1. Activation of TLR2 by a Small Molecule Produced by Staphylococcus epidermidis Increases Antimicrobial Defense against Bacterial Skin Infections 
Yuping Lai, Anna L. Cogen, Katherine A. Radek, Hyun Jeong Park, Daniel T. MacLeod, Anke Leichtle, Allen F. Ryan, Anna Di Nardo, Richard L. Gallo 
Journal of Investigative Dermatology 
Volume 130, Issue 9, Pages (September 2010)
DOI: /jid
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Toxicity of staphylococci to cultured human keratinocytes. (a) The morphology of undifferentiated cultured normal human epidermal keratinocytes (NHEKs) after exposure to a sterile supernatant of culture medium conditioned by the overnight growth of S. aureus Newman or S. epidermidis (b) Sterile filtered whole conditioned medium (0–36μgml−1), the large molecular weight >10kDa dialysis retentate, or the small molecular weight <10kDa ultrafiltrate of conditioned culture medium from two S. aureus strains (S. aureus Newman and S. aureus Rosenbach) and two S. epidermidis strains (S. epidermidis 1457 and S. epidermidis 12228) were used to treat undifferentiated NHEKs for 24hours, followed by lactate dehydrogenase (LDH) assay. Scale bar=50μm. TSB, tryptic soy broth.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Staphylococcus epidermidis induces human β-defensin (hBDs) 2 and 3 in normal human keratinocytes. (a) hBD3 and (b) hBD2 expression of undifferentiated human keratinocytes stimulated by 36μgml−1 of sterile 10kDa ultrafiltrates from nine bacteria conditioned culture media for 24hours. (c) The time- and (d) dose-dependent curves of hBD3 induced by sterile <10kDa products from S. epidermidis cultured media (SECM). (e–g) Immunofluorescent staining of hBD3 induced by 36μgml−1 of SECM in cultured undifferentiated keratinocytes. (e) Rabbit IgG, (f) tryptic soy broth (TSB), and (g) SECM. (h) hBD3 and (i) hBD2 expression of differentiated human keratinocytes stimulated by 36μgml−1 of sterile 10kDa ultrafiltrates from nine bacteria conditioned culture media for 24hours. *P<0.05; **P<0.01; ***P< P-values were determined using one-way analysis of variance (ANOVA) (a, b, h, and i) or two-way ANOVA (c, d). Data shown represent two independent experiments with n=3 per group. Scale bar=10μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Effects of differentiation, live S. epidermidis, and S. epidermidis extract on hBD2 and 3 expression by keratinocytes. The expression of hBD2 and hBD3 by live S. epidermidis in undifferentiated versus differentiated normal human keratinocytes, and the inhibitory effect of S. epidermidis >10kDa fraction on hBD3 induced by SECM in differentiated normal human keratinocytes. (a, b) Colony-forming units, 106, of live S. epidermidis 1457 was added to (a) undifferentiated normal human epidermal keratinocytes (NHEKs) or (b) differentiated NHEKs for 24hours. The expression of human β-defensin 3 (hBD3) and hBD2 was analyzed using real-time RT-PCR. (c) Combining the >10kDa dialysis retentate of S. epidermidis 1457 with the <10kDa ultrafiltrate from the conditioned culture medium (SECM) abrogated the capacity of SECM to induce hBD3. ***P< P-values were determined using two-way analysis of variance (ANOVA). Data shown represent two independent experiments with n=3 per group.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Staphylococcus epidermidis increases antimicrobial activity of undifferentiated keratinocytes against multiple skin pathogens. (a) The expression of human β-defensin 3(hBD3) after treatment with hBD3 short hairpin RNA (shRNA). (b) The growth of S. aureus sa113 and (c) group A Streptococcus (GAS) after exposure to lysates from undifferentiated human keratinocytes pretreated with hBD3 shRNA and then stimulated by sterile <10kDa products from S. epidermidis cultured media (SECM). (d) The growth of S. epidermidis 1457 after exposure to lysates of SECM-treated keratinocytes. (e) The growth of S. aureus sa113 after exposure to lysates of ECCM-treated keratinocytes. (f) The survival of human papillomavirus 5 (HPV5) in HaCaT and (g) vaccinia virus in undifferentiated human keratinocytes after 24-hour SECM pretreatment. *P<0.05; **P<0.01. NS, no significance. P-values were evaluated using two-tailed t-tests (a and f–g) or one-way analysis of variance (ANOVA) (b, c). Data are the mean±SD of triplicate cultures and are representative of two independent experiments.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Staphylococcus epidermidis protects mice from group A Streptococcus (GAS) infection, and Malp2 induces hBDs 2 and 3 against GAS infection in a manner similar to S. epidermidis. (a) Photograph of skin lesions caused by GAS at 3 days after GAS injection. (b) ImageJ analysis of the lesion size of a. (c) Local GAS survival in skin and (d) systemic GAS survival in liver of SECM- and tryptic soy broth (TSB)-pretreated mice. (e) The expression of human β-defensins (hBDs) 2 and 3 by Toll-like receptor 2 (TLR2) ligands in undifferentiated normal human keratinocytes. (f) ImageJ analysis of skin lesion size of Malp2- and phosphate-buffered saline (PBS)-pretreatment mice. (g) The survival of local GAS in skin and (h) systemic survival of GAS in the livers of Malp2- and PBS-pretreatment mice. The injection of SECM, TSB, Malp2, or PBS at 24 and 2hours before live GAS injection did not cause skin lesions. *P<0.05; ***P< P-values were determined using two-tailed t-tests. Data shown are representative of two independent experiments with n=3–6 per group.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Staphylococcus epidermidis activates Toll-like receptor 2 (TLR2) to induce β-defensins. (a) TLR2 expression in undifferentiated normal human keratinocytes after a 24-hour SECM (10μgml−1) stimulation. (b) Human β-defensin 3 (hBD3) expression by SECM (10μgml−1) in undifferentiated normal human epidermal keratinocytes (NHEKs) after TLR2-neutralizing antibody treatment. (c) The expression of hBD2 by SECM (10μgml−1) in undifferentiated NHEKs after TLR2-neutralizing antibody treatment. (d) Murine β-defensin 4 (mBD4) expression induced by 12μg of SECM in wild-type and TLR2-deficient mice. (e) mBD14 expression induced by SECM in vivo. (f) mBD3 expression induced by SECM in vivo. *P<0.05; **P<0.01; ***P< P-values were evaluated using two-tailed t-tests (a), one-way analysis of variance (ANOVA) (b, c), or two-way ANOVA (d). Data shown represent two independent experiments with n=3 (a–c) or n=6 (d–f) per group.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions