Thymol Protects against Aspergillus Fumigatus Keratitis by Inhibiting the LOX-1/IL-1β Signaling Pathway

Abstract

Objective

To explore the anti-inflammatory effects and mechanisms of action of thymol in Aspergillus fumigatus (A. fumigatus) keratitis.

Methods

The minimum inhibitory concentration of thymol against A. fumigatus was detected. To characterize the anti-inflammatory effects of thymol, mouse corneas and human corneal epithelial cells were pretreated with thymol or dimethyl sulfoxide (DMSO) before infection with A. fumigatus spores. Slit-lamp microscopy, immunohistochemistry, myeloperoxidase detection, quantitative real-time polymerase chain reaction, and Western blotting were used to assess infection. Neutrophil and macrophage recruitment, in addition to the secretion of LOX-1 and IL-1β, were quantified to evaluate the relative contribution of thymol to the inflammatory response.

Results

We confirmed that the growth of A. fumigatus was directly inhibited by thymol. In contrast with the DMSO group, there was a lower degree of inflammation in the mouse corneas of the thymol-pretreated group. This was characterized by significantly lower clinical scores, less inflammatory cell infiltration, and lower expression of LOX-1 and IL-1β. Similarly, in vitro experiments indicated that the production of LOX-1 and IL-1β was significantly inhibited after thymol treatment, in contrast with the DMSO-pretreated group.

Conclusion

Our findings demonstrate that thymol exerted a direct fungistatic activity on A. fumigatus. Furthermore, thymol played a protective role in fungal keratitis by inhibiting LOX-1/IL-1β signaling pathway and reducing the recruitment of neutrophils and macrophages.

Keywords

keratitis / Aspergillus fumigatus / thymol / LOX-1 / IL-1β

References

[1] BharathiMJ, RamakrishnanR, MeenakshiR, et al.. Microbial keratitis in South India: influence of risk factors, climate, and geographical variation. Ophthalmic Epidemiol, 2007, 14(2): 61-69

[2] SrinivasanM, ChristineA, CelineG, et al.. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol, 1997, 81: 965-971

[3] PlatoA, HardisonSE, BrownGD. Pattern recognition receptors in antifungal immunity. Semin Immunopathol, 2015, 37(2): 97-106

[4] FigueiredoRT, CarneiroLA, BozzaMT. Fungal surface and innate immune recognition of filamentous fungi. Front Microbiol, 2011, 2: 248

[5] MendaSA, DasM, PanigrahiA, et al.. Association of Postfungal Keratitis Corneal Scar Features With Visual Acuity. JAMA Ophthalmology, 2020, 138(2): 113-118

[6] JengBH. Challenges in the Management of Fungal Keratitis. JAMA Ophthalmology, 2017, 135(6): 525-526

[7] ManciniE, SenatoreF, Del MonteD, et al.. Studies on Chemical Composition, Antimicrobial and Antioxidant Activities of Five Thymus vulgaris L. Essential Oils. Molecules, 2015, 20(7): 12 016-12 028

[8] MarcheseA, OrhanIE, DagliaM, et al.. Antibacterial and antifungal activities of thymol: A brief review of the literature. Food Chemistry, 2016, 210: 402-414

[9] MendesSS, BomfimRR, JesusHC, et al.. Evaluation of the analgesic and anti-inflammatory effects of the essential oil of Lippia gracilis leaves. J Ethnopharmacol, 2010, 129(3): 391-397

[10] WangK, JiangS, PuT, et al.. Antifungal activity of phenolic monoterpenes and structure-related compounds against plant pathogenic fungi. Nat Prod Res, 2019, 33(10): 1423-1430

[11] SharifzadehA, KhosraviAR, ShokriH, et al.. Potential effect of 2-isopropyl-5-methylphenol (thymol) alone and in combination with fluconazole against clinical isolates of Candida albicans, C. glabrata and C. krusei. J Mycol Med, 2018, 28(2): 294-299

[12] MathelaCS, SinghKK, GuptaVK. Synthesis and in vitro antibacterial activity of thymol and carvacrol derivatives. Acta Pol Pharm, 2010, 67(4): 375-380

[13] HaeselerG, MaueD, GrosskreutzJ, et al.. Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol. Eur J Anaesthesiol, 2002, 19: 571-579

[14] AeschbachR, LöligerR, ScottBC, et al.. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem Toxicol, 1994, 32(1): 31-36

[15] WuTG, WilhelmusKR, MitchellBM. Experimental keratomycosis in a mouse model. Invest Ophthalmol Vis Sci, 2003, 44(1): 210-216

[16] ManikandanP, Abdel-HadiA, Randhir Babu SinghY, et al.. Fungal Keratitis: Epidemiology, Rapid Detection, and Antifungal Susceptibilities of Fusarium and Aspergillus Isolates from Corneal Scrapings. BioMed Res Int, 2019, 2019: 6395840

