC-21 macrophages (5x10⁵) and differentiated HL-60 cells (5x10⁵) in HBSS were incubated with rPF4-treated bioparticles for 1 h at 37°C. Ice-cold PBS (400 µl) was added to the cell mixtures, and samples were analyzed using Attune NxT flow cytometer (Thermo Fisher Scientific).

IC-21 cells in DMEM (5x10⁵/0.1 ml) were placed into 96-well plates and allowed to adhere for 1 h at 37°C in 5% CO₂. S. aureus was incubated with different concentrations of rPF4 (0-50 µg/ml) for 1 hour at 37°C, and 10 µl of the mixture containing 10⁶ CFU was added to IC-21 cells and incubated for 1 hour at 37°C. Serial dilutions of supernatants were added to agar plates and incubated overnight at 37°C, and phagocytosis was determined by CFU counting. Cells were washed three times with DMEM to remove nonphagocytosed bacteria, and 100 μg/ml of gentamycin in DMEM was added to the wells to kill the remaining extracellular bacteria. After incubation for 1 hour at 37°C, cells were washed and incubated with 0.2% Triton X-100 using vigorous up and down pipetting for 3 min. Serial dilutions of lysates were added to LB agar plates and incubated overnight at 37°C to count CFUs of intracellular bacteria. In additional experiments, after gentamycin treatment, cells were incubated in DMEM for 24 and 48 h at 37°C in 5% CO₂ before lysing and CFU counting. In selected experiments, adherent cells were incubated in DMEM for 3 hours after gentamicin treatment and then permeabilized with 0.2% Triton X-100 for 3 min. Cells were stained with two different nucleic dyes provided in the LIVE/DEAD BacLight Bacterial Viability kit according to the manufacturer’s instructions to distinguish between live and dead bacteria. Confocal images were obtained with a Leica SP8 Confocal System (Exton, PA) using a 63x/1.4 objective.

A single colony of S. aureus from an agar plate was transferred to LB media and incubated with shaking in the screwcap 15-ml plastic tubes containing 3 ml of medium for 16 h at 37°C. The culture was diluted 20 times in fresh media and grown until the mid-log phase at 37°C. After harvesting bacteria, 10³ CFU was added to the wells containing progressively decreasing concentrations of selected antibiotics or PF4. Plates were incubated for 16 h at 37°C with shaking, and the visible growth of bacteria was evaluated after overnight incubation.

FACS analyses were performed to assess the expression of Mac-1 on the surface of differentiated HL-60 cells and to identify neutrophils and macrophages in the lavage isolated from the inflamed mouse peritoneum. HL60 cells (0.5x10⁶) were incubated with anti-Mac-1 mAb 44a (10 µg/ml) for 30 min at 4°C, followed by Alexa Fluor 647-conjugated secondary antibody. Cells in the peritoneal lavage were washed twice, and resuspended in HBSS. Aliquots (5x10⁵/0.1 ml) were incubated with anti-Mac-1 mAb M1/70 (10 µg/ml), followed by Alexa Fluor 647-conjugated secondary antibody and Alexa Fluor 488-conjugated anti-Ly-6G mAb (5 μg/ml). The expression of epitopes was analyzed using Attune NxT flow cytometer (ThermoFisher).

S. aureus (1x10⁸/ml) in PBS was incubated with rPF4 for 30 min at 22°C and washed twice in PBS. Bacteria then were incubated with rabbit polyclonal anti-PF4 antibody (1:250) for 30 min at 22°C followed by Alexa Fluor 488-conjugated secondary antibody, washed in PBS, and resuspended in 1% paraformaldehyde. Labeled bacteria were deposited on glass coverslips using Cytospin at 2000g for 5 min, and confocal images were acquired using Leica SP8 Confocal System (Exton, PA) using a 60x/1.4 and 100x/1.4 oil objectives.

