SHORT COMMUNICATION 741
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Advances in dermatology and venereology Acta Dermato-Venereologica
Keratinocyte Proliferation and Differentiation on IL-9 Stimulation : An Explorative In vitro Study
Hanna NIEHUES , Jos P . H . SMITS , Diana RODIJK-OLTHUIS , Joost SCHALKWIJK and Ellen H . VAN DEN BOGAARD * Department of Dermatology , Radboud Institute for Molecular Life Sciences , Radboud University Medical Center , Rene Descartesdreef 1 , NL-6525 GL Nijmegen , The Netherlands . * E-mail : Ellen . vandenBogaard @ radboudumc . nl Accepted Feb 20 , 2017 ; Epub ahead of print Feb 22 , 2017
Recent studies have indicated the significance of Th9 cells in a variety of inflammatory and allergy-related diseases ( see Kaplan et al . ( 1 )). Th9 cells are a distinct subset of CD4 + T cells present in the skin , which secrete interleukin ( IL ) -9 ( 2 – 4 ). IL-9 is transiently expressed by skin-tropic and skin-resident Th9 cells to regulate the production of inflammatory cytokines ( 5 ). IL-9 , like IL-4 , is associated with predominantly type 2 immune responses , and aberrant IL-9 expression or signalling in skin is implicated in allergic contact dermatitis ( ACD ) ( 6 ), atopic dermatitis ( AD ) ( 7 – 9 ), and psoriasis ( 5 , 10 ). Targeting of IL-9 or its receptor may therefore be an interesting new therapeutic avenue to be explored . Although the IL-9 receptor ( IL9R ) is expressed by immune cells as well as epithelial cells ( 11 ), the majority of research focuses on the Th9 / IL-9 axis in immune cells . Recent in vitro studies , however , have shown the regulation of IL9R expression in keratinocytes by IL-4 ( 12 ) and increased IL9R expression in basal keratinocytes of psoriatic lesions ( 10 ). Furthermore , IL-9 increased IL-8 ( CXCL8 ) and vascular endothelial growth factor ( VEGF ) secretion by keratinocytes in vitro ( 6 , 7 ). The effects of IL-9 on human keratinocytes with regard to epidermal proliferation , differentiation and host defence are still poorly understood . This study examined the effects of IL-9 on epidermal morphology , proliferation , differentiation and host defence , and studied cytokine-mediated regulation of IL9R expression on keratinocytes , and investigated the potential additive or synergistic effects by IL-9 in Th2-cytokine mediated epidermal responses .
MATERIALS AND METHODS
Human epidermal equivalents ( HEEs ) and monolayer cultures generated from adult primary human keratinocytes were exposed to human recombinant IL-9 or other pro-inflammatory cytokines , as indicated . Epidermal morphology was studied by histology , and gene and protein expression was analysed by quantitative PCR analysis and immunohistochemistry , respectively . For detailed description , see Appendix S1 1 .
RESULTS
First , a dose range of IL-9 was tested on differentiating submerged keratinocyte monolayer cultures and no effect on cell morphology and viability was observed ,
1 https :// www . medicaljournals . se / acta / content / abstract / 10.2340 / 00015555-2643 even at the highest concentration of 500 ng / ml ( data not shown ). For further in depth analysis of keratinocyte proliferation and differentiation , HEEs were exposed to 50 ng / ml human recombinant IL-9 , being a biologically relevant concentration as shown by the induction of CXCL8 expression ( Fig . 1 , ( 10 )). After 72 h of IL-9 stimulation , HEEs showed normal epidermal morphology with a fully stratified epidermis and a well-developed stratum corneum ( Fig . S1a 1 ). The number of proliferating cells , measured with Ki67 staining , and the epidermal thickness was similar to that of control HEEs ( Figs S1a and S2 1 ). Next , we analysed the expression of the major epidermal differentiation proteins , keratin 10 ( K10 ), involucrin ( IVL ), filaggrin ( FLG ) and loricrin ( LOR ) ( Fig . S1b 1 ). For all markers , IL-9 did not alter protein localization or expression levels , nor did it change the expression of epidermal differentiation genes ( Fig . S3 1 ). Also , marker expression for keratinocyte activation or host defence , namely keratin 16 ( K16 ) and SKALP remained unaffected . Human beta defensin 2 ( hBD2 ) is absent in unstimulated HEEs and is also not induced by IL-9 ( Fig . S1c 1 ). This in contrast to the classical Th2 cytokines , IL-4 and IL-13 , which downregulated FLG , LOR and IVL expression ( Fig . S4a 1 ) and induced K16 and SKALP expression ( Fig . S4b 1 ).
The suggested role of IL-9 in atopic diseases , which are largely Th2 driven , led us to investigate the interaction of IL-9 with IL-4 and / or IL-13 . Co-stimulation with IL-9 did not alter the effect of Th2 cytokines on downregulation of epidermal differentiation proteins ( Fig . S5a 1 ) or the induction of inflammatory epidermal markers , K16 and SKALP ( Figs S5b and S6 1 ).
We hypothesized that the absence of significant effects by IL-9 in our study could be due to low IL9R expression ( mean Ct value 34 ) under the conditions described above . IL9R expression appears to be induced in inflammatory processes , and we therefore stimulated keratinocyte monolayers and HEEs with various cytokine combinations and found interferon gamma ( IFN-γ ) to be the main inducer of IL9R expression ( Fig . 2 ). We determined the minimal , but effective , IFN-γ exposure time and observed significantly induced IL9R expression after 6 h of IFN-γ stimulation ( Fig . S7 1 ). Thereafter , HEEs were exposed to IL-9 for 72 h . IFN-γ alone induced the mRNA expression of IVL , and the chemokines CCL5 and CXLC10 , yet no additional effect of IL-9 stimulation was observed on these or other genes analysed ( Fig . S8A 1 ). Similar to previous
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 / 00015555-2643 Acta Derm Venereol 2017 ; 97 : 741 – 742