Staphylococcus aureus Lipoteichoic Acid Damages the Skin Barrier through an IL-1-Mediated Pathway

Abstract

Staphylococcus aureus is a significant bacterial pathogen that may penetrate through the barrier into the epidermis and dermis of the skin. We hypothesized that the S. aureus cell wall product lipoteichoic acid (LTA) may contribute to the development of inflammation and skin barrier defects; however, the effects of LTA in vivo are not well understood. In this study, we examined the effects induced by intradermal S. aureus LTA. We found that keratinocytes in LTA-treated skin were highly proliferative, expressing 10-fold increased levels of Ki67. Furthermore, we observed that LTA caused damage to the skin barrier with substantial loss of filaggrin and loricrin expression. In addition, levels of the IL-1 family of inflammatory cytokines, as well as the neutrophil-attracting chemokines Cxcl1 and Cxcl2, were increased. Concomitantly, we observed significant numbers of neutrophils infiltrating into the epidermis. Finally, we determined that LTA-induced signals were mediated in part through IL-1, because an IL-1 receptor type 1 antagonist ameliorated the effects of LTA, blocking neutrophil recruitment and increasing the expression of skin barrier proteins. In summary, we show that S. aureus LTA alone is sufficient to promote keratinocyte proliferation, inhibit expression of epidermal barrier proteins, induce IL-1 signaling, and recruit cells involved in skin inflammation.

Fig 1.. S.aureus LTA induced inflammatory cytokine expression is mediated by p63 in primary keratinocytes.

Primary human keratinocytes transfected with control or p63 siRNA were treated +/− LTA for 24 hrs and analyzed by RT-PCR for the expression of cytokine and neutrophil chemotactic genes. Notably, LTA induced cytokine expression is absent in p63 siRNA silenced cells. Data are mean ± SEM, n = 3. *P<0.05; **P<0.01; ***P<0.001.

Fig 2.. Intradermal injection of S. aureus LTA induces epidermal thickening and hyperproliferation.

Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) Representative H&E staining of skin showing epidermal thickening upon LTA treatment. Bar = 100 μM. (b) Increased numbers of Ki67+ cells in the epidermis following LTA treatment. Bar = 40 μM. Dashed line represents epidermal-dermal junction. (c) Increased numbers of Keratin 5 (K5) positive cells after LTA treatment indicates keratinocyte involvement in epidermal thickening. (d) Quantitative analysis of Ki67+ cells in the epidermis. Data are mean ± SEM, n = 6, ***P<0.001.

Fig 3.. Neutrophils are recruited to the site of LTA injection.

Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) Representative H&E staining of skin showing neutrophil infiltration into the epidermis following LTA treatment (arrow). Bar = 100 μM. (b) Quantitation by RT-PCR for the neutrophil marker Ly6G, showing a 500-fold increase in neutrophils following LTA treatment. (c) RT-PCR analysis showing that neutrophil attracting chemokines are potently induced by LTA. Data are mean ± SEM, n = 6. **P<0.01; ***P<0.001.

Fig 4.. Neutrophil infiltration is associated with loss of filaggrin and loricrin expression.

Mice were injected ID with PBS or LTA. Skin samples at the site of injection were harvested 48 hours later. Neutrophil infiltration is observed with LTA treatment (H&E). Loss of filaggrin and loricrin staining (red stain at top of epidermis) is associated with neutrophil infiltration. Bar = 200 μM. Dashed line represents epidermal-dermal junction.

Fig 5.. IL-1 cytokines are induced by intradermal LTA.

Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) mRNA was analyzed by rt-PCR for expression of cytokine genes. Data are mean, n = 6. **P<0.01. (b) Staining of skin showing epidermal thickening and increased IL-1b expression in the epidermis upon LTA treatment. Bar = 40 μM. Dashed line represents epidermal-dermal junction.

Fig 6.. The IL-1R1 antagonist blocks LTA induced neutrophil recruitment and increases Flg and Lor expression.

Mice were injected ID with PBS, the IL-1 receptor 1 antagonist (IL-1Ra), LTA, or LTA combined with IL-1Ra. Skin samples at site of injection were harvested 48 hours later. (a) Quantitation by RT-PCR for the neutrophil marker Ly6G, shows that IL-1Ra causes an 80% reduction in neutrophils following LTA treatment. (b and c) RT-PCR analysis shows that LTA induced expression of neutrophil attracting chemokines is reduced by IL-1Ra. The LTA induced reduction of Flg (d) and Lor (e) expression is ameliorated by IL-1Ra. (f) The LTA induced increase in epidermal thickness is unchanged by IL-1Ra. Data are mean ± SEM, n = 6. *P<0.05; **P<0.01.