A multi-platform analysis of human gingival crevicular fluid reveals ferroptosis as a relevant regulated cell death mechanism during the clinical progression of periodontitis

Abstract

Ferroptosis is implicated in the pathogenesis of numerous chronic-inflammatory diseases, yet its association with progressive periodontitis remains unexplored. To investigate the involvement and significance of ferroptosis in periodontitis progression, we assessed sixteen periodontitis-diagnosed patients. Disease progression was clinically monitored over twelve weeks via weekly clinical evaluations and gingival crevicular fluid (GCF) collection was performed for further analyses. Clinical metrics, proteomic data, in silico methods, and bioinformatics tools were combined to identify protein profiles linked to periodontitis progression and to explore their potential connection with ferroptosis. Subsequent western blot analyses validated key findings. Finally, a single-cell RNA sequencing (scRNA-seq) dataset (GSE164241) for gingival tissues was analyzed to elucidate cellular dynamics during periodontitis progression. Periodontitis progression was identified as occurring at a faster rate than traditionally thought. GCF samples from progressing and non-progressing periodontal sites showed quantitative and qualitatively distinct proteomic profiles. In addition, specific biological processes and molecular functions during progressive periodontitis were revealed and a set of hub proteins, including SNCA, CA1, HBB, SLC4A1, and ANK1 was strongly associated with the clinical progression status of periodontitis. Moreover, we found specific proteins - drivers or suppressors - associated with ferroptosis (SNCA, FTH1, HSPB1, CD44, and GCLC), revealing the co-occurrence of this specific type of regulated cell death during the clinical progression of periodontitis. Additionally, the integration of quantitative proteomic data with scRNA-seq analysis suggested the susceptibility of fibroblasts to ferroptosis. Our analyses reveal proteins and processes linked to ferroptosis for the first time in periodontal patients, which offer new insights into the molecular mechanisms of progressive periodontal disease. These findings may lead to novel diagnostic and therapeutic strategies.

References

1. Lamont, R. J., Koo, H.& Hajishengallis, G. The oral microbiota: dynamic communities and host interactions.Nat. Rev. Microbiol. 16, 745-759 (2018).
2. Papapanou, P. N.et al.Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.J. Periodontol. 89, S173-S182 (2018).
3. Torres, A.et al.Proteomic profile of human gingival crevicular fluid reveals specific biological and molecular processes during clinical progression of periodontitis. J. Periodontal. Res. https://doi.org/10.1111/jre.13169(2023).
4. Dixon, S. J.et al.Ferroptosis: An Iron-Dependent Form of Nonapoptotic.Cell Death. Cell 149, 1060-1072 (2012).
5. Amaral, E. P.et al.A major role for ferroptosis in Mycobacterium tuberculosis-induced cell death and tissue necrosis.J. Exp. Med. 216, 556-570 (2019).
6. Dar, H. H.et al.Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium.J. Clin. Investig. 128, 4639-4653 (2018).
7. Pajuelo, D.et al.NAD+ Depletion Triggers Macrophage Necroptosis, a Cell Death Pathway Exploited by Mycobacterium tuberculosis.Cell Rep. 24, 429-440 (2018).
8. Wei, S.et al.Serum irisin levels are decreased in patients with sepsis, and exogenous irisin suppresses ferroptosis in the liver of septic mice.Clin. Transl. Med. 10, e173(2020).
9. do Van, B.et al. Ferroptosis, a newly characterized form of cell death in Parkinson’s disease that is regulated by PKC.Neurobiol. Dis. 94, 169-178 (2016).
10. Hassannia, B., Vandenabeele, P.& vanden Berghe, T. Targeting Ferroptosis to Iron Out Cancer.Cancer Cell 35, 830-849 (2019).
11. Sun, Y.et al.The emerging role of ferroptosis in inflammation.Biomed. Pharmacother. 127, 110108(2020).
12. Weiland, A.et al.Ferroptosis and Its Role in Diverse Brain Diseases.Mol. Neurobiol. 56, 4880-4893 (2019).
13. Boyer, E.et al.Increased transferrin saturation is associated with subgingival microbiota dysbiosis and severe periodontitis in genetic haemochromatosis.Sci. Rep. 8, 15532(2018).
14. Guo, L.-N., Yang, Y.-Z.& Feng, Y.-Z. Serum and salivary ferritin and Hepcidin levels in patients with chronic periodontitis and type 2 diabetes mellitus.BMC Oral. Health 18, 63(2018).
