POS0081 MACROPHAGES DRIVE IL-6 PRODUCTION BY FIBROBLASTS THROUGH GALECTIN 3 IN POLYMYALGIA RHEUMATICA AND GIANT CELL ARTERITIS

Abstract

Background:

Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are closely related autoimmune diseases that often occur together. PMR and GCA may form a spectrum encompassing musculoskeletal and arterial inflammation, respectively. Glucocorticoids remain the mainstay treatment. IL-6 receptor targeting therapies have recently emerged as glucocorticoid-sparing agents in PMR and GCA, thereby highlighting the central role of IL-6 in PMR/GCA. We previously identified fibroblasts as potent sources of IL-6 [1, 2]. However, the mechanisms underlying this aberrant IL-6 production are not fully understood. Galectins, which are glycan-binding proteins, appear to be central regulators in the immune system, with galectin 3 playing a prominent pro-inflammatory role in various autoimmune diseases. While galectin 3 shows little effect on macrophages [3], it may induce expression of IL-6 by fibroblasts [4]. To date, it is unknown whether galectin 3 could contribute to the pathogenic IL-6 response in PMR/GCA.

Objectives:

• 1)To determine serum levels of galectin 3 in patients with PMR or GCA.
• 2)To investigate the expression of galectin 3 in PMR- and GCA-affected tissues.
• 3)To examine the effects of galectin 3 on the production of IL-6 by human fibroblasts.

Methods:

Serum levels of galectin 3 were measured by quantitative enzyme-linked immunosorbent assay (ELISA) in patients with newly diagnosed, treatment-naive PMR (n=28) or GCA (n=29), as well as in age- and sex-matched healthy controls (n=25). Ultrasound-guided biopsies were obtained from the subacromial bursa of patients with active PMR (n=9). Temporal artery biopsies (n=9) and aortic aneurysm tissues (n=10) were obtained from patients with GCA, either at diagnosis or during aortic replacement surgery, respectively. Immunohistochemistry staining was performed for galectin 3 and scored quantitatively. Opal triple immunofluorescence staining for CD68, CD3, and galectin 3 was performed to identify the cellular source of galectins. Monocytes isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (n=3) were differentiated into macrophages by culturing the cells with GM-CSF and M-CSF for 8 days and adding IFN-γ for the last 24 hours, as previously described [5]. These three macrophage-polarizing cytokines are abundant in GCA and PMR affected tissues [5, 6]. Galectin 3 was measured by ELISA in macrophage supernatants. Human aortic adventitial fibroblasts from a healthy donor (n=1) were purchased from PromoCell (Germany). Synovial fibroblasts were isolated from synovial fluid obtained from the subacromial bursa of a patient with active PMR (n=1) and dermal fibroblasts were obtained from the skin of a patient with cancer (n=1). Fibroblasts were cultured with or without galectin 3 for 24 hours. Galectin 3 was measured by ELISA in untreated fibroblasts supernatants. ELISA and qPCR were performed to determine the expression of IL-6 and CCL2, a macrophage attracting chemokine, in the cultured cells. Statistical analysis was performed by the Mann Whitney U test. P values <0.05 were considered statistically significant.

Results:

Serum level of galectin 3 were statistically significantly higher in patients with PMR (median 7.63 ng/ml, range 3.5- 11.87) than in patients with GCA (median 6.06 ng/ml, 0.85- 18.54) and HCs (median 5.80 ng/ml, 2.70- 11.95), whereas serum levels of galectin 3 were similar in GCA patients and HCs. Galectin 3 was expressed in both the PMR-affected bursa tissue and the GCA-affected arterial tissues. The percentage of galectin 3 expressing cells was significantly higher in PMR bursa (median 56%, 28-90) than in GCA-affected temporal artery (median 22%, 0-75) and aorta (median 10%, 0-38). Opal triple staining indicated that infiltrating macrophages, and to a lesser extent T cells, were the major source of galectin 3 in all tissues. In vitro studies confirmed that monocyte-derived macrophages produce galectin 3 when differentiated under the influence of macrophage-polarizing cytokines known to be expressed in the GCA/PMR tissue micro-environment (i.e., GM-CSF, M-CSF and IFN- γ) [5, 6]. Human fibroblasts did not produce galectin 3 in vitro. However, the addition of galectin 3 prominently boosted the production of IL-6 by fibroblasts. Furthermore, it enhanced the production of the macrophage attractant CCL2. Figure 1The hypothetic role of galectin 3 in PMR and GCA. Monocyte-derived macrophages exposed to GM-CSF, M-CSF and IFN-γ produce galectin 3, which steers fibroblasts towards a pro-inflammatory phenotype, as indicated by increased IL-6 and CCL2 production.

Conclusion:

Although galectin 3 is expressed in all GCA/PMR-affected tissues, this glycan-binding protein is most prominently expressed in tissues and serum of patients with PMR. Macrophages are the main source of galectin 3 in the tissues. This glycan-binding protein may contribute to the pathogenic mechanisms of PMR/GCA by enhancing IL-6 production in fibroblasts and promoting macrophage recruitment through the release of CCL2. These findings highlight galectin-3 as a potential critical mediator in the pathogenic crosstalk between macrophages and fibroblasts in PMR/GCA, driving disease progression.

REFERENCES:

[1] Jiemy WF, et al. IL-6 in synovial tissue in polymyalgia rheumatica. Ann Rheum Dis 2023;82(3):440-42. [2] Xu S, et al. Fibroblast activation protein in GCA. Arthritis Care Res 2024;76(9):1322-32. [3] Novak R, et al. Galectin-1 and -3 in human macrophages. Biochim Biophys Acta 2012;1820(9):1383-90. [4] Filer A, et al. Galectin-3 in rheumatoid synovial fibroblasts. Arthritis Rheum 2009;60(6):1604-14. [5] Jiemy WF, et al. Macrophage phenotypes in GCA. Clin Transl Immunol 2020;9(9):e1164. [6] Jiemy WF & Zhang A et al. GM-CSF drives IL-6 in PMR. Ann Rheum Dis 2024;In Press.

Acknowledgements:

We thank the patients that participated in our study.

Disclosure of Interests:

Anqi Zhang: None declared, Wayel Abdulahad: None declared, Shuang Xu: None declared, Clementine Sey: None declared, Elien Hensema: None declared, Peter Heeringa: None declared, Arjan Diepstra: None declared, Sandra Hein: None declared, Elisabeth Brouwer speaker fees from Roche in 2017-2018, outside the submitted work, consulting fees from Roche in 2017-2018, outside the submitted work, William F. Jiemy: None declared, Maria Sandovici consulting fees from AbbVie, outside the submitted work, Kornelis van der Geest received fees from Roche and AbbVie, outside the submitted work. © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.