Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections

Abstract

Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of <10 kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human beta-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.

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 1457. (b) Sterile filtered whole conditioned medium (0–36 μgml⁻¹), the large molecular weight > 10 kDa 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 24 hours, followed by lactate dehydrogenase (LDH) assay. Scale bar = 50 μm. TSB, tryptic soy broth.

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 36 μgml⁻¹ of sterile 10 kDa ultrafiltrates from nine bacteria conditioned culture media for 24 hours. (c) The time- and (d) dose-dependent curves of hBD3 induced by sterile <10 kDa products from S. epidermidis cultured media (SECM). (e–g) Immunofluorescent staining of hBD3 induced by 36 μg ml⁻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 36 μgml⁻¹ of sterile 10 kDa ultrafiltrates from nine bacteria conditioned culture media for 24 hours. *P<0.05; **P<0.01; P<0.001. 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 = 10 μm.

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 > 10 kDa 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 24 hours. The expression of human β-defensin 3 (hBD3) and hBD2 was analyzed using real-time RT-PCR. (c) Combining the > 10 kDa dialysis retentate of S. epidermidis 1457 with the < 10 kDa ultrafiltrate from the conditioned culture medium (SECM) abrogated the capacity of SECM to induce hBD3. P<0.001. P-values were determined using two-way analysis of variance (ANOVA). Data shown represent two independent experiments with n = 3 per group.

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 < 10 kDa 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.

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 2 hours before live GAS injection did not cause skin lesions. *P<0.05; P<0.001. P-values were determined using two-tailed t-tests. Data shown are representative of two independent experiments with n = 3–6 per group.

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 (10 μgml⁻¹) stimulation. (b) Human β-defensin 3 (hBD3) expression by SECM (10 μgml⁻¹) in undifferentiated normal human epidermal keratinocytes (NHEKs) after TLR2-neutralizing antibody treatment. (c) The expression of hBD2 by SECM (10 μgml⁻¹) in undifferentiated NHEKs after TLR2-neutralizing antibody treatment. (d) Murine β-defensin 4 (mBD4) expression induced by 12 μg 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<0.001. 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.