Introduction: The hair follicle bulge has recently been added to a growing list of human tissue compartments that exhibit a complex combination of immunosuppressive mechanisms, termed immune privilege (IP), which appear to restrict immune mediated injury in specific locations. As epithelial hair follicle stem cells (eHFSC) reside in the hair follicle bulge region it is conceivable that these IP mechanisms protect this vital compartment from immune-mediated damage, thereby ensuring the ongoing growth and cyclic regeneration of the hair follicle. Lichen planopilaris (and variants) are inflammatory hair disorders that result in hair follicle destruction and permanent alopecia. Growing evidence suggests that eHFSC destruction is a key factor in the permanent follicle loss seen in these conditions, and that IP collapse may predispose these cells to immune mediated injury. Aims: The overall aim of this project was to generate immunohistomorphometric, gene profiling, and limited functional evidence to probe the 'bulge immune privilege collapse' hypothesis in a carefully selected model disease for inflammation-induced epithelial stem cell death, lichen planopilaris (LPP). Methods: Adult patients with LPP (or variant frontal fibrosing alopecia) were recruited. Biopsies from lesional and non-lesional scalp skin were performed and either snap frozen in liquid nitrogen, fixed in formalin for paraffin embedding or transferred immediately for hair follicle organ culture. Both frozen and paraffin embedded tissue was processed for immunohistochemistry (IHC) analysis using various immune privilege, hair bulge (eHFSC) and immune cell markers. Cultured samples were supplemented with various chemicals know to influence hair follicle immune privilege with analysis performed using IHC. Further, additional paired lesional and non-lesional samples were sectioned horizontally for laser capture microdissection of bulge cells. Following extraction of RNA, reverse transcription and amplification of cDNA from these selected bulge cells; gene expression profiling was performed comparing lesional with non-lesional samples. Selected, differentially regulated genes were validated using IHC and quantitative real-time PCR. Results: Bulge IP collapse is present in active LPP, as suggested by increased expression of MHC class I, β2microglobulin and MHC class II, along with reduced expression of the locally produced immunosuppressant TGFβ2, at both the gene and protein level. Microarray pathway analysis supports these data with the antigen processing canonical pathway being prominently enriched. Cell mediated immune responses are prominent in active disease, suggested by significantly increased numbers of activated and cytotoxic T-cells infiltrating the bulge epithelium, along with greater numbers of mast cells and macrophages in the peri-follicular connective tissue sheath. Bulge cell eHFSC loss is suggested by loss of bulge cell markers (e.g. keratin 15) on IHC, microarray and qPCR, and supported by microarray analysis showing virtually global loss of recognised bulge eHFSC signatures. Hair follicle organ culture experiments confirm bulge IP collapse in a separate cohort of patients, and demonstrate that the pro-inflammatory cytokine, interferon-γ can further collapse IP in the bulge epithelium of cultured hair follicles. Conclusion: These data identify collapse of immune privilege in bulge cells in active LPP, and identify prominent cell-mediated immune responses and loss of eHFSC signatures in active disease. The pro-inflammatory cytokine, interferon-γ also appears to play a prominent role in IP collapse and contributes to immune cell trafficking into affected tissue. Future study is required to ascertain triggering factors of IP collapse and pursue other identified candidates from gene expression analysis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553448 |
| Date | January 2011 |
| Creators | Harries, Matthew |
| Contributors | Paus, Ralf; Griffiths, Christopher |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-immunopathobiology-of-lichen-planopilaris(a72b5c46-fcfd-46c8-a30a-715720233f59).html |
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