Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury

Abstract

The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA) and acts selectively on keratinocytes triggered through Toll-like receptor 3(TLR3). We show that TLR3 activation is required for normal inflammation after injury and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. To our knowledge, these findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.

Figure 1. S. epidermidis inhibits poly(I:C)-induced inflammatory cytokines produced by keratinocytes, and inflammation in mouse skin

(a) Quantification of IL-6 and TNF-α mRNA expression of cultured normal human keratinocytes treated with 36 μg ml⁻¹ of a sterile <10 kDa product of S. epidermidis conditioned culture media (SE) and 10 μg ml⁻¹ of the TLR3 ligand [poly(I:C)] for 24 h. (b) Photographs of the ears of BALB/c mice 24 h after subcutaneous injection with PBS alone, SE alone, poly(I:C) alone or SE and poly(I:C). (c) H&E staining of ears treated as in (b), scale bars represent 100 μm. (d) Quantification of IL-6 and TNF-α expression in tissue from mouse ears treated as in (b). **P <0.01 and *** P <0.001. P-values were determined by Two-tailed t tests. All data are representative of three independent experiments with n = 3–6 per group, and are mean ± SEM.

Figure 1. S. epidermidis inhibits poly(I:C)-induced inflammatory cytokines produced by keratinocytes, and inflammation in mouse skin

(a) Quantification of IL-6 and TNF-α mRNA expression of cultured normal human keratinocytes treated with 36 μg ml⁻¹ of a sterile <10 kDa product of S. epidermidis conditioned culture media (SE) and 10 μg ml⁻¹ of the TLR3 ligand [poly(I:C)] for 24 h. (b) Photographs of the ears of BALB/c mice 24 h after subcutaneous injection with PBS alone, SE alone, poly(I:C) alone or SE and poly(I:C). (c) H&E staining of ears treated as in (b), scale bars represent 100 μm. (d) Quantification of IL-6 and TNF-α expression in tissue from mouse ears treated as in (b). **P <0.01 and *** P <0.001. P-values were determined by Two-tailed t tests. All data are representative of three independent experiments with n = 3–6 per group, and are mean ± SEM.

Figure 1. S. epidermidis inhibits poly(I:C)-induced inflammatory cytokines produced by keratinocytes, and inflammation in mouse skin

(a) Quantification of IL-6 and TNF-α mRNA expression of cultured normal human keratinocytes treated with 36 μg ml⁻¹ of a sterile <10 kDa product of S. epidermidis conditioned culture media (SE) and 10 μg ml⁻¹ of the TLR3 ligand [poly(I:C)] for 24 h. (b) Photographs of the ears of BALB/c mice 24 h after subcutaneous injection with PBS alone, SE alone, poly(I:C) alone or SE and poly(I:C). (c) H&E staining of ears treated as in (b), scale bars represent 100 μm. (d) Quantification of IL-6 and TNF-α expression in tissue from mouse ears treated as in (b). **P <0.01 and *** P <0.001. P-values were determined by Two-tailed t tests. All data are representative of three independent experiments with n = 3–6 per group, and are mean ± SEM.

Figure 1. S. epidermidis inhibits poly(I:C)-induced inflammatory cytokines produced by keratinocytes, and inflammation in mouse skin

(a) Quantification of IL-6 and TNF-α mRNA expression of cultured normal human keratinocytes treated with 36 μg ml⁻¹ of a sterile <10 kDa product of S. epidermidis conditioned culture media (SE) and 10 μg ml⁻¹ of the TLR3 ligand [poly(I:C)] for 24 h. (b) Photographs of the ears of BALB/c mice 24 h after subcutaneous injection with PBS alone, SE alone, poly(I:C) alone or SE and poly(I:C). (c) H&E staining of ears treated as in (b), scale bars represent 100 μm. (d) Quantification of IL-6 and TNF-α expression in tissue from mouse ears treated as in (b). **P <0.01 and *** P <0.001. P-values were determined by Two-tailed t tests. All data are representative of three independent experiments with n = 3–6 per group, and are mean ± SEM.