15. Mukherjee S.The Role of Crevicular Fluid Iron in Periodontal Disease.J. Periodontol. 56, 22-27 (1985).
16. Bains R.& Bains, V. The antioxidant master glutathione and periodontal health.Dent. Res J. 12, 389(2015).
17. Grant M. M., Brock G. R., Matthews J. B.& Chapple, I. L. C. Crevicular fluid glutathione levels in periodontitis and the effect of non-surgical therapy.J. Clin. Periodontol. 37, 17-23 (2010).
18. Wang, Y., Andrukhov, O.& Rausch-Fan, X. Oxidative Stress and Antioxidant System in Periodontitis.Front. Physiol. 8, 910(2017).
19. Xing, L.et al.Fibroblast ferroptosis is involved in periodontitis-induced tissue damage and bone loss.Int Immunopharmacol. 114, 109607(2023).
20. Fu, E.et al.Role of ferroptosis in periodontitis: An animal study in rats.J. Periodontal Res. 58, 1031-1040 (2023).
21. Wong S. L.& Wagner, D. D. Peptidylarginine deiminase 4: a nuclear button triggering neutrophil extracellular traps in inflammatory diseases and aging.FASEB J. 32, 6258-6370 (2018).
22. Li, P.et al.PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.J. Exp. Med. 207, 1853-1862 (2010).
23. Koopman, G.et al.Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.Blood 84, 1415-1420 (1994).
24. Vermes I., Haanen C., Steffens-Nakken, H. & Reutellingsperger, C. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.J. Immunol. Methods 184, 39-51 (1995).
25. Leppilahti J. M., Kallio M. A., Tervahartiala T., Sorsa T.& Mäntylä, P. Gingival Crevicular Fluid Matrix Metalloproteinase-8 Levels Predict Treatment Outcome Among Smokers With Chronic Periodontitis.J. Periodontol. 85, 250-260 (2014).
26. Hernández, M.et al.Associations between matrix metalloproteinase-8 and -14 and myeloperoxidase in gingival crevicular fluid from subjects with progressive chronic periodontitis: a longitudinal study.J. Periodontol. 81, 1644-1652 (2010).
27. Sorsa, T.et al.Analysis of matrix metalloproteinases, especially MMP-8, in gingival crevicular fluid, mouthrinse and saliva for monitoring periodontal diseases.Periodontology 70, 142-163 (2016).
28. Baeza, M.et al.Diagnostic accuracy for apical and chronic periodontitis biomarkers in gingival crevicular fluid: an exploratory study.J. Clin. Periodontol. 43, 34-45 (2016).
29. Tymkiw, K. D.et al.Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis.J. Clin. Periodontol. 38, 219-228 (2011).
30. Hernández, M.et al.MMP-8, TRAP-5, and OPG Levels in GCF Diagnostic Potential to Discriminate between Healthy Patients’, Mild and Severe Periodontitis Sites.Biomolecules 10, 1500(2020).
31. Teles, R.et al.Patterns of periodontal disease progression based on linear mixed models of clinical attachment loss.J. Clin. Periodontol. 45, 15-25 (2018).
32. Teles R. P., Patel M., Socransky S. S.& Haffajee, A. D. Disease Progression in Periodontally Healthy and Maintenance Subjects.J. Periodontol. 79, 784-794 (2008).
33. Teles, R.et al.Modelling changes in clinical attachment loss to classify periodontal disease progression.J. Clin. Periodontol. 43, 426-434 (2016).
34. Papapanou, P. N., Wennström, J. L.& Gröndahl, K. A 10-year retrospective study of periodontal disease progression.J. Clin. Periodontol. 16, 403-411 (1989).
35. Schätzle, M.et al.Clinical course of chronic periodontitis.J. Clin. Periodontol. 30, 909-918 (2003).
36. Gilthorpe, M. S.et al.Unification of the ‘Burst’ and ‘Linear’ Theories of Periodontal Disease Progression: A Multilevel Manifestation of the Same Phenomenon.J. Dent. Res. 82, 200-205 (2003).
37. Bibi, T.et al.Gingival Crevicular Fluid (GCF): A Diagnostic Tool for the Detection of Periodontal Health and Diseases.Molecules 26, 1208(2021).
38. Bostanci N.& Belibasakis, G. N. Gingival crevicular fluid and its immune mediators in the proteomic era.Periodontology 76, 68-84 (2018).
39. Baliban, R. C.et al.Novel protein identification methods for biomarker discovery via a proteomic analysis of periodontally healthy and diseased gingival crevicular fluid samples.J. Clin. Periodontol. 39, 203-212 (2012).
