Background: Epidemiological observational studies investigating the association between cathepsins and autoimmune diseases have shown inconsistent results. Hence, we conducted a Mendelian randomization analysis to assess the potential causal impact of cathepsins on these diseases. Methods: Employing a two-sample Mendelian randomization analysis, we used single nucleotide polymorphisms as instrumental variables to examine the impact of cathepsins on autoimmune diseases. The research comprised univariable and multivariable Mendelian randomization analyses, focusing on individual and combined effects of cathepsins. Statistical techniques included inverse variance weighted method and supplementary methods like MR-Egger for comprehensive assessment. Results: In our Mendelian randomization study, we identified diverse associations between cathepsins and autoimmune diseases. Specifically, cathepsin G was found to significantly increase the risk of myasthenia gravis, while the effects of cathepsin B on rheumatoid arthritis and systemic lupus erythematosus varied. Furthermore, multivariable analysis revealed significant correlations between cathepsins F, G and Z with myasthenia gravis. Importantly, no evidence of reverse causation or horizontal pleiotropy was observed. Conclusion: The study establishes a significant causal relationship between cathepsin G and myasthenia gravis risk.

1. Cao F, Liu YC, Ni QY, et al. Temporal trends in the prevalence of autoimmune diseases from 1990 to 2019. Autoimmunity Reviews. 2023; 22(8): 103359. doi: 10.1016/j.autrev.2023.103359

2. Okada Y, Yamamoto K. Genetics and functional genetics of autoimmune diseases. Seminars in Immunopathology. 2022; 44(1): 1-2. doi: 10.1007/s00281-022-00915-x

3. Olek MJ. Multiple Sclerosis. Annals of Internal Medicine. 2021; 174(6): ITC81-ITC96. doi: 10.7326/aitc202106150

4. Finckh A, Gilbert B, Hodkinson B, et al. Global epidemiology of rheumatoid arthritis. Nature Reviews Rheumatology; 2022.

5. Barber MRW, Drenkard C, Falasinnu T, et al. Global epidemiology of systemic lupus erythematosus. Nature Reviews Rheumatology. 2021; 17(9): 515-532. doi: 10.1038/s41584-021-00668-1

6. Li DP, Han YX, He YS, et al. A global assessment of incidence trends of autoimmune diseases from 1990 to 2019 and predicted changes to 2040. Autoimmunity Reviews. 2023; 22(10): 103407. doi: 10.1016/j.autrev.2023.103407

7. Lenti MV, Rossi CM, Melazzini F, et al. Seronegative autoimmune diseases: A challenging diagnosis. Autoimmunity Reviews. 2022; 21(9): 103143. doi: 10.1016/j.autrev.2022.103143

8. Sechi E, Flanagan EP. Antibody-Mediated Autoimmune Diseases of the CNS: Challenges and Approaches to Diagnosis and Management. Frontiers in Neurology. 2021; 12. doi: 10.3389/fneur.2021.673339

9. Xu F, Fei Z, Dai H, et al. Mesenchymal Stem Cell‐Derived Extracellular Vesicles with High PD‐L1 Expression for Autoimmune Diseases Treatment. Advanced Materials. 2021; 34(1). doi: 10.1002/adma.202106265

10. Cao F, He YS, Wang Y, et al. Global burden and cross-country inequalities in autoimmune diseases from 1990 to 2019. Autoimmunity Reviews. 2023; 22(6): 103326. doi: 10.1016/j.autrev.2023.103326

11. Jiang H, Dong Z, Xia X, et al. Cathepsins in oral diseases: mechanisms and therapeutic implications. Frontiers in Immunology. 2023; 14. doi: 10.3389/fimmu.2023.1203071

12. Pišlar A, Bolčina L, Kos J. New Insights into the Role of Cysteine Cathepsins in Neuroinflammation. Biomolecules. 2021; 11(12): 1796. doi: 10.3390/biom11121796

13. Smyth P, Sasiwachirangkul J, Williams R, et al. Cathepsin S (CTSS) activity in health and disease - A treasure trove of untapped clinical potential. Molecular Aspects of Medicine. 2022; 88: 101106. doi: 10.1016/j.mam.2022.101106

14. Wang Y, Zhao J, Gu Y, et al. Cathepsin H: Molecular characteristics and clues to function and mechanism. Biochemical Pharmacology. 2023; 212: 115585. doi: 10.1016/j.bcp.2023.115585

15. Hook G, Reinheckel T, Ni J, et al. Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders. Pharmacological Reviews. 2022; 74(3): 600-629. doi: 10.1124/pharmrev.121.000527

16. Mustafa A, Elkhamisy F, Arghiani N, et al. Potential crosstalk between pericytes and cathepsins in the tumour microenvironment. Biomedicine & Pharmacotherapy. 2023; 164: 114932. doi: 10.1016/j.biopha.2023.114932

17. Ruiz-Blázquez P, Pistorio V, Fernández-Fernández M, et al. The multifaceted role of cathepsins in liver disease. Journal of Hepatology. 2021; 75(5): 1192-1202. doi: 10.1016/j.jhep.2021.06.031