Figure 2. Inflammation in wounds is dependent on TLR3 and inhibited by S. epidermidis

(a) Quantification of IL-6 and TNF-α production by ELISA in extracts of skin taken from 2 mm surrounding the wound edge 3 d after aseptic injury. (b) H&E staining of skin 2 mm adjacent to mouse wounds of wild-type (Tlr3^+/+) and Tlr3-deficient (Tlr3^−/−) mice treated as in (a). Black scale bars represent 200 μm and white scale represent 25 μm. Arrows designate region of 400X magnification shown in inset. (c) The induction of TNF-α by UVB-irradiated keratinocytes (UVR-cells) in untreated normal human keratinocytes and the blockage of UVR-cell-induced TNF-α by TLR3 siRNAs. (d) SE inhibited TNF-α production stimulated by UVR-cells. (e) RNase treatment decreased the capacity of UVR-cells to induce TNF-α in untreated normal human keratinocytes in culture, but not DNase treatment. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were analyzed by Two-way ANOVA in (a) or One-way ANOVA in (c)–(e). Data are the mean ± SEM. and are representative of two to four independent experiments with n = 4–7 per group.

Figure 2. Inflammation in wounds is dependent on TLR3 and inhibited by S. epidermidis

(a) Quantification of IL-6 and TNF-α production by ELISA in extracts of skin taken from 2 mm surrounding the wound edge 3 d after aseptic injury. (b) H&E staining of skin 2 mm adjacent to mouse wounds of wild-type (Tlr3^+/+) and Tlr3-deficient (Tlr3^−/−) mice treated as in (a). Black scale bars represent 200 μm and white scale represent 25 μm. Arrows designate region of 400X magnification shown in inset. (c) The induction of TNF-α by UVB-irradiated keratinocytes (UVR-cells) in untreated normal human keratinocytes and the blockage of UVR-cell-induced TNF-α by TLR3 siRNAs. (d) SE inhibited TNF-α production stimulated by UVR-cells. (e) RNase treatment decreased the capacity of UVR-cells to induce TNF-α in untreated normal human keratinocytes in culture, but not DNase treatment. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were analyzed by Two-way ANOVA in (a) or One-way ANOVA in (c)–(e). Data are the mean ± SEM. and are representative of two to four independent experiments with n = 4–7 per group.

Figure 2. Inflammation in wounds is dependent on TLR3 and inhibited by S. epidermidis

(a) Quantification of IL-6 and TNF-α production by ELISA in extracts of skin taken from 2 mm surrounding the wound edge 3 d after aseptic injury. (b) H&E staining of skin 2 mm adjacent to mouse wounds of wild-type (Tlr3^+/+) and Tlr3-deficient (Tlr3^−/−) mice treated as in (a). Black scale bars represent 200 μm and white scale represent 25 μm. Arrows designate region of 400X magnification shown in inset. (c) The induction of TNF-α by UVB-irradiated keratinocytes (UVR-cells) in untreated normal human keratinocytes and the blockage of UVR-cell-induced TNF-α by TLR3 siRNAs. (d) SE inhibited TNF-α production stimulated by UVR-cells. (e) RNase treatment decreased the capacity of UVR-cells to induce TNF-α in untreated normal human keratinocytes in culture, but not DNase treatment. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were analyzed by Two-way ANOVA in (a) or One-way ANOVA in (c)–(e). Data are the mean ± SEM. and are representative of two to four independent experiments with n = 4–7 per group.

Figure 2. Inflammation in wounds is dependent on TLR3 and inhibited by S. epidermidis

(a) Quantification of IL-6 and TNF-α production by ELISA in extracts of skin taken from 2 mm surrounding the wound edge 3 d after aseptic injury. (b) H&E staining of skin 2 mm adjacent to mouse wounds of wild-type (Tlr3^+/+) and Tlr3-deficient (Tlr3^−/−) mice treated as in (a). Black scale bars represent 200 μm and white scale represent 25 μm. Arrows designate region of 400X magnification shown in inset. (c) The induction of TNF-α by UVB-irradiated keratinocytes (UVR-cells) in untreated normal human keratinocytes and the blockage of UVR-cell-induced TNF-α by TLR3 siRNAs. (d) SE inhibited TNF-α production stimulated by UVR-cells. (e) RNase treatment decreased the capacity of UVR-cells to induce TNF-α in untreated normal human keratinocytes in culture, but not DNase treatment. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were analyzed by Two-way ANOVA in (a) or One-way ANOVA in (c)–(e). Data are the mean ± SEM. and are representative of two to four independent experiments with n = 4–7 per group.

