Svenja Philipp

The inflammatory eye disease Graves’ orbitopathy (GO) is the main complication of autoimmune Graves’ disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.

Introduction: The inflammatory eye disease Graves’ orbitopathy (GO) is the main complication of Graves’ disease in patients. In previous studies we have shown that hypoxia and HIF-1 dependent pathways could play an important role in the pathogenic process of GO. Hypoxia is known to attract inflammatory cells and therefore maintains inflammation and recruitment of immune cells like macrophages (MQ). However, few is known about the specific contribution of MQ to the progression of orbitopathy. Therefore, we investigated the role and interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. Methods & Results: We analyzed the expression levels of hypoxic marker HIF-1α, MQ marker CD68, proinflammatory cytokine TNFα and recruitment proteins CCL2, CCL5 and CCL20 in fat biopsies of control and GO patients by real-time PCR. We found that HIF-1α, CD68, TNFα, CCL2, CCL5 and CCL20 mRNA expression was increased in the fat tissue of GO patients. Next, we analyzed the cytokine profile of supernatants from fat biopsies with a multiplex ELISA. We could show an enhanced secretion of TNFα, CCL2 and CCL20 only under hypoxia while CCL5 was induced on protein level under normoxia as well as under hypoxia in GO tissue. An immunofluorescence stain of CD68 and TNFα was used to demonstrate the source of TNFα in the orbital tissue. The Immunofluorescence indicated that TNFα secretion occurs in conjunction with CD68 positive MQ. To further, investigate the inflammatory interaction of MQ and OF, we stimulated OF with TNFα or co-cultured them with M1-MQ from a THP-1 cell line under normoxic and hypoxic conditions. We found that OF expressed hypoxic marker HIF-1α, hypoxia target gene VEGF and immune marker ICAM-1 as well as chemokines CCL2, CCL5 and CCL20 most pronounced upon TNFα stimulation and hypoxia. M1-MQ enhanced the induction of HIF-1α and CCL2 in OF in addition to hypoxia alone, whereas proinflammatory inhibitors Etanercept and dexamethasone reduced this effect. Furthermore, we found that OF-macrophage co-culture enhanced adipogenic differentiation and adiponectin secretion under hypoxia. PX-478, a HIF-1α inhibitor, reduced the adipogenic differentiation of OF significantly. Conclusion: In summary, our results show that hypoxia and macrophage-OF interactions have a cumulative effect. The findings indicate that the inflammatory milieu and hypoxic signaling in the orbit are characterized by TNFα positive macrophages, which interact with OF which results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option for GO.

Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are often challenging to differentiate. Furthermore, it is still uncertain how chronic inflammation, such as IgG4-ROD, can lead to mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, we aimed to evaluate the diagnostic value of gene expression analysis to differentiate orbital autoimmune diseases and elucidate genetic overlaps. First, we established a database of NSOI, relapsing NSOI, IgG4-ROD and MALT lymphoma patients of our orbital center (2000–2019). In a consensus process, three typical patients of the above mentioned three groups (mean age 56.4 ± 17 years) at similar locations were selected. Afterwards, RNA was isolated using the RNeasy FFPE kit (Qiagen) from archived paraffin-embedded tissues. The RNA of these 12 patients were then subjected to gene expression analysis (NanoString nCounter^®), including a total of 1364 target genes. The most significantly upregulated and downregulated genes were used for a machine learning algorithm to distinguish entities. This was possible with a high probability (p < 0.0001). Interestingly, gene expression patterns showed a characteristic overlap of lymphoma with IgG4-ROD and NSOI. In contrast, IgG4-ROD shared only altered expression of one gene regarding NSOI. To validate our potential biomarker genes, we isolated the RNA of a further 48 patients (24 NSOI, 11 IgG4-ROD, 13 lymphoma patients). Then, gene expression pattern analysis of the 35 identified target genes was performed using a custom-designed CodeSet to assess the prediction accuracy of the multi-parameter scoring algorithms. They showed high accuracy and good performance (AUC ROC: IgG4-ROD 0.81, MALT 0.82, NSOI 0.67). To conclude, genetic expression analysis has the potential for faster and more secure differentiation between NSOI and IgG4-ROD. MALT-lymphoma and IgG4-ROD showed more genetic similarities, which points towards progression to lymphoma.

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.