40. Tsuchida, S.et al.Proteomic analysis of gingival crevicular fluid for discovery of novel periodontal disease markers.Proteomics 12, 2190-2202 (2012).
41. Fentoğlu, Ö.et al.Evaluation of Lipid Peroxidation and Oxidative DNA Damage in Patients With Periodontitis and Hyperlipidemia.J. Periodontol. 86, 682-688 (2015).
42. Akalιn F. A., Baltacιoğlu E., Alver A.& Karabulut, E. Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis.J. Clin. Periodontol. 34, 558-565 (2007).
43. Borges, I. Jr.et al.Proinflammatory and Oxidative Stress Markers in Patients with Periodontal Disease.Mediators Inflamm. 2007, 1-5 (2007).
44. Yang, W. S.et al.Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl Acad. Sci. 113, 804(2016).
45. Stockwell, B. R.et al.Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.Cell 171, 273-285 (2017).
46. Unal, E.et al.Paraoxonase and Arylesterase Activities, Lipid Profile, and Oxidative Damage in Experimental Ischemic Colitis Model.Gastroenterol. Res. Pr. 2012, 1-5 (2012).
47. Baliban, R. C.et al.Discovery of biomarker combinations that predict periodontal health or disease with high accuracy from GCF samples based on high-throughput proteomic analysis and mixed-integer linear optimization.J. Clin. Periodontol. 40, 131-139 (2013).
48. Figueiredo, R. T.et al.Characterization of Heme as Activator of Toll-like Receptor 4.J. Biol. Chem. 282, 20221-20229 (2007).
49. Dutra F. F.& Bozza, M. T. Heme on innate immunity and inflammation.Front Pharmacol 5, 115(2014).
50. Liu L. Y.,McGregor, N., Wong, B. K. J., Butt, H. & Darby, I. B. The association between clinical periodontal parameters and free haem concentration within the gingival crevicular fluid: a pilot study.J. Periodontal Res. 51, 86-94 (2016).
51. Valko, M.et al.Free radicals and antioxidants in normal physiological functions and human disease.Int J. Biochem. Cell Biol. 39, 44-84 (2007).
52. Collin F.Chemical Basis of Reactive Oxygen Species Reactivity and Involvement in Neurodegenerative Diseases.Int J. Mol. Sci. 20, 2407(2019).
53. Lee C., Lee J., Nam M., Choi Y.& Park, S.-H. Tomentosin Displays Anti-Carcinogenic Effect in Human Osteosarcoma MG-63 Cells via the Induction of Intracellular Reactive Oxygen Species.Int J. Mol. Sci. 20, 1508(2019).
54. Fellner, L.et al.Toll-like receptor 4 is required for α-synuclein dependent activation of microglia and astroglia.Glia 61, 349-360 (2013).
55. Spillantini, M. G.et al.α-Synuclein in Lewy bodies.Nature 388, 839-840 (1997).
56. Xia, Y.et al.Characterization of the human α-synuclein gene: Genomic structure, transcription start site, promoter region and polymorphisms1.J. Alzheimers. Dis. 3, 485-494 (2001).
57. Jakes, R., Spillantini, M. G.& Goedert, M. Identification of two distinct synucleins from human brain.FEBS Lett. 345, 27-32 (1994).
58. Stefanova, N.et al.Toll-Like Receptor 4 Promotes α-Synuclein Clearance and Survival of Nigral Dopaminergic Neurons.Am. J. Pathol. 179, 954-963 (2011).
59. Kim, C.et al.Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia.Nat. Commun. 4, 1562(2013).
60. Lunnon, K.et al.Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer’s disease.Nat. Neurosci. 17, 1164-1170 (2014).
61. De Jager, P. L. et al. Alzheimer’s disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci.Nat. Neurosci. 17, 1156-1163 (2014).
62. Liscovitch N.& French, L. Differential Co-Expression between α-Synuclein and IFN-γ Signaling Genes across Development and in Parkinson’s Disease.PLoS One 9, e115029(2014).
63. Zheng Y., Wang L., Zhang W., Xu H.& Chang, X. Transgenic mice over-expressing carbonic anhydrase I showed aggravated joint inflammation and tissue destruction.BMC Musculoskelet. Disord. 13, 256(2012).
64. Chang, X.et al.Increased expression of carbonic anhydrase I in the synovium of patients with ankylosing spondylitis.BMC Musculoskelet. Disord. 11, 279(2010).