S. aureus grown in LB media was centrifugated at 4000g, washed twice in PBS, and resuspended in 2% glutaraldehyde. Bacteria were deposited onto carbon-coated copper grids (Electron Microscopy Sciences), stained with 2% uranyl acetate for 45 sec, and washed thrice in water. Samples were analyzed using a Talos L120C G2 microscope.

8-weeks old mice were inoculated IP with 100 μl of S. aureus suspensions (5x10⁷ CFU) alone or S. aureus immediately followed by the injection of different concentrations (0-65 µg/mouse) endotoxin-free rPF4. Mice were sacrificed after 24 h, and peritoneal cavities were lavaged with 5 ml of a sterile endotoxin-free solution of PBS containing 5 mM EDTA. Aliquots of the peritoneal lavage were serially diluted and plated on LB agar plates. After 16 hours of incubation at 37°C, colonies on the plates were counted. Data are expressed as a CFU/ml recovered from peritoneal lavage. Peritoneal inflammation was assessed by determining the total number of leukocytes in the peritoneal lavage, and differential cell counts were determined using Wright-stained cytospin preparations.

Mice were inoculated IP with S. aureus (5x10⁸ CFU) without or in combination with endotoxin-free rPF4 (0.6 mg/kg). Animals were monitored for signs of mortality and morbidity every hour for the first 24 hours and then once daily for ten days. The primary endpoint used to assess the progress of infection and PF4 activity was infection-mediated death. The Kaplan-Meier survival plots were prepared to analyze the effect of PF4 on survival.

All data are presented as the mean ± S.D. The statistical differences between the two groups were determined using a Student’s t-test. Multiple comparisons were made using ANOVA followed by Tukey’s or Dunn’s post-test using GraphPad Instat software. Differences were considered significant at P < 0.05.

To serve as an opsonin, bacteria-bound PF4 should be available to the phagocytic receptor Mac-1 expressed on the surface of neutrophils and monocyte/macrophages. In agreement with a previous study (Krauel et al., 2011), nonencapsulated S. aureus, dose-dependently bound Iodine¹²⁵-labeled rPF4. At 10 µg/ml PF4, 0.11 fg of protein (8.5x10⁶ molecules) bound per 1 µm² of bacteria. The lack of the capsule was confirmed by transmission electron microscopy ( Figure 1A ). To examine further the association of PF4 with bacteria, we investigated the localization of rPF4 using confocal microscopy with polyclonal anti-PF4 antibody and secondary antibody conjugated with Alexa Fluor 488. As shown in Figure 1B , S. aureus was surrounded by a layer of rPF4, indicating that PF4 accumulated on the surface. The binding of the antibody to PF4 was specific since bacteria incubated only with the secondary antibody did not show fluorescence ( Figure S1 ). We also examined the localization of rPF4 on the surface of S. aureus grown under conditions that promote capsule formation. Transmission electron microscopy confirmed the presence of a polysaccharide capsule phenotypically similar to previously reported images (Verbrugh et al., 1982; O'Riordan and Lee, 2004; Wen and Zhang, 2015) and a thickness ranging from 115 to 610 nm in various cells ( Figure 1C ). Like nonencapsulated bacteria, rPF4 formed a layer on the surface of the capsule ( Figure 1D ).

Since previous studies that evaluated the ability of PF4 to kill bacteria produced conflicting results (Krijgsveld et al., 2000; Tang et al., 2002), we re-examined the effect of rPF4 on bacteria in the LB medium (pH 7.0) and the MES buffer (pH 5.5). S. aureus was incubated with different concentrations of rPF4 for 1-3 h, and bacterial growth in suspension and on LB agar plates was monitored. As shown in Figures 1E, F , Figure S2 , rPF4 did not affect S. aureus growth at neutral and acidic pH, even at a concentration as high as 100 µg/ml. We also confirmed the lack of bactericidal activity of rPF4 compared with several antibiotics using a minimal inhibitory concentration (MIC) assay ( Figure S3 ). In contrast to rPF4, all tested antibiotics, except ampicillin, were active.

