INVESTIGATIVE REPORT
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ActaDV ActaDV Advances in dermatology and venereology Acta Dermato-Venereologica
The” Alarmins” HMBG1 and IL-33 Downregulate Structural Skin Barrier Proteins and Impair Epidermal Growth
Uffe NYGAARD 1, 2, Ellen H. VAN DEN BOGAARD 3, Hanna NIEHUES 3, Malene HVID 2, Mette DELEURAN 1, Claus JOHANSEN 1 and Christian VESTERGAARD 1
1
Department of Dermatology and Venereology, Aarhus University Hospital, Aarhus University, 2 Department of Clinical Medicine, Aarhus University, Aarhus, Denmark, and 3 Department of Dermatology, Radboud Institute for Molecular Life Sciences, Radboud UMC, Nijmegen, The Netherlands
The epidermal-derived“ alarmins” high-mobility group box 1( HMGB1) protein and interleukin-33( IL-33) are upregulated in patients with atopic dermatitis. However, their capacity as pro-inflammatory cytokines and their derived effects on skin barrier regulation are poorly elucidated. We investigated the impact of HMGB1 and IL-33 on gene transcription, protein expression and epidermal differentiation across 3 distinct keratinocyte in vitro models. Primary keratinocytes from healthy donors were used in submerged monolayer cultures, 3D human epidermis equivalents and 3D human skin equivalents. All keratinocyte models underwent 96-h stimulation with HMGB1( 100 μM) or IL-33( 100 ng / ml) using IL-4( 50 ng / ml) as positive control of regulation and vehicle as negative control. We found that HMGB1 and IL-33 downregulated transcription of several genes from members of the epidermal differentiation complex, including filaggrin. Furthermore, HMGB1 downregulated the expression of the encoded proteins in the upper epidermis. Finally, IL-33 and HMGB1 ultimately led to impaired epidermal growth and maturation. In conclusion, HMGB1 and IL-33 could play a significant role in the atopic dermatitis pathophysiology due to negative regulation of structural proteins, stratum corneum formation and epidermal growth.
Key words: filaggrin; involucrin; loricrin; IL-33; HMGB1; IL- 4; skin; keratinocytes; cell culture; human skin equivalents; atopic dermatitis.
Accepted Oct 12, 2016; Epub ahead of print Oct 14, 2016 Acta Derm Venereol 2017: 97: 305 – 312.
Corr: Uffe Harboe Nygaard, Department of Dermatology and Venereology, Aarhus University Hospital, P. P. Oerumsgade 11, DK-8000 Aarhus, Denmark. E-mail: uffenygaard @ clin. au. dk
The skin is a vital first line of defence between the environment and the human body. Studies indicate that defects in epidermal barrier function contribute to triggering and perpetuating skin inflammation in atopic dermatitis( AD)( 1 – 5). Furthermore, disease activity and barrier function seem to be intimately related, and fluctuations in either can drive the disease( 6). The role of skin barrier dysfunction as an important contributing factor in AD is further substantiated by loss-of-function mutations seen in the filaggrin gene which is a major risk factor for development of AD( 3, 7, 8). Filaggrin is a key protein in epidermal homeostasis and barrier integrity; and through its processing to free amino acids, it facilitates retention of water in the stratum corneum( SC)( natural moisturising factor)( 3, 9). In conjunction with filaggrin, other proteins encoded by genes in the epidermal differentiation complex, including loricrin and involucrin, are indispensable elements of the epidermal barrier( 3, 10). Skin alterations in AD are characterised by increased transepidermal water loss( TEWL) and a defective terminal keratinocyte differentiation entailing reduced levels of filaggrin, involucrin and loricrin( 11 – 13).
Loss-of-function mutations in the filaggrin gene are found in approximately one third of all AD patients and are considered responsible for the decreased skin barrier function, the increased TEWL and increased disease severity. Yet, all patients with AD display a reduced expression of filaggrin and also involucrin and loricrin. This phenomenon is explained in part by the impact of cytokines, e. g. interleukin( IL)-4, IL-13 and IL-25 that reduce the expression of barrier proteins( 12 – 14).
The cytokine IL-33 is a member of the IL-1-family. IL-33 is constitutively expressed in healthy skin and is upregulated in skin of AD patients and in AD-like lesions in mice( 15 – 18). Furthermore, we recently found elevated IL-33 serum levels in children with AD( 19, 20). IL-33 is classified as an alarmin due to the passive release upon cellular stress like allergen stimulation, scratching or non-apoptotic cell death, though these mechanism are still to be fully elucidated( 16, 21, 22). It displays Th2 immunomodulatory effects by inducing IL-5 and IL-13. It also displays a Th1 response via upregulation of IFN-γ( 23). Keratinocytes produce IL-33 and also express its receptor complex consisting of ST2 and IL-1RAcP on their surface( 16, 24). Moreover, IL-33 appears to exert receptor-independent transcriptional effects in the nucleus of epithelial cells( 25), thereby operating as a dualfunction immune mediator similarly to high-mobility group box 1( HMGB1)( 26). HMGB1 is localised in the nucleus in almost all cells and is rapidly mobilised to other sites within the cell, and into the extracellular space in response to a variety of stimuli( 26, 27). Through RAGE and TLR4 signalling, HMGB1 mediates immune responses in non-infectious inflammation and shows
This is an open access article under the CC BY-NC license. www. medicaljournals. se / acta Journal Compilation © 2017 Acta Dermato-Venereologica. doi: 10.2340 / 00015555-2552 Acta Derm Venereol 2017; 97: 305 – 312