Immune Netw.  2015 Dec;15(6):269-277. 10.4110/in.2015.15.6.269.

Withaferin A Inhibits Helicobacter pylori-induced Production of IL-1beta in Dendritic Cells by Regulating NF-kappaB and NLRP3 Inflammasome Activation

• Kim JE
• Lee JY
• Kang MJ
• Jeong YJ
• Choi JA
• Oh SM
• Lee KB
• Park JH

• KMID: 2132701
• DOI: http://doi.org/10.4110/in.2015.15.6.269

Abstract

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. There is evidence that IL-1beta is associated with the development of gastric cancer. Therefore, downregulation of H. pylori-mediated IL-1beta production may be a way to prevent gastric cancer. Withaferin A (WA), a withanolide purified from Withania somnifera, is known to exert anti-inflammatory and anti-tumor effects. In the present study, we explored the inhibitory activity of WA on H. pylori-induced production of IL-1beta in murine bone marrow-derived dendritic cells (BMDCs) and the underlying cellular mechanism. Co-treatment with WA decreased IL-1beta production by H. pylori in BMDCs in a dose-dependent manner. H. pylori-induced gene expression of IL-1beta and NLRP3 (NOD-like receptor family, pyrin domain containing 3) were also suppressed by WA treatment. Moreover, IkappaB-alpha phosphorylation by H. pylori infection was suppressed by WA in BMDCs. Western blot analysis revealed that H. pylori induced cleavage of caspase-1 and IL-1beta, as well as increased procaspase-1 and pro IL-1beta protein levels, and that both were suppressed by co-treatment with WA. Finally, we determined whether WA can directly inhibit ac tivation of the NLRP3 inflammasome. NLRP3 activators induced IL-1beta secretion in LPS-primed macrophages, which was inhibited by WA in a dose-dependent manner, whereas IL-6 production was not affected by WA. Moreover, cleavage of IL-1beta and caspase-1 by NLRP3 activators was also dose-dependently inhibited by WA. These findings suggest that WA can inhibit IL-1beta production by H. pylori in dendritic cells and can be used as a new preventive and therapeutic agent for gastric cancer.

Keyword

Dendritic cells; Helicobacter pylori; Interleukin 1beta; NLRP3 inflammasome; Withaferin A

MeSH Terms

Blotting, Western
Caspase 1
Dendritic Cells*
Down-Regulation
Gastritis
Gene Expression
Helicobacter pylori
Helicobacter*
Humans
Interleukin-1beta
Interleukin-6
Macrophages
NF-kappa B*
Peptic Ulcer
Phosphorylation
Stomach Neoplasms
Withania
Caspase 1
Interleukin-1beta
Interleukin-6
NF-kappa B

Figure

• 

  Figure 1 WA down-regulates H. pylori-mediated IL-1β production and related signaling in immune cells. (A and B) BMDCs were infected with H. pylori at the indicated MOIs (50 in B) for 18 h in the absence or presence of the indicated doses of WA (500 nM in A). The IL-1β levels in culture supernatants were measured by ELISA. (C and D) BMDCs were co-treated with H. pylori (MOI 50) and 500 nM WA for the indicated times, and IL-1β and NLRP3 mRNA expression levels were evaluated by real-time PCR. (E) BMDCs were infected with H. pylori (MOI 50) in the absence or presence of 500 nM of WA or 20 µM of Bay 11 7082. Cellular proteins were harvested at the indicated time points, and the amounts of regular and phospho-form IκB-α and β-actin were determined by western blotting. (F) BMDCs were infected with H. pylori (MOI 50) and treated with various doses of Bay 11-7082 or left untreated for 18 h, and then examined by ELISA. (G) THP-1 cells were also infected with H. pylori at MOI 50 for 18 h in the absence or presence of the indicated doses of WA. The IL-1β levels in culture supernatants were measured by ELISA. Data are shown as the mean±SD of triplicate samples from one representative experiment of three independent experiments (*p<0.5, **p<0.01, and ***p<0.001).

