Upregulation of Toll-Like Receptors (TLRs) 6, 7, and 8 in Keloid Scars
2011; Elsevier BV; Volume: 131; Issue: 10 Linguagem: Inglês
10.1038/jid.2011.163
ISSN1523-1747
AutoresRania Abubaker Bagabir, Farhatullah Syed, Riina Rautemaa, Duncan Angus McGrouther, Ralf Paus, Ardeshir Bayat,
Tópico(s)Cutaneous Melanoma Detection and Management
Resumokeloid scar quantitative real-time RT-PCR Toll-like receptor TO THE EDITOR Keloid scar (KS) is a benign fibroproliferative dermal tumor of unknown origin that develops in genetically susceptible individuals (Shih and Bayat, 2009Shih B. Bayat A. Genetics of keloid scarring.Arch Dermatol Res. 2009; 302: 319-339Crossref Scopus (177) Google Scholar). KS commonly occurs following trauma to the skin, which is likely to facilitate inoculation of infectious agents. Interestingly, it has been repeatedly observed that KS develops after clinical evidence of viral infection, such as chicken pox (Gathse et al., 2003Gathse A. Ibara J.R. Obengui P. et al.Gigantic keloïds after chicken-pox. A case report.Bull Soc Pathol Exot. 2003; 96: 401-402PubMed Google Scholar) and zoster (Manikhas et al., 1986Manikhas M.G. Egorova M.A. Orlova E.V. Keloid scars as a sequel of herpes zoster.Vestn Dermatol Venerol. 1986; 9: 54-55PubMed Google Scholar; Koley et al., 2009Koley S. Saoji V. Salodkar A. Unusual formation of keloids after each episode of recurrent herpes zoster in an HIV positive patient.Indian J Sex Transm Dis. 2009; 30: 109-111Crossref PubMed Scopus (4) Google Scholar). In addition, it has been reported that some skin tumors such as Merkel cell carcinoma are associated with viruses (Masahiro et al., 2009Masahiro S. Reety A. Hyun Jin K. et al.Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors.Int J Cancer. 2009; 125: 1243-1249Crossref PubMed Scopus (301) Google Scholar). On this basis, it has been speculated that KS might be induced by viral infection (Alonso et al., 2008Alonso P.E. Rioja L.F. Pera C. Keloids: a viral hypothesis.Med Hypotheses. 2008; 70: 156-166Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar); nonetheless, there is no experimental evidence in support of this hypothesis. A previous attempt at identifying viral DNA directly in KS was unsuccessful, as it failed to identify human herpesvirus-8 or Epstein–Barr virus DNA in seven keloid cases (Pantanowitz and Duke, 2008Pantanowitz L. Duke W.H. Keloids do not harbor EBV or HHV8.Med Hypotheses. 2008; 70: 704Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). However, it is possible that the experimental technique employed in this study was neither sensitive nor searched for the appropriate viral particle. Thus, not knowing the identity of the viral agent, we have approached this question using an alternative approach. In this pilot study, we asked whether any evidence could be identified that points to a viral encounter by the skin immune system in KS. To this end, we specifically investigated the in situ expression of Toll-like receptors (TLRs) known to recognize single- or double-stranded viral particles, i.e., TLRs 2, 3, and 6-9 (Delaloye et al., 2009Delaloye J. Roger T. Steiner-Tardivel Q.G. et al.Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.PLoS Pathog. 2009; 5: e1000480Crossref PubMed Scopus (261) Google Scholar; O’Neill and Bowie, 2010O’Neill L.A.J. Bowie A.G. Sensing and signaling in antiviral innate immunity.Curr Biol. 2010; 20: R328-R333Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) by fluorescence and assessed by quantitative immunohistomorphometry (Kloepper et al., 2009Kloepper J.E. Koji S. Yusur A.N. et al.Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.Exp Dermatol. 