Tannic acid prevents macrophage-induced pro-fibrotic response in lung epithelial cells via suppressing TLR4-mediated macrophage polarization

  • Ayyanar Sivanantham
  • Dhamotharan Pattarayan
  • Nandhine Rajasekar
  • Adithi Kannan
  • Lakshmanan Loganathan
  • Ramalingam Bethunaickan
  • Santanu Kar Mahapatra
  • Rajaguru Palanichamy
  • Karthikeyan Muthusamy
  • Subbiah RajasekaranEmail author
Original Research Paper



Polarized macrophages induce fibrosis through multiple mechanisms, including a process termed epithelial-to-mesenchymal transition (EMT). Mesenchymal cells contribute to the excessive accumulation of fibrous connective tissues, leading to organ failure. This study was aimed to investigate the effect of tannic acid (TA), a natural dietary polyphenol on M1 macrophage-induced EMT and its underlying mechanisms.


First, we induced M1 polarization in macrophage cell lines (RAW 264.7 and THP-1). Then, the conditioned-medium (CM) from these polarized macrophages was used to induce EMT in the human adenocarcinomic alveolar epithelial (A549) cells. We also analysed the role of TA on macrophage polarization.


We found that TA pre-treated CM did not induce EMT in epithelial cells. Further, TA pre-treated CM showed diminished activation of MAPK in epithelial cells. Subsequently, TA was shown to inhibit LPS-induced M1 polarization in macrophages by directly targeting toll-like receptor 4 (TLR4), thereby repressing LPS binding to TLR4/MD2 complex and subsequent signal transduction.


It was concluded that TA prevented M1 macrophage-induced EMT by suppressing the macrophage polarization possibly through inhibiting the formation of LPS-TLR4/MD2 complex and blockage of subsequent downstream signal activation. Further, our findings may provide beneficial information to develop new therapeutic strategies against chronic inflammatory diseases.


EMT LPS M1 macrophages Mesenchymal cells Tannic acid TLR4 



This work was supported by Ramalingaswami re-entry fellowship (BT/RLF/Re-entry/36/2013). S. R. is the recipient of Ramalingaswami re-entry fellowship from the Department of Biotechnology (DBT), Government of India. This study was also supported in part by the Department of Science and Technology (DST; Award No: YSS/2014/000125) (to S. R.), Government of India. The first author (A. S.) gratefully acknowledges the support of Indian Council of Medical Research (ICMR), New Delhi, India for the award of ICMR‐Senior Research Fellowship (SRF; Award No: 45/03/2018‐BMS/PHA/OL). The infrastructure of Department of Biotechnology, Anna University, BIT-campus is supported by the Department of Science and Technology-Fund for Improvement of S and T Infrastructure in Universities and Higher Educational Institutions (DST-FIST).

Author contributions

SR has conceptualized, designed the experiments, acquired financial support, supervised the study, and wrote the manuscript; AS performed most of the experiments; DP and NR involved in the acquisition of data; AS, DP and SR analysed data and interpreted the results; AK and SKM participated in cytokine analysis; LL and KM participated in computational analysis; RB participated in flow cytometry analysis; RB, SKM and RP contributed reagents/materials/analysis tools.

Compliance with ethical standards

Conflict of interest

The authors report no conflict of interest.

Supplementary material

11_2019_1282_MOESM1_ESM.pdf (108 kb)
Supplementary material 1 (PDF 108 kb)