Since many antimicrobial peptides contain amphipathic α-helices that can insert into the plasma membrane, they exert not only bactericidal but also a cytotoxic effect on host cells (Dijksteel et al., 2021). PF4 has the α-helix at the C-terminus, including residues 57 through 70, and a synthetic 13-residue peptide spanning the C-terminus of PF4 can kill bacteria, presumably by disrupting their membranes (Darveau et al., 1992). Therefore, we examined the ability of rPF4 to perturb the plasma membrane by determining the leakage of calcein dye from various host cells, including leukocytes isolated from the peritoneal lavage, cultured IC-21 macrophages, and human U937 monocytic cells. rPF4 did not cause calcein leakage even at a concentration as high as 80 µg/ml (10 µM) ( Figure 2 ). In contrast, the cathelicidin peptide LL-37, a potent α-helical antimicrobial peptide, dose-dependently increased calcein leakage from all cells.

The finding that Mac-1 dislikes negatively charged residues and surfaces (Lishko et al., 2004; Podolnikova et al., 2015a) suggests that the bacterial surface displaying negatively charged molecules may resist phagocytosis by causing the repulsion of Mac-1. To explore this possibility, in the initial experiments, we tested adhesion of Mac-1-expressing cells to surfaces coated with a negatively charged mammalian polysaccharide heparan sulfate, which is structurally similar to bacterial heparosan (Li et al., 2013) and served as a model negatively-charged surface. A standard approach to test the interaction between Mac-1 and its ligands is using HEK293 cells stably transfected with Mac-1 (Mac-1-HEK293) to examine their ability to bind immobilized ligands (Yakubenko et al., 2002; Lishko et al., 2004). These cells failed to attach to plastic coated with increasing concentrations (1-100 µg/ml) of heparan sulfate ( Figure 3A ). Although cells attached to some extent to the uncoated plastic (~20%), which is known to support Mac-1-mediated adhesion (Yakubenko et al., 2002), only a few cells adhered to heparan sulfate. Mac-1-HEK293 cells remained round on these surfaces, a hallmark of weak adhesion ( Figures 3D, E ). Also, no adhesion of isolated peritoneal macrophages and IC-21 macrophage cell line to heparan sulfate was detected (not shown). Treatment of heparan sulfate-coated surfaces with rPF4 resulted in its binding as determined using an anti-PF4 antibody ( Figure 3B ). rPF4-coated surfaces supported dose-dependent adhesion of Mac-1-HEK293 cells, IC-21, and peritoneal macrophages ( Figure 3C ). Furthermore, cells could spread on rPF4-coated surfaces ( Figures 3D, E ). These data suggest that the inability of Mac-1 on macrophages to bind the negatively charged surfaces can afford bacteria protection, allowing them to evade phagocytosis, and the binding of rPF4 can remove the antiphagocytic shield.

To determine the effect of rPF4 on phagocytosis of S. aureus, we performed phagocytic assays using various populations of Mac-1-expressing leukocytes. Since the phagocytic function of neutrophils is essential for resolving microbial infections (Nathan, 2006), and Mac-1 is involved in the binding of PF4 by neutrophils (Lishko et al., 2018), we initially examined the effect of rPF4 on ingestion of S. aureus by HL60 cells, a neutrophil-like cell line. HL60 cells were differentiated into granulocytes (dHL-60) by culturing in DMSO, and expression of Mac-1 was verified by flow cytometry ( Figure 4A ). The effect of rPF4 on phagocytosis was assessed using S. aureus bioparticles conjugated with a pH-sensitive pHrodo dye, which emits fluorescence only after phagocytosis in the acidic conditions of phagolysosomal compartments (Guo et al., 2015). Figures 4B, C show that rPF4 dose-dependently increased phagocytosis of S. aureus bioparticle by dHL-60 cells.