Figure 2. Inflammation in wounds is dependent on TLR3 and inhibited by S. epidermidis

(a) Quantification of IL-6 and TNF-α production by ELISA in extracts of skin taken from 2 mm surrounding the wound edge 3 d after aseptic injury. (b) H&E staining of skin 2 mm adjacent to mouse wounds of wild-type (Tlr3^+/+) and Tlr3-deficient (Tlr3^−/−) mice treated as in (a). Black scale bars represent 200 μm and white scale represent 25 μm. Arrows designate region of 400X magnification shown in inset. (c) The induction of TNF-α by UVB-irradiated keratinocytes (UVR-cells) in untreated normal human keratinocytes and the blockage of UVR-cell-induced TNF-α by TLR3 siRNAs. (d) SE inhibited TNF-α production stimulated by UVR-cells. (e) RNase treatment decreased the capacity of UVR-cells to induce TNF-α in untreated normal human keratinocytes in culture, but not DNase treatment. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were analyzed by Two-way ANOVA in (a) or One-way ANOVA in (c)–(e). Data are the mean ± SEM. and are representative of two to four independent experiments with n = 4–7 per group.

Figure 3. Staphylococcal LTA inhibits poly(I:C)-induced TNF-α

(a) Quantification of TNF-α in culture media of human keratinocytes after treatment with PBS or TLR-ligands alone (white bars), or in combination with 10 μg ml⁻¹ of poly(I:C) (black bars); LTA-SA (10 μg ml⁻¹), LPS (100 ng ml⁻¹), Flagellin (50 ng ml⁻¹). (b) Keratinocyte TNF-α stimulated with poly(I:C) (10 μg ml⁻¹) combined with a panel of TLR2 ligands LTA-SE, LTA-SA, LTA-SF, LTA-BS, Zymosan, PGN-EK, PGN-SA (10 μg ml⁻¹), Malp-2 (100 ng ml⁻¹), Pam3CSK4, FSL-1 (1 μg ml⁻¹). (c) Antibody to S. epidermidis LTA prevented SE from suppressing poly(I:C)-induced TNF-α in keratinocytes. (d) Quantification of TNF-α from keratinocytes treated with 10 μg ml⁻¹ of poly(I:C) and 5 μg ml⁻¹ of synthetic LTA containing alanines (LTA 2 D-alanines and LTA 3 D-alanines), or only the anchor (LTA-anchor 2F), or containing N-acetylglucosamine (LTA 2 GlcNac and LTA 5 NacGlc). (e) Quantification of TNF-α from keratinocytes stimulated by 10 μg ml⁻¹ of poly(I:C) with a sterile <10 kDa product of S. aureus Sa113 parental strain conditioned culture media (SA wt; 36 μg ml⁻¹), or a similar product of S. aureus Sa113 dltA mutant (SA dltA; 36 μg ml⁻¹). ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by One-way ANOVA. Data are the mean ± SEM of triplicates stimulations and are representative of two to four independent experiments.

Figure 3. Staphylococcal LTA inhibits poly(I:C)-induced TNF-α

(a) Quantification of TNF-α in culture media of human keratinocytes after treatment with PBS or TLR-ligands alone (white bars), or in combination with 10 μg ml⁻¹ of poly(I:C) (black bars); LTA-SA (10 μg ml⁻¹), LPS (100 ng ml⁻¹), Flagellin (50 ng ml⁻¹). (b) Keratinocyte TNF-α stimulated with poly(I:C) (10 μg ml⁻¹) combined with a panel of TLR2 ligands LTA-SE, LTA-SA, LTA-SF, LTA-BS, Zymosan, PGN-EK, PGN-SA (10 μg ml⁻¹), Malp-2 (100 ng ml⁻¹), Pam3CSK4, FSL-1 (1 μg ml⁻¹). (c) Antibody to S. epidermidis LTA prevented SE from suppressing poly(I:C)-induced TNF-α in keratinocytes. (d) Quantification of TNF-α from keratinocytes treated with 10 μg ml⁻¹ of poly(I:C) and 5 μg ml⁻¹ of synthetic LTA containing alanines (LTA 2 D-alanines and LTA 3 D-alanines), or only the anchor (LTA-anchor 2F), or containing N-acetylglucosamine (LTA 2 GlcNac and LTA 5 NacGlc). (e) Quantification of TNF-α from keratinocytes stimulated by 10 μg ml⁻¹ of poly(I:C) with a sterile <10 kDa product of S. aureus Sa113 parental strain conditioned culture media (SA wt; 36 μg ml⁻¹), or a similar product of S. aureus Sa113 dltA mutant (SA dltA; 36 μg ml⁻¹). ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by One-way ANOVA. Data are the mean ± SEM of triplicates stimulations and are representative of two to four independent experiments.

