Lysozyme Protects Against Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Inflammation in Human Corneal Epithelial Cells

Yinting Song, Haokun Zhang, Yanfang Zhu, Xiao Zhao, Yi Lei, Wei Zhou, Jinguo Yu, Xue Dong, Xiaohong Wang, Mei Du, Hua Yan, Yinting Song, Haokun Zhang, Yanfang Zhu, Xiao Zhao, Yi Lei, Wei Zhou, Jinguo Yu, Xue Dong, Xiaohong Wang, Mei Du, Hua Yan

Abstract

Purpose: The purpose of this study was to investigate the effects of lysozyme, an antimicrobial enzyme found in tears that protects the eye against pathogens, on pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through corneal epithelial cells.

Methods: The expression of the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease (TMPRSS2) in human corneal epithelial cells (HCECs) was measured by RT-PCR and Western blotting. The altered expression of the pro-inflammatory molecules induced by spike protein and lysozyme was analyzed by RT-PCR. Cell toxicity was tested by CCK8 assay. The cell entry of SAR-CoV-2 in HCECs and primary rabbit corneal epithelial cells (RbCECs) was detected by luciferase assay.

Results: ACE2 and TMPRSS2 were highly expressed in HCECs. The spike proteins of SARS-CoV-2 stimulated a robust inflammatory response in HCECs, characterized by increased secretion of pro-inflammatory molecules, including IL-6, TNF-α, iNOS, and MCP-1, and pretreatment with lysozyme in HCECs markedly decreased the production of proinflammatory molecules induced by spike proteins. In addition, the inflammatory cytokine TNF-α enhanced the entry of SARS-CoV-2 into HCECs, which can be mitigated by pretreatment with lysozyme.

Conclusions: In this study, we analyzed the susceptibility of human corneal epithelial cells to SARS-CoV-2 infection and suggested the protective effects of lysozyme on SARS-CoV-2 infection.

Conflict of interest statement

Disclosure: Y. Song, None; H. Zhang, None; Y. Zhu, None: X. Zhao, None; Y. Lei, None; W. Zhou, None; J. Yu, None; X. Dong, None; X. Wang, None; M. Du, None; H. Yan, None

Figures

Figure 1.
Figure 1.
Analysis of the gene and protein expression of receptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human corneal epithelial cells (HCECs). The mRNA expression of angiotensin-converting enzyme 2 (ACE2) (A) and TMPRSS2 (B) in ARPE-19, A-549, and HCECs are detected by RT-PCR. (C) Western blot analysis of ACE2 and TMPRSS2 expression in a different cell line. (D,E) Quantification of ACE2 and TMPRSS2 protein expression. Values are mean ± SD from at least three independent experiments. *P < 0.5; **P < 0.1; ***P < 0.001 versus control group using one-way analysis of variance (ANOVA).
Figure 2.
Figure 2.
Lysozyme reduced viral entry into HCECs in a dose-dependent manner. (A) Measurement of cytotoxicity by the CCK8 assay in HCECs treated with various doses of lysozyme for 24 hours. (B) Western blot analysis of the efficiency of ACE2 overexpression. (C) Measurement of cell entry by luciferase assay in HCECs treated with ACE2 overexpressing lentivirus and different dose lysozyme (5 mg/mL and 10 mg/mL). Values are mean ± SD from at least three independent experiments. *P < 0.5; **P < 0.1; ***P < 0.001 versus control group using one-way ANOVA.
Figure 3.
Figure 3.
The inflammatory response induced by spike protein of SARS-CoV2 in HCECs. (A–F) mRNA expression of various inflammatory cytokines, including TNF-α (A), IL-6 (B), MCP-1 (C), iNOS (D), IL-8 (E), and IL-1β (F) in HCEC cells treated with 1 µg/mL spike protein detected by RT-PCR. Values are mean ± SD from at least three independent experiments. *P < 0.5; **P < 0.1; ***P < 0.001 versus control group using the t-test.
Figure 4.
Figure 4.
Lysozyme attenuates the elevated secretion of inflammatory molecules induced by spike proteins. (A–F) Changes in mRNA expression of various inflammatory cytokines, including TNF-α (A), IL-6 (B), MCP-1 (C), iNOS (D), IL-8 (E), and IL-1β (F) in HCECs pretreated with lysozyme (2 hours or 0 hours before stimulation) detected by RT-PCR. (G–L) Detection of inflammatory cytokines, including TNF-α (G), IL-6 (H), MCP-1 (I), iNOS (J), IL-8 (K), and IL-1β (L) in HCECs pretreated with lysozyme by ELISA. Values are mean ± SD from at least three independent experiments. *P < 0.5; **P < 0.1; ***P < 0.001 versus control group using one-way ANOVA. #P < 0.5; ##P < 0.1; ###P < 0.001 versus spike group using one-way ANOVA.
Figure 5.
Figure 5.
Lysozyme attenuates the cell entry of SARS-CoV-2 under inflammatory conditions. (A) Measurement of cell entry by luciferase assay in HCECs treated with control, pseudotyped SARS-CoV-2, ACE2 overexpressing lentivirus, TNF-α (10 ng/mL) and lysozyme (10 mg/mL). (B) Western blot analysis of ACE2 and TMPRSS2 protein expression in HCEC and primary rabbit corneal epithelial cells (RbCECs). (C) Measurement of cell entry by luciferase assay in RbCECs treated with control, pseudotyped SARS-CoV-2, ACE2 overexpressing lentivirus, TNF-α (10 ng/mL) and lysozyme (10 mg/mL). Values are mean ± SD from at least three independent experiments. *P < 0.5; **P < 0.1; ***P < 0.001 versus control group using one-way ANOVA.