• 

  Figure 2 WA reduces the levels of pro and cleaved forms of caspase-1 and IL-1β induced by H. pylori in BMDCs. BMDCs were treated with LPS (100 ng/mL), ATP (5 mM), H. pylori (MOI 50), and WA (500 nM), individually or in combination, as indicated. Pro and cleaved forms of caspase-1 and IL-1β were detected by western blot analysis. β-actin was used as a control for the loading volume.

• 

  Figure 3 WA inhibits the production of IL-1β, but not IL-6, by ATP, nigericin, and monosodium urate crystals in BMDMs. BMDMs were primed with LPS (1 µg/mL) for 6 h and subsequently treated with indicated doses of WA or Bay 11-7082 (10 µM). The cells were further incubated with ATP (2 mM) and nigericin (10 µM) for 40 min, or with MSU (200 µg/mL) for 4 h. The levels of IL-1β (A-C) and IL-6 (D-F) in culture supernatants were determined by ELISA.

• 

  Figure 4 Caspase-1 activation and IL-1β maturation by NLRP3 activators are suppressed by WA in BMDMs. BMDMs were primed with LPS (1 µg/mL) for 6 h and subsequently treated with the indicated doses of WA or Bay 11-7082 (10 µM). The cells were then incubated with ATP (2 mM) and nigericin (10 µM) for 40 min, or with MSU (200 µg/mL) for 4 h. Pro and cleaved forms of caspase-1 and IL-1β (A-C) was detected by western blot analysis. β-actin was used as a control for the loading volume.

Reference

1. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med. 2002; 347:1175–1186.

2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61:69–90.

3. Fucci L, Eusebi LH, Bazzoli F. Gastric cancer, Helicobacter pylori infection and other risk factors. World J Gastrointest Oncol. 2010; 2:342–347.

4. Crabtree JE. Gastric mucosal inflammatory responses to Helicobacter pylori. Aliment Pharmacol Ther. 1996; 10:Suppl 1. 29–37.

5. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood. 1996; 87:2095–2147.

6. Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J. 1994; 8:1314–1325.

7. Leemans JC, Cassel SL, Sutterwala FS. Sensing damage by the NLRP3 inflammasome. Immunol Rev. 2011; 243:152–162.

8. Yang J, Hu Z, Xu Y, Shen J, Niu J, Hu X, Guo J, Wei Q, Wang X, Shen H. Interleukin-1B gene promoter variants are associated with an increased risk of gastric cancer in a Chinese population. Cancer Lett. 2004; 215:191–198.

9. Palli D, Saieva C, Luzzi I, Masala G, Topa S, Sera F, Gemma S, Zanna I, D'Errico M, Zini E, Guidotti S, Valeri A, Fabbrucci P, Moretti R, Testai E, del GG, Ottini L, Matullo G, Dogliotti E, Gomez-Miguel MJ. Interleukin-1 gene polymorphisms and gastric cancer risk in a high-risk Italian population. Am J Gastroenterol. 2005; 100:1941–1948.

10. Kumar S, Kumar A, Dixit VK. Evidences showing association of interleukin-1B polymorphisms with increased risk of gastric cancer in an Indian population. Biochem Biophys Res Commun. 2009; 387:456–460.

11. Tu S, Bhagat G, Cui G, Takaishi S, Kurt-Jones EA, Rickman B, Betz KS, Penz-esterreicher M, Bjorkdahl O, Fox JG, Wang TC. Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice. Cancer Cell. 2008; 14:408–419.

12. Shigematsu Y, Niwa T, Rehnberg E, Toyoda T, Yoshida S, Mori A, Wakabayashi M, Iwakura Y, Ichinose M, Kim YJ, Ushijima T. Interleukin-1beta induced by Helicobacter pylori infection enhances mouse gastric carcinogenesis. Cancer Lett. 2013; 340:141–147.

13. Tschopp J, Schroder K. NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? Nat Rev Immunol. 2010; 10:210–215.

14. Hitzler I, Sayi A, Kohler E, Engler DB, Koch KN, Hardt WD, Muller A. Caspase-1 has both proinflammatory and regulatory properties in Helicobacter infections, which are differentially mediated by its substrates IL-1beta and IL-18. J Immunol. 2012; 188:3594–3602.

