J Korean Med Sci.  2023 Mar;38(12):e91. 10.3346/jkms.2023.38.e91.

Tiotropium Bromide Improves Neutrophilic Asthma by Recovering Histone Deacetylase 2 Activity

• An TJ
• Kim JH
• Hur J
• Park CK
• Lim JU
• Kim S
• Rhee CK
• Yoon HK

• KMID: 2541039
• DOI: http://doi.org/10.3346/jkms.2023.38.e91

Abstract

Background
The value of tiotropium bromide (TIO) in neutrophilic asthma was meaningful in previous study. We hypothesized that TIO’s mechanism of action is associated with histone deacetylase 2 (HDAC2) activity, which is key for controlling the transcription of inflammatory cytokines and usually downregulated in neutrophilic asthma.
Methods
The effects of TIO and dexamethasone (DEX) on HDAC2 activity, nuclear factor kappa B (NF-κB), and C-X-C motif chemokine ligand 1 (CXCL1) were evaluated in neutrophilic asthma mouse model (C57BL, 6-week-old). An in-vitro study was conducted using primary human bronchial/tracheal epithelial (HBE) cells from asthma patients. Western blot analyses were performed for phospho-phospholipase Cγ-1 (PLCγ-1) and inositol trisphosphate (IP₃ ) receptors (IP₃ R) with treating lipopolysaccharide (LPS) and TIO.
Results
Ovalbumin was used to induce eosinophilic inflammation in this study. After neutrophilic asthma was induced by LPS (O+L group), HDAC2 activity was diminished with increased NF-κB activity and CXCL1 compared to the control group. TIO significantly improved NF-κB activity, CXCL1, and HDAC2 activity compared with the O+L group in in-vivo study (P < 0.05, each). Western blot analyses showed that LPS treated HBE cells from asthma patients increased PLCγ-1 and diminished IP₃ receptor levels. After TIO treatment, recovery of IP₃ R and improved PLCγ-1 levels were observed.
Conclusion
These results support the hypothesis that TIO modulates inflammation by recovering HDAC2 activity from the acetylcholine-stimulated inflammation cascade in neutrophilic asthma. The detailed inflammation cascade of recovering HDAC2 activity by TIO might be associated with PLCγ-1-IP₃-IP₃R mediated intracellular calcium ion pathway.

Keyword

Asthma; Neutrophils; Tiotropium bromide; Histone Deacetylases 2

Figure

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  Fig. 1 Schematic flow of the acute neutrophilic asthma mouse model. The mice were sensitized with OVA on days 0 and 7. OVA was challenged intranasally on days 14, 15, 21, 22, and 23. Neutrophilic inflammation was induced by intranasal LPS which was administered on days 18, 21, and 23. Dexamethasone was intraperitoneally administered on days 14, 17, 20, and 23. Tiotropium bromide was inhaled on days 21, 22, and 23.OVA = ovalbumin, LPS = lipopolysaccharides.

• 

  Fig. 2 Schematic flow of in-vitro model for acute neutrophilic asthma. Primary human bronchial/tracheal epithelial Cell: Asthma (PCS-300-011) (ATCC, Manassas, VA, USA) was cultivated. After 24 hours of culture, Tiotropium bromide was administered to the culture medium (50 pM and 50 μM). After 1 hour, LPS (10 mg/mL) was stimulated for 5 minutes. After 48 hours, the cells were harvested for analyses.LPS = lipopolysaccharide.

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  Fig. 3 Measurement of nuclear factor kappa B activity and C-X-C motif chemokine ligand 1. Proinflammatory cytokines, such as NF-κB and CXCL1, were measured and compared among groups. Tests were conducted in four groups: control, O+L, O+L+D, and O+L+T. (A) The O+L group showed elevated NF-κB activity compared to that of the control (P < 0.001). The O+L+T group showed improvement in NF-κB activity compared to that of the O+L group (P < 0.001), which was not observed in the O+L+D group. (B) The BAL CXCL1 levels were higher in the O+L group than in the control group (P < 0.01). The O+L+T group showed lower BAL CXCL1 levels than those observed in the O+L group (P < 0.05), which was not observed in the O+L+D group. O+L vs. all (*P < 0.05, **P < 0.01, ***P < 0.001).O = ovalbumin, L = lipopolysaccharides, D = dexamethasone, T = Tiotropium bromide, NF-κB = nuclear factor kappa B, CXCL1 = C-X-C motif chemokine ligand 1, BAL = bronchoalveolar lavage.

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  Fig. 4 Measurement of histone deacetylase 2 activity. HDAC2 activity was measured. The tests were conducted in four groups: control, O+L, O+L+D, and O+L+T. It was decreased in the O+L group compared to that in the control group (P < 0.001). This did not improve after dexamethasone treatment. Unlike dexamethasone, Tiotropium bromide recovered HDAC2 activity compared to that of the O+L group (P < 0.01). O+L vs. all (**P < 0.01, ***P < 0.001).HDAC2 = histone deacetylase 2, O = ovalbumin, L = lipopolysaccharides, D = dexamethasone, T = Tiotropium bromide.

