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Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina
Purpose:
We established S100A9 as a myeloid-derived damage-associated molecular pattern (DAMPs) protein associated with increasing severity of diabetic retinopathy (DR) in type 2 diabetic subjects. The present study investigates the retinal localization, expression, and mechanisms of action for S100A9 in the young obese Ossabaw pig retina.
Methods:
Retinae from Ossabaw pigs fed a Western diet for 10 weeks were evaluated for S100 and inflammatory mediator expression using quantitative PCR and Western blot. Double immunohistochemistry was performed to identify the cellular sources of S100A9 in the pig retina. Primary pig retinal microglial cells (pMicroglia) were examined for S100A9 production. S100A9-induced responses were also investigated, and inhibitor studies elucidated the mechanism of action via the NLRP3 inflammasome. A specific inhibitor, Paquinimod (ABR-215757), was administered in vitro to assess the rescue of S100A9-induced NLRP3 inflammasome activation in pMicroglia.
Results:
The expression of the S100 family in the obese Ossabaw pig retina showed a significant elevation of S100A9, consistent with increased levels of circulating S100A9. Moreover, the retina had elevated levels of inflammatory mediators IL-6, IL-8, MCP-1, IL-1β and NLRP3. Retinal microglia in obese Ossabaw were activated and accompanied by an increased expression of intracellular S100A9. pMicroglia isolated from pig retina transformed from ramified to amoeboid state when activated with LPS and produced high S100A9 transcript and protein levels. The S100A9 protein, in turn, further activated pMicroglia by heightened production of S100A9 transcripts and secretion of pro-inflammatory IL-1β protein. Inhibition of TLR4 with TAK242 and NLRP3 with MCC950 attenuated the production of IL-1β during S100A9 stimulus. Finally, pre-treatment with Paquinimod successfully reduced S100A9-driven increases of glycosylated-TLR4, NLRP3, ASC, Caspase-1, and IL-1β production.
Conclusion:
We demonstrated that microglial-derived S100A9 perpetuates pro-inflammatory responses via the NLRP3 inflammasome in the retina of young Western-diet-fed Ossabaw pigs exhibiting diabetic retinopathy.
Insights
Microglia-derived S100A9 protein exacerbates inflammation in diabetic retinopathy by activating the NLRP3 inflammasome in the retina. This study in Ossabaw pigs reveals a key mechanism driving disease progression.
Area of Science:
- Ophthalmology
- Immunology
- Molecular Biology
Background:
- Diabetic retinopathy (DR) is a severe complication of diabetes, linked to myeloid-derived damage-associated molecular patterns (DAMPs). S100A9 protein has been associated with increasing DR severity in type 2 diabetes.
- Understanding the role of S100A9 in the retina is crucial for developing targeted therapies for DR.
Purpose of the Study:
- To investigate the retinal localization, expression, and mechanisms of action of S100A9 in the obese Ossabaw pig retina, a model for type 2 diabetes and DR.
- To elucidate the role of S100A9 in microglial activation and inflammatory mediator production in the context of DR.
Main Methods:
- Quantitative PCR and Western blot analysis of retinal S100 and inflammatory mediator expression in Ossabaw pigs fed a Western diet.
- Double immunohistochemistry to identify S100A9-expressing cells, with a focus on retinal microglia.
- In vitro studies using primary pig retinal microglial cells (pMicroglia) to assess S100A9 production, activation, and response to S100A9 stimulation, including inflammasome pathway analysis and inhibitor studies.
Main Results:
- Elevated S100A9 expression was observed in the retinas of obese Ossabaw pigs, correlating with increased circulating S100A9 levels and heightened expression of inflammatory mediators (IL-6, IL-8, MCP-1, IL-1β, NLRP3).
- Retinal microglia were activated and showed increased intracellular S100A9. Activated pMicroglia produced high levels of S100A9 and secreted pro-inflammatory IL-1β.
- S100A9 stimulation of pMicroglia led to further S100A9 production and IL-1β secretion, mediated via TLR4 and the NLRP3 inflammasome pathway. Paquinimod treatment reduced S100A9-induced activation of NLRP3 inflammasome components.
Conclusions:
- Microglial-derived S100A9 plays a significant role in perpetuating pro-inflammatory responses in the retina through NLRP3 inflammasome activation.
- These findings highlight S100A9 as a potential therapeutic target for managing diabetic retinopathy by modulating microglial activation and inflammasome pathways.
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