Nonviable Staphylococcus aureus and its peptidoglycan stimulate macrophage recruitment, angiogenesis, fibroplasia, and collagen accumulation in wounded rats

We have previously shown that local application at the time of operation of Staphylococcus aureus, nonviable S. aureus, its cell wall, or S. aureus peptidoglycan accelerates wound healing. We hypothesized that this effect is due to both direct and indirect mechanisms, among which is an increase in the inflammatory response to wounding, resulting in an increase in macrophages, angiogenesis, and fibroblasts. Twenty-seven Sprague-Dawley male rats were anesthetized, and two 7-cm paravertebral skin incisions were made. Four polyvinyl alcohol sponges, two on each side, containing either 100 microliter of isotonic saline or 0.5 mg of nonviable S. aureus or S. aureus peptidoglycan in 100-microliter saline were implanted subcutaneously. Nonviable S. aureus or S. aureus peptidoglycan (860 microgram/cm incision) in 200-microliter saline were inoculated into the incisions at closure. The rats ate a commercial rat chow and drank tap water ad libitum throughout. After days 3 and 7 postwounding, rats were euthanized, and tissues were examined for immunohistochemical features of reparative tissue using ED-1, Factor VIII, and vimentin antibodies, markers for monocyte/macrophages, endothelial cells, and mesenchymal cells (including fibroblasts), respectively. Incisions treated with nonviable S. aureus or S. aureus peptidoglycan showed more macrophages along and deep in the wound tract 7 days postoperatively. Nonviable S. aureus or S. aureus peptidoglycan-treated sponges were surrounded and penetrated by much larger capsules of reparative tissue than saline-treated sponges at both 3 and 7 days. Neutrophil influx was much greater in nonviable S. aureus or S. aureus peptidoglycan-treated sponges, especially in central regions, and there were many more ED-1-stained macrophages in distinct geographic locations, specifically, the more peripheral-cortical areas. Some clustering of macrophages occurred around areas of invasion by reparative tissue into the surrounding subcutaneous fat and within the interstices of the sponges at the interface between reparative tissue and acute inflammatory cells. In contrast, saline-treated sponge reparative tissue had significantly fewer macrophages, much thinner and flimsy reparative tissue, with proportionately fewer macrophages clustering centrally. There were many more mesenchymal cells (notably fibroblasts) and new blood vessels and much more reparative collagen in the nonviable S. aureus or S. aureus peptidoglycan-treated sponges. We conclude that local application of nonviable S. aureus or S. aureus peptidoglycan at wounding induces an increased number and alteration in location of macrophages, increased influx (or proliferation) of mesenchymal cells (notably fibroblasts), and increased angiogenesis and reparative collagen accumulation, as well as increasing the overall acute inflammatory response to wounding.