An Early Wave of Macrophage Infiltration Intertwined with Antigen-Specific Proinflammatory T Cells and Browning of Adipose Tissue Characterizes the Onset of Orbital Inflammation in a Mouse Model of Graves' Orbitopathy
Abstract
Background: Graves' orbitopathy (GO) is an autoimmune-driven manifestation of Graves' disease (GD) where pathogenic autoantibodies to the thyrotropin receptor (TSHR) activate orbital fibroblasts/preadipocytes in the orbital tissue to induce inflammation and extracellular matrix deposition. Since there are significant limitations to study immunological and proinflammatory mediator expression in early and during disease progression in GO patients, we used our experimental mouse model to elucidate early pathogenic processes.
Methods: We have developed a robust mouse model of GD/GO induced by electroporation immunization of plasmid encoding human TSHR A-subunit, comprising multiple injections over a course of 15 weeks to fully recapitulate the orbital pathology. In this study, we investigated kinetics of GO development in the model by serial analyses of immunological and cellular parameters during course of orbital inflammation.
Results: Pathogenic anti-TSHR antibodies with thyroid-stimulating properties developed early after the second immunization step with concomitant induction of hyperthyroidism. Examination of orbital tissue showed an early wave of macrophage infiltration followed subsequently by CD3+ T cells into the orbital tissue. Examination of antigen-specific T cell activity using recombinant human A-subunit protein showed high CD8+ T cell proliferation during this early phase of disease onset, whereas effector CD4+ T cells and CD25+FOXP3+ regulatory T cells (Tregs) were downregulated. The early phase of disease was also characterized by abundant presence of proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Moreover, as the disease progressed, there was significant increase in browning of orbital fat tissue, which may be dependent on the proinflammatory milieu and/or the increased thyroid hormone levels during the established hyperthyroid status.
Conclusions: This work revealed early infiltration of macrophages in the orbital region and induction of pathogenic anti-TSHR antibodies during disease onset in the model. This was followed subsequently by influx of CD8+ T cells specific for TSHR coupled with reduction in Tregs and substantial increase in brown adipose tissue. These new insights into the development of orbital inflammation in the model have implications for testing new therapeutic regimens by targeting macrophage function during early phases of orbital inflammation in the model.
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References
1. Davies TF, Andersen S, Latif R, Nagayama Y, Barbesino G, Brito M, Eckstein AK, Stagnaro-Green A, Kahaly GJ 2020 Graves' disease. Nat Rev Dis Primers 6:52.
2. Dik WA, Virakul S, van Steensel L 2016 Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves' ophthalmopathy. Exp Eye Res 142:83–91.
3. Krieger CC, Place RF, Bevilacqua C, Marcus-Samuels B, Abel BS, Skarulis MC, Kahaly GJ, Neumann S, Gershengorn MC 2016 TSH/IGF-1 receptor cross talk in Graves' ophthalmopathy pathogenesis. J Clin Endocrinol Metab 101:2340–2347.
4. Wiersinga WM, Smit T, van der Gaag R, Koornneef L 1988 Temporal relationship between onset of Graves' ophthalmopathy and onset of thyroidal Graves' disease. J Endocrinol Invest 11:615–619.
5. Eckstein AK, Plicht M, Lax H, Neuhauser M, Mann K, Lederbogen S, Heckmann C, Esser J, Morgenthaler NG 2006 Thyrotropin receptor autoantibodies are independent risk factors for Graves' ophthalmopathy and help to predict severity and outcome of the disease. J Clin Endocrinol Metab 91:3464–3470.
6. Kahaly GJ, Diana T, Kanitz M, Frommer L, Olivo PD 2020 Prospective trial of functional thyrotropin receptor antibodies in Graves disease. J Clin Endocrinol Metab 105:e1006–e1014.
7. Stohr M, Oeverhaus M, Lytton SD, Horstmann M, Zwanziger D, Moller L, Stark A, Fuhrer-Sakel D, Bechrakis N, Berchner-Pfannschmidt U, Banga JP, Philipp S, Eckstein A 2021 Predicting the relapse of hyperthyroidism in treated Graves' disease with orbitopathy by serial measurements of TSH-receptor autoantibodies. Horm Metab Res 53:235–244.
8. Stohr M, Oeverhaus M, Lytton SD, Horstmann M, Zwanziger D, Moller L, Stark A, Fuhrer-Sakel D, Bechrakis N, Berchner-Pfannschmidt U, Banga JP, Philipp S, Eckstein A 2021 Predicting the course of Graves' orbitopathy using serially measured TSH-receptor autoantibodies by automated binding immunoassays and the functional bioassay. Horm Metab Res 53:435–443.
9. Pappa A, Lawson JM, Calder V, Fells P, Lightman S 2000 T cells and fibroblasts in affected extraocular muscles in early and late thyroid associated ophthalmopathy. Br J Ophthalmol 84:517–522.
