Title: Identification of Staphylococcal Protein A in Infected Atopic Dermatitis Lesions
Abstract: atopic dermatitis lipoteichoic acid staphylococcal protein A TO THE EDITOR Staphylococcus aureus infection is a known trigger for skin inflammation and can modulate immune responses. Atopic dermatitis (AD), a chronic inflammatory pruritic skin disease, affects 10–20% of children and 1–3% of adults (De Benedetto et al., 2009De Benedetto A. Agnihothri R. McGirt L.Y. et al.Atopic dermatitis: a disease caused by innate immune defects?.J Invest Dermatol. 2009; 129: 14-30Crossref PubMed Scopus (234) Google Scholar). Owing to the loss of skin integrity by scratching, as well as decreased levels of antimicrobial peptides in comparison with normal skin or other inflammatory diseases such as psoriasis (Ong et al., 2002Ong P.Y. Ohtake T. Brandt C. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1549) Google Scholar; Leung, 2003Leung D.Y. Infection in atopic dermatitis.Curr Opin Pediatr. 2003; 15: 399-404Crossref PubMed Scopus (133) Google Scholar), patients with AD are particularly susceptible to staphylococcal skin infections, which can further worsen their skin disease (Bieber, 2008Bieber T. Atopic dermatitis.N Engl J Med. 2008; 358: 1483-1494Crossref PubMed Scopus (1419) Google Scholar). Studies have suggested several underlying mechanisms for staphylococcus-mediated inflammation, which include production of inflammatory cytokines following either direct infection of keratinocytes or immune cells by the bacteria, or indirectly by bacterial products (Travers et al., 2001Travers J.B. Norris D.A. Leung D.Y. The keratinocyte as a target for staphylococcal bacterial toxins.J Invest Dermatol Symp Proc Soc Invest Dermatol. 2001; 6: 225-230Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar; Leung, 2003Leung D.Y. Infection in atopic dermatitis.Curr Opin Pediatr. 2003; 15: 399-404Crossref PubMed Scopus (133) Google Scholar; Sasaki et al., 2003Sasaki T. Kano R. Sato H. et al.Effects of staphylococci on cytokine production from human keratinocytes.Br J Dermatol. 2003; 148: 46-50Crossref PubMed Scopus (38) Google Scholar; Baker, 2006Baker B.S. The role of microorganisms in atopic dermatitis.Clin Exp Immunol. 2006; 144: 1-9Crossref PubMed Scopus (211) Google Scholar). We have demonstrated previously that lipoteichoic acid (LTA), a Gram-positive bacterial lipoprotein, may have an important role in the ability of S. aureus to exacerbate AD lesions (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In the present study, we report that staphylococcal protein A (SPA) could also contribute to this process. SPA is a 40–60kDa bacteria surface protein. It binds to the Fc region of IgG via interaction with the heavy chain, which disrupts opsonization and phagocytosis, contributing to the virulence of S. aureus (Foster, 2005Foster T.J. Immune evasion by staphylococci.Nat Rev. 2005; 3: 948-958Google Scholar). Studies have shown that SPA can also activate the tumor necrosis factor receptor-1, which leads to NF-κB and activator protein 1 (AP-1) activation and subsequent production of cytokines and chemokines, promoting the inflammatory response in staphylococcal pneumonia (Gomez et al., 2004Gomez M.I. Lee A. Reddy B. et al.Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1.Nat Med. 2004; 10: 842-848Crossref PubMed Scopus (290) Google Scholar). Although SPA has potent biological effects, a potential pathologic role for this staphylococcal protein or the amounts found within infected skin lesions remain largely unknown. To assess the SPA levels in infected AD lesions, we enrolled a total of 89 children (aged 3 months to 6 years) with clinically impetiginized AD diagnosed using the criteria of Hanifen and Rajka. Experiments in our protocol were approved by the Indiana University Institutional Review Committee, and adhered to the Helsinki Guidelines, including using written, informed patient consent in these human studies. (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Sixty-three patients returned for their second visit following a 2-week regimen of oral antibiotics (cephalexin or clindamycin if allergic to cephalosporins or penicillins) and topical corticosteroids. The experimental procedures were previously described (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). At each visit, subjects underwent a clinical assessment of a clinically infected lesion of dermatitis using the eczema area and severity index score (Hanifin et al., 2001Hanifin J.M. Thurston M. Omoto M. et al.The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI Evaluator Group.Exp Dermatol. 