Chloroquine regulates the lipopolysaccharide-induced inflammatory response in RAW264.7 cells

Main Article Content

Natsuki Ota
Shoya Endo
Kouki Honma
Kuninori Iwayama
Hiroshi Yamashita
Ryosuke Tatsunami
Keisuke Sato

Keywords

autophagy, chloroquine, inflammatory cytokine

Abstract

Introduction and objectives: Macrophage-induced inflammation plays a key role in defense against injury and harmful pathogens. Autophagy and the inflammatory response are associated; however, the relationship between the autophagy pathway and lipopolysaccharide (LPS)- induced inflammatory responses remains unknown. We aimed to determine the effect of autophagy on the LPS-induced myeloid differentiation factor 88 (MyD88)/nuclear transcription factor kB (NF-kB) pathway-mediated inflammatory response in RAW264.7 cells.


Materials and Methods: To determine the effect of autophagy on the LPS-induced inflammatory response, using various in vitro assays, we determined the effect of autophagy inhibitors and inducers on the inflammatory response in RAW264.7 cells.


Results: Chloroquine (CQ), an autophagy inhibitor, suppressed pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor α (TNFα) in LPS-stimulated RAW264.7 cells. CQ also affected inflammatory mediators such as myeloid differentiation factor 88 and NF-kB in LPS-stimulated RAW264.7 cells.


Conclusion: This study demonstrated that CQ regulates the LPS-induced inflammatory response in RAW264.7 cells. We propose that targeting the regulation of pro-inflammatory cytokine levels and inflammatory mediators using CQ is a promising therapeutic approach for preventing inflammatory injury. CQ serves as a potential therapeutic target for treating various inflammatory diseases.

Abstract 100 | PDF Downloads 141 HTML Downloads 0 XML Downloads 3

References

1. Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 2005;4(3):281-6. 10.2174/1568010054022024

2. Zhang X, Xue C, Xu Q, Zhang Y, Li H, Li F, Liu Y, Guo C. Caprylic acid suppresses inflammation via TLR4/NF-κB signaling and improves atherosclerosis in ApoE-deficient mice. Nutr Metab. 2019;16:40. 10.1186/s12986-019-0359-2

3. Mazgaeen L, Gurung P. Recent advances in lipopolysaccharide recognition systems. Int J Mol Sci. 2020;21(2):379. 10.3390/ijms21020379

4. Negishi H, Fujita Y, Yanai H, Sakaguchi S, Ouyang X, Shinohara M, Takayanagi H, Ohba Y, Taniguchi T, Honda K. Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor--dependent gene induction program. Proc Natl Acad Sci USA. 2006;103(41):15136-41. 10.1073/pnas.0607181103

5. Sharif O, Bolshakov VN, Raines S, Newham P, Perkins ND. Transcriptional profiling of the LPS-induced NF-kappaB response in macrophages. BMC Immunol. 2007;8:1. 10.1186/1471-2172-8-1

6. Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2:17023. 10.1038/sigtrans.2017.23

7. Kany S, Vollrath JT, Relja B. Cytokines in inflammatory disease. Int J Mol Sci. 2019;20(23):6008. 10.3390/ijms20236008

8. Elinav E, Nowarski R, Thaiss CA, Hu B, Jin C, Flavell RA. Inflammation-induced cancer: crosstalk between tumors, immune cells, and microorganisms. Nat Rev Cancer. 2013;13(11):759-71. 10.1038/nrc3611

9. Donath MY. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat Rev Drug Discov. 2014;13(6):465-76. 10.1038/nrd4275

10. Yang Y, Klionsky DJ. Autophagy and disease: unanswered questions. Cell Death Differ. 2020;27(3):858-871. 10.1038/s41418-019-0480-9

11. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature. 2011;20;469(7330):323-35. 10.1038/nature09782

12. Deretic V, Levine B. Autophagy balances inflammation in innate immunity. Autophagy. 2018;14(2):243-251. 10.1080/15548627.2017.1402992

13. Cao Y, Chen J, Ren G, Zhang Y, Tan X, Yang L. Punicalagin Prevents Inflammation in LPS-induced RAW264.7 macrophages by inhibiting FoxO3a/autophagy signaling pathway. Nutrients. 2019;11(11):2794. 10.3390/nu11112794

14. Sakai J, Cammarota E, Wright JA, Cicuta P, Gottschalk RA, Li N, Fraser IDC, Bryant CE. Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα expression requires TRIF and MyD88. Sci Rep. 2017;7(1):1428. 10.1038/s41598-017-01600-y

15. Medzhitov R. Inflammation 2010: new adventures of an old flame. Cell. 2010;140(6):771-6. 10.1016/j.cell.2010.03.006

16. Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol. 2007;147(2):227-35. 10.1111/j.1365-2249.2006.03261.x

17. Chen L, Deng H, Cui H, Fang J, Zuo Z, Deng J, Li Y, Wang X, Zhao L. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget. 2017 Dec 14;9(6):7204-7218. 10.18632/oncotarget.23208

18. Shin C, Ito Y, Ichikawa S, Tokunaga M, Sakata-Sogawa K, Tanaka T. MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses. Sci Rep. 2017;7:46097. 10.1038/srep46097

19. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013;496(7446):445-55. 10.1038/nature12034

20. Hirayama D, Iida T, Nakase H. The Phagocytic Function of Macrophage-Enforcing Innate Immunity and Tissue Homeostasis. Int J Mol Sci. 2017;19(1):92. 10.3390/ijms19010092

21. Crayne CB, Albeituni S, Nichols KE, Cron RQ. The Immunology of Macrophage Activation Syndrome. Front Immunol. 2019;10:119. 10.3389/fimmu.2019.00119

22. Otsuka R, Seino KI. Macrophage activation syndrome and COVID-19. Inflamm Regen. 2020;40:19. 10.1186/s41232-020-00131-w

23. Lerkvaleekul B, Vilaiyuk S. Macrophage activation syndrome: early diagnosis is key. Open Access Rheumatol. 2018;10:117-128. 10.2147/OARRR.S151013

24. Carpino G, Nobili V, Renzi A, De Stefanis C, Stronati L, Franchitto A, Alisi A, Onori P, De Vito R, Alpini G, Gaudio E. Macrophage Activation in Pediatric Nonalcoholic Fatty Liver Disease (NAFLD) Correlates with Hepatic Progenitor Cell Response via Wnt3a Pathway. PLoS One. 2016;11(6):e0157246. 10.1371/journal.pone.0157246

25. Fairweather D, Rose NR. Inflammatory heart disease: a role for cytokines. Lupus. 2005;14(9):646-51. 10.1191/0961203305lu2192oa

26. Porcherie A, Cunha P, Trotereau A, Roussel P, Gilbert FB, Rainard P, et al. Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Vet Res. 2012;43(1):14. 10.1186/1297-9716-43-14

27. Sacks D, Baxter B, Campbell BCV, Carpenter JS, Cognard C, Dippel D, et al. Multisociety consensus quality improvement revised consensus statement for endovascular therapy of acute ischemic stroke. Int J Stroke. 2018;13(6):612-32. 10.1177/1747493018778713

28. Taciak B, Białasek M, Braniewska A, Sas Z, Sawicka P, Kiraga Ł, Rygiel T, Król M. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLoS One. 2018;13(6):e0198943. 10.1371/journal.pone.0198943

29. Elisia I, Pae HB, Lam V, Cederberg R, Hofs E, Krystal G. Comparison of RAW264.7, human whole blood and PBMC assays to screen for immunomodulators. J Immunol Methods. 2018;452:26-31. 10.1016/j.jim.2017.10.004

30. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009;1(6):a001651. 10.1101/cshperspect.a001651

31. Shao J, Li Y, Wang Z, Xiao M, Yin P, Lu Y, et al. 7b, a novel naphthalimide derivative, exhibited anti-inflammatory effects via targeted-inhibiting TAK1 following down-regulation of ERK1/2-and p38 MAPK-mediated activation of NF-κB in LPS-stimulated RAW264.7 macrophages. Int Immunopharmacol. 2013;17(2):216-28. 10.1016/j.intimp.2013.06.00

32. Sugiyama K, Muroi M, Kinoshita M, Hamada O, Minai Y, Sugita-Konishi Y, Kamata Y, Tanamoto K. NF-κB activation via MyD88-dependent Toll-like receptor signaling is inhibited by trichothecene mycotoxin deoxynivalenol. J Toxicol Sci. 2016;41(2):273-9. 10.2131/jts.41.273

33. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO, Goode J, Lin P, Mann N, Mudd S, Crozat K, Sovath S, Han J, Beutler B. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature. 2003;424(6950):743-8. 10.1038/nature01889

34. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science. 2003;301(5633):640-3. 10.1126/science.1087262

35. Sugiyama K, Muroi M, Tanamoto K. A novel TLR4-binding peptide that inhibits LPS-induced activation of NF-kappaB and in vivo toxicity. Eur J Pharmacol. 2008;594(1-3):152-6. 10.1016/j.ejphar.2008.07.037

36. Kitchens RL, Ulevitch RJ, Munford RS. Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway. J Exp Med. 1992;176(2):485-94. 10.1084/jem.176.2.485

37. Drion CM, van Scheppingen J, Arena A, Geijtenbeek KW, Kooijman L, van Vliet EA, Aronica E, Gorter JA. Effects of rapamycin and curcumin on inflammation and oxidative stress in vitro and in vivo-in search of potential anti-epileptogenic strategies for temporal lobe epilepsy. J Neuroinflammation. 2018;15(1):212. 10.1186/s12974-018-1247-9

38. Weichhart T, Haidinger M, Katholnig K, Kopecky C, Poglitsch M, Lassnig C, Rosner M, Zlabinger GJ, Hengstschläger M, Müller M, Hörl WH, Säemann MD. Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells. Blood. 2011;117(16):4273-83. 10.1182/blood-2010-09-310888

39. Rainsford KD, Parke AL, Clifford-Rashotte M, Kean WF. Therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases. Inflammopharmacology. 2015;23(5):231-69. 10.1007/s10787-015-0239-y

40. Shippey EA, Wagler VD, Collamer AN. Hydroxychloroquine: An old drug with new relevance. Cleve Clin J Med. 2018;85(6):459-467. 10.3949/ccjm.85a.17034