PurposeMacular edema (ME) is a leading cause of visual loss in a range of retinal diseases and despite the use of antivascular endothelial growth factor (anti-VEGF) agents, its successful treatment remains a major clinical challenge. Based on the indirect clinical evidence that interleukin-6 (IL-6) is a key additional candidate mediator of ME, we interrogated the effect of IL-6 on blood–retinal barrier (BRB) integrity in vitro.MethodsHuman retinal pigment epithelial cell (ARPE-19) and human retinal microvascular endothelial cell (HRMEC) monolayers were used to mimic the outer and inner BRB, respectively. Their paracellular permeability was assessed by measuring the passive permeation of 40 kDa fluorescein isothiocyanate (FITC)-dextran across confluent cells in the presence of IL-6. Transendothelial/epithelial electrical resistance (TEER) then was measured and the distribution of the tight junction protein ZO-1 was assessed by immunofluorescence using confocal microscopy.ResultsTreatment with IL-6 for 48 hours significantly increased the diffusion rate of FITC-dextran, decreased TEER, and disrupted the distribution of ZO-1 in ARPE-19 cells, which constitutively express the IL-6 transmembrane receptor, and this was reversed with IL-6R blockade. In contrast, IL-6 did not affect the paracellular permeability, TEER, or ZO-1 distribution in HRMECs.ConclusionsThese in vitro data support the hypothesis that IL-6 reversibly disrupts the integrity of ARPE-19 cells, but it does not affect HRMECs.Translational RelevanceIL-6 is a candidate therapeutic target in the treatment of outer BRB driven ME.

Background: Corticosteroids remain the first-line treatment for patients with immune thrombocytopenia (ITP). However, 20% to 30% of patients do not respond to treatment at tolerable doses. This variation in corticosteroid efficacy is replicated in other autoimmune diseases and may have an adaptive immune basis. Objective: To test the hypothesis that CD4 + T-cell responses to corticosteroids are different in patients with clinically defined corticosteroid refractory ITP. Methods: In this prospective cohort study, CD4 + T cells from patients with ITP were cultured in the presence or absence of dexamethasone (Dex). Intracellular cytokine expression was then quantified by flow cytometry and compared with patients' clinical response to corticosteroid treatment. A control cohort of patients with autoimmune uveitis was also studied to evaluate whether our findings were limited to ITP or are potentially generalizable across autoimmune diseases. Results: The ratio of interleukin (IL)-10 to IL-17 expression following CD4 + T cell culture with Dex was able to discriminate between ITP patients with a clinically defined complete (n = 33), partial (n = 12) or nonresponse (n = 11) to corticosteroid treatment (P = .002). These findings were replicated in patients with autoimmune uveitis (complete response n = 14, nonresponse n = 22; P = .01). Conclusions: There is a relative abrogation of IL-10 and persistence of IL-17 expression in the CD4 + T cells of patients who clinically fail corticosteroid therapy. This observation has potential to inform both our mechanistic understanding of the action of corticosteroids in the treatment of ITP, and as a biomarker for steroid refractory disease, with potential application across a range of hematological and nonhematological conditions.

Immune thrombocytopenia (ITP) is thought to result from an aberrant adaptive autoimmune response, involving autoantibodies, B and T lymphocytes, directed at platelets and megakaryocytes. Previous reports have demonstrated skewed CD4 + T-helper subset distribution and enhanced production of pro-inflammatory cytokines such as interleukin 17A and interferon gamma. The role of monocytes (MCs) in ITP is less widely described, but innate immune cells have a role in shaping CD4 + T-cell phenotypes. Glucocorticoids (GCs) are commonly used for first-line ITP treatment and modulate a broad range of immune cells including T cells and MCs. Using multiparameter flow cytometry analysis, we demonstrate the expansion of intermediate MCs (CD14 ++ CD16 +) in untreated patients with newly diagnosed ITP, with these cells displaying a pro-inflammatory phenotype, characterised by enhanced expression of CD64 and CD80. After 2 weeks of prednisolone treatment (1 mg/kg daily), the proportion of intermediate MCs reduced, with enhanced expression of the anti-inflammatory markers CD206 and CD163. Healthy control MCs were distinctly different than MCs from patients with ITP before and after GC treatment. Furthermore, the GC-induced phenotype was not observed in patients with chronic ITP receiving thrombopoietin receptor agonists. These data suggest a role of MCs in ITP pathogenesis and clinical response to GC therapy.

CD4 + T cell mediated uveitis is conventionally treated with systemic immunosuppressive agents, including corticosteroids and biologics targeting key inflammatory cytokines. However, their long-term utility is limited due to various side effects. Here, we investigated whether DNA methylation inhibitor zebularine can target CD4 + T cells and control intraocular inflammation. Our results showed that zebularine restrained the expression of inflammatory cytokines IFN-γ and IL-17 in both human and murine CD4 + T cells in vitro . Importantly, it also significantly alleviated intraocular inflammation and retinal tissue damage in the murine experimental autoimmune uveitis (EAU) model in vivo , suggesting that the DNA methylation inhibitor zebularine is a candidate new therapeutic agent for uveitis.