Acta Dermato-Venereologica 98-8CompleteContent | Page 12

762 INVESTIGATIVE REPORT Toll-like Receptor 3 Agonist, Polyinosinic-polycytidylic Acid, Upregulates Carbonic Anhydrase II in Human Keratinocytes* Bani Kaur SURI 1 , Navin Kumar VERMA 1# and Artur SCHMIDTCHEN 1–3# Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, 2 Department of Biomedical Sciences, Copenhagen Wound Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark, and 3 Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden # These authors contributed equally. 1 Carbonic anhydrases are ubiquitously expressed enzy- mes that reversibly hydrate carbon dioxide to bicar- bonate and protons. While the main function of carbo- nic anhydrases is to regulate pH and osmotic balance, their involvement in other physiological processes remains to be explored. This study analysed changes in mRNA and protein levels of carbonic anhydrase II in human primary keratinocytes treated with various toll-like receptor agonists and cytokines. A significant upregulation of carbonic anhydrase I I at the mRNA and protein levels was observed upon treatment with polyinosinic-polycytidylic acid, a toll-like receptor 3 agonist. Furthermore, in agreement with the increased expression of carbonic anhydrase II in atopic derma- titis skin, carbonic anhydrase II was upregulated by the Th2 cytokines interleukins -4 and -13. In conclu- sion, these results suggest a potential role of carbonic anhydrase II in Th2-dependent and toll-like receptor 3-induced pathways in inflammatory skin conditions. Key words: carbonic anhydrase II, keratinocytes, TLR3, poly(I:C), Th2 cytokines, inflammation. Accepted May 4, 2018; Epub ahead of print May 8, 2018 Acta Derm Venereol 2018; 98: 762–765. Corr: Bani Kaur Suri, Lee Kong Chian School of Medicine, Nanyang Tech- nological University, Experimental Medicine Building, 59 Nanyang Drive, Singapore 636921, Singapore. E-mail: [email protected] S kin forms the first line of defence and thus is an im- portant site for immune response. Toll-like receptors (TLRs) constitute a family of signal transducing proteins, which sense the environment and activate downstream pathways related to inflammation, antiviral response, maintenance of homeostasis and regeneration (1). While TLRs play a crucial role in the cutaneous innate immune system, cytokines form part of the adaptive immune response by inducing and executing immune and inflam- matory responses. Carbonic anhydrases (CAs) catalyse the inter-con- version of carbon dioxide and bicarbonates with the release of a proton (H + ). Due to this fundamental, yet crucial reaction, CAs are involved in regulating pH, osmotic and electrolytic balance across kingdoms (2). *The Editor-in-Chief (AS) has not had responsibility for this article; it has been handled fully by the former Editor-in-Chief, who made the decision to accept it. doi: 10.2340/00015555-2963 Acta Derm Venereol 2018; 98: 762–765 SIGNIFICANCE Carbonic anhydrases (CAs) reversibly convert carbon diox- ide and water to bicarbonate and protons, thereby regula- ting pH and osmotic balance in different tissues. However, their role in skin health is unclear. Increased levels of a CA isoform, CA II, were found in human keratinocytes upon treatment with a toll-like receptor 3 (TLR3) agonist, poly- inosinic-polycytidylic acid (poly(I:C)), and Th2 cytokines, IL-4 and IL-13. The TLR3 pathway is implicated in antiviral and wound responses, and Th2 cytokines are involved in eczema. This suggests that CA II has a role in inflammatory skin conditions. Of the 15 CAs identified in humans, very few have been reported in keratinocytes and even fewer have their functions characterized. Carbonic anhydrase II (CA II), first discovered in red blood cells, has also been reported in keratinocytes. Although involvement of CA II in regulating pH in the skin has been hypothesized, its role is still unclear (3). To investigate the regulation of CA II in the context of host pathogen interaction and the ensuing immune response, an expression study was conducted for CA II in human keratinocytes using a panel of TLR agonists and cytokines. METHODS Adult normal human epidermal keratinocytes (NHEK) (ATCC, Manassas, USA) were cultured in dermal cell basal media, supp- lemented with keratinocyte growth kit, as per the ATCC recom- mendations. NHEK cells in plates were treated with TLR 1–9 agonists (InvivoGen, San Diego, USA) and cytokines (Milteny Biotec, Gladbach, Germany) for 12 h. Quantitative real-time PCR (qRT-PCR) was conducted for studying changes in CA2 gene ex- pression upon treatment. Primers used were 5’-CCTGAATCCTT- GGATTACTGGAC-3’ and 5’-TCCCCATTGAAGTTAAGTT- TACG-3’ for CA2 and 5’-TCGACAATGGCAGCATCTAC-3’ and 5’-ATCCGTCTCCACAGACAAGG-3’ for the housekeeping gene RPLP0. Protein levels were evaluated by Western immunoblot- ting using CA II (ab124687, Abcam, Cambridge, UK) and RPLP0 (11290-2-AP, Proteintech, Chicago, USA) antibodies. Unbiased protein expression was quantified using an automated microscopy- based High Content Analysis (HCA) system (IN Cell Investigator 2200, GE Healthcare, New York, USA). High magnification ima- ges were captured to visualize the localization of CA II protein using confocal laser scanning microscope (LSM800, Zeiss, Jena, Germany). All experiments were repeated at least 3 times. This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2018 Acta Dermato-Venereologica.