Inflammatory response of cultured macrophages in treatment-naive patients with systemic sclerosis

Background. Сhronic inflammation is one of the main factors in the progression of systemic sclerosis (SSc). Macrophages activated via a proinflammatory pathway can be considered as major participants in the maintaining of system chronic lowgrade inflammation.

The aim of this study was to evaluate the inflammatory response and of macrophages in patients with systemic sclerosis to reveal the most significant inflammatory mediators in pathogenesis of this disease.

Materials and methods. The study included 34 treatment-naive SSc patients and 17 controls. Macrophages were obtained by culturing peripheral blood monocytes. The macrophage response was analyzed by deviations in the parameters of basal, lipopolysaccharide (LPS) stimulated, and restimulated secretion of the tumor necrosis factor α (TNF-α), interleukin (IL) 1β, C-C motif ligand 2 (CCL2), and IL-8 by cultured macrophages in SSc patients compared to the control group. The levels of basal and LPS-stimulated secretion were assessed on day 1. The second LPS stimulation was performed on day 7 to assess the cell response to repeated stimulation (restimulated secretion) after the first stimulation to characterize the resistance of the macrophage immune response. Cell resistance (tolerance) was calculated as the ratio of secretion during repeated stimulation to LPS-stimulated secretion. Concentrations of TNF-α, IL-1β, CCL2, and IL-8 cytokines in the culture fluid were determined out using an enzyme-linked immunosorbent assay.

Results. Basal and restimulated secretion of all studied cytokines was significantly higher in the SSc group compared to the control group; LPS-stimulated secretion was statistically significantly higher in the SSс group only for IL-1β. Impaired resistance of the immune tolerance of macrophages to CCL2 was detected in 50% of treatment-naive SSc patients.

Conclusions. The results of the study demonstrate a pro-inflammatory response of macrophages with increased levels of basal and restimulated secretion of TNF-α, IL-1β, CCL2 and IL-8, as well as impaired tolerance of the immune response of macrophages in treatment-naive SSc patients in relation to the secretion of CCL2. These data indicate the active participation of CCL2 in the development of chronic inflammation in SSc that can be considered as a target for the development of new therapeutic approaches for SSc.

1. Volkmann ER, Andréasson K, Smith V. Systemic sclerosis. Lancet. 2023;401(10373):304-318. doi: 10.1016/S0140-6736(22)01692-0

2. Moore DF, Steen VD. Racial disparities in systemic sclerosis. Rheum Dis Clin North Am. 2020;46(4):705-712. doi: 10.1016/J.RDC.2020.07.009

3. Coffey CM, Radwan YA, Sandhu AS, Crowson CS, Bauer PR, Matteson EL, et al. Epidemiology and trends in survival of systemic sclerosis in Olmsted county (1980–2018): A population-based study. J Scleroderma Relat Disord. 2021;6(3):264-270. doi: 10.1177/23971983211026853

4. Li JX. Secular trends in systemic sclerosis mortality in the United States from 1981 to 2020. Int J Environ Res Public Health. 2022;19(22):15088. doi: 10.3390/ijerph192215088

5. Bazsó A, Szodoray P, Shoenfeld Y, Kiss E. Biomarkers reflecting the pathogenesis, clinical manifestations, and guide therapeutic approach in systemic sclerosis: A narrative review. Clin Rheumatol. 2024;43:3055-3072. doi: 10.1007/s10067-024-07123-y

6. Truchetet ME, Brembilla NC, Chizzolini C. Current concepts on the pathogenesis of systemic sclerosis. Clin Rev Allergy Immunol. 2023;64(3):262-283. doi: 10.1007/s12016-021-08889-8

7. Trombetta AC, Soldano S, Contini P, Tomatis V, Ruaro B, Paolino S, et al. A circulating cell population showing both M1 and M2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement. Respir Res. 2018;19(1):186. doi: 10.1186/s12931-018-0891-z

8. Cutolo M, Soldano S, Smith V. Pathophysiology of systemic sclerosis: Current understanding and new insights. Expert Rev Clin Immunol. 2019;15(7):753-764. doi: 10.1080/1744666X.2019.1614915

9. Peng Y, Zhou M, Yang H, Qu R, Qiu Y, Hao J, et al. Regulatory mechanism of M1/M2 macrophage polarization in the development of autoimmune diseases. Mediators Inflamm. 2023;2023:8821610. doi: 10.1155/2023/8821610

10. Yang S, Zhao M, Jia S. Macrophage: Key player in the pathogenesis of autoimmune diseases. Front Immunol. 2023;14:1080310. doi: 10.3389/fimmu.2023.1080310

11. Bekkering S, Blok BA, Joosten LA, Riksen NP, van Crevel R, Netea MG. In vitro experimental model of trained innate immunity in human primary monocytes. Clin Vaccine Immunol. 2016;23(12):926-933. doi: 10.1128/CVI.00349-16

12. Simpson E. Medawar’s legacy to cellular immunology and clinical transplantation: A commentary on Billingham, Brent and Medawar (1956) ‘Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance.’ Philos Trans R Soc Lond B Biol Sci. 2015;370(1666):20140382. doi: 10.1098/RSTB.2014.0382

13. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance’ of foreign cells. Nature. 1953;172:603-606.

14. Murphy K. Autoimmunity and transplantation. In: Murphy K, Weaver C (eds). Janeway’s Immunobiology. 2015. URL: https://www.numerade.com/books/chapter/autoimmunity-and-transplantation/ (Accessed: 1st November 2024).

