International Journal of Molecular Sciences Review Is There Evidence for IGF1R-Stimulating Abs in Graves’ Orbitopathy Pathogenesis? Christine C. Krieger , Susanne Neumann and Marvin C. Gershengorn * Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, Bethesda, MD 20892, USA; [email protected] (C.C.K.); [email protected] (S.N.) * Correspondence: [email protected]; Tel.: +1-301-451-6305 Received: 31 July 2020; Accepted: 3 September 2020; Published: 8 September 2020 Abstract: In this review, we summarize the evidence against direct stimulation of insulin-like growth factor 1 receptors (IGF1Rs) by autoantibodies in Graves’ orbitopathy (GO) pathogenesis. We describe a model of thyroid-stimulating hormone (TSH) receptor (TSHR)/IGF1R crosstalk and present evidence that observations indicating IGF1R’s role in GO could be explained by this mechanism. We evaluate the evidence for and against IGF1R as a direct target of stimulating IGF1R antibodies (IGF1RAbs) and conclude that GO pathogenesis does not involve directly stimulating IGF1RAbs. We further conclude that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO and maintain that the TSHR should remain a major target for further development of a medical therapy for GO in concert with drugs that target TSHR/IGF1R crosstalk. Keywords: IGF1R; TSHR; receptor crosstalk; Graves’ orbitopathy; IGF1R antibodies; TSHR antibodies; Graves’ orbital fibroblasts; hyaluronan 1. Introduction Graves’ orbitopathy (GO) (also termed Graves’ ophthalmopathy, thyroid-associated ophthalmopathy or thyroid eye disease) has been under intense study with a goal to not only understand its pathogenesis but also to aid in the development of medical therapies. GO pathophysiology appears to involve activation of receptors for thyrotropin (thyroid-stimulating hormone, TSH) and insulin-like growth factor 1 (IGF1) on fibroblasts/preadipocytes in orbital tissue (OFs) [1]. Under normal physiological conditions, TSH receptors (TSHRs), which are G protein-coupled receptors (GPCRs or seven transmembrane-spanning receptors) and IGF1 receptors (IGF1Rs), which are receptor tyrosine kinases (RTKs), are cell-surface proteins that are activated by their respective ligands that, in turn, are themselves under tight regulation. Persistent activation of GPCRs and RTKs is involved in several diseases. For example, activating mutations of TSHRs and IGF1Rs have been shown to cause hyperthyroidism [2] and tumor formation [3], respectively. In autoimmune diseases, such as Graves’ disease (GD), autoantibodies have been found in the circulation that activate receptors consistently. Lack of physiologic regulation leads to overstimulation and disease [4,5]. In GO, a consensus has emerged that there are stimulating autoantibodies that bind to and directly activate TSHRs (TSAbs) on OFs [4,6,7]. However, it remains controversial as to whether there are autoantibodies that bind to and directly activate IGF1Rs (stimulating IGF1RAbs) [8,9]. It appears that cell-mediated immunity plays a role in GO, and it is possible that there are TSHRs and IGF1Rs on lymphocytes that may be involved; however, these receptors would have to be activated by the antibodies. As noted above, a number of studies have found that both TSHRs and IGF1Rs are involved in GO pathogenesis [1,10]. These studies have been performed primarily in vitro using OFs from GO patients (GOFs) or fibroblast-like cells isolated from the blood of GO patients (so-called fibrocytes) [9]. Of note, Int. J. Mol. Sci. 2020, 21, 6561; doi:10.3390/ijms21186561 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6561 2 of 13 the majority of these studies did not attempt to show direct activation of IGF1Rs but instead relied on the ability of immunoglobulins from GO patients (GO-Igs) to either replicate some of the post-receptor effects of IGF1 or have their effects inhibited by IGF1R antagonists. In only three previous reports were effects of GO-Igs on the IGF1R studied [11–13] (see below). An alternative possibility for the involvement of IGF1Rs in GO pathogenesis is that IGF1R involvement results not from stimulating IGF1RAbs but from crosstalk of IGF1Rs with TSHRs that are activated by TSAbs [11]. In this review, we first contrast what is known about TSHR autoantibodies in GD hyperthyroidism and GO to what has been found regarding IGF1R autoantibodies. We describe a model of TSHR/IGF1R crosstalk and present evidence that IGF1R’s role in GO could be explained by this mechanism. Then, we evaluate the evidence for and against IGF1R as a direct target of stimulating IGF1RAbs in GO pathogenesis. Our conclusion that GO pathogenesis does not involve directly stimulating IGF1RAbs