  1. 1.
    Gilbane AJ, Denton CP, Holmes AM. Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells. Arthritis Res Ther. 2013;15:215.CrossRefGoogle Scholar
  2. 2.
    Ramming A, Dees C, Distler JH. From pathogenesis to therapy-Perspective on treatment strategies in fibrotic diseases. Pharmacol Res. 2015;100:93–100.CrossRefGoogle Scholar
  3. 3.
    Willis BC, duBois RM, Borok Z. Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc. 2006;3:377–82.CrossRefGoogle Scholar
  4. 4.
    Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341–50.CrossRefGoogle Scholar
  5. 5.
    Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15:178–96.CrossRefGoogle Scholar
  6. 6.
    Stone RC, Pastar I, Ojeh N, Chen V, Liu S, Garzon KI, et al. Epithelial-mesenchymal transition in tissue repair and fibrosis. Cell Tissue Res. 2016;365:495–506.CrossRefGoogle Scholar
  7. 7.
    Braga TT, Agudelo JS, Camara NO. Macrophages during the fibrotic process: M2 as friend and foe. Front Immunol. 2015;25:602.Google Scholar
  8. 8.
    Cao Q, Harris DC, Wang Y. Macrophages in kidney injury, inflammation, and fibrosis. Physiology (Bethesda). 2015;30:183–94.Google Scholar
  9. 9.
    Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol. 2013;229:176–85.CrossRefGoogle Scholar
  10. 10.
    Shi J, Li Q, Sheng M, Zheng M, Yu M, Zhang L. The role of TLR4 in M1 macrophage-induced epithelial-mesenchymal transition of peritoneal mesothelial cells. Cell Physiol Biochem. 2016;40:1538–48.CrossRefGoogle Scholar
  11. 11.
    Scalbert A, Manach C, Morand C, Rémésy C, Jiménez L. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr. 2005;45:287–306.CrossRefGoogle Scholar
  12. 12.
    Salminen JP, Karonen M. Chemical ecology of tannins and other phenolics: we need a change in approach. Funct Ecol. 2011;25:325–38.CrossRefGoogle Scholar
  13. 13.
    Pattarayan D, Sivanantham A, Krishnaswami V, Loganathan L, Palanichamy R, Natesan S, et al. Tannic acid attenuates TGF-β1-induced epithelial-to-mesenchymal transition by effectively intervening TGF-β signaling in lung epithelial cells. J Cell Physiol. 2018;233:2513–25.CrossRefGoogle Scholar
  14. 14.
    Pattarayan D, Sivanantham A, Bethunaickan R, Palanichamy R, Rajasekaran S. Tannic acid modulates fibroblast proliferation and differentiation in response to pro-fibrotic stimuli. J Cell Biochem. 2018;119:6732–42.CrossRefGoogle Scholar
  15. 15.
    Sivanantham A, Pattarayan D, Bethunaickan R, Kar A, Mahapatra SK, Thimmulappa RK, et al. Tannic acid protects against experimental acute lung injury through down-regulation of TLR4 and MAPK. J Cell Physiol. 2019;234:6463–76.CrossRefGoogle Scholar
  16. 16.
    Chai WM, Wei QM, Deng WL, Zheng YL, Chen XY, Huang Q, et al. Anti-melanogenesis properties of condensed tannins from Vigna angularis seeds with potent antioxidant and DNA damage protection activities. Food Funct. 2019;10:99–111.CrossRefGoogle Scholar
  17. 17.
    Koval A, Pieme CA, Queiroz EF, Ragusa S, Ahmed K, Blagodatski A, et al. Tannins from Syzygium guineense suppress Wnt signaling and proliferation of Wnt-dependent tumors through a direct effect on secreted Wnts. Cancer Lett. 2018;435:110–20.CrossRefGoogle Scholar
  18. 18.
    Gourlay G, Constabel CP. Condensed tannins are inducible antioxidants and protect hybrid polar against oxidative stress. Tree Physiol. 2019;39:345–55.CrossRefGoogle Scholar
  19. 19.
    Helmy IM, Azim AM. Efficacy of ImageJ in the assessment of apoptosis. Diag Pathol. 2012;7:15.CrossRefGoogle Scholar
  20. 20.
    Wang Y, Su L, Morin MD, Jones BT, Whitby LR, Surakattula MM, et al. TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS. Proc Natl Acad Sci USA. 2016;113:E884–93.CrossRefGoogle Scholar