10. Pappa A, Calder V, Ajjan R, Fells P, Ludgate M, Weetman AP, Lightman S 1997 Analysis of extraocular muscle-infiltrating T cells in thyroid-associated ophthalmopathy (TAO). Clin Exp Immunol 109:362–369.
11. Smith TJ 2005 Insights into the role of fibroblasts in human autoimmune diseases. Clin Exp Immunol 141:388–397.
12. Rotondo Dottore G, Torregrossa L, Caturegli P, Ionni I, Sframeli A, Sabini E, Menconi F, Piaggi P, Sellari-Franceschini S, Nardi M, Latrofa F, Vitti P, Marcocci C, Basolo F, Marino M 2018 Association of T and B cells infiltrating orbital tissues with clinical features of Graves orbitopathy. JAMA Ophthalmol 136:613–619.
13. Moshkelgosha S, So PW, Deasy N, Diaz-Cano S, Banga JP 2013 Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation. Endocrinology 154:3008–3015.
14. Banga JP, Moshkelgosha S, Berchner-Pfannschmidt U, Eckstein A 2015 Modeling Graves' orbitopathy in experimental Graves' disease. Horm Metab Res 47:797–803.
15. Berchner-Pfannschmidt U, Moshkelgosha S, Diaz-Cano S, Edelmann B, Gortz GE, Horstmann M, Noble A, Hansen W, Eckstein A, Banga JP 2016 Comparative assessment of female mouse model of Graves' orbitopathy under different environments, accompanied by proinflammatory cytokine and T-cell responses to thyrotropin hormone receptor antigen. Endocrinology 157:1673–1682.
16. Eckstein A, Philipp S, Goertz G, Banga JP, Berchner-Pfannschmidt U 2020 Lessons from mouse models of Graves' disease. Endocrine 68:265–270.
17. Schluter A, Flogel U, Diaz-Cano S, Gortz GE, Stahr K, Oeverhaus M, Plohn S, Mattheis S, Moeller LC, Lang S, Bechrakis NE, Banga JP, Eckstein A, Berchner-Pfannschmidt U 2018 Graves' orbitopathy occurs sex-independently in an autoimmune hyperthyroid mouse model. Sci Rep 8:13096.
18. Moshkelgosha S, Verhasselt HL, Masetti G, Covelli D, Biscarini F, Horstmann M, Daser A, Westendorf AM, Jesenek C, Philipp S, Diaz-Cano S, Banga JP, Michael D, Plummer S, Marchesi JR, Eckstein A, Ludgate M, Berchner-Pfannschmidt U, consortium I 2021 Modulating gut microbiota in a mouse model of Graves' orbitopathy and its impact on induced disease. Microbiome 9:45.
19. Plohn S, Hose M, Schluter A, Michel L, Diaz-Cano S, Hendgen-Cotta UB, Banga JP, Bechrakis NE, Hansen W, Eckstein A, Berchner-Pfannschmidt U 2019 Fingolimod improves the outcome of experimental Graves' disease and associated orbitopathy by modulating the autoimmune response to the thyroid-stimulating hormone receptor. Thyroid 29:1286–1301.
20. Schluter A, Horstmann M, Diaz-Cano S, Plohn S, Stahr K, Mattheis S, Oeverhaus M, Lang S, Flogel U, Berchner-Pfannschmidt U, Eckstein A, Banga JP 2018 Genetic immunization with mouse thyrotrophin hormone receptor plasmid breaks self-tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy. Clin Exp Immunol 191:255–267.
21. Zhang M, Ding X, Wu LP, He MQ, Chen ZY, Shi BY, Wang Y 2021 A promising mouse model of Graves' orbitopathy induced by adenovirus expressing thyrotropin receptor A subunit. Thyroid 31:638–648.
22. Park S, Park DY, Kim J, Woo KI, Kim YD, Han J, Chung TY, Cha HS, Lim DH 2020 Enhanced orbital adipogenesis in a mouse model of T-cell-mediated autoimmunity, zymosan A-treated SKG mice: implications for Graves' ophthalmopathy. Sci Rep 10:7329.
23. Chen MH, Chen MH, Liao SL, Chang TC, Chuang LM 2008 Role of macrophage infiltration in the orbital fat of patients with Graves' ophthalmopathy. Clin Endocrinol (Oxf) 69:332–337.
24. Eckstein AK, Quadbeck B, Tews S, Mann K, Kruger C, Mohr CH, Steuhl KP, Esser J, Gieseler RK 2004 Thyroid associated ophthalmopathy: evidence for CD4(+) gammadelta T cells; de novo differentiation of RFD7(+) macrophages, but not of RFD1(+) dendritic cells; and loss of gammadelta and alphabeta T cell receptor expression. Br J Ophthalmol 88:803–808.
25. Pawlowski P, Reszec J, Eckstein A, Johnson K, Grzybowski A, Chyczewski L, Mysliwiec J 2014 Markers of inflammation and fibrosis in the orbital fat/connective tissue of patients with Graves' orbitopathy: clinical implications. Mediators Inflamm 2014:412158.
