experimental-autoimmune-uveitis-an-intraocular-inflammatory-mouse

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

University College London

Isocitrate dehydrogenase (IDH) genes 1 and 2 are frequently mutated in acute myeloid leukaemia (AML), low-grade glioma, cholangiocarcinoma (CC) and chondrosarcoma (CS). For AML, low-grade glioma and CC, mutant IDH status is associated with a DNA hypermethylation phenotype, implicating altered epigenome dynamics in the aetiology of these cancers. Here we show that the IDH variants in CS are also associated with a hypermethylation phenotype and display increased production of the oncometabolite 2-hydroxyglutarate, supporting the role of mutant IDH-produced 2-hydroxyglutarate as an inhibitor of TET-mediated DNA demethylation. Meta-analysis of the acute myeloid leukaemia, low-grade glioma, cholangiocarcinoma and CS methylation data identifies cancer-specific effectors within the retinoic acid receptor activation pathway among the hypermethylated targets. By analysing sequence motifs surrounding hypermethylated sites across the four cancer types, and using chromatin immunoprecipitation and western blotting, we identify the transcription factor EBF1 (early B-cell factor 1) as an interaction partner for TET2, suggesting a sequence-specific mechanism for regulating DNA methylation. 

Meta-analysis of IDH-mutant cancers identifies EBF1 as an interaction partner for TET2.

The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.

T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

Experimental autoimmune uveitis (EAU), in which CD4 Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4 T cells in vitro, downregulating levels of Th17 cells. Additionally, the uveitogenic capacity of Th17 cells to transfer EAU was abrogated by BET inhibitors in an adoptive transfer model. In human CD4 T cells, a 5-d exposure to BET inhibitors was accompanied by a significant downregulation of Th17-associated genes IL-17A, IL-22, and retinoic acid-related orphan receptor γt. However, in vitro, the inhibitors had no effect on already polarized Th17 cells. The key finding is that, in response to BET inhibitors, Th17-enriched cultures developed a regulatory phenotype, upregulated FOXP3 expression and IL-10 secretion, and lost pathogenicity in vivo. We conclude that BET targeting of Th17 cells is a potential therapeutic opportunity for a wide range of inflammatory and autoimmune diseases, including uveitis.

Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.

The most prevalent form of bone cancer is osteosarcoma (OS), which is associated with poor prognosis in case of metastases formation. Mice harbouring liver kinase B1 (LKB1) develop osteoblastoma-like tumours. Therefore, we asked whether loss of LKB1 gene has a role in the pathogenesis of human OS.

Post-translational regulation contributes to the loss of LKB1 expression through SIRT1 deacetylase in osteosarcomas.

Immunopathogenic roles for both Th1 (CD4 IFN-γ ) and Th17 (CD4 IL-17A ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4 T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Functionally distinct IFN-γ IL-17A Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B (LTB) and inhibiting complement component 5 (C5) activation. Nomacopan has been shown to provide therapeutic benefit in experimental autoimmune uveitis (EAU). Longer acting forms of nomacopan were more efficacious in mouse EAU models, and the long-acting variant that inhibited only LTB was at least as effective as the long-acting variant that inhibited both C5 and LTB, preventing structural damage to the retina and a significantly reducing effector T helper 17 cells and inflammatory macrophages. Increased levels of LTB and C5a (produced upon C5 activation) were detected during disease progression. Activated retinal lymphocytes were shown to express LTB receptors (R) in vitro and in inflamed draining lymph nodes. Levels of LTBR-expressing active/inflammatory retinal macrophages were also increased. Within the draining lymph node CD4 T-cell population, 30% expressed LTBR following activation in vitro, whereas retinal infiltrating cells expressed LTBR and C5aR. Validation of expression of those receptors in human uveitis and healthy tissues suggests that infiltrating cells could be targeted by inhibitors of the LTB-LTB4 receptor 1 (BLT1) pathway as a novel therapeutic approach. This study provides novel data on intraocular LTB and C5a in EAU, their associated receptor expression by retinal infiltrating cells in mouse and human tissues, and in attenuating EAU via the dual inhibitor nomacopan.

Leukotriene B and Its Receptor in Experimental Autoimmune Uveitis and in Human Retinal Tissues: Clinical Severity and LTB Dependence of Retinal Th17 Cells.

The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

Retinal vascular diseases have distinct, complex and multifactorial pathogeneses yet share several key pathophysiological aspects including inflammation, vascular permeability and neovascularisation. In non-infectious posterior uveitis (NIU), retinal vasculitis involves vessel leakage leading to retinal enlargement, exudation, and macular oedema. Neovascularisation is not a common feature in NIU, however, detection of the major angiogenic factor-vascular endothelial growth factor A (VEGF-A)-in intraocular fluids in animal models of uveitis may be an indication for a role for this cytokine in a highly inflammatory condition. Suppression of VEGF-A by directly targeting the leukotriene B4 (LTB4) receptor (BLT1) pathway indicates a connection between leukotrienes (LTs), which have prominent roles in initiating and propagating inflammatory responses, and VEGF-A in retinal inflammatory diseases. Further research is needed to understand how LTs interact with intraocular cytokines in retinal inflammatory diseases to guide the development of novel therapeutic approaches targeting both inflammatory mediator pathways.

Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis.

Non-infectious uveitis (NIU) is an inflammatory eye disease initiated via CD4 T-cell activation and transmigration, resulting in focal retinal tissue damage and visual acuity disturbance. Cell adhesion molecules (CAMs) are activated during the inflammatory process to facilitate the leukocyte recruitment cascade. Our review focused on CAM-targeted therapies in experimental autoimmune uveitis (EAU) and NIU. We concluded that CAM-based therapies have demonstrated benefits for controlling EAU severity with decreases in immune cell migration, especially via ICAM-1/LFA-1 and VCAM-1/VLA-4 (integrin) pathways. P-selectin and E-selectin are more involved specifically in uveitis related to vasculitis. These therapies have potential clinical applications for the development of a more personalized and specific treatment. Localized therapies are the future direction to avoid serious systemic side effects.

Article	Year
Meta-analysis of IDH-mutant cancers identifies EBF1 as an interaction partner for TET2. Nature communications\| PubMed ID: 23863747	2013
T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex. Cell reports\| PubMed ID: 27292648	2016
Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 28039300	2017
Post-translational regulation contributes to the loss of LKB1 expression through SIRT1 deacetylase in osteosarcomas. British journal of cancer\| PubMed ID: 28632727	2017
Functionally distinct IFN-γ IL-17A Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response. European journal of immunology\| PubMed ID: 32652562	2020
Leukotriene B and Its Receptor in Experimental Autoimmune Uveitis and in Human Retinal Tissues: Clinical Severity and LTB Dependence of Retinal Th17 Cells. The American journal of pathology\| PubMed ID: 33159884	2021
Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis. Journal of neuroinflammation\| PubMed ID: 33602234	2021
Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis. Cells\| PubMed ID: 33671954	2021
Adhesion Molecule Targeted Therapy for Non-Infectious Uveitis. International journal of molecular sciences\| PubMed ID: 35008929	2022

Malihe Eskandarpour

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