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  Fig. 5 Western blot analysis for a phospho-phospholipase Cγ-1 and inositol trisphosphate receptor. We used western blot analyses (A) to detect the levels of p-PLCγ-1 (B) and IP3R (C). These experiments were conducted in four groups: control, LPS, LPS-TIO (50 pM), and LPS-TIO (50 μM). Band intensity was quantified with image software (Multi Gauge, Version 3.0, Fujifilm Life Science, Tokyo, Japan). The p-PLCγ-1 expression ratio was increased and the IP3R expression ratio was reduced in the LPS group. Along with the increase in TIO concentration from 50 pM to 50 μM, those ratios were relatively recovered as in the control group.p-PLCγ-1 = phospho-phospholipase Cγ-1, IP3R = inositol trisphosphate receptor, LPS = lipopolysaccharide, TIO = Tiotropium bromide.

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  Fig. 6 Proposal of possible mechanism of p-PLCγ-1-IP3-IP3R pathway controlled by TIO in neutrophilic asthma. ACh induces airway inflammation by attaching to its receptor in bronchial epithelial cells. It triggers phosphorylation of PLCγ-1, which leads to an increased level of IP3. It binds to IP3R and IP3-favor calcium channel in the ER. Ionized calcium ions are depleted in the ER. Then, HDAC2 is deactivated by phosphorylation, and NF-κB transcription is triggered in the nucleus. TIO blocks the ACh receptor, which is the highest level of controlling inflammation in neutrophilic asthma. It is mediated by the p-PLCγ-1-IP3-IP3R pathway.ACh = Acetylcholine, PLCγ-1 = phospholipase Cγ-1, IP3 = inositol trisphosphate, IP3R = inositol trisphosphate receptor, ER = endoplasmic reticulum, HDAC2 = histone deacetylase 2, NF-κB = nuclear factor kappa B, TIO = Tiotropium bromide.

Reference

1. Gibson PG, Foster PS. Neutrophilic asthma: welcome back! Eur Respir J. 2019; 54(5):1901846. PMID: 31699782.

2. Ray A, Kolls JK. Neutrophilic inflammation in asthma and association with disease severity. Trends Immunol. 2017; 38(12):942–954. PMID: 28784414.

3. Kang N, Song WJ. Discovering biomarkers of neutrophilic asthma: a clinician’s perspective. Allergy Asthma Immunol Res. 2022; 14(1):1–4. PMID: 34983102.

4. Bullone M, Carriero V, Bertolini F, Folino A, Mannelli A, Di Stefano A, et al. Elevated serum IgE, oral corticosteroid dependence and IL-17/22 expression in highly neutrophilic asthma. Eur Respir J. 2019; 54(5):1900068. PMID: 31439682.

5. An TJ, Rhee CK, Kim JH, Lee YR, Chon JY, Park CK, et al. Effects of macrolide and corticosteroid in neutrophilic asthma mouse model. Tuberc Respir Dis (Seoul). 2018; 81(1):80–87. PMID: 29332324.

6. Kobayashi Y, Bossley C, Gupta A, Akashi K, Tsartsali L, Mercado N, et al. Passive smoking impairs histone deacetylase-2 in children with severe asthma. Chest. 2014; 145(2):305–312. PMID: 24030221.

7. Gunawardhana LP, Gibson PG, Simpson JL, Powell H, Baines KJ. Activity and expression of histone acetylases and deacetylases in inflammatory phenotypes of asthma. Clin Exp Allergy. 2014; 44(1):47–57. PMID: 24355018.

8. Lai T, Wu M, Zhang C, Che L, Xu F, Wang Y, et al. HDAC2 attenuates airway inflammation by suppressing IL-17A production in HDM-challenged mice. Am J Physiol Lung Cell Mol Physiol. 2019; 316(1):L269–L279. PMID: 30407865.

9. An TJ, Kim JH, Park CK, Yoon HK. Tiotropium bromide has a more potent effect than corticosteroid in the acute neutrophilic asthma mouse model. Tuberc Respir Dis (Seoul). 2022; 85(1):18–24. PMID: 34727490.

10. Hamelmann E, Vogelberg C, Szefler SJ. Tiotropium for the treatment of asthma in adolescents. Expert Opin Pharmacother. 2017; 18(3):305–312. PMID: 28110558.

11. Reddel HK, Bacharier LB, Bateman ED, Brightling CE, Brusselle GG, Buhl R, et al. Global Initiative for Asthma Strategy 2021: executive summary and rationale for key changes. Eur Respir J. 2021; 59(1):2102730. PMID: 34667060.

12. Anzalone G, Gagliardo R, Bucchieri F, Albano GD, Siena L, Montalbano AM, et al. IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: effect of Tiotropium. Life Sci. 2016; 152:107–116. PMID: 27038884.

