Korean J Physiol Pharmacol.  2021 Mar;25(2):111-118. 10.4196/kjpp.2021.25.2.111.

27-Hydroxycholesterol induces macrophage gene expression via LXR-dependent and -independent mechanisms

• Kim BY
• Son Y
• Cho Hr
• Lee D
• Eo SK
• Kim K

• KMID: 2512954
• DOI: http://doi.org/10.4196/kjpp.2021.25.2.111

Abstract

27-Hydroxycholesterol (27OHChol) exhibits agonistic activity for liver X receptors (LXRs). To determine roles of the LXR agonistic activity in macrophage gene expression, we investigated the effects of LXR inhibition on the 27OHChol-induced genes. Treatment of human THP-1 cells with GSK 2033, a potent cell-active LXR antagonist, results in complete inhibition in the transcription of LXR target genes (such as LXRα and ABCA1) induced by 27OHChol or a synthetic LXR ligand TO 901317. Whereas expression of CCL2 and CCL4 remains unaffected by GSK 2033, TNF-α expression is further induced and 27OHChol-induced CCL3 and CXCL8 genes are suppressed at both the transcriptional and protein translation levels in the presence of GSK 2033. This LXR antagonist downregulates transcript levels and surface expression of CD163 and CD206 and suppresses the transcription of CD14, CD80, and CD86 genes without downregulating their surface levels. GSK 2033 alone had no effect on the basal expression levels of the aforementioned genes. Collectively, these results indicate that LXR inhibition leads to differential regulation of 27-hydroxycholesterolinduced genes in macrophages. We propose that 27OHChol induces gene expression and modulates macrophage functions via LXR-dependent and -independent mechanisms.

Keyword

Gene expression; Liver X receptors; Macrophage; 27-Hydroxycholesterol

Figure

• 

  Fig. 1 Inhibitory effects of GSK 2033 on the transcription and protein translation of the LXRα and ABCA1 genes. THP-1 cells were serum-starved overnight and treated with GSK 2033 (1 μM) for 2 h. Cells were further stimulated with 27OHChol (2.5 μg/ml) or TO 901317 (1 μM) for 48 h in RPMI media supplemented with 10% FBS. The levels of LXRα (A) or ABCA1 (B) gene transcripts were assessed by real-time PCR. Data are expressed as the means ± standard deviation (n = 3 replicates for each group). LXRs, liver X receptors; FBS, fetal bovine serum. ***p < 0.001 vs. control; ###p < 0.001 vs. 27OHChol or TO 901317; #p < 0.05 vs. 27OHChol or TO 901317. (C) LXRα and ABCA1 proteins were detected by Western blot analysis. Data represent a representative experiment (from 3 independent experiments).

• 

  Fig. 2 Differential effects of GSK 2033 on 27OHChol-induced expression of C-C chemokines. After serum-starvation overnight, THP-1 cells were treated with GSK 2033 (1 μM) for 2 h and stimulated with 27OHChol (2.5 μg/ml) for 48 h in RPMI media supplemented with 10% FBS. (A) Levels of CCL2, CCL3, and CCL4 gene transcripts were assessed by real-time PCR. (B) The amounts of CCL2, CCL3, and CC4 proteins secreted in culture media were quantified by ELISA. Data are expressed as the means ± standard deviation (n = 3 replicates for each group). The results are representative of 3 independent experiments. FBS, fetal bovine serum. ***p < 0.001 vs. control; *p < 0.05 vs. control; ###p < 0.001 vs. 27OHChol.

• 

  Fig. 3 Opposite effects of GSK 2033 on CXCL8 and TNF-α expression. Serum-starved THP-1 cells were treated with GSK 2033 (1 μM) for 2 h and stimulated for 48 h with 27OHChol (2.5 μg/ml). (A) Transcript levels of the CXCL8 and TNF-α genes were assessed by real-time PCR. (B) The amounts of CXCL8 and TNF-α proteins secreted in culture media were quantified by ELISA. Data are expressed as the means ± standard deviation (n = 3 replicates for each group). The results are representative of 3 independent experiments. The results are representative of 3 independent experiments. ***p < 0.001 vs. control; ###p < 0.001 vs. 27OHChol.

• 

  Fig. 4 Suppressive effects of GSK 2033 on M2 surface makers. Following serum-starvation, THP-1 cells were treated with GSK 2033 (1 μM) for 2 h and stimulated with 27OHChol (2.5 μg/ml) for 48 h. (A) Transcript levels of the DC163 and CD206 genes were assessed by real-time PCR. Data are expressed as the means ± standard deviation (n = 3 replicates for each group). (B) After immunostaining of CD163 and CD206 with fluorophore-conjugated Abs, cells were analyzed by flow cytometry. The results are representative of 3 independent experiments. ***p < 0.001 vs. control; ### p < 0.001 vs. 27OHChol.

