TO THE EDITOR Recent clin. trials have established a central role for the T helper type 2 (Th2) cytokines IL-4 and IL-13 in the pathol. of atopic dermatitis (AD) (Beck et al., 2014).Although blockade of the IL-4 and IL-13 receptors causes significant clearing of skin lesions, a direct effect of Th2 cytokines on wound healing processes has not yet been demonstrated.Furthermore, AD patients are frequently affected by infection with the pathogen, Staphylococcus aureus (Boguniewicz and Leung, 2011).Elevated levels of staphylococcal products are frequently found on the skin of affected patients (Travers et al., 2010), and these products may affect the wound healing process as well.A primary event in skin healing is induction of matrix metalloproteinases (MMPs) (Inoue et al., 1995).MMPs-1, -9, and -10 are expressed at the leading edge of the wound (Inoue et al., 1995; Rechardt et al., 2000; Turchi et al., 2003) and are required for keratinocyte migration into the damaged area (Pilcher et al., 1997; Agren, 1999).Inhibition of MMP function effectively blocks keratinocyte migration and wound healing (Mirastschijski et al., 2002b).However, overexpression of MMPs has been reported in skin diseases and can inhibit wound closure as well (Saarialho-Kere et al., 1994; Mirastschijski et al., 2002a).In this study, we determined the effects of staphylococcal products and AD-associated Th2 cytokines on MMP expression and on keratinocyte migration.We first examined the effect of bacterial products on MMP levels.Real-time PCR and ELISAs were used to quantify mRNA and protein expression of MMP-1, -9, and -10.We found that these MMPs were significantly induced by exposure to staphylococcal lipoteichoic acid (LTA) (Figure 1a and Supplementary Figure S1 online).Peptidoglycan also modestly induced these MMPs.In contrast, neither E. coli-derived lipopolysaccharide nor the staphylococcal toxins S. aureus enterotoxin B and Toxic Shock Syndrome Toxin were able to induce expression of MMPs (Figure 1a).We also found that LTA-induced events are dependent upon expression of the Toll-like receptor signaling adaptor mol., MyD88, as a MyD88 peptide inhibitor completely blocked gene expression of MMP-1, -9, and -10 (Supplementary Figure S2 online).We next determined the effect of Th2 cytokines on MMP productionWe found that basal level expression of MMP-9 was inhibited by Th2 cytokines (Figure 1b and c).LTA-induced expression of MMP-1, -9, and -10 was also significantly inhibited by Th2 cytokines.(Figure 1b and c).These results demonstrate that Th2 cytokines interfere with both basal and LTA-induced MMP expression.For comparison, tumor necrosis factor-α (TNF-α) does not interfere with MMP expression (Supplementary Figure S3 online), as previously described (Han et al., 2001).Expression of tissue inhibitor of metalloproteinase (TIMPs) was also examinedIn contrast to MMPs, TIMP levels were not substantially altered by LTA or Th2 cytokines (Supplementary Figure S4 online).We next focused on determining the mol. events induced by Th2 cytokines that influence MMP gene expression.Signal transducer and activator of transcription 6 (STAT6) is a transcription factor activated by ligation of the IL-4 and IL-13 receptors (Albanesi et al., 2007).We therefore used small interfering RNA (siRNA) directed against STAT6 to determine whether Th2 cytokines signal through STAT6 to modulate MMP levels.Figure 2a demonstrates that basal MMP-9 expression is inhibited by Th2 cytokines in control but not in STAT6 siRNA-treated cells.Furthermore, the Th2-mediated inhibition of LTA-induced MMP expression was no longer observed in STAT6 siRNA-treated keratinocytes (Figure 2a).The increased expression of MMPs in STAT6 knockdown cells was significant.Therefore, we conclude that the inhibition of MMP expression by Th2 cytokines is dependent upon STAT6.As MMPs coordinate epithelial wound healing by enabling cell detachment and migration on collagen (Pilcher et al., 1997), we further investigated whether Th2 cytokines inhibited ''wound'' closure in a monolayer of human keratinocytes grown on a collagen matrix.Using an in vitro wound scratch assay, we find that cells treated with media alone but disrupted by the scratch migrated into the depleted area (Figure 2b).In contrast, pre-treatment with Th2 cytokines inhibited the rate of keratinocyte migration compared with control keratinocytes (Figure 2b and c).Possibly because of the endogenous activation of MMPs at the leading edge (Pilcher et al., 1997; Turchi et al., 2003), we did not observe an additive effect of LTA on wound closure in a scratch assay (Figure 2b and c).However, we do observe that Th2 cytokines have a dominant inhibitory effect, blocking migration in all cases.For comparison, the cytokine TNF-α did not inhibit the closure of keratinocyte monolayers (Supplementary Figure S3 online), as previously described (Eyerich et al., 2009).Consistent with the observed effects on MMP expression, the inhibition of migration mediated by Th2 cytokines was ablated in Stat6 siRNA-treated cells (Figure 2c).Therefore, Th2 inhibition of migration related to wound closure is also dependent on STAT6.A critical role for Th2 cytokines in AD skin disease is emerging.Here we directly demonstrate that Th2 cytokines inhibit MMP expression and keratinocyte migration, both essential components of the wound healing process.There is, however, a paradoxical effect of MMPs in wound healing.Although MMPs are required for normal migration leading to wound closure, overexpression is a key feature of chronic wounds and skin disease.It therefore remains possible that overexpression of MMPs induced by LTA may contribute to skin disease as well.Recent studies have evaluated MMP expression in AD skin (Esaki et al., 2015).Using micro-dissection techniques, AD skin was sectioned into dermal and epidermal components.MMP-1, a gene induced by staphylococcal LTA, was identified as the most prominently upregulated gene in the dermis.As AD lesions are frequently infected with S. aureus, it seems possible that LTA-induced overexpression of MMPs may be a contributing factor in disease.In contrast, increases in MMP-1, -9, or -10 levels in the epidermis of lesional AD skin were not reported (in a list of the top 25 most upregulated genes).However, this may be a consequence of the inhibitory effects of Th2 cytokines.We propose that Th2 cytokines, as well as staphylococcal LTA, may contribute to delayed wound healing and pathol. associated with AD by deregulating MMP production and altering cell migration.