will be based, in part, on negative data. Although a negative hypothesis cannot be conclusively proved, we reason that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO. 2. The Role of TSHR and IGF1R Autoantibodies in GO Pathogenesis Although autoantibodies are a major contributing factor to the development of GO, the mechanism through which this occurs is unclear. One hypothesis proposes that shedding of the A-subunit of TSHR leads to the initial autoimmune response including generation of TSAbs. In this scenario, recently reviewed by Rapoport et al. [14], TSHR A-subunits are cleaved from the extra-cellular domain and released into the lymphatic system. Because of its highly glycosylated state, the A-subunit is taken up by antigen-presenting cells. Resulting B-cell proliferation and maturation may lead to the direct cause of GD and GO. This hypothesis is supported by recent efforts to produce animal models for GD and GO [15,16] (reviewed in [17]), which used the human TSHR A-subunit to generate TSAbs in mice. McLachlan et al. further demonstrated that mouse TSHR A-subunit, which was less glycosylated than human, was unable to induce production of TSAbs. These and similar works were instrumental in our current understanding of GD and GO. To our knowledge, no group has attempted to create an animal model of GO using IGF1R as an immunogenic agent. It stands to reason that if GO were a disease induced by IGF1RAbs, this strategy could be employed to create an experimental system of significant value. Such an animal model could aid in understanding GO pathogenesis. It would also facilitate testing of future therapies targeting IGF1R, if IGF1RAbs were the source of orbitopathy. However, as helpful as this animal model may be, the work in human patients investigating the putative role of IGF1RAbs in GO pathogenesis is still in beginning stages. Studying autoantibodies in patients is far from straightforward. Even after isolating antibodies for their specific antigen, they are polyclonal, and their function ranges from stimulating, to neutral, to antagonizing. Antibody populations also change during the progression of disease, as has been documented for TSHR antibodies in GD and GO patients [18,19]. Nevertheless, development of assays that assess function as well as measure titer of TSHR antibodies has greatly aided our ability to use TSHR antibodies as biomarkers of GD and GO (reviewed in [20]). In contrast, investigation and characterization of IGF1RAbs from GO patients have not seen similar progress. Evidence for IGF1RAbs in GO patients was first reported by Weightman et al. in 1993 [21]. In 2013, two contemporary studies set the stage for the still unresolved controversy as to the prevalence and function of IGF1RAbs in GO patients. Minich et al. reported the presence of IGF1RAbs in a subset of GO patients and further characterized the biological function of those IGF1RAbs, finding them to be antagonistic [12]. In contrast, Varewijk et al. found that GO patients with high TSAb titers were more likely to have stimulatory IGF1RAbs [13]. These reports conflicted on the function of IGF1RAbs but employed different methods, precluding direct comparisons. More recently, Marino et al. [22] and Lanzolla et al. [23], both with the University Hospital of Pisa, have measured serum IGF1RAbs in GD and GO patients using a newly available commercial assay for Int. J. Mol. Sci. 2020, 21, 6561 3 of 13 IGF1R antibodies. Unlike the assay used by Varewijk et al., the sandwich ELISA used by Marino and colleagues had the advantage of directly measuring circulating IGF1RAbs and distinguishing them from other antibodies [22]. First, Marino et al. compared the incidence of IGF1RAbs in patients with GD (hyperthyroid), patients with autoimmune thyroiditis (hypothyroid), and healthy subjects (no past or present thyroid disease). Within the GD group, 54/80 subjects had GO. Patients with GD were more likely than other groups to have IGF1RAbs positive sera, however, the presence of IGF1RAbs was not significantly different in patients with GO compared to GD patients without GO. Within the patients with IGF1RAb positive sera, Marino et al. next compared IGF1RAb concentrations amongst groups. Again, GD patients had higher concentrations, but no significant difference was found between GD patients with or without GO. Surprisingly, IGF1RAb concentration negatively correlated with GO severity. The authors inferred those IGF1RAbs were antagonistic, though this was not tested in a functional assay. Lanzolla et al. more deeply investigated possible differences between GD patients with or without GO by measuring serum IGF1RAbs in the same