15. Jeljeli M, Riccio LGC, Doridot L, Chêne C, Nicco C, Chouzenoux S, et al. Trained immunity modulates inflammation-induced fibrosis. Nat Commun. 2019;10(1):5670. doi: 10.1038/s41467-019-13636-x

16. Kalashnikova SA, Polyakova LV. The use of bacterial lipopolysaccharide for pathological processes modeling in biomedical research (literature review). Journal of New Medical Technologies. 2017;24(2):209-219 (In Russ.). doi: 10.12737/article_5947d50a4ddf68.91843258

17. Perosa F, Prete M, Di Lernia G, Ostuni C, Favoino E, Valentini G. Anti-centromere protein A antibodies in systemic sclerosis: Significance and origin. Autoimmun Rev. 2016;15(1):102-109. doi: 10.1016/J.AUTREV.2015.10.001

18. Tiniakou E, Crawford J, Darrah E. Insights into origins and specificities of autoantibodies in systemic sclerosis. Curr Opin Rheumatol. 2021;33(6):486-494. doi: 10.1097/BOR.0000000000000834

19. Murdaca G, Spanò F, Contatore M, Guastalla A, Puppo F. Potential use of TNF-α inhibitors in systemic sclerosis. Immunotherapy. 2014;6(3):283-289. doi: 10.2217/imt.13.173

20. Xu D, Mu R, Wei X. The roles of IL-1 family cytokines in the pathogenesis of systemic sclerosis. Front Immunol. 2019;10:2025. doi: 10.3389/fimmu.2019.02025

21. Lin C, Jiang Z, Cao L, Zou H, Zhu X. Role of NLRP3 inflammasome in systemic sclerosis. Arthritis Res Ther. 2022;24(1):196. doi: 10.1186/s13075-022-02889-5

22. Carvalheiro T, Horta S, van Roon JAG, Santiago M, Salvador MJ, Trindade H, et al. Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients. Inflamm Res. 2018;67(2):169-177. doi: 10.1007/s00011-017-1106-7

23. Rudnik M, Hukara A, Kocherova I, Jordan S, Schniering J, Milleret V, et al. Elevated fibronectin levels in profibrotic CD14+ monocytes and CD14+ macrophages in systemic sclerosis. Front Immunol. 2021;12:642891. doi: 10.3389/fimmu.2021.642891

24. Codullo V, Baldwin HM, Singh MD, Fraser AR, Wilson C, Gilmour A, et al. An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis. Ann Rheum Dis. 2011;70(6):1115-1121. doi: 10.1136/ard.2010.137349

25. Al-Adwi Y, Westra J, van Goor H, Burgess JK, Denton CP, Mulder DJ. Macrophages as determinants and regulators of fibrosis in systemic sclerosis. Rheumatology (Oxford). 2023;62(2):535-545. doi: 10.1093/rheumatology/keac410

26. Distler JH, Akhmetshina A, Schett G, Distler O. Monocyte chemoattractant proteins in the pathogenesis of systemic sclerosis. Rheumatology (Oxford). 2009;48(2):98-103. doi: 10.1093/rheumatology/ken401

27. Netea MG, Quintin J, van der Meer JW. Trained immunity: A memory for innate host defense. Cell Host Microbe. 2011;9(5):355-361. doi: 10.1016/j.chom.2011.04.006

28. Badii M, Gaal O, Popp RA, Crișan TO, Joosten LAB. Trained immunity and inflammation in rheumatic diseases. Joint Bone Spine. 2022;89(4):105364. doi: 10.1016/j.jbspin.2022.105364

29. Bhandari R, Ball MS, Martyanov V, Popovich D, Schaafsma E, Han S, et al. Profibrotic activation of human macrophages in systemic sclerosis. Arthritis Rheumatol. 2020;72(7):1160-1169. doi: 10.1002/art.41243

30. Antonelli A, Ferri C, Fallahi P, Ferrari SM, Giuggioli D, Colaci M, et al. CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosis – A longitudinal study. Rheumatology (Oxford). 2008;47(1):45-49. doi: 10.1093/rheumatology/kem313

31. Hasegawa M, Fujimoto M, Matsushita T, Hamaguchi Y, Takehara K, Sato S. Serum chemokine and cytokine levels as indicators of disease activity in patients with systemic sclerosis. Clin Rheumatol. 2011; 30(2): 231-237. doi: 10.1007/s10067-010-1610-4

32. Kania G, Rudnik M, Distler O. Involvement of the myeloid cell compartment in fibrogenesis and systemic sclerosis. Nat Rev Rheumatol. 2019;15(5):288-302. doi: 10.1038/S41584-019-0212-Z

33. Zanin-Silva DC, Van Kooten N, Papadimitriou T, Dorst D, Walgreen B, Vitters E, et al. Monocytes drive myofibroblast contraction in a 3D skin model used to understand fibrosis in systemic sclerosis Ann Rheum Dis. 2024;83(1):1907-1908.

34. Meli M, Gitzelmann G, Koppensteiner R, Amann-Vesti BR. Predictive value of nailfold capillaroscopy in patients with Raynaud’s phenomenon. Clin Rheumatol. 2006;25(2):153-158. doi: 10.1007/S10067-005-1146-1

35. Spencer-Green G. Outcomes in primary Raynaud phenomenon: A meta-analysis of the frequency, rates, and predictors of transition to secondary diseases. Arch Intern Med. 1998;158(6):595-600. doi: 10.1001/ARCHINTE.158.6.595

36. Bissell LA, Abignano G, Emery P, Del Galdo F, Buch MH. Absence of scleroderma pattern at nail fold capillaroscopy valuable in the exclusion of scleroderma in unselected patients with Raynaud’s phenomenon. BMC Musculoskelet Disord. 2016;17(1):262-283. doi: 10.1186/s12891-016-1206-5