2009; 19: 305-312Crossref PubMed Scopus (100) Google Scholar). These expressions were followed-up by quantitative real-time RT-PCR (QRT-PCR; see Supplementary Table S1 online for primer sequences and gene bank accession numbers). The expression of these TLRs was compared with carefully defined sites (Supplementary Figure S1 online) within and immediately outside the keloid lesion (“intralesional”: core of keloid, “perilesional”: keloid's margin, and “extralesional”: normal uninvolved skin adjacent to KSs), obtained from 13 KS patients (see demographic data in Supplementary Table S2 online). This was compared with normal skin (nine patients) from the vicinity of a normal scar (patients with no history of raised scarring). As shown in Figure 1a and b, immunoreactivity for TLR-8 was significantly increased (P<0.05) in intra- and perilesional sites of KS (see Supplementary Figure S2 online for representative images) relative to normal dermis. The increased intensity of TLR-8 immunoreactivity was associated with scattered positive cells within the intra- and perilesional sites of KS, whereas no TLR-positive cells were observed in extralesional KS or normal skin (Supplementary Table S3 online). This protein expression pattern corresponded well with the substantial upregulation in the TLR-8 mRNA in intra- and perilesional sites of KS, as measured by QRT-PCR with 4.2- and 2.1-fold change, respectively, relative to normal skin (Figure 2). Although there was no obvious increase in TLR-8 protein expression in situ in extralesional skin of KS patients (Figure 1a and b), transcription of TLR-8 was increased by 2.5-fold change compared with normal control skin (Figure 2).Figure 2Toll-like receptor (TLR) transcription in keloid scar. Fold change of transcripts of TLRs 2, 3, and 6–9 in keloid scar (KS)-defined lesional sites relative to normal skin (a) and a schematic model illustrating the increased TLR expression at KS-defined sites (b). Primers for TLRs were designed by and purchased from Roche Diagnostics, West Sussex, UK (Supplementary Table S1 online). mRNA was extracted from full-thickness tissue derived from defined KS sites (n=7) and normal skin (n=7). In all, 2μg of mRNA was reverse transcribed and quantitative real-time RT-PCR reactions were carried out. The cycle threshold (Ct) values were normalized to the geometric mean of two housekeeping genes (SDHA (succinate dehydrogenase complex subunit A) and RPL32 (60S ribosomal protein L32)) and analyzed by fold change (2−ΔΔCT) relative to normal skin. Error bars represent mean±SEM. *P<0.05 and **P≤0.01.View Large Image Figure ViewerDownload (PPT) With respect to the immunoreactivity of TLR-2, -3, -6, -7, and -9, no significant immunoreactivity differences were observed in the dermis of KS lesion compared with control dermis (Figure 1a and b; and Supplementary Figure S2 online). The transcript steady-state level of TLR-9 within the keloid lesion was significantly upregulated (intralesional P=0.02 and perilesional P=0.04) relative to normal skin (Figure 2). However, TLR-9 protein expression was not significantly changed (Figure 1a–c). TLR-6 transcription was slightly upregulated in intralesional sites, whereas TLR-7 transcription was upregulated across the different keloid lesional sites (Figure 2). There was a significant increase in TLR-6 (P=0.01) and TLR-7 (P≤0.02) immunoreactivity in situ when the epidermis only was compared between KS and normal control skin (Figure 1c, Supplementary Figure S1 online). In the epidermis, TLR-2 and TLR-3 gene expressions were upregulated within KS, whereas the in situ immunoreactivity was decreased (Figures 1 and 2). The antibody specificity of TLRs 6–8 was also confirmed by using the corresponding blocking peptide for each TLR (Supplementary Figure S4 online). As these findings suggest a heightened responsiveness of the skin innate immune system toward classical viral infection-associated TLR ligands (Delaloye et al., 2009Delaloye J. Roger T. Steiner-Tardivel Q.G. et al.Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.PLoS Pathog. 2009; 5: e1000480Crossref PubMed Scopus (261) Google Scholar; O’Neill and Bowie, 2010O’Neill L.A.J. Bowie A.G. Sensing and signaling in antiviral innate immunity.Curr Biol. 2010; 20: R328-R333Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), this preliminary study offers indirect support for the viral hypothesis of KS pathogenesis (Alonso et al., 2008Alonso P.E. Rioja L.F. Pera C. Keloids: a viral hypothesis.Med Hypotheses. 