[17] YangH, WangQ, HanL, et al.. Nerolidol inhibits the LOX-1/IL-1beta signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea. Int Immunopharmacol, 2020, 80: 106118

[18] Rose-NussbaumerJ, PrajnaNV, KrishnanT, et al.. Mycotic Ulcer Treatment Trial, Risk factors for low vision related functioning in the Mycotic Ulcer Treatment Trial: a randomised trial comparing natamycin with voriconazole. Br J Ophthalmol, 2016, 100(7): 929-932

[19] BaeYS, RheeMS. Short-Term Antifungal Treatments of Caprylic Acid with Carvacrol or Thymol Induce Synergistic 6-Log Reduction of Pathogenic Candida albicans by Cell Membrane Disruption and Efflux Pump Inhibition. Cell Physiol Biochem, 2019, 53(2): 285-300

[20] BragaPC, CuliciM, AlfieriM, et al.. Thymol inhibits Candida albicans biofilm formation and mature biofilm. Int J Antimicrob Agents, 2008, 31(5): 472-477

[21] ZhangM, GeJ, YuX. Transcriptome Analysis Reveals the Mechanism of Fungicidal of Thymol Against Fusarium oxysporum f. sp. niveum. Curr Microbiol, 2018, 75(4): 410-419

[22] KasperL, KonigA, KoenigPA, et al.. The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. Nat Commun, 2018, 9(1): 4260

[23] GolbahariS, Abtahi FroushaniSM. Synergistic benefits of Nicotine and Thymol in alleviating experimental rheumatoid arthritis. Life Sci, 2019, 239: 117037

[24] EftekhariM, HoseinsalariA, MansourianM, et al.. Trachyspermum ammi (L.) Sprague, superb essential oil and its major components on peptic ulcers: in vivo combined in silico studies. DARU J Pharm Sci, 2019, 27(1): 317-327

[25] YaoL, HouG, WangL, et al.. Protective effects of thymol on LPS-induced acute lung injury in mice. Microb Pathog, 2018, 116: 8-12

[26] HindLE, HuttenlocherA. Neutrophil Reverse Migration and a Chemokinetic Resolution. Dev Cell, 2018, 47(4): 404-405

[27] SerhanCN, SavillJ. Resolution of inflammation: the beginning programs the end. Nat Immunol, 2005, 6(12): 1191-1197

[28] BoffD, CrijnsH, TeixeiraMM, et al.. Neutrophils: beneficial and harmful cells in septic arthritis. Int J Mol Sci, 2018, 19(2): 468

[29] NiuL, LiuX, MaZ, et al.. Fungal keratitis: Pathogenesis, diagnosis and prevention. Microb Pathog, 2020, 138: 103802

[30] SerhanCN, BrainSD, BuckleyCD, et al.. Resolution of inflammation: state of the art, definitions and terms. FASEB J, 2007, 21(2): 325-332

[31] HuJZ, HuYF, ChenSK, et al.. Role of activated macrophages in experimental Fusarium solani keratitis. Exp Eye Res, 2014, 129: 57-65

[32] ZhangQ, LiuJ, MaL, et al.. Wnt5a is involved in LOX-1 and TLR4 induced host inflammatory response in peri-implantitis. J Periodontal Res, 2020, 55(2): 199-208

[33] ZhangQ, LiuJ, MaL, et al.. LOX-1 is involved in TLR2 induced RANKL regulation in peri-implantitis. Int Immunopharmacol, 2019, 77: 105956

[34] LiC, ZhaoGQ, CheCY, et al.. The Role of LOX-1 in Innate Immunity to Aspergillus fumigatus in Corneal Epithelial Cells. Invest Ophthalmol Vis Sci, 2015, 56(6): 3593-3603

[35] CheCY, LiC, LinJ, et al.. Wnt5a contributes to dectin-1 and LOX-1 induced host inflammatory response signature in Aspergillus fumigatus keratitis. Cell Signal, 2018, 52: 103-111

[36] PalomoJ, DietrichD, MartinP, et al.. The interleukin (IL)-1 cytokine family—Balance between agonists and antagonists in inflammatory diseases. Cytokine, 2015, 76(1): 25-37

[37] ZhaoGQ, HuM, LiC, et al.. Osteopontin contributes to effective neutrophil recruitment, IL-1beta production and apoptosis in Aspergillus fumigatus keratitis. Immunol Cell Biol, 2018, 96(4): 401-412

[38] ChengM, LinJ, LiC, et al.. Wedelolactone suppresses IL-1beta maturation and neutrophil infiltration in Aspergillus fumigatus keratitis. Int Immunopharmacol, 2019, 73: 17-22

[39] PearlmanE, SunY, RoyS, et al.. Host defense at the ocular surface. Int Rev Immunol, 2013, 32(1): 4-18