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KIMURA OCULAR IMMUNOLOGY LABORATORY
Autoimmune mechanisms in a new spontaneous model of Sjögren’s syndrome
J. DeVoss, N. LeClair, C. Meagher, K. Johannes, M.S. Anderson and E.C. Strauss
Autoimmunity results from a breakdown in tolerance to self-antigens; moreover, a complex mix of genetic and environmental factors is also considered to play a role in this process. For example, genetic mapping suggests that multiple loci appear to be involved with a predisposition for manifesting an autoimmune disease. In addition, these genetic loci presumably interact with the environment, and this dynamic interplay facilitates the breakdown of tolerance and the development of autoimmunity. As a result of the complexity of these processes, it has been difficult to investigate the mechanisms mediating autoimmune disease. Although rare, autoimmune disease resulting from defects in a single gene can provide an invaluable opportunity to acquire fundamental insights into the mechanisms initiating and sustaining autoimmunity. One such disease is autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also referred to as autoimmune polyendocrinopathy syndrome type I. This syndrome is characterized by adrenal insufficiency, hypoparathyroidism, and mucocutaneous candidiasis. In addition to the APECED syndrome, these patients also develop severe Sjögren’s syndrome (SS) with keratoconjunctivitis sicca and ocular surface inflammation (Figure 1). APECED is a monogenic syndrome that results from mutations in the autoimmune regulator (Aire) gene. The Aire gene is primarily expressed in thymic medullary epithelial and monocyte-derived cells. Within these cells, Aire controls the expression of ectopic antigens and plays a pivotal role in central tolerance. Aire exerts its influence on central tolerance by upregulating ectopic antigens, which are then processed and presented to developing thymocytes (Figure 2). Thymocytes that bind to the self-antigens with high affinity will undergo apoptosis, culling the immune repertoire of potentially self-reactive cells. In Aire-deficient mice, and presumably in APECED patients, the lack of Aire expression results in an inability to present these self-antigens to developing thymocytes. In this environment, self-reactive thymocytes survive, resulting in a population of mature, autoreactive T cells capable of mediating autoimmune disease. Our investigations in Aire-deficient mice clearly show that these autoreactive T cells migrate to the lacrimal gland and salivary gland and induce foci of spontaneous autoimmune exocrinopathy consistent with SS (Figure 3). This phenotype can be transferred to immunodeficient mice by lymphocytes from Aire-deficient mice further demonstrating the autoimmune nature of this spontaneous disease model (Figure 4). The autoimmune infiltrate promotes severe functional impairment of the exocrine glands resulting in progressive and profound ocular surface pathology and impaired salivary secretion. Furthermore, multiplex screenings with lacrimal and salivary gland protein extracts show that the autoimmune response is both oligoclonal and specific. With mass spectrometry analysis, we have identified potential biomarkers and target autoantigens in these exocrine glands; genes for these candidate autoantigens are Aire regulated. Our results suggest that SS autoimmunity is mediated by both cellular and humoral responses. We anticipate the results from our studies will facilitate the development of target-specific therapies for SS patients. Finally, translational studies have been initiated to study and validate candidate biomarkers and autoantigens in Sjögren’s patients.
Figure 1. Severe keratoconjunctivitis sicca and ocular
surface inflammatory changes in an APECED patient.
Figure 2. Model of Aire expression of tissue-specific genes in the thymus showing that negative selection of autoreactive thymocytes is critical to prevent autoimmunity.
Figure 3. Immunohistochemistry of lacrimal glands from Aire-deficient mice showing foci (brown staining) of infiltration of CD4+, CD8+, and IgD+ lymphocytes (control = 2O alone ).
Figure 4. Adoptive transfer of lymphocytes from Aire-deficient mice to immunodeficient RAG mice showing foci (brown staining) of predominantly CD4+ and CD8+ lymphocytic infiltrate; a few IgD+ B cells were also observed (control = 2O alone).
