Immune Netw.  2015 Apr;15(2):100-109. 10.4110/in.2015.15.2.100.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) Modulates Th2 Immunity through Attenuation of IL-4 Expression

• Yoon SY
• Kang HB
• Ko YE
• Shin SH
• Kim YJ
• Sohn KY
• Han YH
• Chong S
• Kim JW

• KMID: 2168035
• DOI: http://doi.org/10.4110/in.2015.15.2.100

Abstract

Controlling balance between T-helper type 1 (Th1) and T-helper type 2 (Th2) plays a pivotal role in maintaining the biological rhythm of Th1/Th2 and circumventing diseases caused by Th1/Th2 imbalance. Interleukin 4 (IL-4) is a Th2-type cytokine and often associated with hypersensitivity-related diseases such as atopic dermatitis and allergies when overexpressed. In this study, we have tried to elucidate the function of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) as an essential modulator of Th1/Th2 balance. EC-18 has showed an inhibitory effect on the production of IL-4 in a dose-dependent manner. RT-PCR analysis has proved EC-18 affect the transcription of IL-4. By analyzing the phosphorylation status of Signal transducer and activator of transcription 6 (STAT6), which is a transcriptional activator of IL-4 expression, we discovered that EC-18 induced the decrease of STAT6 activity in several stimulated cell lines, which was also showed in STAT6 reporter analysis. Co-treatment of EC-18 significantly weakened atopy-like phenotypes in mice treated with an allergen. Collectively, our results suggest that EC-18 is a potent Th2 modulating factor by regulating the transcription of IL-4 via STAT6 modulation, and could be developed for immune-modulatory therapeutics.

Keyword

EC-18; Th2 modulator; IL-4; STAT6

MeSH Terms

Animals
Cell Line
Dermatitis, Atopic
Hypersensitivity
Interleukin-4*
Mice
Phenotype
Phosphorylation
STAT6 Transcription Factor
Interleukin-4
STAT6 Transcription Factor

Figure

• 

  Figure 1 Structure of EC-18.

• 

  Figure 2 EC-18 reduced antigeninduced IL-4 mRNA transcripts in RBL-2H3 cells. RBL-2H3 cells were treated with anti-DNP-IgE (50 ng/ml) for 12 h followed by stimulation with DNP-HSA antigen (25 ng/ml) for 2 h, and NK-92 cells were treated with PMA (10 ng/ml) for 24 h, respectively. EC-18 was pretreated from 0.01 to 10 µg/ml both in RBL-2H3 (A) and NK-92 (C). As positive controls, STAT6 inhibitor (AS 1517499) and STAT1 inhibitor (Fludarabine) was pretreated in RBL-2H3 (B) and NK-92 (D), respectively. After incubation for 2 h, mRNA was isolated from each group of cells and RT-PCR was carried out. As an internal control, GAPDH was used.

• 

  Figure 3 EC-18 decreased the levels of IL-4. Secreted IL-4 and IFN-γ were evaluated using ELISA. IL-4 and IFN-γ were produced using the DNP-HSA antigen (25 ng/ml) and PMA (10 ng/ml) in RBL-2H3 (A, B) and NK-92 (C, D) cells, respectively. EC-18 was pretreated from 0.001 to 10 µg/ml in RBL-2H3 (A) and from 0.001 to 10 µg/ml in NK-92 (C). As positive controls, the STAT6 inhibitor (AS 1517499) and STAT1 inhibitor (Fludarabine) were pretreated in RBL-2H3 (B) and NK-92 (D), respectively. After 12 hours of cell culture, culture supernatants were harvested and the produced IL-4 and IFN-γ were quantitated by ELISA. Each bar represents the mean±SD. Significance (p<0.001), indicated by **, is the EC-18-treated cells versus the antigen only-treated cells.

• 

  Figure 4 EC-18 dephosphorylated activated STAT6. Phosphorylation of STAT6 was examined using antiphospho STAT6 in the U937 (A), A549 (C), and Jurkat (D) cell lysates treated with EC-18 with concentrations from 0.01 to 10 µg/ml. Phosphorylation of STAT6 was induced by 10 ng/ml of the IL-4 treatment. (B) STAT1 phosphorylation was checked out in the cell lysate treated EC-18 with concentrations from 0.01 to 10 µg/ml. The activation of STAT1 was induced by 10 ng of the IFN-γ treatment in the U937 cells. Dephosphorylation of STAT1 and STAT6 was examined at 15 min after stimulation with IFN-γ and IL-4, respectively, in the EC-18 pretreated cells.

• 

  Figure 5 EC-18 inhibited the transcriptional activity for STAT6, but not for STAT1. One µg of pGL4- STAT6-Luc-hygro or pGL4-STAT1- Luc-hygro vector was transfected into HEK293 (A, B) and A549 (C, D) cells, and the transfected cells were pretreated with EC-18 (0.01-10 µg/ml) at the indicated concentrations and incubated for 24 h, followed by stimulation with 10 ng/ml of IL-4 and IFN-γ for an additional incubation period of 6 h. STAT6 and STAT1 promoter activities were assessed using the Dual-GloTM Luciferase assay system. Each bar represents the mean±SD. Significant values are represented by *(p<0.05) and **(p<0.001). It is a comparison of the EC-18- treated cells to IL-4 alone.