References

    1. Zhu N, Zhang D, Wang W, et al. .. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382: 727–733.
    1. Zhou P, Yang XL, Wang XG, et al. .. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579: 270–273.
    1. Lei C, Qian K, Li T, et al. .. Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nature Communications. 2020; 11: 2070.
    1. Röltgen K, Powell AE, Wirz OF, et al. .. Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Sci Immunol. 2020; 5: eabe0240.
    1. Wu P, Duan F, Luo C, et al. .. Characteristics of Ocular Findings of Patients With Coronavirus Disease 2019 (COVID-19) in Hubei Province, China. JAMA Ophthalmol. 2020; 138: 575–578.
    1. Ma C, Nikiforov A, De Geyter N, Dai X, Morent R, Ostrikov KK.. Future antiviral polymers by plasma processing. Prog Polymer Sci. 2021; 118: 101410.
    1. Seah IYJ, Anderson DE, Kang AEZ, et al. .. Assessing Viral Shedding and Infectivity of Tears in Coronavirus Disease 2019 (COVID-19) Patients. Ophthalmology. 2020; 127: 977–979.
    1. Xia J, Tong J, Liu M, Shen Y, Guo D.. Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection. J Med Virol. 2020; 92: 589–594.
    1. Fleming A, Allison VD.. Observations on a Bacteriolytic Substance (“Lysozyme”) Found in Secretions and Tissues. Br J Exp Pathol. 1922; 3: 252–260.
    1. Garreis F, Fau-Gottschalt M, Gottschalt M, Fau-Paulsen FP, Paulsen FP.. Antimicrobial peptides as a major part of the innate immune defense at the ocular surface. Dev Ophthalmol. 2010; 45: 16–22.
    1. McDermott AM. Antimicrobial compounds in tears. Exp Eye Res. 2013; 117: 53–61.
    1. Harding CJ, Huwiler SG, Somers H, et al. .. A lysozyme with altered substrate specificity facilitates prey cell exit by the periplasmic predator Bdellovibrio bacteriovorus. Nature Communications. 2020; 11: 4817.
    1. Nguyen GTH, Tran TN, Podgorski MN, Bell SG, Supuran CT, Donald WA.. Nanoscale Ion Emitters in Native Mass Spectrometry for Measuring Ligand-Protein Binding Affinities. ACS Central Science. 2019; 5: 308–318.
    1. Villa TG, Feijoo-Siota L, Rama JLR, Ageitos JM.. Antivirals against animal viruses. Biochem Pharmacol. 2017; 133: 97–116.
    1. Małaczewska J, Kaczorek-Łukowska E, Wójcik R, Siwicki AK.. Antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus. BMC Veterinary Res. 2019; 15: 318.
    1. Hanstock HG, Edwards JP, Walsh NP.. Tear Lactoferrin and Lysozyme as Clinically Relevant Biomarkers of Mucosal Immune Competence. Front Immunol. 2019; 10: 1178.
    1. McDermott AM. Antimicrobial compounds in tears. Exp Eye Res. 2013; 117: 53–61.
    1. Saari KM, Aine E, Posz A, Klockars M.. Lysozyme content of tears in normal subjects and in patients with external eye infections. Graefes Arch Clin Exp Ophthalmol. 1983; 221: 86–88.
    1. Narayanan S, Redfern RL, Miller WL, Nichols KK, McDermott AM.. Dry eye disease and microbial keratitis: is there a connection? Ocular Surf. 2013; 11: 75–92.
    1. Lee JS, Koh JY, Yi K, et al. .. Single-cell transcriptome of bronchoalveolar lavage fluid reveals sequential change of macrophages during SARS-CoV-2 infection in ferrets. Nature Communications. 2021; 12: 4567.
