Non-caveolar Caveolin-1 in Müller glia mediates LPS-induced NF-κB signaling and IL-6 secretion

Abstract

Purpose : Müller glia are the major glial cell type in the retina and are responsible for maintaining retinal neuronal health. Müller glia express the signature protein for caveolae, Caveolin1 (Cav1) outside of the caveolae (i.e., “Non-caveolar Cav1”). They also express little or no Cavin1/PTRF, a protein required to sequester Cav1 into caveolae. Dependent on cellular context, Cav1 can promote or suppress Toll-like receptor4 (TLR4) responses. Here, we show that Non-caveolar Cav1 expressed in Müller glia promotes pro-inflammatory responses by mediating LPS-induced NF-κB signaling. We also show that tumor necrosis factor (TNF), a cytokine upregulated by neural injury induces Cavin1/PTRF and TLR4 in Müller glia.

Methods : To investigate the immunomodulatory role of Non-caveolar Cav1, we silenced Cav1 using Cav1 specific shRNA or sequestered Cav1 into Caveolae by over-expressing Cavin1/PTRF by Adenoviral transduction in MIO-M1 Müller glia. We interrogated NF-κB signaling upon TLR4 stimulation with LPS by Western blots and immunocytochemistry. We quantified IL-6 secretion by ELISA in Cav1-silenced or Cavin1/PTRF-transduced cells with or without LPS stimulation. Using Western blotting, we assayed for TNFα-induced TLR4 and Cavin1/PTRF protein expression in MIO-M1 glia. We evaluated the expression of caveolae and TLR4 signaling components in publicly available single cell RNAseq datasets.

Results : LPS stimulation of MIO-M1 Müller glia induces the phosphorylation of IKK, degradation of IκB and NF-κB/p65 nuclear localization. Both Cav1 silencing and Cavin1/PTRF overexpression in MIO-M1 Müller glia significantly reduced LPS-induced IKK phosphorylation and NF-κB/p65 nuclear localization. Also, both Cav1 silencing and Cavin1/PTRF overexpression significantly suppress LPS-induced IL-6 secretion in MIO-M1 Müller glia. Treatment of MIO-M1 Müller glia with TNF-α resulted in time dependent induction of TLR-4 and Cavin1-PTRF expression.

Conclusions : Our results suggest that Non-Caveolar Cav1 in Müller glia enhance TLR4-mediated NF-κB signaling and cytokine expression. These results also support the idea that suppression of TLR4-mediated inflammatory signaling by Cavin1/PTRF is mediated by sequestration of Cav1 into caveolae. TNF-α induction of caveolar and TLR4 proteins in Müller glia could provide new insight into how these cells respond to neurotoxic insults in the retina, in-vivo.

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