• 

  Fig. 5 Differential effects of GSK 2033 on expression of M1 surface markers. Serum-starved THP-1 cells were treated with GSK 2033 (1 μM) for 2 h, followed by stimulation with 27OHChol (2.5 μg/ml) for 48 h. (A) Transcript levels of the CD14, CD80, and CD86 genes were assessed by real-time PCR. Data are expressed as the means ± standard deviation (n = 3 replicates for each group). (B) After immunostaining of CD14, CD80, and CD86 with fluorophore-conjugated Abs, cells were analyzed by flow cytometry. The results are representative of 3 independent experiments. ***p < 0.001 vs. control; **p < 0.01 vs. control; *p < 0.05 vs. control; ###p < 0.001 vs. 27OHChol; #p < 0.05 vs. 27OHChol.

Reference

1. Schroepfer GJ Jr. 2000; Oxysterols: modulators of cholesterol metabolism and other processes. Physiol Rev. 80:361–554. DOI: 10.1152/physrev.2000.80.1.361. PMID: 10617772.

2. Brown AJ, Jessup W. 1999; Oxysterols and atherosclerosis. Atherosclerosis. 142:1–28. DOI: 10.1016/S0021-9150(98)00196-8. PMID: 9920502.

3. Iuliano L. 2011; Pathways of cholesterol oxidation via non-enzymatic mechanisms. Chem Phys Lipids. 164:457–468. DOI: 10.1016/j.chemphyslip.2011.06.006. PMID: 21703250.

4. Carpenter KL, Taylor SE, van der Veen C, Williamson BK, Ballantine JA, Mitchinson MJ. 1995; Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development. Biochim Biophys Acta. 1256:141–150. DOI: 10.1016/0005-2760(94)00247-V. PMID: 7766691.

5. Garcia-Cruset S, Carpenter KL, Guardiola F, Stein BK, Mitchinson MJ. 2001; Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions. Free Radic Res. 35:31–41. DOI: 10.1080/10715760100300571. PMID: 11697115.

6. Heo W, Kim SM, Eo SK, Rhim BY, Kim K. 2014; FSL-1, a Toll-like receptor 2/6 agonist, induces expression of interleukin-1α in the presence of 27-hydroxycholesterol. Korean J Physiol Pharmacol. 18:475–480. DOI: 10.4196/kjpp.2014.18.6.475. PMID: 25598661. PMCID: PMC4296036.

7. Kim SM, Lee SA, Kim BY, Bae SS, Eo SK, Kim K. 2013; 27-Hydroxycholesterol induces recruitment of monocytic cells by enhancing CCL2 production. Biochem Biophys Res Commun. 442:159–164. DOI: 10.1016/j.bbrc.2013.11.052. PMID: 24269812.

8. Lee J, Kim BY, Son Y, Giang DH, Lee D, Eo SK, Kim K. 2019; 4'-O-Methylalpinumisoflavone inhibits the activation of monocytes/macrophages to an immunostimulatory phenotype induced by 27-hydroxycholesterol. Int J Mol Med. 43:2177–2186. DOI: 10.3892/ijmm.2019.4135.

9. Son Y, Kim SM, Lee SA, Eo SK, Kim K. 2013; Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells. Biochem Biophys Res Commun. 438:161–168. DOI: 10.1016/j.bbrc.2013.07.046. PMID: 23876312.

10. Kim SM, Kim BY, Eo SK, Kim CD, Kim K. 2015; 27-Hydroxycholesterol up-regulates CD14 and predisposes monocytic cells to superproduction of CCL2 in response to lipopolysaccharide. Biochim Biophys Acta. 1852:442–450. DOI: 10.1016/j.bbadis.2014.12.003. PMID: 25497142.

11. Kim SM, Kim BY, Lee SA, Eo SK, Yun Y, Kim CD, Kim K. 2014; 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes. Toxicol Appl Pharmacol. 274:462–470. DOI: 10.1016/j.taap.2013.12.007. PMID: 24370436.

12. Fu X, Menke JG, Chen Y, Zhou G, MacNaul KL, Wright SD, Sparrow CP, Lund EG. 2001; 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J Biol Chem. 276:38378–38387. DOI: 10.1074/jbc.M105805200. PMID: 11504730.

13. Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ. 2006; Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell. 126:789–799. DOI: 10.1016/j.cell.2006.06.049. PMID: 16923397. PMCID: PMC6211849.

14. Ignatova ID, Angdisen J, Moran E, Schulman IG. 2013; Differential regulation of gene expression by LXRs in response to macrophage cholesterol loading. Mol Endocrinol. 27:1036–1047. DOI: 10.1210/me.2013-1051. PMID: 23686114. PMCID: PMC3706843.

15. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf DJ. 1995; LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev. 9:1033–1045. DOI: 10.1101/gad.9.9.1033. PMID: 7744246.