    1. Haabeth OAW, Lohmeyer JJK, Sallets A, et al. .. An mRNA SARS-CoV-2 Vaccine Employing Charge-Altering Releasable Transporters with a TLR-9 Agonist Induces Neutralizing Antibodies and T Cell Memory. ACS Central Science. 2021; 7: 1191–1204.
    1. Tseng SH, Lam B, Kung YJ, et al. .. A novel pseudovirus-based mouse model of SARS-CoV-2 infection to test COVID-19 interventions. J Biomed Sci. 2021; 28: 34.
    1. Chu H, Hu B, Huang X, et al. .. Host and viral determinants for efficient SARS-CoV-2 infection of the human lung. Nature Communications. 2021; 12: 134.
    1. Chen TF, Chang YC, Hsiao Y, et al. .. DockCoV2: a drug database against SARS-CoV-2. Nucleic Acids Res. 2021; 49: D1152–D1159.
    1. Rao S, Lau A, So HC.. Exploring Diseases/Traits and Blood Proteins Causally Related to Expression of ACE2, the Putative Receptor of SARS-CoV-2: A Mendelian Randomization Analysis Highlights Tentative Relevance of Diabetes-Related Traits. Diabetes Care. 2020; 43: 1416–1426.
    1. Rossi ÁD, de Araújo JLF, de Almeida TB, et al. .. Association between ACE2 and TMPRSS2 nasopharyngeal expression and COVID-19 respiratory distress. Sci Rep. 2021; 11: 9658.
    1. Wang Y, Fan Y, Huang Y, et al. .. TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2. Cellular Signalling. 2021; 85: 110064.
    1. Petrovszki D, Walter FR, Vigh JP, et al. .. Penetration of the SARS-CoV-2 Spike Protein across the Blood-Brain Barrier, as Revealed by a Combination of a Human Cell Culture Model System and Optical Biosensing. Biomedicines. 2022; 10: 188.
    1. Suzuki YJ, Gychka SG.. SARS-CoV-2 Spike Protein Elicits Cell Signaling in Human Host Cells: Implications for Possible Consequences of COVID-19 Vaccines. Vaccines. 2021; 9: 36.
    1. Patra T, Meyer K, Geerling L, et al. .. SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells. PLoS Pathogens. 2020; 16: e1009128.
    1. Petruk G, Puthia M, Petrlova J, et al. .. SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity. J Molec Cell Biol. 2020; 12: 916–932.
    1. Zhou Y, Duan C, Zeng Y, et al. .. Ocular Findings and Proportion with Conjunctival SARS-COV-2 in COVID-19 Patients. Ophthalmology. 2020; 127: 982–983.
    1. Dettorre G, Diamantis N, Loizidou A, et al. .. 319O The systemic pro-inflammatory response identifies cancer patients with adverse outcomes from SARS-CoV-2 infection. Ann Oncol. 2020; 31: S1366.
    1. Boudewijns R, Thibaut HJ, Kaptein SJF, et al. .. STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters. Nature Communications. 2020; 11: 5838–5838.
    1. Mann ER, Menon M, Knight SB, et al. .. Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19. Sci Immunol. 2020; 5: eabd6197.
    1. Lee JS, Park S, Jeong HW, et al. .. Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19. Sci Immunol. 2020; 5: eabd1554.
    1. Jover E, Matilla L, Garaikoetxea M, et al. .. Beneficial Effects of Mineralocorticoid Receptor Pathway Blockade against Endothelial Inflammation Induced by SARS-CoV-2 Spike Protein. Biomedicines. 2021; 9: 639.
    1. Wrobel AG, Benton DJ, Xu P, et al. .. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat Struct Mol Biol. 2020; 27: 763–767.