13. Wollin L, Pieper MP. Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD. Pulm Pharmacol Ther. 2010; 23(4):345–354. PMID: 20362689.

14. Park CK, An TJ, Kim JH, Rhee CK, Yoon HK. Synergistic effect of roflumilast with dexamethasone in a neutrophilic asthma mouse model. Clin Exp Pharmacol Physiol. 2022; 49(6):624–632. PMID: 35181901.

15. Holownia A, Mroz RM, Skopinski T, Kielek A, Kolodziejczyk A, Chyczewska E, et al. Tiotropium increases cytosolic muscarinic M3 receptors and acetylated H3 histone proteins in induced sputum cells of COPD patients. Eur J Med Res. 2010; 15(Suppl 2):64–67. PMID: 21147623.

16. Neri T, Scalise V, Passalacqua I, Sanguinetti C, Lombardi S, Pergoli L, et al. Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells. Sci Rep. 2019; 9(1):11631. PMID: 31406171.

17. Berridge MJ. The inositol trisphosphate/calcium signaling pathway in health and disease. Physiol Rev. 2016; 96(4):1261–1296. PMID: 27512009.

18. Kania E, Roest G, Vervliet T, Parys JB, Bultynck G. IP₃ receptor-mediated calcium signaling and its role in autophagy in cancer. Front Oncol. 2017; 7:140. PMID: 28725634.

19. Kalchiem-Dekel O, Yao X, Levine SJ. Meeting the challenge of identifying new treatments for type 2-low neutrophilic asthma. Chest. 2020; 157(1):26–33. PMID: 31525357.

20. Nair P, Surette MG, Virchow JC. Neutrophilic asthma: misconception or misnomer? Lancet Respir Med. 2021; 9(5):441–443. PMID: 33577751.

21. Wang M, Gao P, Wu X, Chen Y, Feng Y, Yang Q, et al. Impaired anti-inflammatory action of glucocorticoid in neutrophil from patients with steroid-resistant asthma. Respir Res. 2016; 17(1):153. PMID: 27852250.

22. Global Strategy for Asthma Management and Prevention. Updated 2023. Accessed March 4, 2023. www.ginasthma.org .

23. Brusselle GG, Vanderstichele C, Jordens P, Deman R, Slabbynck H, Ringoet V, et al. Azithromycin for prevention of exacerbations in severe asthma (AZISAST): a multicentre randomised double-blind placebo-controlled trial. Thorax. 2013; 68(4):322–329. PMID: 23291349.

24. Gibson PG, Yang IA, Upham JW, Reynolds PN, Hodge S, James AL, et al. Efficacy of azithromycin in severe asthma from the AMAZES randomised trial. ERJ Open Res. 2019; 5(4):00056-2019.

25. Higham A, Cadden P, Southworth T, Rossall M, Kolsum U, Lea S, et al. Leukotriene B4 levels in sputum from asthma patients. ERJ Open Res. 2016; 2(4):00088-2015. PMID: 28053970.

26. Bhatt L, Roinestad K, Van T, Springman EB. Recent advances in clinical development of leukotriene B4 pathway drugs. Semin Immunol. 2017; 33:65–73. PMID: 29042030.

27. Wechsler ME, Laviolette M, Rubin AS, Fiterman J, Lapa e Silva JR, Shah PL, et al. Bronchial thermoplasty: long-term safety and effectiveness in patients with severe persistent asthma. J Allergy Clin Immunol. 2013; 132(6):1295–1302. PMID: 23998657.

28. Barnes PJ. Similarities and differences in inflammatory mechanisms of asthma and COPD. Breathe. 2011; 7(3):229–238.

29. Barnes PJ. Histone deacetylase-2 and airway disease. Ther Adv Respir Dis. 2009; 3(5):235–243. PMID: 19812111.

30. Butler CA, McQuaid S, Taggart CC, Weldon S, Carter R, Skibinski G, et al. Glucocorticoid receptor β and histone deacetylase 1 and 2 expression in the airways of severe asthma. Thorax. 2012; 67(5):392–398. PMID: 22156779.

31. Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, Barnes PJ, et al. Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression. J Exp Med. 2006; 203(1):7–13. PMID: 16380507.

32. Pelaia G, Vatrella A, Busceti MT, Gallelli L, Calabrese C, Terracciano R, et al. Pharmacologic rationale underlying the therapeutic effects of tiotropium/olodaterol in COPD. Ther Clin Risk Manag. 2015; 11:1563–1572. PMID: 26504398.

33. Cassambai S, Mee CJ, Renshaw D, Hussain A. Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart. Toxicol Appl Pharmacol. 2019; 384:114778. PMID: 31618660.

34. Saleem H, Tovey SC, Riley AM, Potter BV, Taylor CW. Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues. PLoS One. 2013; 8(2):e58027. PMID: 23469136.