Perturbation of cutaneous homeostasis including immune dysregulation and skin barrier dysfunction evokes skin disorders. In this study, we examined the effect of Eucalyptus citriodora (Euc-c) extract on cytokine production, cell proliferation and cell differentiation in HaCaT cells to elucidate its influence on cutaneous homeostasis. Euc-c suppressed significantly LPS-induced IL-6 and TNF-a-induced IL-8 production from HaCaT cells. Conversely IL-1ra production was significantly enhanced by Euc-c. The expressions of IVL, CERS3 and CERS4, keratinocyte differentiation markers, were upregulated to 3.1, 2.8 and 2.7-fold respectively by Euc-c treatment, compared to the control, while the proliferation was downregulated. The lipid contents in Euc-c-treated cells tended to increase, compared with non-treated cells. To explore the underlying mechanism of these effect, we next performed siRNA experiments against PPAR-b/d. Euc-c enhanced PPAR-b/d mRNA expression to 3.25-fold, while PPAR-b/d mRNA expression in transfected cells was suppressed. The expressions of IVL, CERS3 and CERS4 in transfected cells were suppressed to 1.48, 0.82 and 0.72-fold respectively, concomitant with suppression of PPAR-b/d mRNA expression. These results indicated that Euc-c exerts anti-inflammatory effects and regulates keratinocyte differentiation via the modulation of PPAR-b/d pathway. Therefore, the application of Euc-c is expected to exert beneficial effect on skin disorders evoked by perturbation of skin homeostasis.

1. Menona GK, Clearyb GW, Lanec ME. The structure and function of the stratum corneum. Intl J Pharm 2012; 435(1):3-9 doi: 10.1016/j.ijpharm.2012.06.005

2. Ansel J, Perry P, Brown J, et al. Cytokine modulation of keratinocyte cytokines. J Invest Dermatol 1990; 94(6):101s-107s

3. Elias PM. Stratum corneum defensive functions: An integrated view. J Invest Dermatol 2005; 125(2):183-200

4. Jungersted JM, Hellgren LI, Høgh JK, et al. Ceramides and barrier function in healthy skin. Acta Derm Venereol 2010; 90(4):350-353 doi: 10.2340/00015555-0894

5. Sandilands A, Sutherland C, Irvine AD, et al. Filaggrin in the frontline: role in skin barrier function and disease. J Cell Sci 2009; 122(9):1285-1294 doi:10.1242/jcs.033969

6. Koch PJ, de Viragh PA, Scharer E, et al. Lessons from loricrin-deficient mice: Compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J Cell Biol 2000; 151(2):389-400 doi: 10.1083/jcb.151.2.389

7. Elsholz F, Harteneck C, Muller W, et al. Calcium - a central regulator of keratinocyte differentiation in health and disease. Eur J Dermatol 2014; 24(6): 650-661 doi:10.1684/ejd.2014.2452

8. Hänel KH, Cornelissen C, Lüscher B, et al. Cytokines and the Skin Barrier. Int. J Mol Sci 2013; 14(4): 6720-6745 doi:10.3390/ijms14046720

9. Grone A. Keratinocytes and cytokines. Vet Immunol Immunopathol 2002; 88(1-2): 1-12

10. Issemann I, Green S. Activation of a member of the steroid receptor superfamily by peroxisome proliferator. Nature 1990; 347(6294): 645-649 doi:10.1038/347645a0

11. Sher T, Hua-Fang Y, McBride O, et al. cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry 1993; 32(21): 5598-5604 doi:10.1021/bi00072a015

12. Kliewer SA, Umesono K, Nooman DJ, et al. Convergence of 9-cis retinoic acid and peroxisome proliferator signaling pathways through heterodimer formation of their receptors. Nature 1992; 358(6389): 771-774 doi:10.1038/358771a0

13. Lamichane S, Lamichane BD, Kwon SM. Pivotal roles of peroxisome proliferator-activated receptors (PPARs) and their signal cascade for cellular and whole-body energy homeostasis. Int J Mol Sci 2018; 19: 949, 2018 doi:10.3390/ijms19040949

14. Rivier M, Safonova I, Lebrun P, et al. Differential expression of peroxisome proliferator-activated receptor subtypes during the differentiation of human keratinocytes. J Invest Dermatol 1998; 111(6):1116-1121 doi:10.1046/j.1523-1747.1998.00439.x

15. Aioi A. Peroxisome proliferator-activated receptors (PPARs) activation as therapeutic targets in skin inflammation. Trends in Immunother 2018; 2(4): 1-14. doi:10.24294/ti.v2i4.1063

16. Sertznig P, Seifert M, Tilgen W, Reichrath J. Peroxisome proliferator-activated receptors (PPARs) and the human skin: Importance of PPARs in skin physiology and dermatologic diseases. Am J Clin Dermatol 2008; 9(1): 15-31

17. Sertznig P, Reichrath J. Peroxisome proliferator-activated receptors (PPARs) in dermatology. Dermato-Encrinol 2011; 3(3):130-135 doi:10.4161/derm.3.3.15025