2001; 10: 11-18Crossref PubMed Scopus (670) Google Scholar). Lesional wash fluid was collected, aliquoted and stored at -80°C until use. S. aureus, S. epidermidis (coagulase-negative staphylococcus) and group A streptococcus colonies were quantified by limiting dilution assay. Quantitative measurement of SPA was performed using ELISA (Assay Designs, Ann Arbor, MI). Measurements of LTA and cytokines IL-6, IL-8 and tumor necrosis factor-α were done as previously described (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The levels of SPA, LTA and cytokines in wash fluid were first calculated based on the area of the chamber (ngcm−2), and then converted to volume (ngcm−3) based on the estimation of 0.1cm effective epidermal thickness. S. aureus was detected from clinically impetiginized AD lesions in 88.8% (79/89) of the subjects at their first visit. Treatment with antibiotics resulted in decreased amounts of S. aureus on the lesional skin samples and improvement of the lesional eczema area and severity index scores (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Measurable amounts of SPA were found in 91.0% (81/89) of the samples during the first visit and in 55.6% (35/63) during the second visit. Of note, 64.0% (57/89) of the first-visit samples but only 9.5% (6/63) of the second-visit samples had SPA levels >0.5ngcm−3. As depicted in Figure 1a, there was a strong positive correlation between amounts of SPA and staphylococcal bacterial colony-forming unit (visit 1: r=0.71, P<0.0001; visit 2: r=0.45, P=0.0002). In addition, the level of SPA was positively correlated with the lesional eczema area and severity index score at visit 1 (visit 1: r=0.36, P=0.0005; visit 2: r=0.15, P=0.23) (Figure 1b). S. aureus infection or SPA itself has been shown to induce the pro-inflammatory cytokine tumor necrosis factor-α in primary human keratinocytes (Ezepchuk et al., 1996Ezepchuk Y.V. Leung D.Y. Middleton M.H. et al.Staphylococcal toxins and protein A differentially induce cytotoxicity and release of tumor necrosis factor-alpha from human keratinocytes.J Invest Dermatol. 1996; 107: 603-609Crossref PubMed Scopus (93) Google Scholar; Aufiero et al., 2007Aufiero B. Guo M. Young C. et al.Staphylococcus aureus induces the expression of tumor necrosis factor-alpha in primary human keratinocytes.Int J Dermatol. 2007; 46: 687-694Crossref PubMed Scopus (13) Google Scholar). Our previous studies indicated that biologically relevant levels of pro-inflammatory cytokines can be measured in many wash fluid specimens derived from clinically impetiginized AD lesions (Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). As shown in Figure 1c, the level of SPA was positively correlated with the amounts of tumor necrosis factor-α for the first-visit samples (r=0.41, P<0.0001). Moreover, there was a positive correlation between SPA and other pro-inflammatory cytokines, including IL-6 and IL-8 (data not shown). The dose of SPA needed to induce cutaneous reactions in human skin has been reported to be in the ngml−1 range (White and Noble, 1985White M.I. Noble W.C. The cutaneous reaction to staphylococcal protein A in normal subjects and patients with atopic dermatitis or psoriasis.Br J Dermatol. 1985; 113: 179-183Crossref PubMed Scopus (25) Google Scholar). Therefore, pharmacologically active (that is, >10ngcm−3) levels of SPA are commonly encountered (in 30% of the first-visit samples) in subjects with impetiginized AD lesions. These data suggested that SPA might contribute to the worsening of AD lesions by S. aureus infection. In fact, it has been shown that topical application of SPA after destroying skin barrier function with a detergent can induce AD-like skin inflammation in mice (Terada et al., 2006Terada M. Tsutsui H. Imai Y. et al.Contribution of IL-18 to atopic-dermatitis-like skin inflammation induced by Staphylococcus aureus product in mice.Proc Natl Acad Sci USA. 2006; 103: 8816-8821Crossref PubMed Scopus (91) Google Scholar). However, it is also possible that the increased skin inflammation might be simply caused by other coexisting bacterial products such as LTA and staphylococcal toxins that have been implicated in the worsening of AD in response to infection (Ezepchuk et al., 1996Ezepchuk Y.V. Leung D.Y. Middleton M.H. et al.Staphylococcal toxins and protein A differentially induce cytotoxicity and release of tumor necrosis factor-alpha from human keratinocytes.J Invest Dermatol. 1996; 107: 603-609Crossref PubMed Scopus (93) Google Scholar; Travers et al., 2001Travers J.B. Norris D.A. Leung D.Y. The keratinocyte as a target for staphylococcal bacterial toxins.J Invest Dermatol Symp Proc Soc Invest Dermatol. 2001; 6: 225-230Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Travers et al., 2010Travers J.B. Kozman A. Mousdicas N. et al.Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.J Allergy Clin Immunol. 2010; 125 (e141–2): 146-152Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Indeed, we observed a strong positive correlation between SPA and LTA among first-visit samples (r=0.55, P<0.0001) (Figure 1d). It also remains unclear at this point how SPA induces skin inflammation, for example, the cell type(s) and receptor(s) being involved. Further investigations are warranted to elucidate the role of SPA in the pathogenesis of S. aureus-mediated worsening of AD. This research was supported in part by grants from the Riley Memorial Association, National Institutes of Health grants HL62996 (JBT) and U19 AI070448 (MHK and JBT), and a Veteran's Administration Merit Award (JBT).