Virginia L. Calder

Several regulatory B-cell (Breg) subsets have been implicated in autoimmune diseases. We investigated Breg subsets in non-infectious uveitis (NIU) and in mouse models of experimental autoimmune uveitis (EAU). Blood samples from NIU patients with active disease (n = 13), in remission (n = 37) and healthy controls (n = 10) were immunophenotyped for CD19+CD24hiCD38lo memory B cells, CD19+CD24hiCD38hi-immature Bregs (iBregs), and CD19+CD38intCD24int-mature Bregs. In NIU, levels of peripheral blood iBregs and serum interleukin-10 (IL-10) were decreased in active disease vs. remission (P < .01). In quiescent NIU, iBregs were isolated and, when added at increasing ratios to autologous CD3+ T cells, downregulated T-cell proliferation. In an EAU model, at peak disease, CD19+CD1dhiCD5+ B10, CD19+CD5+ B1, CD19+CD21hiCD24hiCD23+ transitional two marginal-zone precursor (T2-MZP) and CD19+CD21hiCD23− marginal-zone B (MZB) Breg subsets were analysed both phenotypically and functionally. In EAU spleens, all Breg subsets investigated were detectable, but the IL-10–expressing B10-Breg subset was immunosuppressive and inversely correlated with disease severity (P = .048, R2 = 0.47). Blood-derived IL-10–expressing B10 and B1 Bregs were increased in both adjuvant controls and EAU but were significantly reduced in clinically milder EAU. In dissociated retinal cells, only the B10-Breg subset was detectable and increased in EAU. Selective Breg subsets may be immunosuppressive, with iBregs in NIU and IL-10–expressing B10 Bregs in EAU.

Purpose of review: Atopic dermatitis (AD) is a chronic inflammatory skin disease involving Th2 cytokine-driven inflammation. AD patients are at risk of developing conjunctivitis, including atopic keratoconjunctivitis (AKC), a sight-threatening chronic allergic eye disease involving both Th2 and Th1/17 cytokine-driven inflammation. In AD, dupilumab is highly effective by inhibiting interleukin (IL)-4 and IL-13. However, some AD patients develop dupilumab-associated ocular surface disease (DAOSD), an AKC-like disease. There are no biomarkers to predict who will develop DAOSD. The purpose of this review is to highlight recent findings in AD and AKC suggesting different immunopathogenic mechanisms are involved. Recent findings: A recent proteomics study of tear fluids identified raised inflammatory markers (n = 31) in AD patients with DAOSD (n = 22) and a shift towards a Th1/17 profile. Alternative biologics have been investigated for treating moderate-to-severe AD which have fewer ocular side-effects. The inhibitory effects of dupilumab cause an associated upregulation of IL-33 which could lead to an AKC-like disease. A recent therapeutic approach in AD via regulatory T cells suggests a novel treatment for those at risk of DAOSD. Summary: Ocular side-effects of dupilumab suggest that the immunopathogenic pathways in moderate-to-severe AD and AKC are not the same and, for DAOSD, might require different treatment approaches.

Several clinical subtypes of uveitis exist yet specific immunopathogenic mechanisms involved remain unclear. Ex vivo studies are limited by lack of fresh retinal biopsies and studies have relied on aqueous humour or peripheral blood, which may not directly reflect disease. The aim of this review is to compare the various in vivo models and review their contributions to our understanding of disease processes. These models, although unable to reflect all clinical signs, have provided insight into the contribution of genes and molecules, characterisation of effector T-cells, cell trafficking into retinal tissues, the contribution of tissue-resident myeloid cells and the mechanism(s) of action of several anti-inflammatory compounds. In vivo uveitis models have provided an excellent resource with which to study the molecular and cellular processes involved. Recent refinements in models, improved imaging, and the application of omics have greatly increased the number of readouts and translational opportunities. Future approaches with in vitro models will also be discussed.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFβ-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

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.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4⁺ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4⁺FoxP3⁺CD25⁺ regulatory T cells (Tregs) were known to suppress function of effector CD4⁺ T cells and contribute to resolution of disease. It has been recently reported that some CD4⁺ T-cell subsets demonstrate shared phenotypes with another CD4⁺ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4⁺ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

Background: 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. Methods: Mice (female; B10.RIII or C57Bl/6; aged 6–8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4⁺ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. Results: There was a significant reduction in clinical and histological scores in GW₁₀- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45⁺ leukocytes infiltrating the retinae and vitreous fluids in the treated GW₁₀ group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW₁₀-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C⁺ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW₁₀ treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. Conclusions: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4⁺ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of 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.