Abstract
Purpose :
Soluble immunomodulating factors produced by the retinal pigment epithelial cells (RPE) alter the response to pro-inflammatory signals by inducing anti-inflammatory activity in macrophages. We assayed the macrophages to see if this alternative activation is RPE mediating innate immune memory.
Methods :
Conditioned media (CM) containing RPE soluble factors were generated by incubating RPE-eyecups in serum-free media for 24hrs. The RPE-eyecups were made from the eyes of C57BL/6 naive mice and mice with active experimental autoimmune uveitis (EAU). The RPE-eyecups are the intact RPE monolayer, choroid, and sclera of the posterior eye segment. Resting peritoneal macrophages were collected from naive C57BL/6 mice and treated with CM for 24 hours. The cultures of CM-treated macrophages were washed, fresh serum-free media was added, and the macrophages were allowed to rest for 48 hours. Endotoxin (LPS 1 µg/ml) was added, and 24 hours later, the culture supernatant was assayed for TNF-α and IL-10 by ELISA. In another set of experiments, the monocytic cell line RAW 264.7 was treated with α-MSH instead of RPE eyecup conditioned and assayed for nitric oxide generation by Griess reagent after the 2 days of rest and LPS stimulation.
Results :
When stimulated with LPS in comparison to unstimulated macrophages, the macrophages not pre-treated with CM significantly (P < 0.001) produced TNF-α but not IL-10. Pretreatment with CM of naive RPE eyecups had lowered but not significantly affected LPS-stimulated TNF-α production by the macrophages; however, the LPS-stimulation significantly (P < 0.001) induced elevated IL-10 production. In comparison, macrophages pretreated with CM from EAU RPE eyecups eyes had no significant change in TNF-α production but were suppressed in IL-10 to a level not significantly different from the background of untreated LPS-stimulated macrophages. The macrophages pretreated with α-MSH were significantly (P < 0.05) suppressed in LPS-stimulated nitric oxide generation in comparison to the stimulated macrophages not pre-treated.
Conclusions :
The results demonstrate that the soluble immunomodulating factors of RPE, potentially involving α-MSH, can impart a selective innate immune memory that shifts toward promoting anti-inflammatory activity. Therefore, RPE have the potential to mediate innate immune memory to maintain immune suppression to proinflammatory signals.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.