TNFα secreted by DNA-sensing in macrophage compromises retinal pigment epithelial barrier function

Abstract

Purpose : While chronic inflammation plays a role in age-related macular degeneration (AMD), characterization of the stressors involved and their impact on disease remains incomplete. Previously, we demonstrated that retinal pigment epithelial (RPE) cells directly respond to RNA but not DNA due to lack of the DNA sensor cGAS. In this study, we utilized a macrophage and RPE co-culture system to study RPE response to DNA through cell-cell interactions.

Methods : Monocytes derived from human blood were differentiated into macrophages by GM-CSF or M-CSF (100 ng/ml) for 7 days. iCell-RPE, an iPS-derived RPE cell line (FUJIFILM Cellular Dynamics), were seeded (200 k cells/cm²) in transmembrane wells and cultured for 3 weeks until maturation. For co-culture, macrophages (500 k cells/ml) were treated and seeded into the basal compartment of a tissue culture plate, or to the basal compartment of a cellZscope2 24-well insert for transepithelial resistance (TER) measurements. Cells were exposed to dsDNA-EC or 3p-hpRNA. Gene expression changes and protein levels were quantified by qPCR and ELISA, respectively. Neutralizing antibodies against IFNβ or TNFα were applied to the apical and basal compartments to block cytokine effects.

Results : In a macrophage-RPE co-culture system, type I IFN response, e.g. ISG15 (22.6±3.66 ng/ml vs 1.2±0.13 ng/ml in veh control, Figure 1A), and loss of TER (41.4±11.7% reduction) were observed in RPE after DNA treatment. IFNβ neutralization did not prevent DNA-induced RPE barrier function loss, suggesting other secreted factors are involved. Analysis of the macrophage secretome post-DNA exposure revealed the induction of pro-inflammatory cytokines IL-18, IL-6, TNFα, and IL-12 (p<0.05). In addition to IFNβ, direct application of TNFα significantly disrupted RPE barrier function (p<0.05). Use of anti-TNFα neutralizing antibody prevented DNA-induced RPE TER loss in the co-culture system (Figure 1B).

Conclusions : We find that DNA-induced macrophage activation promotes RPE’s IFN response and barrier function disruption. Investigation of secreted cytokines following macrophages activation by DNA sensing indicates that neutralization of macrophage-derived TNFα, but not IFNβ, is sufficient to block the RPE secondary response and rescue barrier function. These results highlight the significant role of heterotypical cellular interactions in para-inflammatory diseases such as AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.