65. Bertoldi, C.et al.Non-bacterial protein expression in periodontal pockets by proteome analysis.J. Clin. Periodontol. 40, 573-582 (2013).
66. Bogdan A. R., Miyazawa M., Hashimoto K.& Tsuji, Y. Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease.Trends Biochem. Sci. 41, 274-286 (2016).
67. Sun, X.et al.HSPB1 as a novel regulator of ferroptotic cancer cell death.Oncogene 34, 5617-5625 (2015).
68. Chen, H.et al.Heat shock protein 27 downregulates the transferrin receptor 1-mediated iron uptake.Int. J. Biochem. Cell Biol. 38, 1402-1416 (2006).
69. Koppula, P., Zhuang, L.& Gan, B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy.Protein Cell 12, 599-620 (2021).
70. Kang, Y. P.et al. Non-canonical Glutamate-Cysteine Ligase Activity Protects against Ferroptosis. Cell Metab. 33, 174-189.e7 (2021).
71. Tonetti, M. S., Greenwell, H.& Kornman, K. S. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition.J. Clin. Periodontol. 45, S149-S161 (2018).
72. Gupta, S., Chhina, S.& Arora, S. A. A systematic review of biomarkers of gingival crevicular fluid: Their predictive role in diagnosis of periodontal disease status.J. Oral. Biol. Craniofac Res. 8, 98-104 (2018).
73. Zhao Y., Li J., Guo W., Li H.& Lei, L. Periodontitis-level butyrate-induced ferroptosis in periodontal ligament fibroblasts by activation of ferritinophagy.Cell Death Discov. 6, 119(2020).
74. Naruishi K.& Nagata, T. Biological effects of interleukin-6 on Gingival Fibroblasts: Cytokine regulation in periodontitis.J. Cell Physiol. 233, 6393-6400 (2018).
75. Hajishengallis G.& Korostoff, J. M. Revisiting the Page & Schroeder model: the good, the bad and the unknowns in the periodontal host response 40 years later.Periodontology 75, 116-151 (2017).
76. Denton C. P.& Ong, V. H. Interleukin-6 and Related Proteins as Biomarkers in Systemic Sclerosis.J. Scleroderma Relat. Disord. 2, S13-S19 (2017).
77. Liu, J., Wang, Y.& Ouyang, X. Beyond Toll-Like Receptors: Porphyromonas gingivalis Induces IL-6, IL-8, and VCAM-1 Expression Through NOD-Mediated NF-κB and ERK Signaling Pathways in Periodontal Fibroblasts.Inflammation 37, 522-533 (2014).
78. Sundararaj, K. P.et al.Interleukin-6 Released from Fibroblasts Is Essential for Up-regulation of Matrix Metalloproteinase-1 Expression by U937 Macrophages in Coculture.J. Biol. Chem. 284, 13714-13724 (2009).
79. Bartold P. M.& Haynes, D. R. Interleukin-6 production by human gingival fibroblasts.J. Periodontal. Res. 26, 339-345 (1991).
80. Boström, E. A.et al.Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines.PLoS One 10, e0134608(2015).
81. Naruishi, K.et al.Role of soluble interleukin-6 receptor in inflamed gingiva for binding of interleukin-6 to gingival fibroblasts.J. Periodontal. Res. 34, 296-300 (1999).
82. Scheres, N.et al.Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis.J. Periodontal. Res. 46, 407-416 (2011).
83. Takashiba, S., Naruishi, K.& Murayama, Y. Perspective of Cytokine Regulation for Periodontal Treatment: Fibroblast Biology.J. Periodontol. 74, 103-110 (2003).
84. Kinsella, M. G., Bressler, S. L.& Wight, T. N. The Regulated Synthesis of Versican, Decorin, and Biglycan: Extracellular Matrix Proteoglycans That Influence Cellular Phenotype.Crit. Rev. Eukaryot. Gene Expr. 14, 203-234 (2004).
85. Dong, Y., Zhong, J.& Dong, L. The Role of Decorin in Autoimmune and Inflammatory Diseases.J. Immunol. Res. 2022, 1-11 (2022).
86. Liu, J.et al.DCN released from ferroptotic cells ignites AGER-dependent immune responses.Autophagy 18, 2036-2049 (2022).
87. Frey H., Schroeder N., Manon-Jensen, T., Iozzo, R. V. & Schaefer, L. Biological interplay between proteoglycans and their innate immune receptors in inflammation.FEBS J. 280, 2165-2179 (2013).
