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RNase 7 Protects Healthy Skin from Staphylococcus aureus Colonization

      TO THE EDITOR
      The Gram-positive bacterium Staphylococcus aureus is an important pathogen that causes various skin infections (
      • Miller L.G.
      • Kaplan S.L.
      Staphylococcus aureus: a community pathogen.
      ). However, healthy skin is usually not infected by S. aureus, despite the high carrier rates in the normal population (
      • Noble W.C.
      Skin bacteriology and the role of Staphylococcus aureus in infection.
      ). This suggests that the cutaneous defense system has the capacity to effectively control the growth of S. aureus. There is increasing evidence that antimicrobial proteins are important effectors of the cutaneous defense system (
      • Harder J.
      • Gläser R.
      • Schröder J.M.
      Human antimicrobial proteins effectors of innate immunity.
      ). A recent study reported that keratinocytes contribute to cutaneous innate defense against S. aureus through the production of human β-defensin-3 (
      • Kisich K.O.
      • Howell M.D.
      • Boguniewicz M.
      • et al.
      The constitutive capacity of human keratinocytes to kill Staphylococcus aureus is dependent on beta-defensin 3.
      ). In addition to human β-defensin-3,, other antimicrobial proteins may also participate in cutaneous defense against S. aureus. One candidate is RNase 7, a potent antimicrobial ribonuclease that is highly expressed in healthy skin (
      • Harder J.
      • Schröder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ;
      • Köten B.
      • Simanski M.
      • Gläser R.
      • et al.
      RNase 7 contributes to the cutaneous defense against Enterococcus faecium.
      ).
      To investigate the hypothesis that RNase 7 may contribute to protect healthy skin from S. aureus colonization, we first incubated natural RNase 7 isolated from stratum corneum skin extracts (
      • Harder J.
      • Schröder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ) with S. aureus (ATCC 6538). In concordance with our initial report about RNase 7 (
      • Harder J.
      • Schröder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ), we verified that RNase 7 exhibited a high killing activity against S. aureus (lethal dose of 90% =3–6μgml−1).
      Recently, we reported a moderate induction of RNase 7 mRNA expression in primary keratinocytes treated with heat-killed S. aureus (
      • Harder J.
      • Schröder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ). To assess the induction of RNase 7 by S. aureus in the context of more physiological conditions, we used human skin explants and incubated them for 2, 6, and 20hours with living S. aureus (the use of skin material for this study was approved by the ethical committee of the University Kiel (A 104/06) in accordance with the Declaration of Helsinki Principles). As shown in Figure 1, only 2hours of incubation with living S. aureus significantly induced the secretion of RNase 7 on the skin surface as measured by an RNase 7-specific ELISA (
      • Köten B.
      • Simanski M.
      • Gläser R.
      • et al.
      RNase 7 contributes to the cutaneous defense against Enterococcus faecium.
      ). This indicates a fast release of preformed material. The reduced secretion after 20hours could be a result of cytotoxic effects from S. aureus, as well as of a decreased viability of skin explants. Secretion of RNase 7 after 2hours was enhanced by increasing concentrations of S. aureus (Supplementary Figure S1 online).
      Figure thumbnail gr1
      Figure 1Induced secretion of RNase 7 on the skin surface on treatment with living S. aureus. Defined areas (0.8cm2) of skin explants derived from plastic surgery were incubated with or without approximately 1,000 colony-forming units of S. aureus (ATCC 6538) in 100μl of sodium phosphate buffer. After 2, 6, and 20hours, the concentration of secreted RNase 7 was determined by ELISA. Stimulation with S. aureus for 2hours revealed a significant induction as compared with the unstimulated control after 2hours (*P<0.05, Student's t-test; n.s.=not significant). Data shown are means of triplicates of five skin explants derived from five donors.
      To further assess the physiological relevance of RNase 7 in cutaneous defense against S. aureus, we investigated whether the killing activity of skin extracts derived from stratum corneum was inhibited through blocking of the antimicrobial activity of RNase 7 by RNase 7-specific antibodies as previously described (
      • Köten B.
      • Simanski M.
      • Gläser R.
      • et al.
      RNase 7 contributes to the cutaneous defense against Enterococcus faecium.
      ). First, we analyzed whether RNase 7 antibodies neutralized the antibacterial activity of RNase 7 against S. aureus. For this purpose, we tested the activity of RNase 7 against S. aureus in an antibacterial microdilution assay in the presence of RNase 7 antibodies. This approach revealed that the application of RNase 7 antibodies completely blocked the S. aureus-killing activity of RNase 7. In contrast, the antimicrobial activity was not inhibited when equivalent concentrations of irrelevant antibodies (derived from goat preimmune serum;
