difference antimicrobial activity may be due to the conditions under which activity was assayed or the differences in d-toxin primary sequence (glutamine at position 3 or the addition of a threonine at position 24 in the S. aureus d-toxin). Several phenol soluble modulins (PSMs) produced by S. epidermidis, S. aureus d-toxin derivatives, and Staphylococcus haemolyticus gonococcal growth inhibitor exhibit antimicrobial properties and activity. The S. epidermidis PSMs have also been shown to enhance the antimicrobial activity of LL-37 on Group A Streptococcus (GAS)[15]. Previous studies have similarly reported that host AMPs act in synergy to kill bacteria [16]. Specifically, LL-37 and hBD2 have been shown to synergistically kill Group B Streptococcus in vitro [17]. Moreover, host AMPs have been shown to act synergistically with an antimicrobial peptide produced by L. lactis to inhibit E. coli [18]. Thus, we sought to determine whether the antimicrobial d-toxin, also known as PSMc, produced by the resident cutaneous microbe, S. epidermidis, could interact with the host antimicrobials leading to greater pathogen inhibition and enhancement of the host’s innate immune system. Here, we demonstrate that the antimicrobial PSMs contribute to host innate immunity through interacting with and amplifying the cutaneous antimicrobial response. These data illustrate a novel means by which d-toxin contributes to the innate immune system, pointing towards a beneficial role for S. epidermidis on the skin’s surface. Results d-Toxin Is Deposited on the Skin and Binds to Neutrophil Extracellular Traps Phenol soluble modulins are multifunctional and can act to enhance virulence when invasive [19], or as antimicrobials when PSMs Cooperate with Host AMPs in direct contact with pathogens such as GAS. To further evaluate the relevance of PSMs as surface antimicrobials on human skin we first determined if d-toxin was detectable on normal human skin. Immunohistochemistry demonstrated that d-toxin is abundantly detectable in the normal epidermis, hair follicle and sparsely in the dermis (Figure 1a). Similar staining was observed in a second skin sample from a different individual and staining was confirmed with a second custom-made anti-d-toxin antibody (data not shown). It is unclear if the two different antibodies recognize different epitopes, as the commercially available epitope is not known. Next, since injured skin rapidly accumulates neutrophils at sites of infection and injury, and these cells act in part to protect the skin through the formation of neutrophil extracellular traps (NETs) containing antimicrobial peptides [6,20], we evaluated if d- toxin from surface S. epidermidis could interact with NETs and contribute to their activity. d-toxin was added to PMA-induced neutrophil extracellular traps (NETs) in culture. Addition of d- toxin to these cells showed that d-toxin bound to the NETs and colocalized with cathelicidin endogenously released from the neutrophil (Figure 1b). The specifity of the antibody for d-toxin is demonstrated by lack of staining of normal human keratinocytes in the absence of d-toxin but positive staining in the presence of d- toxin (Figure 1c). As the high isoelectric point of this peptide predicts that this association with NETs could occur through DNA binding, d-toxin association with DNA was next directly evaluated using tryptophan spectroscopy. In buffer alone, d-toxin’s trypto- phan emits maximally at 341 nm. In the presence of neutrophil DNA, the maximal emission shifted to 331 nm (Figure 1d). The blue shift caused by the presence of neutrophil DNA suggested a direct association with d-toxin. Finally, in addition to interacting with NETs, we sought to determine if d-toxin could also induce Figure 1. d-toxin is deposited in the skin by S. epidermidis and binds neutrophil extracellular traps. a, normal healthy human skin stained for d-toxin, showed deposition in the epidermis and dermis. Inset is 406magnification of d-toxin in dermis. Bar represents 50 mm. This is a single specimen representative of two. Nuclei are labeled with DAPI (blue) and d-toxin is labeled with Alexa fluor 488 (green). The far left panel depicts the IgG control for the anti d-toxin staining. b, d-toxin was added to neutrophil extracellular traps (NETs), which were subsequently stained for LL-37 and d-toxin. Staining shows colocalization of antimicrobial peptides along DNA strands. Bar represents 20 mm. Nuclei are labeled with DAPI (blue), d-toxin is labeled with Alexa fluor 488 (green), and LL-37 is labeled with Alexa fluor 568 (red). The far left panel depicts the IgG control for the anti d-toxin and anti LL-37 staining. c, primary keratinocytes incubated with (+ d-toxin) and without (-d-toxin) d-toxin then anti d-toxin staining evaluated. Nuclei are stained with DAPI (blue) and d-toxin is labeled with Alexa fluor 568 (red). d, tryptophan spectroscopy of d-toxin in the presence and absence of neutrophil DNA. doi:10.1371/journal.pone.0008557.g001 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8557