We next examined the effect of rPF4 on phagocytosis by mouse neutrophils recruited to the peritoneum by TG injection. Since both types of myeloid leukocytes, neutrophils and monocytes, migrate into the peritoneum during the first hours of sterile inflammation induced by TG injection (Henderson et al., 2001), we used flow cytometry to distinguish the populations of cells in a 4-hour lavage. Neutrophils were identified as Ly6G⁺,CD11b⁺, and monocyte/macrophages as Ly6G^-,CD11b⁺ cells ( Figure 4D ). The gated cell populations were then used to detect pHrodo staining in the presence of rPF4 ( Figure 4E ). rPF4 increased phagocytosis of S. aureus bioparticles by both cell types in a dose-dependent manner ( Figure 4E ). More cells in the population of Ly6G⁺,CD11b⁺ neutrophils phagocytosed S. aureus bioparticles in the absence of rPF4 than Ly6G^-,CD11b⁺ monocyte/macrophages ( Figure 4F ). However, although not statistically significant, Ly6G^-,CD11b⁺ cells tended to exhibit higher phagocytic activity with increasing concentrations of rPF4 than Ly6G⁺,CD11b⁺ cells (3.1 vs. 1.7-fold) ( Figure 4G ).

To further examine the effect of rPF4 on cells of the monocyte/macrophage origin, we used a mouse peritoneum-derived macrophage cell line IC-21 and peritoneal mouse resident and inflammatory macrophages. As shown in Figures 5A, B , rPF4 augmented phagocytosis of pHrodo-coupled S. aureus bioparticles by adherent IC-21 macrophages in a dose-dependent manner, and quantification indicated that at 40 µg/ml, rPF4 increased phagocytosis by ~4-fold compared to nontreated cells. Also, at this concentration, rPF4 increased phagocytosis of bioparticles by suspended IC-21 cells by ~5-fold ( Figures 5C, D ).

Resident peritoneal leukocytes comprised of ~60% macrophages and ~40% B lymphocytes (Ghosn et al., 2010; Podolnikova et al., 2016) were obtained by lavage from the unstimulated mouse peritoneum. Recent studies demonstrated that bacterial entry into the peritoneum induces rapid macrophage adherence (Zhang et al., 2019). Therefore, we measured phagocytosis using cells adherent to plastic after removing nonadherent B lymphocytes. At 40 µg/ml, rPF4 enhanced phagocytosis by adherent resident macrophages by ~10-fold ( Figures 5E, F ). Since the composition and phenotype of macrophages in the peritoneal cavity change in response to infectious stimuli (Ghosn et al., 2010; Cassado Ados et al., 2015), we also measured phagocytosis by inflammatory macrophages obtained 3 days after intraperitoneal TG injection. These cells originate from blood monocytes that infiltrate the peritoneum and gradually differentiate into the cells termed small peritoneal macrophages (Ghosn et al., 2010). Adherent inflammatory macrophages also exhibited augmented phagocytosis of S. aureus bioparticles in the presence of rPF4 ( Figures 5G, H ), although the effect of rPF4 was lesser than on resident macrophages. These results indicate that rPF4 significantly increases phagocytosis of S. aureus bioparticles by both neutrophils and macrophages.

Since S. aureus bioparticles may not fully recapitulate the properties of live bacteria, we have examined the effect of rPF4 on phagocytosis of live S. aureus. In addition, we compared phagocytosis of nonencapsulated and encapsulated bacteria. As shown in Figure 6A , in the absence of rPF4, resident peritoneal macrophages ingested 1.7 ± 0.05-fold more nonencapsulated than encapsulated S. aureus. rPF4 enhanced phagocytosis of both strains, albeit to different extents. In particular, ~2.8 times more encapsulated bacteria were ingested by macrophages in the presence than in the absence of rPF4 compared to ~1.8 times for nonencapsulated bacteria.

We have also examined whether phagocytosis of live S. aureus was mediated by Mac-1. We previously demonstrated that Mac-1-deficient macrophages minimally ingested E. coli, and adding rPF4 did not enhance phagocytosis, indicating that the effect was specific for Mac-1 (Lishko et al., 2018). In line with these data, only a small number of S. aureus was phagocytosed by Mac-1-deficient macrophages, and rPF4 did not increase their phagocytosis by these cells ( Figure 6B ).

