Thesis defense Lude Moekotte

Thursday, September 26, 2024 at 12:15 PM - Thursday, September 26, 2024 at 1:15 PM
Thesis defense Lude Moekotte

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Inherited retinal degenerations (IRDs) were long considered untreatable, but recently considerable advances have been made in genetic therapies aimed to correct the causal mutations in genes. Through better understanding of the pathophysiology of IRDs and insights in factors influencing the disease course, these approaches may be improved and halt or reverse visual decline of this blinding family of conditions. There is emerging evidence suggesting that the immune system contributes to the progression of the disease. This thesis contributes to this body of knowledge by investigating the role of the immune system in IRDs.

We outlined the state-of-the-art and the tools for disease management of retinitis pigmentosa (RP). RP is the most common form of IRD, and characterized by degeneration of rods, followed by the degeneration of cones. RP is caused by pathogenic variants in around 100 genes, and clinically highly heterogeneous, likely due to the involvement of additional yet poorly understood environmental and (epi)genetic disease modifiers. There is a wide range of ocular comorbidities associated with RP, such as cataract, cystoid macular edema (CME), retinal detachment, uveitis, and glaucoma. Because molecular studies have shown that RP patients exhibit elevated levels of pro-inflammatory cytokines and chemokines in their eyes, it has been hypothesized that inflammatory pathways contribute to the development of these comorbidities. Such pro-inflammatory profiles were shown to be more pronounced in patients with a younger age of onset of symptoms and in patients with an overall lower visual function. Because cataract is also associated with younger age of disease onset, we speculate that it may be related to increased eye inflammation. CME occurs frequently in RP patients, especially in patients carrying mutations in the Crumbs Homolog 1 (CRB1) gene. Furthermore, CRB1-IRDs are more often associated with uveitis than other types of RP, suggesting an increased susceptibility to immunological abnormalities, as supported by disease models and immunohistochemical studies. Breakdown of the blood-retinal barrier and autoimmunity have been hypothesized to be part of the pathophysiology of CME in RP patients. This is supported by the fact that retinal autoantibodies have been found more frequently in IRD patients with CME compared to IRD patients without CME. Our findings are also in agreement with this, where we detected a disbalance in antibody positive IgA+CD24+ and IgG+CD27+ B cells in CRB1-IRD patients. These findings support the hypothesis of blood-retina barrier breakdown and inflammatory processes that deteriorate retinal function. Regarding treatment, Carbonic anhydrase inhibitors (CAI), anti-vascular endothelial growth factor (anti-VEGF) injections, and intravitreal injection of a dexamethasone implant seem to have positive effect on the symptoms of CME in RP patients, and systemic usage of CAIs hold a superior effect over local topical usage of CAIs. There are also various investigational treatment modalities, one of which are genetic therapies, which should start in early disease stages. Currently, one genetic therapy, designed to correct mutations in the RPE65 gene, has been approved for clinical practice, and several others are currently in clinical trials. There are, however, several immune-mediated adverse reactions associated with these gene therapies. A better understanding of the underlying mechanisms of inflammation can help develop immunoregulatory treatments that minimize side effects.

Previous literature described that CRB1-IRDs are associated with more immunological abnormalities than other types of RP. We therefore studied the alterations in molecular pathways interacting with or forming the immune system in CRB1-IRD patients. We used flow cytometry in peripheral blood samples of a cohort of 33 patients with a CRB1-IRD and 32 healthy controls to phenotype the composition of immune cell subsets. We discovered an expansion of CD4+ T cells and CD8+ T cells carrying tissue homing markers on their cell surface, such as sialyl Lewis X antigen (or CLA) and CD103, as well as a decrease of plasmacytoid dendritic cells (pDCs), in patients with a CRB1-IRD compared to controls. The increased frequency of circulating tissue homing-positive T cells, such as sialyl Lewis X antigen, correlate with disease severity in other autoimmune conditions, such as psoriasis. T cell activation induces the expression of homing receptors that bind ligands on peripheral vasculature and allow migration into organs and tissues, affecting immune regulation and response within tissues. The upregulation of T cells with CD103 is particularly interesting, because while it is also found on T cells resident to eye tissues, such as the cornea and retina, CD103 is primarily found in T cells that home towards the mucosa of the gut. IRD models support the hypothesis that gut dysbiosis exacerbates retinal degeneration by promoting inflammatory-related pathways, and in chapter 5 we found compelling evidence of an altered gut metabolism fueling inflammation in patients with a CRB1-IRD. We showed that patients systemically have lower concentrations of secondary bile acids, which are known to have neuroprotective properties and protect against photoreceptor degeneration. Some secondary bile acids, such as deoxycholic acid (DCA) and their derivatives, are known to have a regulatory (i.e., anti-inflammatory) effect on T cell activation. Several commensal residents of the human gut microbiome are required to metabolize secondary bile acids from primary bile acids, produced in the liver. This makes it likely that in our cohort of patients changes in the composition of these essential microbial colonies contribute to decreased concentration of secondary bile acids, which may result in more unchecked T cell activation in response to eye tissue damage. One mechanism could be similar to uveitis, where the secondary bile acid DCA directly influences dendritic cell activation and decreases the severity of eye inflammation. While such mechanistic studies with dendritic cells in CRB1-IRDs are currently lacking, in chapter 3, we discovered a decrease of plasmacytoid dendritic cells (pDCs) in patients with a CRB1-IRD. pDCs are antigen presenting cells that have been described to be decreased in circulation in numerous inflammatory conditions, including uveitis. The results of these molecular studies suggest that changes in gut commensal-dependent metabolites, dendritic cells, and T cells interact in CRB1-IRDs. It is conceivable that patients with a CRB1-IRD exhibit changes in T cell activation due to changes in their gut microbiome, which in turn exacerbates their immune responses to deteriorating eye tissues.

We show that patients with a CRB1-IRD have elevated levels of cholesteryl esters, a class of lipids formed from esterification by neurotoxic cholesterol, commonly observed as a protective mechanism to prevent further neurodegeneration. The presence of cholesteryl esters at sites of neurodegeneration suggests that esterification occurs as a protective process. We also observed elevated levels of a related class of lipids involved in cholesterol biosynthesis; the glycerophospholipids, which can have both pro- and anti-inflammatory effects. Perhaps patients have an imbalance of lipids such as glycerophospholipids, increasing cholesterol, in turn leading to esterification of cholesterol into cholesteryl esters. Functional studies are needed to elucidate causality and determine their role in the biology of CRB1-IRDs.

We determined the plasma proteome of patients with a CRB1-IRD and controls. Concentration of various plasma complement factors were increased in patients, and pathway enrichment analysis revealed that one of the most enriched pathways was the complement cascade. Our findings of elevated complement factor H (CFH) and complement factor H related proteins (CFHR) support earlier findings of complement system involvement in retinal degenerations. CFH/CFHR proteins are involved in the regulation of complement component C3, a complement system component important in the activation of microglia and photoreceptor cell injury. Earlier studies show an association between single nucleotide polymorphisms (SNPs) in the CFH gene and retinal diseases, including age-related macular degeneration (AMD) and multifocal choroiditis. SNPs in CFH are strongly correlated with plasma levels of complement factors encoded by genes in the CFH gene cluster. The CRB1 and CFH/CFHR genes are in close proximity and recombination signatures in the general population suggest that linkage between CRB1 and CFH/CFHR variants may be possible. This could mean that some CRB1 pathogenic variants may be associated with specific genotypes of CFH. Genome-wide association studies of CRB1-IRDs would provide an exciting opportunity to identify further pathways that influence this disease, since other immune genes may also be involved.