18. Stoka V, Vasiljeva O, Nakanishi H, et al. The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer. International Journal of Molecular Sciences. 2023; 24(21): 15613. doi: 10.3390/ijms242115613

19. Behl T, Chadha S, Sehgal A, et al. Exploring the role of cathepsin in rheumatoid arthritis. Saudi Journal of Biological Sciences. 2022; 29(1): 402-410. doi: 10.1016/j.sjbs.2021.09.014

20. Shibamura-Fujiogi M, Yuki K, Hou L. Cathepsin L regulates pathogenic CD4 T cells in experimental autoimmune encephalomyelitis. International Immunopharmacology. 2021; 93: 107425. doi: 10.1016/j.intimp.2021.107425

21. Tato M, Kumar SV, Liu Y, et al. Cathepsin S inhibition combines control of systemic and peripheral pathomechanisms of autoimmune tissue injury. Scientific Reports. 2017; 7(1). doi: 10.1038/s41598-017-01894-y

22. Li J, Chen Z, Kim G, et al. Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence. Science Advances. 2023; 9(28). doi: 10.1126/sciadv.adf3924

23. Toomey CB, Cauvi DM, Hamel JC, et al. Cathepsin B Regulates the Appearance and Severity of Mercury-Induced Inflammation and Autoimmunity. Toxicological Sciences. 2014; 142(2): 339-349. doi: 10.1093/toxsci/kfu189

24. Dheilly E, Battistello E, Katanayeva N, et al. Cathepsin S Regulates Antigen Processing and T Cell Activity in Non-Hodgkin Lymphoma. Cancer Cell. 2020; 37(5): 674-689.e12. doi: 10.1016/j.ccell.2020.03.016

25. Schwenck J, Maurer A, Fehrenbacher B, et al. Cysteine-type cathepsins promote the effector phase of acute cutaneous delayed-type hypersensitivity reactions. Theranostics. 2019; 9(13): 3903-3917. doi: 10.7150/thno.31037

26. Galibert M, Wartenberg M, Lecaille F, et al. Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S. European Journal of Medicinal Chemistry. 2018; 144: 201-210. doi: 10.1016/j.ejmech.2017.12.012

27. Vidak E, Javoršek U, Vizovišek M, et al. Cysteine Cathepsins and Their Extracellular Roles: Shaping the Microenvironment. Cells. 2019; 8(3): 264. doi: 10.3390/cells8030264

28. Zhang TP, Li HM, Leng RX, et al. Plasma levels of adipokines in systemic lupus erythematosus patients. Cytokine. 2016; 86: 15-20. doi: 10.1016/j.cyto.2016.07.008

29. Kim J, Ahn M, Choi Y, et al. Upregulation of Cathepsins in Olfactory Bulbs Is Associated with Transient Olfactory Dysfunction in Mice with Experimental Autoimmune Encephalomyelitis. Molecular Neurobiology. 2020; 57(8): 3412-3423. doi: 10.1007/s12035-020-01952-z

30. Sasawatari S, Okamura T, Kasumi E, et al. The Solute Carrier Family 15A4 Regulates TLR9 and NOD1 Functions in the Innate Immune System and Promotes Colitis in Mice. Gastroenterology. 2011; 140(5): 1513-1525. doi: 10.1053/j.gastro.2011.01.041

31. Suyama M, Koike M, Asaoka D, et al. Increased Immunoreactivity of Cathepsins in the Rat Esophagus under Chronic Acid Reflux Esophagitis. Journal of Histochemistry & Cytochemistry. 2014; 62(9): 645-660. doi: 10.1369/0022155414542300

32. Emdin CA, Khera AV, Kathiresan S. Mendelian Randomization. JAMA. 2017; 318(19): 1925. doi: 10.1001/jama.2017.17219

33. Lawlor DA, Harbord RM, Sterne JAC, et al. Mendelian randomization: Using genes as instruments for making causal inferences in epidemiology. Statistics in Medicine. 2008; 27(8): 1133-1163. doi: 10.1002/sim.3034

34. Davies NM, Holmes MV, Davey Smith G. Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ; 2018.

35. Sun BB, Maranville JC, Peters JE, et al. Genomic atlas of the human plasma proteome. Nature. 2018; 558(7708): 73-79. doi: 10.1038/s41586-018-0175-2

36. Burgess S, Davey Smith G, Davies NM, et al. Guidelines for performing Mendelian randomization investigations. Wellcome Open Research. 2019; 4: 186. doi: 10.12688/wellcomeopenres.15555.1

37. Bowden J, Del Greco M F, Minelli C, et al. Improving the accuracy of two-sample summary-data Mendelian randomization: moving beyond the NOME assumption. International Journal of Epidemiology. 2018; 48(3): 728-742. doi: 10.1093/ije/dyy258

38. Bowden J, Davey Smith G, Burgess S. Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. International Journal of Epidemiology. 2015; 44(2): 512-525. doi: 10.1093/ije/dyv080