Molecular and cellular mechanisms in inflammatory angiogenesis
C. Meagher, M. Nakanishi, W.C. Greene and E.C. Strauss
Angiogenesis, the formation of new from preexisting blood vessels, is a complex, multistep process that involves expression of cytokines and intracellular adhesion molecules, recruitment of leukocytes, expression of growth factors, extracellular matrix remodeling, endothelial cell migration and proliferation, and vessel formation. Pathologic angiogenesis is a critical component of tumorigenesis, chronic systemic inflammatory disorders such as rheumatoid arthritis, and several sight-threatening eye diseases including age-related macular degeneration and corneal inflammatory angiogenesis. VEGF-A has emerged as the preeminent growth factor promoting pathologic angiogenesis. Two other members of the VEGF family, VEGF-C and VEGF-D, have been shown to be essential in pathologic lymphangiogenesis, a process mediating tumor metastasis and immune rejection of corneal allografts. The concept of modulating angiogenesis dependent diseases represents a powerful approach to treatment. However, the results from clinical trials targeting angiogenic growth factor activity have not always replicated the remarkable efficacy shown in preclinical model studies. Insights from cancer investigations suggest the timing of anti-angiogenic therapy appears to be critical as early therapeutic intervention is associated with more favorable outcomes. Moreover, with disease progression, expansion of the spectrum of proangiogenic factors may contribute to treatment resistance and require multiple anti-angiogenic agents to effect results. A potential complementary or alternative strategy to specific inhibition of angiogenic and lymphangiogenic growth factors or their receptors may reside in selective targeting of proximal signaling master regulators that control and coordinate angiogenesis and lymphangiogenesis. Accumulating evidence suggests a major integration and codependence of inflammation with the processes of pathologic angiogenesis and lymphangiogenesis. However, the underlying signaling mechanism initiating and promoting the coordinate regulation of angiogenesis, lymphangiogenesis, and inflammation remains an open question.
The IKK2/NF-kB signaling pathway functions as a master regulator of inflammatory responses and serves as an essential link between inflammation and tumorigenesis. We investigated the hypothesis that IKK2 plays a critical and essential role in the coordinate regulation of inflammatory angiogenesis and lymphangiogenesis. In a novel mouse model of pathologic vascularization, our results show that selective inhibition of IKK2 with a small molecule antagonist prevents the onset of VEGF-A induced inflammatory angiogenesis and VEGF-C/VEGF-D stimulated lymphangiogenesis (Figure 5). Moreover, active inflammatory angiogenic and lymphangiogenic responses are rapidly arrested following selective inhibition of IKK2. These results were confirmed in mice with genetic deficiency of the p50 subunit of NF-kB. Our studies indicate that IKK2 initiates and controls VEGF-A induced angiogenesis and VEGF-D stimulated lymphangiogenesis through c-fos mediated AP-1 activity, and VEGF-C is controlled directly by IKK2/NF-kB (Figure 6). Our findings demonstrate that IKK2 is an essential upstream regulatory link coordinating the processes of inflammatory angiogenesis and lymphangiogenesis. These results suggest the intriguing possibility that IKK2 inhibition may represent a novel therapeutic strategy for preventing or arresting pathologic angiogenesis and lymphangiogenesis in diseases characterized by these processes. In ongoing research, we have recently identified the essential cellular and cytokine inflammatory axis promoting inflammatory angiogenesis and lymphangiogenesis.
Figure 5. Selective inhibition of IKK2 prevents inflammatory angiogenesis and lymphangiogenesis. Appearance of representative corneas from three groups of mice: no cautery control at 72 hours treated with vehicle (left panel), post-cauterization at 72 hours treated with vehicle (center panel), and post-cauterization at 72 hours treated with the selective IKK2 inhibitor MLN-1145 (right panel). Corneal flat mounts assessed by immunofluorescent staining of blood vessels (bv) with CD31 (FITC, green) and lymphatic vessels (lv) with LYVE-1 (Cy3, red); Corneal histology sections stained with hematoxylin and eosin from representative mice.
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