  21. 21.
    Maestro Suite, 2017, Version11.4.011, Schrodinger, LLC, NY.Google Scholar
  22. 22.
    Loganathan L, Muthusamy K. Investigation of drug interaction potentials and binding modes on direct renin inhibitors. A computational modeling studies. Lett Drug Des Discov. 2019;16:919–38.CrossRefGoogle Scholar
  23. 23.
    Kawasaki K, Nogawa H, Nishijima M. Identification of mouse MD-2 residues important for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 antibodies, and for conferring LPS and taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis. J Immunol. 2003;170:413–20.CrossRefGoogle Scholar
  24. 24.
    Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature. 2009;458:1191–5.CrossRefGoogle Scholar
  25. 25.
    Santos AFM, Macedo LJA, Chaves MH, Castaneda ME, Merkoci A, Lima FCA, et al. Hybrid self-assembled materials constituted by ferromagnetic nanoparticles and tannic acid: a theoretical and experimental investigation. J Braz Chem Soc. 2016;27:727–34.Google Scholar
  26. 26.
    Kong J, Yu S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin. 2007;39:549–59.CrossRefGoogle Scholar
  27. 27.
    Deng YR, Liu WB, Lian ZX, Li X, Hou X. Sorafenib inhibits macrophage-mediated epithelial-mesenchymal transition in hepatocellular carcinoma. Oncotarget. 2016;7:38292–305.Google Scholar
  28. 28.
    Wu KQ, Muratore CS, So EY, Sun C, Dubielecka PM, Reginato AM, et al. M1 macrophage-induced endothelial-to-mesenchymal transition promotes infantile hemangioma regression. Am J Pathol. 2017;187:2102–11.CrossRefGoogle Scholar
  29. 29.
    Yeung OW, Lo CM, Ling CC, Qi X, Geng W, Li CX, et al. Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma. J Hepatol. 2015;62:607–16.CrossRefGoogle Scholar
  30. 30.
    Li Q, Lv LL, Wu M, Zhang XL, Liu H, Liu BC. Dexamethasone prevents monocyte-induced tubular epithelial-mesenchymal transition in HK-2 cells. J Cell Biochem. 2003;114:632–8.CrossRefGoogle Scholar
  31. 31.
    Borthwick LA, Corris PA, Mahida R, Walker A, Gardner A, Suwara M, et al. TNFα from classically activated macrophages accentuates epithelial to mesenchymal transition in obliterative bronchiolitis. Am J Transplant. 2013;13:621–33.CrossRefGoogle Scholar
  32. 32.
    Yamada M, Kuwano K, Maeyama T, Hamada N, Yoshimi M, Nakanishi Y, et al. Dual-immunohistochemistry provides little evidence for epithelial-mesenchymal transition in pulmonary fibrosis. Histochem Cell Biol. 2008;129:453–62.CrossRefGoogle Scholar
  33. 33.
    Marmai C, Sutherland RE, Kim KK, Dolganov GM, Fang X, Kim SS, et al. Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2011;301:L71–8.CrossRefGoogle Scholar
  34. 34.
    Liang H, Gu Y, Li T, Zhang Y, Huangfu L, Hu M, et al. Integrated analyses identify the involvement of microRNA-26a in epithelial-mesenchymal transition during idiopathic pulmonary fibrosis. Cell Death Dis. 2014;5:e1238.CrossRefGoogle Scholar
  35. 35.
    Tanjore H, Xu XC, Polosukhin VV, Degryse AL, Li B, Han W, et al. Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. Am J Respir Crit Care Med. 2009;180:657–65.CrossRefGoogle Scholar
  36. 36.
    Inai T, Kobayashi J, Shibata Y. Claudin-1 contributes to the epithelial barrier function in MDCK cells. Eur J Cell Biol. 1999;78:849–55.CrossRefGoogle Scholar
  37. 37.
    Shiozaki A, Bai XH, Shen-Tu G, Moodley S, Takeshita H, Fung SY, et al. Claudin 1 mediates TNFα-induced gene expression and cell migration in human lung carcinoma cells. PLoS One. 2012;7:e38049.CrossRefGoogle Scholar
  38. 38.
    Fortier AM, Asselin E, Cadrin M. Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation. J Biol Chem. 2013;288:11555–71.CrossRefGoogle Scholar