Staphylococci can survive within phagocytes (Garzoni and Kelley, 2009; Fraunholz and Sinha, 2012). To examine whether the PF4-induced augmentation of phagocytosis impacts the intracellular killing of rPF4-coated S. aureus in the phagolysosome, we determined the survival of internalized bacteria by quantifying CFUs of viable bacteria using the opsonophagocytic assay. Adherent IC-21 macrophages were incubated for 1 hour with nonencapsulated S. aureus (MOI 1:20) preincubated or not with rPF4. Counting CFUs of nonphagocytosed bacteria and subtracting this number from the initial input showed that ~1.7- and ~2.8-fold fewer bacteria remained in the supernatant after treatment of bacteria with 25 and 50 µg/ml rPF4, respectively, compared to control nontreated bacteria ( Figure 7A ). After removing nonphagocytosed bacteria, adherent macrophages were treated with gentamicin for 1 h to remove additional extracellular bacteria, followed by cell lysis and quantitation of viable intracellular bacteria. The number of viable intracellular bacteria paralleled the augmentation of phagocytosis by rPF4 ( Figure 7B ). In particular, ~2.1 and ~3.4-fold greater numbers were found in macrophages that ingested rPF4-treated bacteria in the presence of 25 µg/ml and 50 µg/ml, respectively, than nontreated bacteria. Also, as detected by the LIVE/DEAD BacLight assay, which allows differentiating between live and dead bacteria, an increased number of viable bacteria remained in macrophages 3 h after gentamicin treatment ( Figure 7C ). However, after 48 hours, the number of colonies was drastically reduced to ≤0.1% for both treated and nontreated bacteria ( Figure 7B , Figure S4 ), suggesting that the augmentation of phagocytosis by rPF4 did not compromise the ability of macrophages to carry out the effective intracellular killing. Hence, although treatment with PF4 resulted in an early increase of viable bacteria in macrophages, the numbers decreased to the same levels as for nontreated bacteria after 48 h, indicating that the intracellular killing eliminated significantly more rPF4-coated bacteria.

To assess the effect of rPF4 on bacterial clearance, mice were infected with 5x10⁷ CFU of S. aureus, followed immediately by an IP injection of rPF4. Bacterial counts in the peritoneal lavage were assessed 24 h after infection, and saline served as vehicle control. As shown in Figure 8A , rPF4 enhanced bacteria clearance. The effect of rPF4 was dose-dependent with an IC₅₀ of 5.8 ± 1.2 µg/mouse, corresponding to 0.29 ± 0.06 mg/kg. At ≥10 µg/mouse (65 µg/mouse maximal testable concentration), rPF4 reduced live bacteria in the peritoneal lavage by >90%. Analysis of cell numbers in the lavage and examination of cytospin preparations of lavage fluid from infected mice showed that whereas S. aureus induced a substantial influx of neutrophils and monocyte/macrophages in the peritoneum, their numbers in mice treated with 13 µg/ml rPF4 were reduced by ~2-fold (2.3 ± 0.1 x10⁶/ml vs. 1.4 ± 0.04 x10⁶/ml for neutrophils and 2.3 ± 0.1 x10⁶/ml vs. 0.89± 0.02 x10⁶/ml for macrophages) ( Figure 8B ). rPF4 injected alone slightly decreased neutrophil migration, but this difference was not statistically significant compared to the PBS control. In addition, rPF4 effectively reduced infection caused by 5x10⁷ CFU MRSA with efficacy at the same dose as for antibiotic-susceptible strain ( Figure 8C ).

The increased bacterial clearance in the presence of rPF4 translated into a significant difference in survival profile relative to nontreated mice. rPF4 treatment substantially improved survival from a sub-lethal dose of bacterial inoculum (5x10⁸) compared to vehicle control ( Figure 8D ).