• 

  Figure 6 EC-18 decreased the plasma levels of IL-4 and IgE in DNCB-induced atopy-like dermatitis mice. EC-18 was administrated orally to the mice every day for 18 days and then sensitized with 0.2% DNCB 6 times and 1% DNCB 1 time over 7 days and then challenged with 0.2% DNCB for 5 times every 2 days. After the collection of serum from mice at Day 0, Day 4, Day 14, and Day 18, the plasma IL-4 and IgE levels were analyzed. Each bar represents the mean±SD. Significance (p<0.05), indicated by *, is in the EC-18 -treated group versus the control group treated with olive oil.

Reference

1. Kleiman R, Miller RW, Earle FR, Wolff IA. Optically active aceto-triglycerides of oil from Euonymus verrucosus seed. Lipids. 1966; 1:286–287.

2. Myher JJ, Kuksis A, Marai L, Sandra P. Identification of the more complex triacylglycerols in bovine milk fat by gas chromatography-mass spectrometry using polar capillary columns. J Chromatogr. 1988; 452:93–118.

3. Limb JK, Kim YH, Han SY, Jhon GJ. Isolation and characterization of monoacetyldiglycerides from bovine udder. J Lipid Res. 1999; 40:2169–2176.

4. Yang HO, Kim SH, Cho SH, Kim MG, Seo JY, Park JS, Jhon GJ, Han SY. Purification and structural determination of hematopoietic stem cell-stimulating monoacetyldiglycerides from Cervus nippon (deer antler). Chem Pharm Bull (Tokyo). 2004; 52:874–878.

5. Lee TS, Yook JS, Lee JS, Yoo CH, Lee JC, Lee CM, Lee WH. Preparation of glycerol derivatives and intermediates therefor. KR 10-0789323. 2005.

6. Lee TS, Yook JS, Lee JS, Yoo CH, Lee JC, Lee CM, Lee WH. Preparation method of 1-palmitoyl-3-acetylglycerol, and preparation method of 1-palmitoyl-2-linoleoyl-3-acetylglycerol using same. WO 2013/043009 A2. 2013.

7. Yang HO, Park JS, Cho SH, Yoon JY, Kim MG, Jhon GJ, Han SY, Kim SH. Stimulatory effects of monoacetyldiglycerides on hematopoiesis. Biol Pharm Bull. 2004; 27:1121–1125.

8. Kim MH, Chang HM, Kim TW, Lee SK, Park JS, Kim YH, Lee TY, Jang SJ, Suh CW, Lee TS, Kim SH, Lee SG. EC-18, a synthetic monoacetyldiacylglyceride, inhibits hematogenous metastasis of KIGB-5 biliary cancer cell in hamster model. J Korean Med Sci. 2009; 24:474–480.

9. Hong JJ, Koh Y, Park JS, Jung HD, Kim SH, Lee TS, Badellino MM. Enteral administration of a synthetic monoacetyldiglyceride improves survival in a murine model of abdominal sepsis. J Trauma. 2010; 68:62–68.

10. Shin IS, Shin NR, Jeon CM, Kwon OK, Sohn KY, Lee TS, Kim JW, Ahn KS, Oh SR. EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetylglycerol), attenuates the asthmatic response in an aluminum hydroxide/ovalbumin-induced model of asthma1. Int Immunopharmacol. 2014; 18:116–123.

11. Coffman RL. Converging discoveries: the first reports of IL-4. J Immunol. 2013; 190:847–848.

12. Van Dyken SJ, Locksley RM. Interleukin-4- and interleukin-13-mediated alternatively activated macrophages: roles in homeostasis and disease. Annu Rev Immunol. 2013; 31:317–343.

13. Sehra S, Yao Y, Howell MD, Nguyen ET, Kansas GS, Leung DY, Travers JB, Kaplan MH. IL-4 regulates skin homeostasis and the predisposition toward allergic skin inflammation. J Immunol. 2010; 184:3186–3190.

14. Hatano Y, Adachi Y, Elias PM, Crumrine D, Sakai T, Kurahashi R, Katagiri K, Fujiwara S. The Th2 cytokine, interleukin-4, abrogates the cohesion of normal stratum corneum in mice: implications for pathogenesis of atopic dermatitis. Exp Dermatol. 2013; 22:30–35.

15. Bao L, Shi VY, Chan LS. IL-4 regulates chemokine CCL26 in keratinocytes through the Jak1, 2/Stat6 signal transduction pathway: Implication for atopic dermatitis. Mol Immunol. 2012; 50:91–97.