Figure 3. Staphylococcal LTA inhibits poly(I:C)-induced TNF-α

(a) Quantification of TNF-α in culture media of human keratinocytes after treatment with PBS or TLR-ligands alone (white bars), or in combination with 10 μg ml⁻¹ of poly(I:C) (black bars); LTA-SA (10 μg ml⁻¹), LPS (100 ng ml⁻¹), Flagellin (50 ng ml⁻¹). (b) Keratinocyte TNF-α stimulated with poly(I:C) (10 μg ml⁻¹) combined with a panel of TLR2 ligands LTA-SE, LTA-SA, LTA-SF, LTA-BS, Zymosan, PGN-EK, PGN-SA (10 μg ml⁻¹), Malp-2 (100 ng ml⁻¹), Pam3CSK4, FSL-1 (1 μg ml⁻¹). (c) Antibody to S. epidermidis LTA prevented SE from suppressing poly(I:C)-induced TNF-α in keratinocytes. (d) Quantification of TNF-α from keratinocytes treated with 10 μg ml⁻¹ of poly(I:C) and 5 μg ml⁻¹ of synthetic LTA containing alanines (LTA 2 D-alanines and LTA 3 D-alanines), or only the anchor (LTA-anchor 2F), or containing N-acetylglucosamine (LTA 2 GlcNac and LTA 5 NacGlc). (e) Quantification of TNF-α from keratinocytes stimulated by 10 μg ml⁻¹ of poly(I:C) with a sterile <10 kDa product of S. aureus Sa113 parental strain conditioned culture media (SA wt; 36 μg ml⁻¹), or a similar product of S. aureus Sa113 dltA mutant (SA dltA; 36 μg ml⁻¹). ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by One-way ANOVA. Data are the mean ± SEM of triplicates stimulations and are representative of two to four independent experiments.

Figure 3. Staphylococcal LTA inhibits poly(I:C)-induced TNF-α

(a) Quantification of TNF-α in culture media of human keratinocytes after treatment with PBS or TLR-ligands alone (white bars), or in combination with 10 μg ml⁻¹ of poly(I:C) (black bars); LTA-SA (10 μg ml⁻¹), LPS (100 ng ml⁻¹), Flagellin (50 ng ml⁻¹). (b) Keratinocyte TNF-α stimulated with poly(I:C) (10 μg ml⁻¹) combined with a panel of TLR2 ligands LTA-SE, LTA-SA, LTA-SF, LTA-BS, Zymosan, PGN-EK, PGN-SA (10 μg ml⁻¹), Malp-2 (100 ng ml⁻¹), Pam3CSK4, FSL-1 (1 μg ml⁻¹). (c) Antibody to S. epidermidis LTA prevented SE from suppressing poly(I:C)-induced TNF-α in keratinocytes. (d) Quantification of TNF-α from keratinocytes treated with 10 μg ml⁻¹ of poly(I:C) and 5 μg ml⁻¹ of synthetic LTA containing alanines (LTA 2 D-alanines and LTA 3 D-alanines), or only the anchor (LTA-anchor 2F), or containing N-acetylglucosamine (LTA 2 GlcNac and LTA 5 NacGlc). (e) Quantification of TNF-α from keratinocytes stimulated by 10 μg ml⁻¹ of poly(I:C) with a sterile <10 kDa product of S. aureus Sa113 parental strain conditioned culture media (SA wt; 36 μg ml⁻¹), or a similar product of S. aureus Sa113 dltA mutant (SA dltA; 36 μg ml⁻¹). ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by One-way ANOVA. Data are the mean ± SEM of triplicates stimulations and are representative of two to four independent experiments.