    1. Bortolotti D, Gentili V, Rizzo S, Rotola A, Rizzo R.. SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway. Cells. 2020; 9: 1975.
    1. Ferrari R, Callerio C, Podio G.. Antiviral activity of lysozyme. Nature. 1959; 183: 548.
    1. Lee-Huang S, Huang PL, Sun Y, et al. .. Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Natl Acad Sci USA. 1999; 96: 2678–2681.
    1. Latorre J, Lluch A, Ortega FJ, et al. .. Adipose tissue knockdown of lysozyme reduces local inflammation and improves adipogenesis in high-fat diet-fed mice. Pharmacol Res. 2021; 166: 105486.
    1. Avisar R, Menaché R, Shaked P, Rubinstein J, Machtey I, Savir H.. Lysozyme content of tears in patients with Sjögren's syndrome and rheumatoid arthritis. Am J Ophthalmol. 1979; 87: 148–151.
    1. Berra M, Galperín G, Berra F, et al. .. Tear Lysozyme in Sjögren´s syndrome, Meibomian gland dysfunction, and non-dry-eye. Arquivos Brasileiros de Oftalmologia. 2021; 85: 103–108.
    1. Eylan E, Ronen D, Romano A, Smetana O.. Lysozyme tear level in patients with herpes simplex virus eye infection. Invest Ophthalmol Vis Sci. 1977; 16: 850–853.
    1. Erickson OF, Hatlen R, Berg M.. Industrial tear study; filter-paper electrophoresis of tears, with results of an industrial study from 1,000 specimens sent by mail. Am J Ophthalmol. 1959; 47: 499–508; discussion 508-499.
    1. Okawada N, Mizoguchi I, Ishiguro T.. Effects of photochemical air pollution on the human eye–concerning eye irritation, tear lysozyme and tear pH. Nagoya J Med Sci. 1979; 41: 9–20.
    1. Watson RR, Reyes MA, McMurray DN.. Influence of malnutrition on the concentration of IgA, lysozyme, amylase and aminopeptidase in children's tears. Proc Soc Exp Biol Med. 1978; 157: 215–219.
    1. Jiang Z, Zhang H, Gao J, et al. .. ACE2 Expression Is Upregulated in Inflammatory Corneal Epithelial Cells and Attenuated by Resveratrol. Invest Ophthalmol Vis Sci. 2021; 62: 25.
    1. Smith JC, Sausville EL, Girish V, et al. .. Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract. Developmental Cell. 2020; 53: 514–529.e513.
    1. Kang WS, Jung E, Kim J.. Aucuba japonica Extract and Aucubin Prevent Desiccating Stress-Induced Corneal Epithelial Cell Injury and Improve Tear Secretion in a Mouse Model of Dry Eye Disease. Molecules (Basel, Switzerland). 2018; 23: 2599.
    1. Ji H, Zhu Y, Zhang Y, et al. .. The Effect of Dry Eye Disease on Scar Formation in Rabbit Glaucoma Filtration Surgery. Int J Mol Sci. 2017; 18: 1150.
    1. Lam H, Bleiden L, de Paiva CS, Farley W, Stern ME, Pflugfelder SC.. Tear cytokine profiles in dysfunctional tear syndrome. Am J Ophthalmol. 2009; 147: 198–205.e191.
    1. Zhu L, Shen J, Zhang C, et al. .. Inflammatory cytokine expression on the ocular surface in the Botulium toxin B induced murine dry eye model. Molec Vis. 2009; 15: 250–258.
    1. Zhuang MW, Cheng Y, Zhang J, et al. .. Increasing host cellular receptor-angiotensin-converting enzyme 2 expression by coronavirus may facilitate 2019-nCoV (or SARS-CoV-2) infection. J Med Virol. 2020; 92: 2693–2701.

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