18. Sadlon AE, Lamson DW. Immune-modifying and antimicrobial effects of eucalyptus oil and simple inhalation devices. Altern Med Rev 2010; 15(1):33-47

19. Gbenou JD, Ahounou JF, Akakpo HB, et al. Phytochemical composition of Cymbopogon citratus and Eucalyptus citriodora essential oils and their anti-inflammatory and analgesic properties on Wistar rats. Mol Biol Rep 2013; 40(2):1127-1134 doi: 10.1007/s11033-012-2155-1

20. Singh HP, Kaur S, Negi K, et al. Assessment of in vitro antioxidant activity of essential oil of Eucalyptus citriodora (lemon-scented Eucalypt; Myrtaceae) and its major constituents. Food Sci Tech 2012; 48 (2) 237-241 doi:10.1016/j.lwt.2012.03.019

21. Sepúlveda-Ariasa JC. Velozab A, Escobar LM, et al. Anti-inflammatory effects of the main constituents and epoxides derived from the essential oils obtained from Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora. J Essential Oil Res 2013; 25(3):186-193 doi: 10.1080/10412905.2012.751556

22. Duh PD, Chen ZT, Lee SW, et al. Antiproliferative activity and apoptosis induction of Eucalyptus citriodora resin and its major bioactive compound in melanoma B16F10 cells. J Agric Food Chem 2012; 60(32):7866-7872 doi: 10.1021/jf301068z

23. Camp D, Davis RA, Campitelli M, et al. Drug-like Properties: Guiding Principles for the Design of Natural Product Libraries. J Nat Prod2012; 75(1):72-81 doi: 1.1021/np200687v

24. Camp D, Campitelli M, Carroll AR, et al. Front Loading Natural Product Screening Libraries for log P: Background, Development and Implementation. Chem Biodiversity, 2013; 10(4):524-537

25. Deyrieux AF, Wilson VG. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology 2007; 54(2):77-83 doi: 10.1007/s10616-007-9076-1

26. Westergaard M, Henningsen J, Svendsen ML. et al. Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid. J. Invest. Dermatol. 2001; 116(5):702–712 doi:10.1046/j.1523-1747.2001.01329.x

27. Boukamp P, Petrussevska RT, Breitkreutz D, et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988; 106(3):761-771

28. Micallef L, Belaubre F, Pinon A, et al. Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes. Exp Dermatol 2009; 18(2):143-151 doi:10.1111/j.1600-0625.2008.00775.x

29. Straus DS, Glass CK. Anti-inflammatory actions of PPAR ligands: new insights on cellular and molecular mechanisms. Trends Immunol 2007; 28(12):551-558 doi: 10.1016/j.it.2007.09.003

30. Devchand PR, Keller H, Peters JM, et al. The PPAR-leukotriene B4 pathway to inflammation control, Nature 1996; 384:39–43 doi:10.1038/384039a0

31. Jiang C, Ting AT, Sees B. PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391:83-86 doi:10.1038/34184

32. Staels B, Koenig W, Habib A, R. et al. Activation of human aortic smooth-muscle cells is inhibited by PPAR alpha but not by PPAR gamma activators. Nature 1998; 393:790–793 doi:10.1038/31701

33. Marx N, Sukhova GK, Collins T, et al. PPAR alpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 1999; 99(24):3125–3131

34. Welch JS, Ricote M, Akiyama TE, et al. PPARγ and PPARγ negatively regulate specific subsets of lipopolysaccharide and IFNγ target genes in macrophages. Proc Natl Acad Sci USA 2003; 100(11):6712–6717 doi:10.1073/pnas.1031789100

35. Ding G, Cheng L, Qin Q, et al. PPAR modulates lipopolysaccharide-induced TNF- inflammation signaling in cultured cardiomyocytes. J Mol Cell Cardiol 2006; 40(6):821–828 doi:10.1016/j.yjmcc.2006.03.422

36. Natarajan C, Bright JJ. Peroxisome proliferator-activated receptor-gamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation. Genes and Immunity 2002; 3(2):59–70 doi:10.1038/sj/gene/6363832

37. Gervois P, Mansouri RM. PPARα as a therapeutic target in inflammation-associated diseases. Expert Opin Ther Targets 2012; 16(11):1113-1125 doi:10.1517/14728222.2012.715633

38. Kuribayashi S, Xu X, Ishii S, et al. A novel thiazolidinediones MCC-555 down-regulates tumor necrosis factor--induced expression of vascular adhesion molecule-1 in vascular endothelial cells. Atherosclerosis 2005; 182(1):71-77 doi: 10.1016/j.atherosclerosis.2005.02.004

39. Tyrone E, James PG, Zineh WI. Modulatory effect of fenofibrate on endothelia production of neutrophil chemokines IL-8 and ENA-78. Cardiovasc Drugs Ther 2012; 26(2):95-99 doi:10.1007/s10557-011-6368-7

40. Ricote M, Glass CK. PPARs and molecular mechanism of transcription. Biochim Biophys Acta 2007; 1771(8):926-936 doi:10.1016/j.bbalip.2007.02.013