Figure 3. Staphylococcal LTA inhibits poly(I:C)-induced TNF-α

(a) Quantification of TNF-α in culture media of human keratinocytes after treatment with PBS or TLR-ligands alone (white bars), or in combination with 10 μg ml⁻¹ of poly(I:C) (black bars); LTA-SA (10 μg ml⁻¹), LPS (100 ng ml⁻¹), Flagellin (50 ng ml⁻¹). (b) Keratinocyte TNF-α stimulated with poly(I:C) (10 μg ml⁻¹) combined with a panel of TLR2 ligands LTA-SE, LTA-SA, LTA-SF, LTA-BS, Zymosan, PGN-EK, PGN-SA (10 μg ml⁻¹), Malp-2 (100 ng ml⁻¹), Pam3CSK4, FSL-1 (1 μg ml⁻¹). (c) Antibody to S. epidermidis LTA prevented SE from suppressing poly(I:C)-induced TNF-α in keratinocytes. (d) Quantification of TNF-α from keratinocytes treated with 10 μg ml⁻¹ of poly(I:C) and 5 μg ml⁻¹ of synthetic LTA containing alanines (LTA 2 D-alanines and LTA 3 D-alanines), or only the anchor (LTA-anchor 2F), or containing N-acetylglucosamine (LTA 2 GlcNac and LTA 5 NacGlc). (e) Quantification of TNF-α from keratinocytes stimulated by 10 μg ml⁻¹ of poly(I:C) with a sterile <10 kDa product of S. aureus Sa113 parental strain conditioned culture media (SA wt; 36 μg ml⁻¹), or a similar product of S. aureus Sa113 dltA mutant (SA dltA; 36 μg ml⁻¹). ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by One-way ANOVA. Data are the mean ± SEM of triplicates stimulations and are representative of two to four independent experiments.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 4. Staphylococcal LTA induces TRAF1 to inhibit TNF-α

(a) Western blot of TRAF1 in cultured human keratinocytes stimulated by LTA-SA for various times. The predicted molecular weight of TRAF1 is ca. 52 kDa. GAPDH was used as endogenous control. (b) Quantification of IL-6 and TNF-α of skin extracts from C57BL/6 Traf1-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for ELISA. (c) Western blot with antibody raised against the N-terminus of human TRAF1 in keratinocyte extract treated with SE and poly(I:C) for the indicated time periods. The predicted molecular weight of N-TRAF1 is ca. 22 kDa. (d) Western blot showing that caspase 8 inhibitor prevented the cleavage of TRAF1 in cultured human keratinocytes. (e) Quantification of Traf1 mRNA expression in skin of germ-free mice and conventional mice. CV: conventional mice; GF: Germ-free mice. (f) Quantification of Traf1 mRNA expression induced by LTA-SA in skin of wild-type, Tlr2^−/− and Myd88^−/− mice. * P <0.05, ** P <0.01 and *** P <0.001. n.s. no significance. P-values were determined by using Two-way ANOVA in (b) and (f), and Two-tailed t tests in (e). Data are mean ± SEM, and are representative of two independent experiments with n = 3–7 per group.

Figure 5. TLR2 is required for LTA to inhibit skin inflammation after injury or injection of poly(I:C)

(a) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for real-time RT-PCR. (b) H&E staining of skin in Tlr2-deficient mice treated as in (a). The scale bars represent 200 μm. (c) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice stimulated by subcutaneous injection of poly(I:C). Some ears were preinjected with LTA-SA, or PBS 2 h prior to injection of poly(I:C). * P <0.05 and ** P <0.01. n.s. no significance. P-values were evaluated by Two-way ANOVA. Data are the mean ± SEM of n = 5–7 and are representative of three independent experiments.

Figure 5. TLR2 is required for LTA to inhibit skin inflammation after injury or injection of poly(I:C)

(a) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for real-time RT-PCR. (b) H&E staining of skin in Tlr2-deficient mice treated as in (a). The scale bars represent 200 μm. (c) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice stimulated by subcutaneous injection of poly(I:C). Some ears were preinjected with LTA-SA, or PBS 2 h prior to injection of poly(I:C). * P <0.05 and ** P <0.01. n.s. no significance. P-values were evaluated by Two-way ANOVA. Data are the mean ± SEM of n = 5–7 and are representative of three independent experiments.

Figure 5. TLR2 is required for LTA to inhibit skin inflammation after injury or injection of poly(I:C)

(a) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice and wild-type controls preinjected by PBS or LTA-SA 24 h and 2 h prior to wounding. 2 mm of skin around the wound edges was collected for real-time RT-PCR. (b) H&E staining of skin in Tlr2-deficient mice treated as in (a). The scale bars represent 200 μm. (c) Quantification of IL-6 and TNF-α mRNA expression in skin from C57BL/6 Tlr2-deficient mice stimulated by subcutaneous injection of poly(I:C). Some ears were preinjected with LTA-SA, or PBS 2 h prior to injection of poly(I:C). * P <0.05 and ** P <0.01. n.s. no significance. P-values were evaluated by Two-way ANOVA. Data are the mean ± SEM of n = 5–7 and are representative of three independent experiments.