  39. 39.
    Grund EM, Kagan D, Tran CA, Zeitvogel A, Starzinski-Powitz A, Nataraja S, et al. Tumor necrosis factor-alpha regulates inflammatory and mesenchymal responses via mitogen-activated protein kinase kinase, p38, and nuclear factor kappaB in human endometriotic epithelial cells. Mol Pharmacol. 2008;73:1394–404.CrossRefGoogle Scholar
  40. 40.
    Xiao J, Gong Y, Chen Y, Yu D, Wang X, Zhang X, et al. IL-6 promotes epithelial-to-mesenchymal transition of human peritoneal mesothelial cells possibly through the JAK2/STAT3 signaling pathway. Am J Physiol Renal Physiol. 2017;313:F310–8.CrossRefGoogle Scholar
  41. 41.
    Lee SO, Yang X, Duan S, Tsai Y, Strojny LR, Keng P, et al. IL-6 promotes growth and epithelial-mesenchymal transition of CD133+ cells of non-small cell lung cancer. Oncotarget. 2016;7:6626–38.Google Scholar
  42. 42.
    Li S, Lu J, Chen Y, Xiong N, Li L, Zhang J, et al. MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial-mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells. Cell Mol Immunol. 2017;14:621–30.CrossRefGoogle Scholar
  43. 43.
    Lee CH, Wu CL, Shiau AL. Toll-like receptor 4 signaling promotes tumor growth. J Immunother. 2010;33:73–82.CrossRefGoogle Scholar
  44. 44.
    Sujitha S, Dinesh P, Rasool M. Berberine modulates ASK1 signaling mediated through TLR4/TRAF2 via upregulation of miR-23a. Toxicol Appl Pharmacol. 2018;359:34–46.CrossRefGoogle Scholar
  45. 45.
    Rahimifard M, Maqbool F, Moeini-Nodeh S, Niaz K, Abdollahi M, Braidy N, et al. Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation. Ageing Res Rev. 2017;36:11–9.CrossRefGoogle Scholar
  46. 46.
    Joh EH, Gu W, Kim DH. Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4 in NF-kB and MAPK pathways. Biochem Pharmacol. 2012;84:40.CrossRefGoogle Scholar
  47. 47.
    Zeng KW, Yu Q, Liao LX, Song FJ, Lv HN, Jiang Y, et al. Anti-neuroinflammatory effect of MC13, a novel coumarin compound from condiment murraya, through inhibiting lipopolysaccharide-Induced TRAF6-TAK1-NF-κB, P38/ERK MAPKS and Jak2-Stat1/Stat3 pathways. J Cell Biochem. 2015;116:1286–99.CrossRefGoogle Scholar
  48. 48.
    Wang Y, Shan X, Dai Y, Jiang L, Chen G, Zhang Y, et al. Curcumin analog L48H37 prevents lipopolysaccharide-induced TLR4 signaling pathway activation and sepsis via targeting MD2. J Pharmacol Exp Ther. 2015;353:539–50.CrossRefGoogle Scholar
  49. 49.
    Kim SY, Koo JE, Seo YJ, Tyagi N, Jeong E, Choi J, et al. Suppression of Toll-like receptor 4 activation by caffeic acid phenethyl ester is mediated by interference of LPS binding to MD2. Br J Pharmacol. 2013;168:1933–45.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Ayyanar Sivanantham
    • 1
  • Dhamotharan Pattarayan
    • 1
  • Nandhine Rajasekar
    • 1
  • Adithi Kannan
    • 2
  • Lakshmanan Loganathan
    • 3
  • Ramalingam Bethunaickan
    • 4
  • Santanu Kar Mahapatra
    • 2
  • Rajaguru Palanichamy
    • 1
  • Karthikeyan Muthusamy
    • 3
  • Subbiah Rajasekaran
    • 1
    • 5
    Email author
  1. 1.Department of Biotechnology, BIT-CampusAnna UniversityTiruchirappalliIndia
  2. 2.Centre for Research in Infectious Diseases (CRID), School of Chemical and BiotechnologySASTRA Deemed To Be UniversityThanjavurIndia
  3. 3.Pharmacogenomics and CADD Lab, Department of BioinformaticsAlagappa UniversityKaraikudiIndia
  4. 4.Department of ImmunologyICMR-National Institute for Research in TuberculosisChennaiIndia
  5. 5.Department of Biochemistry, ICMR-National Institute for Research in Environmental Health, Kamla Nehru Hospital BuildingGandhi Medical College CampusBhopalIndia

Personalised recommendations