41. Stienstra R, Mandard S, Tan NS, et al. The Interleukin-1 receptor antagonist is a direct target gene of PPAR- in liver. J Hepatol 2007; 46 (5):869-877 doi:10.1016/j.jhep.2006.11.019

42. Kim HJ, Kim MY, Hwang JS, et al. PPAR inhibits IL-1-stimulated proliferation and migration of vascular smooth muscle cells via up-regulation of IL-1Ra. Cell Mol Life Sci 2010; 67(12):2119-2130 doi:10.1007/s00018-010-0328-4

43. Romanowska M, Yacoub N, Seidel H, et al. PPAR- enhances keratinocyte proliferation in psoriasis and induces hparin-binding EGF-like growth factor. J Investigative Dermatol 2008; 128(1):110–124 doi:10.1038/sj.jid.5700943

44. Lianga P, Jiang B, Yanga X, et al. The role of peroxisome proliferator-activated receptor-/ in epidermal growth factor-induced HaCaT cell proliferation. Exp Cell Res 2008; 314(17):3142-3151 doi:10.1016/j.yexcr.2008.06.013

45. Kim DJ, Bility MT, Billin AN, et al. PPAR/ selectively induces differentiation and inhibits cell proliferation. Cell Death Differ 2006; 13(1):53-60 doi:10.1038/sj.cdd.4401713

46. Burdick AD, Bility MT, Girroir EE, et al. Ligand activation of peroxisome proliferator-activated receptor-/ (PPAR/) inhibits cell growth of human N/TERT-1 keratinocytes. Cell Signalling 2007; 19 (6) 1163-1171 doi: 10.1016/j.cellsig.2006.12.007

47. Levy M, Futerman AH. Mammalian ceramide synthases. IUBMB Life, 2010; 62(5):347-356 doi: 10.1002/iub.319

48. Mizutani Y, Kihara A, Chiba H, et al. 2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length. J Lipid Res 49(11):2356-2364 doi:10.1194/jlr.M800158-JLR200

49. Kima B, Kima JE, Kim HS. Caffeic acid induces keratinocyte differentiation by activation of PPAR-. J Pharm Pharmacol 2014; 66(1):84-92 doi:10.1111/jphp.12159

50. Mao-Qiang M, Fowler AJ, Schmuth M, et al. Peroxisome-proliferator-activated receptor (PPAR)- activation stimulates keratinocyte differentiation. 2004; J Invest Dermatol 123(2):305 –312 doi:10.1111/j.0022-202X.2004.23235.x

51. Finegold KR. The role of epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid Res 2007; 48(2):2531-2546 doi: 10.1194/jlr.R700013-JLR200

52. Man MQ, Choi EH, Schmuth M, et al. Basis for improved permeability barrier homeostasis induced by PPAR and LXR activators: Liposensors stimulate lipid synthesis, lamellar body secretion and post-secretory lipid processing. J Invest Dermatol 2006; 126(2):386-392 doi:10.1038/sj.jid.5700046

53. Schmuth M, Haqq CM, Cairns WJ, et al. Peroxisome proliferator-activated receptor (PPAR)-/ stimulates differentiation and lipid accumulation in keratinocytes. J Invest Dermatol 2004; 122(4):971-983 doi:10.1111/j.0022-202X.2004.22412.x

54. Chon SH, Tannahill R, Yao X, et al. Keratinocyte differentiation and upregulation of ceramide synthesis induced by an oat lipid extract via the activation of PPAR pathways. Exp Dermatol 2015; 24(4):290-295 doi:10.1111/exd.12658

55. Kima MS, Pyun HB, Hwanga JK. Panduratin A, an activator of PPAR- suppresses the development of oxazolone-induced atopic dermatitis-like symptoms in hairless mice. Life Sci 2014; 100(1):45-54 doi:10.1016/j.lfs.2014.01.076

56. Hatano Y, Man MQ, Uchida Y, et al. Murine atopic dermatitis responds to peroxisome proliferator-activated receptors α and β/δ (but not γγ) and liver X receptor activators. J Allergy Clin Immunol 2010; 125(1):160-169 doi:10.1016/j.jaci.2009.06.049

57. Michalik L, Desvergne B, Tan NS, et al. Impaired skin wound healing in peroxisome proliferator–activated receptor (PPAR)α and PPARβ mutant mice. J Cell Biol 2001; 154(4):799-814 doi:10.1083/jcb.200011148

58. Borland MG, Yao PL, Kehres EM, et al. PPAR and PPAR inhibit melanoma tumorigenicity by modulating inflammation and apoptosis. Toxicol Sci 2017; 159(2):436-448 doi: 10.1093/toxsci/kfx147

59. Ham SA. Yoo T, Hwang JS, et al. Ligand-activated PPAR modulates the migration and invasion of melanoma cells by regulating Snail expression. Am J Cancer Res 2014; 4(6):674-682

60. Jung YR, Lee EK, Kim DJ, et al. Upregulation of collagen expression via PPAR activation in aged skin by magnesium Lithospermate B from Salvia miltiorrhiza. J Nat Prod 2015; 78(8):2110-2115 doi:10.1021/acs.jnatprod.5b00348