2008; 70: 156-166Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The increased epidermal protein expression for TLR-6 and -7 in KS is noteworthy, as keloid keratinocytes may have a role in stimulating keloid fibroblast activity (Lim et al., 2002Lim I.J. Phan T.T. Bay B.H. et al.Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner.Am J Physiol Cell Physiol. 2002; 283: C212-C222Crossref PubMed Scopus (104) Google Scholar). Although a previous study has failed to demonstrate TLR-7 expression in normal human keratinocytes (Lebre et al., 2006Lebre M.C. van der Aar A.M.G. van Baarsen L. et al.Human keratinocytes express functional Toll-like receptor 3, 4, 5, and 9.J Invest Dermatol. 2006; 127: 331-341Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), TLR-7 expression has recently been detected in normal and virally infected human keratinocytes (Ku et al., 2008Ku J.K. Kwon H.J. Kim M.Y. et al.Expression of Toll-like receptors in verruca and molluscum contagiosum.J Korean Med Sci. 2008; 23: 307-314Crossref PubMed Scopus (35) Google Scholar). This is well in line with our current results. The markedly increased upregulation of the TLR-8 gene and protein levels in keloid dermis may reflect the possible encounter of intradermal immunocytes, such as dermal dendrocytes, and/or macrophages with virus-associated ligands (Heydtmann, 2009Heydtmann M. Macrophages in hepatitis B and hepatitis C virus infections.J Virol. 2009; 83: 2796-2802Crossref PubMed Scopus (60) Google Scholar). Viruses such as influenza virus, parechovirus 1, and modified vaccinia virus Ankara have been identified as triggers of antiviral pathways through activation of TLRs 6–8 (Triantafilou et al., 2005Triantafilou K. Vakakis E. Orthopoulos G. et al.TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1.Eur J Immunol. 2005; 35: 2416-2423Crossref PubMed Scopus (72) Google Scholar; Delaloye et al., 2009Delaloye J. Roger T. Steiner-Tardivel Q.G. et al.Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.PLoS Pathog. 2009; 5: e1000480Crossref PubMed Scopus (261) Google Scholar). However, it is uncertain whether viral ligands are responsible for the observed upregulation of TLR-6, -7, and -8 in KS. It also remains to be further investigated whether non-viral ligands of TLRs 6–8 may influence keloid etiopathogenesis by stimulating TLR receptors. Given that viral infection is among the most potent stimuli for upregulating TLRs (Alonso et al., 2008Alonso P.E. Rioja L.F. Pera C. Keloids: a viral hypothesis.Med Hypotheses. 2008; 70: 156-166Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; Delaloye et al., 2009Delaloye J. Roger T. Steiner-Tardivel Q.G. et al.Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.PLoS Pathog. 2009; 5: e1000480Crossref PubMed Scopus (261) Google Scholar), it is reasonable to systematically explore the involvement of either viral or non-viral stimuli in keloid pathogenesis. Existing KS organ culture models may be utilized to investigate whether antagonizing TLRs 6–8-mediated signaling and/or downregulation of these receptors may impact on keloid progression and resolution. NHS local and regional ethical approval as well as institutional approval from University Hospital South Manchester was obtained for carrying out this research. All patients and controls gave full ethically approved informed consent. The study was carried out with strict adherence to the Declaration of Helsinki protocols. We thank Professor Werner Müller for his expert advice. This study was partially supported by a grant from the Ministry of Higher Education in Saudi Arabia. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Download .pdf (6.06 MB) Help with pdf files Supplementary Information
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