      • Köten B.
      • Simanski M.
      • Gläser R.
      • et al.
      RNase 7 contributes to the cutaneous defense against Enterococcus faecium.
      ) were used (data not shown). Having established that RNase 7-specific antibodies neutralized the antimicrobial effect of RNase 7 against S. aureus, we used this approach to test the functional role of RNase 7 in cutaneous defense against S. aureus. To this end, we first incubated a stratum corneum skin extract with S. aureus. The skin extract was diluted to a ratio of 1:200 in 25μl of sodium phosphate buffer (pH 7.4) without or with RNase 7-blocking antibody (4mgml−1) or an equivalent concentration of an irrelevant antibody. This dilution leads to a final concentration of 6μgml−1 RNase 7 in the assay system, which corresponds to the determined range of the lethal dose90 of RNase 7 against S. aureus (see above). After 45minutes, 25μl of S. aureus (ATCC 6538) in sodium phosphate buffer (pH 7.4) containing 2% tryptone soya broth was added and incubated for 3hours. Killing activity was analyzed by plating serial dilutions of the incubation mixture and determining colony-forming units the following day. The skin extract exhibited a potent S. aureus-killing activity, as incubation of S. aureus with the skin extract resulted in a complete killing of S. aureus (Figure 2a). The application of RNase 7-blocking antibodies to the skin extract before inoculation with S. aureus resulted in an outgrowth of S. aureus of approximately 35% as compared with the growth control (Figure 2a). In contrast, an irrelevant antibody did not inhibit the killing activity of the skin extract (Figure 2a). These data indicate an important contribution of RNase 7 to the S. aureus-killing activity of the skin extracts. Other skin-derived antimicrobial proteins may be responsible for the S. aureus-killing activity, which remained after blocking the antimicrobial activity of RNase 7.
      Figure thumbnail gr2
      Figure 2RNase 7 contributes to the killing activity of human skin against S. aureus. (a) Stratum corneum extracts were incubated without (subcutaneous (s.c.)) or with RNase 7-blocking antibody (s.c.+R7-specific Ab) or an irrelevant antibody (s.c.+irrelevant Ab). After 45minutes, S. aureus was added and colony-forming units (CFUs) were determined after 3hours of incubation. Data shown are means±SD of a representative result of three independent experiments. (b) Defined areas (0.8cm2) of skin explants were incubated without (skin control) or with RNase 7 antibody (skin+R7-specific Ab) or with an equivalent concentration of an irrelevant antibody (skin+irrelevant Ab). After 30minutes, S. aureus was added and CFU were determined after 2hours of incubation. Data show means±SD of triplicate experiments of the same donor. A representative result of three independent experiments from three donors is shown. Student's t-test was used for statistics.
      To obtain additional insight into the functional role of RNase 7 in cutaneous defense against S. aureus, we studied the role of RNase 7 using human skin explants. In this experimental ex vivo model, S. aureus was applied to defined areas (0.8cm2) of the surface of skin explants. Preincubation of skin samples with 1.5mgml−1 RNase 7-blocking antibody in 100μl of sodium phosphate buffer (pH 7.4) led to a significant outgrowth of S. aureus as compared with pretreatment of skin with an equivalent concentration of irrelevant antibody (Figure 2b). These data further indicate a physiological relevance of RNase 7 for the control of S. aureus growth on the skin surface.
      In summary, data present herein show that skin infected with living S. aureus responds with an increased release of RNase 7, which contributes to limit the growth of S. aureus. These data are in concordance with a recently published study reporting that lower RNase 7 expression in healthy skin is associated with a higher risk of S. aureus skin infection (
      • Zanger P.
      • Holzer J.
      • Schleucher R.
      • et al.
      Constitutive expression of the antimicrobial peptide RNase 7 is associated with Staphylococcus aureus infection of the skin.
      ). Future studies have to evaluate whether skin infections caused by S. aureus may be associated with an impaired expression or function of RNase 7.

      ACKNOWLEDGMENTS

      We thank H Hinrichs, E Jeske, C Martensen-Kerl, and C Wilgus for excellent technical assistance. This study was supported by a grant from the Federal Ministry of Education and Research (BMBF, Skin Staph) to R Gläser and J Harder and by a Heisenberg program of the Deutsche Forschungsgemeinschaft (DFG) to J Harder.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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