88. Järvinen T. A.H. & Ruoslahti, E. Targeted Antiscarring Therapy for Tissue Injuries.Adv. Wound Care 2, 50-54 (2013).
89. Edwards I. J., Xu H., Wright M. J.& Wagner, W. D. Interleukin-1 upregulates decorin production by arterial smooth muscle cells.Arterioscler Thromb. 14, 1032-1039 (1994).
90. Edwards, I. J., Wagner, W. D.& Owens, R. T. Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells.Am. J. Pathol. 136, 609-621 (1990).
91. Mauviel A., Santra M., Chen Y. Q., Uitto J.& Iozzo, R. V. Transcriptional Regulation of Decorin Gene Expression.J. Biol. Chem. 270, 11692-11700 (1995).
92. Kawamoto, A.et al.Expression of decorin in inflamed human gingival tissue.J. Osaka Dent. Univ. 35, 93-97 (2001).
93. Kawamoto, A., Okazaki, J.& Komasa, Y. Expression of decorin mRNA and protein in human gingival fibroblasts induced by interleukin-1β.J. Osaka Dent. Univ. 2, 145-153 (2002).
94. Pan, S.et al.Identification of ferroptosis, necroptosis, and pyroptosis-associated genes in periodontitis-affected human periodontal tissue using integrated bioinformatic analysis.Front. Pharmacol. 13, 1098851(2023).
95. Linkermann A., Stockwell B. R., Krautwald S.& Anders, H.-J. Regulated cell death and inflammation: an auto-amplification loop causes organ failure.Nat. Rev. Immunol. 14, 759-767 (2014).
96. Galluzzi, L.et al.Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.Cell Death Differ. 25, 486-541 (2018).
97. Jiang Y., Zhou X., Cheng L.& Li, M. The Impact of Smoking on Subgingival Microflora: From Periodontal Health to Disease.Front. Microbiol. 11, 66(2020).
98. Morozumi T., Kubota T., Sato T., Okuda K.& Yoshie, H. Smoking cessation increases gingival blood flow and gingival crevicular fluid.J. Clin. Periodontol. 31, 267-272 (2004).
99. Stein S. H., Wendell K. J., Pabst M.& Scarbecz, M. Profiling gingival crevicular fluid from smoking and non-smoking chronic periodontitis patients.J. Tenn. Dent. Assoc. 86, 20-24 (2006).
100. Jafri, Z., Bhardwaj, A.& Sawai, M. & Sultan, N. Influence of female sex hormones on periodontium: A case series.J. Nat. Sci. Biol. Med. 6, 146(2015).
101. Hajishengallis G.& Chavakis, T. Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities.Nat. Rev. Immunol. 21, 426-440 (2021).
102. Hernández M., Martínez B., Tejerina J. M., Valenzuela M. A.& Gamonal, J. MMP-13 and TIMP-1 determinations in progressive chronic periodontitis.J. Clin. Periodontol. 34, 729-735 (2007).
103. Williams, D. W.et al. Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity. Cell 184, 4090-4104.e15 (2021).
104. Hao, Y.et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01767-y (2023).
105. The UniProt Consortium. UniProt: the universal protein knowledgebase.Nucleic Acids Res. 45, D158-D169 (2017).
106. Ge, S. X., Jung, D.& Yao, R. ShinyGO: a graphical gene-set enrichment tool for animals and plants.Bioinformatics 36, 2628-2629 (2020).
107. Szklarczyk, D.et al.The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible.Nucleic Acids Res. 45, D362-D368 (2017).
108. Shannon, P.et al.Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks.Genome Res. 13, 2498-2504 (2003).
109. Chin, C.-H.et al.cytoHubba: identifying hub objects and sub-networks from complex interactome.BMC Syst. Biol. 8, S11(2014).
110. Fontaine J.-F., Priller F., Barbosa-Silva, A. & Andrade-Navarro, M. A. Génie: literature-based gene prioritization at multi genomic scale.Nucleic Acids Res. 39, W455-W461 (2011).
111. Zhou, N.et al.FerrDb V2: update of the manually curated database of ferroptosis regulators and ferroptosis-disease associations.Nucleic Acids Res. 51, D571-D582 (2023).
112. Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences.Nucleic Acids Res. 50, D543-D552 (2022).
113. Deutsch, E. W.et al.The ProteomeXchange consortium at 10 years: 2023 update.Nucleic Acids Res. 51, D1539-D1548 (2023).
114. Higdon R.& Kolker, E. A predictive model for identifying proteins by a single peptide match.Bioinformatics 23, 277-280 (2007).