15. Semper RP, Mejias-Luque R, Gross C, Anderl F, Muller A, Vieth M, Busch DH, Prazeres da CC, Ruland J, Gross O, Gerhard M. Helicobacter pylori-induced IL-1beta secretion in innate immune cells is regulated by the NLRP3 inflammasome and requires the cag pathogenicity island. J Immunol. 2014; 193:3566–3576.

16. Kim DJ, Park JH, Franchi L, Backert S, Nunez G. The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL-1beta production in Helicobacter pylori infected dendritic cells. Eur J Immunol. 2013; 43:2650–2658.

17. Maitra R, Porter MA, Huang S, Gilmour BP. Inhibition of NFkappaB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation. J Inflamm (Lond). 2009; 6:15.

18. Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, Zhang L, Hanson AD, Conner BP, Rougas J, Pribluda VS. Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis. 2004; 7:115–122.

19. Vyas AR, Singh SV. Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. AAPS J. 2014; 16:1–10.

20. Hahm ER, Singh SV. Withaferin A-induced apoptosis in human breast cancer cells is associated with suppression of inhibitor of apoptosis family protein expression. Cancer Lett. 2013; 334:101–108.

21. Oh JH, Lee TJ, Park JW, Kwon TK. Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 2647 cells. . Eur J Pharmacol. 2008; 599:11–17.

22. Celada A, Gray PW, Rinderknecht E, Schreiber RD. Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J Exp Med. 1984; 160:55–74.

23. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999; 223:77–92.

24. Vanden BW, Sabbe L, Kaileh M, Haegeman G, Heyninck K. Molecular insight in the multifunctional activities of Withaferin A. Biochem Pharmacol. 2012; 84:1282–1291.

25. Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 2001; 107:7–11.

26. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013; 13:397–411.

27. Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Memet S, Huerre MR, Coyle AJ, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol. 2004; 5:1166–1174.

28. Kang MJ, Song EJ, Kim BY, Kim DJ, Park JH. Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1alpha-dependent pathway. Helicobacter. 2014; 19:476–483.

29. He Y, Franchi L, Nunez G. TLR agonists stimulate Nlrp3-dependent IL-1beta production independently of the purinergic P2X7 receptor in dendritic cells and in vivo. J Immunol. 2013; 190:334–339.

30. Juliana C, Fernandes-Alnemri T, Wu J, Datta P, Solorzano L, Yu JW, Meng R, Quong AA, Latz E, Scott CP, Alnemri ES. Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome. J Biol Chem. 2010; 285:9792–9802.

31. El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature. 2000; 404:398–402.

32. Taguchi A, Ohmiya N, Shirai K, Mabuchi N, Itoh A, Hirooka Y, Niwa Y, Goto H. Interleukin-8 promoter polymorphism increases the risk of atrophic gastritis and gastric cancer in Japan. Cancer Epidemiol Biomarkers Prev. 2005; 14:2487–2493.

33. Hamajima N, Naito M, Kondo T, Goto Y. Genetic factors involved in the development of Helicobacter pylori-related gastric cancer. Cancer Sci. 2006; 97:1129–1138.

34. Kai H, Kitadai Y, Kodama M, Cho S, Kuroda T, Ito M, Tanaka S, Ohmoto Y, Chayama K. Involvement of proinflammatory cytokines IL-1beta and IL-6 in progression of human gastric carcinoma. Anticancer Res. 2005; 25:709–713.

35. Rad R, Brenner L, Krug A, Voland P, Mages J, Lang R, Schwendy S, Reindl W, Dossumbekova A, Ballhorn W, Wagner H, Schmid RM, Bauer S, Prinz C. Toll-like receptor-dependent activation of antigen-presenting cells affects adaptive immunity to Helicobacter pylori. Gastroenterology. 2007; 133:150–163.

36. Necchi V, Manca R, Ricci V, Solcia E. Evidence for transepithelial dendritic cells in human H. pylori active gastritis. Helicobacter. 2009; 14:208–222.

37. Rad R, Ballhorn W, Voland P, Eisenacher K, Mages J, Rad L, Ferstl R, Lang R, Wagner H, Schmid RM, Bauer S, Prinz C, Kirschning CJ, Krug A. Extracellular and intracellular pattern recognition receptors cooperate in the recognition of Helicobacter pylori. Gastroenterology. 2009; 136:2247–2257.