39. Coss SL, Zhou D, Chua GT, et al. The complement system and human autoimmune diseases. Journal of Autoimmunity. 2023; 137: 102979. doi: 10.1016/j.jaut.2022.102979

40. Xiao ZX, Miller JS, Zheng SG. An updated advance of autoantibodies in autoimmune diseases. Autoimmunity Reviews. 2021; 20(2): 102743. doi: 10.1016/j.autrev.2020.102743

41. Eggenhuizen PJ, Ng BH, Ooi JD. Treg Enhancing Therapies to Treat Autoimmune Diseases. International Journal of Molecular Sciences. 2020; 21(19): 7015. doi: 10.3390/ijms21197015

42. Schett G, Mackensen A, Mougiakakos D. CAR T-cell therapy in autoimmune diseases. Lancet. 2023; 402(10416): 2034-2044. doi: 10.1016/S0140-6736(23)01126-1

43. t Hart BA. A Tolerogenic Role of Cathepsin G in a Primate Model of Multiple Sclerosis: Abrogation by Epstein–Barr Virus Infection. Archivum Immunologiae et Therapiae Experimentalis. 2020; 68(4). doi: 10.1007/s00005-020-00587-1

44. Khan M, Carmona S, Sukhumalchandra P, et al. Cathepsin G Is Expressed by Acute Lymphoblastic Leukemia and Is a Potential Immunotherapeutic Target. Frontiers in Immunology. 2018; 8. doi: 10.3389/fimmu.2017.01975

45. Luo L, Chen H, Xie K, et al. Cathepsin B serves as a potential prognostic biomarker and correlates with ferroptosis in rheumatoid arthritis. International Immunopharmacology. 2024; 128: 111502. doi: 10.1016/j.intimp.2024.111502

46. Panwar P, Andrault PM, Saha D, et al. Immune regulatory and anti‐resorptive activities of tanshinone IIA sulfonate attenuates rheumatoid arthritis in mice. British Journal of Pharmacology. 2024; 181(24): 5009-5027. doi: 10.1111/bph.17312

47. Su Y, Han Y, Choi HS, et al. Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis. Biomedicine & Pharmacotherapy. 2024; 171: 116153. doi: 10.1016/j.biopha.2024.116153

48. Song T, Yao L, Zhu A, et al. Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma. International Journal of Nanomedicine. 2024; 19: 8059-8070. doi: 10.2147/ijn.s455633

49. Chen H, Wan L, Qiu Y, et al. Microplastics exposure induced and exacerbated the development of systemic lupus erythematosus in mice. Science of The Total Environment. 2024; 909: 168586. doi: 10.1016/j.scitotenv.2023.168586

50. Kawato Y, Fukahori H, Nakamura K, et al. Development of a novel Poly (I: C)-induced murine model with accelerated lupus nephritis and examination of the therapeutic effects of mycophenolate mofetil and a cathepsin S inhibitor. European Journal of Pharmacology. 2023; 938: 175440. doi: 10.1016/j.ejphar.2022.175440

51. Xie Z, Zhao M, Yan C, et al. Cathepsin B in programmed cell death machinery: mechanisms of execution and regulatory pathways. Cell Death & Disease. 2023; 14(4). doi: 10.1038/s41419-023-05786-0

52. Ma H, Ou Z lin, Alaeiilkhchi N, et al. MiR-223 enhances lipophagy by suppressing CTSB in microglia following lysolecithin-induced demyelination in mice. Lipids in Health and Disease. 2024; 23(1). doi: 10.1186/s12944-024-02185-y

53. Fettucciari K, Marguerie F, Fruganti A, et al. Clostridioides difficile toxin B alone and with pro-inflammatory cytokines induces apoptosis in enteric glial cells by activating three different signalling pathways mediated by caspases, calpains and cathepsin B. Cellular and Molecular Life Sciences. 2022; 79(8). doi: 10.1007/s00018-022-04459-z

54. Mishiro T, Nakano S, Takahara S, et al. Relationship between cathepsin B and thrombin in rheumatoid arthritis. The Journal of Rheumatology. 2004; 31(7): 1265-73.

55. Tong B, Wan B, Wei Z, et al. Role of cathepsin B in regulating migration and invasion of fibroblast-like synoviocytes into inflamed tissue from patients with rheumatoid arthritis. Clinical and Experimental Immunology. 2014; 177(3): 586-597. doi: 10.1111/cei.12357

56. Kala M, Chen C, McLachlan SM, et al. Cathepsin S is not crucial to TSHR processing and presentation in a murine model of Graves’ disease. Immunology. 2005; 116(4): 532-540. doi: 10.1111/j.1365-2567.2005.02255.x

57. Wu Y, Li Q, Lou Y, et al. Cysteine cathepsins and autoimmune diseases: A bidirectional Mendelian randomization. Medicine. 2024; 103(43): e40268. doi: 10.1097/md.0000000000040268

58. Yang H, Kala M, Scott BG, et al. Cathepsin S Is Required for Murine Autoimmune Myasthenia Gravis Pathogenesis. The Journal of Immunology. 2005; 174(3): 1729-1737. doi: 10.4049/jimmunol.174.3.1729