TNFα induced by DNA-sensing in macrophage compromises retinal pigment epithelial (RPE) barrier function

Abstract

Increasing evidence suggests that chronic inflammation plays an important role in the pathogenesis of age-related macular degeneration (AMD); however, the precise pathogenic stressors and sensors, and their impact on disease progression remain unclear. Several studies have demonstrated that type I interferon (IFN) response is activated in the retinal pigment epithelium (RPE) of AMD patients. Previously, we demonstrated that human RPE cells can initiate RNA-mediated type I IFN responses through RIG-I, yet are unable to directly sense and respond to DNA. In this study, we utilized a co-culture system combining primary human macrophage and iPS-derived RPE to study how each cell type responds to nucleic acids challenges and their effect on RPE barrier function in a homotypic and heterotypic manner. We find that DNA-induced macrophage activation induces an IFN response in the RPE, and compromises RPE barrier function via tight-junction remodeling. Investigation of the secreted cytokines responsible for RPE dysfunction following DNA-induced macrophages activation indicates that neutralization of macrophage-secreted TNFα, but not IFNβ, is sufficient to rescue RPE morphology and barrier function. Our data reveals a novel mechanism of intercellular communication by which DNA induces RPE dysfunction via macrophage-secreted TNFa, highlighting the complexity and potential pathological relevance of RPE and macrophage interactions.

Use of machine learning for quantification of retinal pigment epithelium tight junctions improves assay sensitivity

“…Dose-response curves for the functional and phenotypic response as a function of TNFα concentration at 24 hours is shown in Figure 5, with an IC 50 of 0.13 ng/mL for TER and EC 50 of 1.23 ng/mL and 1.09 ng/mL for the cell and TJ classifiers, respectively. These results indicate that the functional TER and phenotypic ZO-1 both capture RPE barrier function defects induced by TNFα, which aligns with previous observations (Twarog et al, 2023). Additionally, the machine learning approach allows for reliable quantification of the phenotypic response.…”

Double-stranded RNA induces retinal pigment epithelium cell degeneration and inflammation

“…In 3p-hpRNA-injected animals, ultrastructural evidence of photoreceptor outer segment (POS) degeneration and necrosis, RPE alterations (hypertrophy, increased phagosomes) and inflammatory cell infiltration were observed (Figure 7A-B), validating that RIG-I activation was detrimental for retinal health. The observation of inflammatory cell infiltration was particularly interesting, in conjunction with POS degeneration, due to our previous reports detailing the degenerative influence of macrophage activation on retinal health [14]. Because secretion of IFNβ can cause recruitment and activation of phagocytes such as macrophages [24], we measured macrophage activation though IBA1 expression after 3p-hpRNA transfection and found a significant increase in expression (Figure 7C).…”

“…Exposure to intracellular dsRNA led to increased expression of IFNβ, indicating a type I interferon response in ARPE-19 and iPS-RPE, and increased susceptibility to a degenerative phenotype [6]. IFNβ secretion promotes lymphocyte recruitment and activation, which can lead to the secretion of inflammatory and pro-degenerative cytokines such as TNFα [14]. Additionally, autocrine IFNβ signaling can initiate an autoregulatory feedback loop, as indicated by increased ISG15 expression [6].…”

“…While these primary RIG-I mediated effects are acute, several indicators point to lingering inflammatory phenotype, such as recruitment and activation of macrophages. Previous work from our group has found that macrophages could contribute to RPE and photoreceptor degeneration [14]. Clearance of dsRNA may be a rapidly occurring process, and as indicated by the work exploring DICER1, may protect cells from long-term detrimental effects [16].…”

Emerging Co‐Assembled and Sustained Released Natural Medicinal Nanoparticles for Multitarget Therapy of Choroidal Neovascularization

“…Considering that oxidative damage and inflammatory response during AMD could lead to the damage and degeneration of RPE layer to affect retinal homeostasis, [42,43] we further investigated the protection effect of L/N NPs to RPE cells against oxidative damage and inflammatory. Briefly, human retinal pigment epithelial (ARPE-19) cells were cultured in the presence of H 2 O 2 (100 × 10 −6 m) to mimic the environment with reactive oxygen species, and then different formulations including LNPs, NE, LNPs+NE, and L/N NPs with the concentration of lutein and nintedanib at 0.5 × 10 −6 m were added.…”