16. Zuercher WJ, Buckholz RG, Campobasso N, Collins JL, Galardi CM, Gampe RT, Hyatt SM, Merrihew SL, Moore JT, Oplinger JA, Reid PR, Spearing PK, Stanley TB, Stewart EL, Willson TM. 2010; Discovery of tertiary sulfonamides as potent liver X receptor antagonists. J Med Chem. 53:3412–3416. DOI: 10.1021/jm901797p. PMID: 20345102.

17. Kim SM, Jang H, Son Y, Lee SA, Bae SS, Park YC, Eo SK, Kim K. 2013; 27-hydroxycholesterol induces production of tumor necrosis factor-alpha from macrophages. Biochem Biophys Res Commun. 430:454–459. DOI: 10.1016/j.bbrc.2012.12.021. PMID: 23246833.

18. Hong C, Walczak R, Dhamko H, Bradley MN, Marathe C, Boyadjian R, Salazar JV, Tontonoz P. 2011; Constitutive activation of LXR in macrophages regulates metabolic and inflammatory gene expression: identification of ARL7 as a direct target. J Lipid Res. 52:531–539. DOI: 10.1194/jlr.M010686. PMID: 21187453. PMCID: PMC3035689.

19. Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. 2003; Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med. 9:213–219. DOI: 10.1038/nm820. PMID: 12524534.

20. Fowler AJ, Sheu MY, Schmuth M, Kao J, Fluhr JW, Rhein L, Collins JL, Willson TM, Mangelsdorf DJ, Elias PM, Feingold KR. 2003; Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models: liver-X-receptor-specific inhibition of inflammation and primary cytokine production. J Invest Dermatol. 120:246–255. DOI: 10.1046/j.1523-1747.2003.12033.x. PMID: 12542530.

21. Heine G, Dahten A, Hilt K, Ernst D, Milovanovic M, Hartmann B, Worm M. 2009; Liver X receptors control IgE expression in B cells. J Immunol. 182:5276–5282. DOI: 10.4049/jimmunol.0801804. PMID: 19380774.

22. Park MC, Kwon YJ, Chung SJ, Park YB, Lee SK. 2010; Liver X receptor agonist prevents the evolution of collagen-induced arthritis in mice. Rheumatology (Oxford). 49:882–890. DOI: 10.1093/rheumatology/keq007. PMID: 20159908.

23. Terasaka N, Hiroshima A, Koieyama T, Ubukata N, Morikawa Y, Nakai D, Inaba T. 2003; T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice. FEBS Lett. 536:6–11. DOI: 10.1016/S0014-5793(02)03578-0. PMID: 12586329.

24. Bruhn KW, Marathe C, Maretti-Mira AC, Nguyen H, Haskell J, Tran TA, Vanchinathan V, Gaur U, Wilson ME, Tontonoz P, Craft N. 2010; LXR deficiency confers increased protection against visceral Leishmania infection in mice. PLoS Negl Trop Dis. 4:e886. DOI: 10.1371/journal.pntd.0000886. PMID: 21103366. PMCID: PMC2982826.

25. Korf H, Vander Beken S, Romano M, Steffensen KR, Stijlemans B, Gustafsson JA, Grooten J, Huygen K. 2009; Liver X receptors contribute to the protective immune response against Mycobacterium tuberculosis in mice. J Clin Invest. 119:1626–1637. DOI: 10.1172/JCI35288. PMID: 19436111. PMCID: PMC2689129.

26. Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O'connell RM, Cheng G, Saez E, Miller JF, Tontonoz P. 2004; LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell. 119:299–309. DOI: 10.1016/j.cell.2004.09.032. PMID: 15479645.

27. Töröcsik D, Baráth M, Benko S, Széles L, Dezso B, Póliska S, Hegyi Z, Homolya L, Szatmári I, Lányi A, Nagy L. 2010; Activation of liver X receptor sensitizes human dendritic cells to inflammatory stimuli. J Immunol. 184:5456–5465. DOI: 10.4049/jimmunol.0902399. PMID: 20410489.

28. Zhou Y, Yoshida S, Kubo Y, Yoshimura T, Kobayashi Y, Nakama T, Yamaguchi M, Ishikawa K, Oshima Y, Ishibashi T. 2017; Different distributions of M1 and M2 macrophages in a mouse model of laser-induced choroidal neovascularization. Mol Med Rep. 15:3949–3956. DOI: 10.3892/mmr.2017.6491. PMID: 28440413. PMCID: PMC5436148.

29. Marengo B, Bellora F, Ricciarelli R, De Ciucis C, Furfaro A, Leardi R, Colla R, Pacini D, Traverso N, Moretta A, Pronzato MA, Bottino C, Domenicotti C. 2016; Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages. Biofactors. 42:80–92. DOI: 10.1002/biof.1243. PMID: 26669587.