26. Pawlowski P, Wawrusiewicz-Kurylonek N, Eckstein A, Reszec J, Luczynski W, Johnson K, Kretowski A, Bakunowicz-Lazarczyk A, Gorska M, Szamatowicz J, Chyczewski L, Mysliwiec J 2015 Disturbances of modulating molecules (FOXP3, CTLA-4/CD28/B7, and CD40/CD40L) mRNA expressions in the orbital tissue from patients with severe graves' ophthalmopathy. Mediators Inflamm 2015:340934.
27. Zhang L, Baker G, Janus D, Paddon CA, Fuhrer D, Ludgate M 2006 Biological effects of thyrotropin receptor activation on human orbital preadipocytes. Invest Ophthalmol Vis Sci 47:5197–5203.
28. Zhang L, Evans A, von Ruhland C, Draman MS, Edkins S, Vincent AE, Berlinguer-Palmini R, Rees DA, Haridas AS, Morris D, Tee AR, Ludgate M, Turnbull DM, Karpe F, Dayan CM 2020 Distinctive features of orbital adipose tissue (OAT) in Graves' orbitopathy. Int J Mol Sci 21:9145.
29. Fassbender J, Holthoff HP, Li Z, Ungerer M 2019 Therapeutic effects of short cyclic and combined epitope peptides in a long-term model of Graves' disease and orbitopathy. Thyroid 29:258–267.
30. Noh JY, Hamada N, Inoue Y, Abe Y, Ito K, Ito K 2000 Thyroid-stimulating antibody is related to Graves' ophthalmopathy, but thyrotropin-binding inhibitor immunoglobulin is related to hyperthyroidism in patients with Graves' disease. Thyroid 10:809–813.
31. Kampmann E, Diana T, Kanitz M, Hoppe D, Kahaly GJ 2015 Thyroid stimulating but not blocking autoantibodies are highly prevalent in severe and active thyroid-associated orbitopathy: a prospective study. Int J Endocrinol 2015:678194.
32. Goh SY, Ho SC, Seah LL, Fong KS, Khoo DH 2004 Thyroid autoantibody profiles in ophthalmic dominant and thyroid dominant Graves' disease differ and suggest ophthalmopathy is a multiantigenic disease. Clin Endocrinol (Oxf) 60:600–607.
33. Enzmann DR, Donaldson SS, Kriss JP 1979 Appearance of Graves' disease on orbital computed tomography. J Comput Assist Tomogr 3:815–819.
34. Vaidya B, Shenton BK, Stamp S, Miller M, Baister E, Andrews CD, Dickinson AJ, Perros P, Kendall-Taylor P 2005 Analysis of peripheral blood T-cell subsets in active thyroid-associated ophthalmopathy: absence of effect of octreotide-LAR on T-cell subsets in patients with thyroid-associated ophthalmopathy. Thyroid 15:1073–1078.
35. Matsuzawa K, Izawa S, Okura T, Fujii S, Matsumoto K, Shoji K, Nakamura R, Sumi K, Fujioka Y, Yoshida A, Shigemasa C, Kato M, Yamamoto K, Taniguchi S 2016 Implications of FoxP3-positive and -negative CD4(+) CD25(+) T cells in Graves' ophthalmopathy. Endocr J 63:755–764.
36. Al-Ansari F, Lahooti H, Stokes L, Edirimanne S, Wall J 2018 Correlation between thyroidal and peripheral blood total T cells, CD8(+) T cells, and CD8(+) T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves' ophthalmopathy. Endocr Res 43:264–274.
37. Slowik M, Urbaniak-Kujda D, Bohdanowicz-Pawlak A, Kapelko-Slowik K, Dybko J, Wolowiec D, Jazwiec B, Daroszewski J 2012 CD8+CD28-lymphocytes in peripheral blood and serum concentrations of soluble interleukin 6 receptor are increased in patients with Graves' orbitopathy and correlate with disease activity. Endocr Res 37:89–95.
38. Shen J, Li Z, Li W, Ge Y, Xie M, Lv M, Fan Y, Chen Z, Zhao D, Han Y 2015 Th1, Th2, and Th17 cytokine involvement in thyroid associated ophthalmopathy. Dis Markers 2015:609593.
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Copyright 2022, Mary Ann Liebert, Inc., publishers.
History
Published online: 17 March 2022
Published in print: March 2022
Published ahead of print: 11 January 2022
Published ahead of production: 13 November 2021
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Authors' Contributions
Idea: U.B.-P., A.E., and J.P.S.B. Experimental design: S.P., U.B.-P., and J.P.S.B. Performance of experiments: S.P., M.Hor., M.Hos., A.D., C.J., and U.F. Analysis and interpretation of data: S.P., M.Hos., G.-E.G., W.H., N.B., J.P.S.B., A.E., and U.B.-P. Article writing: S.P. and J.P.S.B. Revising the article: all authors.
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No competing financial interests exist.
Funding Information
This study was supported by funding from the Deutsche Forschungsgemeinschaft (DFG Grant No. BE1377/5-1 to U.B.-P., A.E., J.P.S.B.).
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