Modulation of the local neutrophil response by a novel hyaluronic acid-binding peptide reduces bacterial burden during staphylococcal wound infection

Abstract

Novel approaches targeting the host's immune response to treat Staphylococcus aureus infections have significant potential to improve clinical outcomes, in particular during infection with antibiotic-resistant strains. The hyaluronic acid-binding peptide (HABP) PEP35 was assessed for its ability to treat S. aureus infections using a clinically relevant murine model of surgical wound infection. PEP35 demonstrated no direct antimicrobial activity against a range of antibiotic-susceptible and antibiotic-resistant clinical isolates of Staphylococcus aureus. However, when this peptide was administered at the onset of infection and up to 4 h postchallenge with a methicillin-susceptible (MSSA) or a methicillin-resistant (MRSA) strain of S. aureus, it significantly reduced the bacterial burden at the wound infection site. PEP35 reduced the tissue bacterial burden by exclusively modulating the local neutrophil response. PEP35 administration resulted in a significant early increase in local CXCL1 and CXCL2 production, which resulted in a more rapid influx of neutrophils to the infection site. Importantly, neutrophil influx was not sustained after treatment with PEP35, and administration of PEP35 alone did not induce a local inflammatory response. The immunomodulatory effects of PEP35 on CXC chemokine production were TLR2 and NF-κB dependent. We propose a novel role for a HABP as an innate immunomodulator in the treatment of MSSA and MRSA surgical wound infection through enhancement of the local CXC chemokine-driven neutrophil response.

FIG. 1.

PEP35 promotes early CXC chemokine production and associated neutrophil recruitment at the S. aureus wound infection site. (A) Surgical wounds were established in WT mice and infected with S. aureus strain PS80 (10² CFU) alone or in combination with PEP35 (100 μg) or a control peptide. The tissue levels of CXC chemokines CXCL1 and CXCL2 were measured at 6 and 24 h, respectively, postchallenge; the tissue levels of the CC chemokine CCL2 were also measured at 6 and 24 h postchallenge; and the tissue levels of proinflammatory cytokine IL-1β were measured at 6 h (n = 5 to 20 mice per group). (B) Wound tissue was excised at 24 h after induction of infection, formalin fixed, and embedded in paraffin. Tissue sections were H&E stained to visualize inflammatory infiltration around the wound suture site (two representative sections [1 and 2] of n = 6 to 7 individual mice per group are shown). (C) Tissue sections were scored, and the bar represents median values. (D) Wound tissue was also excised at 24 h and homogenized, and the tissue MPO levels were quantified (n = 10 to 20 mice per group). For all of the data presented the median is represented by the horizontal bar within the box. The upper and lower boundaries represent the 25th to 75th percentiles of the data, and whiskers represent the 10th and 90th percentiles of the data.

FIG. 2.

PEP35 does not result in sustained neutrophil influx at the S. aureus wound infection site. (A) Surgical wounds were established in WT mice and infected with S. aureus strain PS80 (10² CFU) alone or in combination with PEP35 (100 μg) or a control peptide. Wound tissue was excised on day 5 after the induction of infection, formalin fixed, and embedded in paraffin. Tissue sections were H&E stained to visualize inflammatory infiltration around the wound suture site (representative sections of n = 5 individual mice per group). (B) Wound tissue was also excised on day 5 and homogenized, and the tissue MPO levels were quantified (n = 12 to 14 mice per group). The median is represented by the horizontal bar within the box. The upper and lower boundaries represent the 25th to 75th percentiles of the data, and whiskers represent the 10th and 90th percentiles of the data.

FIG. 3.

Administration of PEP35 up to 4 h postinfection enhances the local neutrophil response and promotes bacterial clearance. Surgical wounds were established in WT mice and infected with S. aureus strain PS80 (10² CFU) alone. At the indicated time points postinfection, PEP35 (100 μg) or PBS was administered by intramuscular injection directly into the wound site. CXCL1 (A) and CXCL2 (B) were measured in the wound tissue at 6 and 24 h, respectively, after bacterial challenge. (C) To access neutrophil influx to the infection site, wound tissue was excised at 24 h and homogenized, and the tissue MPO levels were quantified. For data presented in panels A and B, the median is represented by the horizontal bar within the box. The upper and lower boundaries represent the 25th to 75th percentiles of the data, and whiskers represent the 10th and 90th percentiles of the data (n = 5 to 15 mice per group). (D) Wound tissue was also excised on day 3, and the total tissue bacterial burden was quantitated by plate counts. The results are expressed as the log CFU per gram of tissue; the median is indicated by a bar (n = 10 to 20 mice per group).

FIG. 4.

PEP35 enhances the local neutrophil response and promotes bacterial clearance during HA-MRSA surgical wound infection. (A) Surgical wounds were established in WT mice and in mice infected with S. aureus strain USA100 (10² CFU) alone or in combination with PEP35 (100 μg). Tissue levels of CXCL1 were measured at 6 h postchallenge (n = 10 mice per group). (B) Wound tissue was excised at 24 h and homogenized, and the tissue MPO levels were quantified (n = 8 mice per group). For the data in panels A and B, the median is represented by a horizontal bar within the box. The upper and lower boundaries represent the 25th to 75th percentiles of the data, and whiskers represent the 10th and 90th percentiles of the data. (C) Wound tissue was also excised on day 3, and the total tissue bacterial burden was quantitated by plate counts. The results are expressed as the log CFU per gram of tissue, and the median is indicated by a bar (n = 10 mice per group).

FIG. 5.

PEP35 has a dose-dependent differential effect on CXC chemokine production by primary peritoneal macrophages. In vitro cultures of purified WT murine peritoneal macrophages (10⁵ cells) were stimulated with heat-killed S. aureus strain PS80 (10⁵ CFU/ml) in the presence or absence of increasing concentrations of PEP35 for 24 h. CXCL1 (A) and CXCL2 (B) levels in the supernatant were measured by ELISA (upper panel). Macrophages were also stimulated with increasing concentrations of PEP35 alone in the absence of S. aureus. CXC chemokine levels were measured in the supernatant at 24 h (lower panel). The results represent the means ± the standard error of the mean (n = 10 individual experiments). In vitro cultures of purified WT murine peritoneal macrophages (10⁵ cells) were incubated with the NF-κB inhibitor Bay 11-7082 (4 or 2 μM) for 1 h prior to stimulation with heat-killed S. aureus strain PS80 (10⁴ CFU/ml) in the presence or absence of PEP35 (1 μg/ml) for 24 h. CXC chemokine levels in the supernatant were measured by ELISA (C). The results are expressed as the fold increase induced by PEP35 compared to that induced by the no-PEP35 treatment and represent the pooled data for five individual experiments.

FIG. 6.

The immunomodulatory effects of PEP35 in vivo are partially TLR2 dependent. (A) Surgical wounds were established in TLR2^−/− mice and infected with S. aureus strain PS80 (10² CFU) alone or in combination with PEP35 (100 μg) or a control peptide. The tissue levels of CXCL1 were measured at 6 h postchallenge (n = 8 mice per group). (B) Wound tissue was also excised at 24 h and homogenized, and the tissue MPO levels were quantified (n = 8 mice per group). For all data presented, the median is represented by the horizontal bar within the box. The upper and lower boundaries represent the 25th to 75th percentiles of the data, whiskers represent the 10th and 90th percentiles of the data. (C) Wound tissue was excised on day 3 postchallenge, and the total tissue bacterial burden was quantitated by plate counts. The results are expressed as the log CFU per gram of tissue, and the median is indicated by a bar (n = 8 mice per group).