16. Brandt EB, Sivaprasad U. Th2 Cytokines and Atopic Dermatitis. J Clin Cell Immunol. 2011; 2.

17. Zhu N, Gong Y, Chen XD, Zhang J, Long F, He J, Xia JW, Dong L. Association between the polymorphisms of interleukin-4, the interleukin-4 receptor gene and asthma. Chin Med J (Engl). 2013; 126:2943–2951.

18. Nie W, Zang Y, Chen J, Xiu Q. Association between interleukin-4 receptor alpha chain (IL4RA) I50V and Q551R polymorphisms and asthma risk: an update meta-analysis. PLoS One. 2013; 8:e69120.

19. Schuijs MJ, Willart MA, Hammad H, Lambrecht BN. Cytokine targets in airway inflammation. Curr Opin Pharmacol. 2013; 13:351–361.

20. Egawa M, Mukai K, Yoshikawa S, Iki M, Mukaida N, Kawano Y, Minegishi Y, Karasuyama H. Inflammatory monocytes recruited to allergic skin acquire an anti-inflammatory M2 phenotype via basophil-derived interleukin-4. Immunity. 2013; 38:570–580.

21. Burton OT, Darling AR, Zhou JS, Noval-Rivas M, Jones TG, Gurish MF, Chatila TA, Oettgen HC. Direct effects of IL-4 on mast cells drive their intestinal expansion and increase susceptibility to anaphylaxis in a murine model of food allergy. Mucosal Immunol. 2013; 6:740–750.

22. Kaplan MH, Schindler U, Smiley ST, Grusby MJ. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity. 1996; 4:313–319.

23. Schoenborn JR, Wilson CB. Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol. 2007; 96:41–101.

24. Hu X, Ivashkiv LB. Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases. Immunity. 2009; 31:539–550.

25. Wick KR, Berton MT. IL-4 induces serine phosphorylation of the STAT6 transactivation domain in B lymphocytes. Mol Immunol. 2000; 37:641–652.

26. Wu F, Li H, Jin L, Li X, Ma Y, You J, Li S, Xu Y. Deer antler base as a traditional Chinese medicine: a review of its traditional uses, chemistry and pharmacology. J Ethnopharmacol. 2013; 145:403–415.

27. Gilbey A, Perezgonzalez JD. Health benefits of deer and elk velvet antler supplements: a systematic review of randomised controlled studies. N Z Med J. 2012; 125:80–86.

28. Mushaben EM, Kramer EL, Brandt EB, Khurana Hershey GK, Le Cras TD. Rapamycin attenuates airway hyperreactivity, goblet cells, and IgE in experimental allergic asthma. J Immunol. 2011; 187:5756–5763.

29. Geha RS, Jabara HH, Brodeur SR. The regulation of immunoglobulin E class-switch recombination. Nat Rev Immunol. 2003; 3:721–732.

30. Cookson W. The immunogenetics of asthma and eczema: a new focus on the epithelium. Nat Rev Immunol. 2004; 4:978–988.

31. Li L, Lee HH, Bell JJ, Gregg RK, Ellis JS, Gessner A, Zaghouani H. IL-4 utilizes an alternative receptor to drive apoptosis of Th1 cells and skews neonatal immunity toward Th2. Immunity. 2004; 20:429–440.

32. Liew FY. T(H)1 and T(H)2 cells: a historical perspective. Nat Rev Immunol. 2002; 2:55–60.

33. Yang XO, Zhang H, Kim BS, Niu X, Peng J, Chen Y, Kerketta R, Lee YH, Chang SH, Corry DB, Wang D, Watowich SS, Dong C. The signaling suppressor CIS controls proallergic T cell development and allergic airway inflammation. Nat Immunol. 2013; 14:732–740.

34. Knosp CA, Carroll HP, Elliott J, Saunders SP, Nel HJ, Amu S, Pratt JC, Spence S, Doran E, Cooke N, Jackson R, Swift J, Fitzgerald DC, Heaney LG, Fallon PG, Kissenpfennig A, Johnston JA. SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses. J Exp Med. 2011; 208:1523–1531.

35. Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010; 11:889–896.

36. Coffman RL. Origins of the T(H)1-T(H)2 model: a personal perspective. Nat Immunol. 2006; 7:539–541.

37. Sykes A, Edwards MR, Macintyre J, del Rosario A, Bakhsoliani E, Trujillo-Torralbo MB, Kon OM, Mallia P, McHale M, Johnston SL. Rhinovirus 16-induced IFN-alpha and IFN-beta are deficient in bronchoalveolar lavage cells in asthmatic patients. J Allergy Clin Immunol. 2012; 129:1506–1514.

38. Baraldo S, Contoli M, Bazzan E, Turato G, Padovani A, Marku B, Calabrese F, Caramori G, Ballarin A, Snijders D, Barbato A, Saetta M, Papi A. Deficient antiviral immune responses in childhood: distinct roles of atopy and asthma. J Allergy Clin Immunol. 2012; 130:1307–1314.

39. Huber JP, Ramos HJ, Gill MA, Farrar JD. Cutting edge: Type I IFN reverses human Th2 commitment and stability by suppressing GATA3. J Immunol. 2010; 185:813–817.