Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway

Abstract

Inflammation and oxidative stress are common factors involved in the pathogenesis of retinal diseases, such as aged-related macular degeneration (AMD) and diabetic retinopathy (DR). Autophagy is a catabolic process essential to cell survival in response to stress. This process is highly active in retinal pigment epithelium (RPE) cells. Our previous findings demonstrated that lipopolysaccharide (LPS) induces an inflammatory response of RPE cells that implies classical phospholipases D (PLD1 and 2) activation, cyclooxygenase-2 (COX-2) expression, prostaglandin E 2 (PGE 2 ) production and reduced cell viability. In this work, we studied the autophagic process and its modulation by the PLD pathway in D407 and ARPE-19 RPE cells exposed to LPS. LPS (10 μg/ml or 25 μg/ml) exposure for 24 h increased light chain 3B-II (LC3B-II) content (an autophagy marker) and LC3B-positive punctate structures in both RPE cell lines studied. Next, the drug bafilomycin A 1 (BAF, 50 nM) was used to block the autophagic flux. In cells pre-incubated with BAF, LC3B-II and sequestosome 1 (SQSTM1/p62) levels and autophagosome-like structures were increased by LPS, demonstrating that the inflammatory injury increases the autophagic process in RPE cells. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595) or PLD2 (VU0285655-1) inhibitors prior to LPS addition. Under control condition, LC3B-positive punctate structures were increased in cells pre-incubated with PLD2 inhibitor while with PLD1 inhibitor were increased in cells exposed to LPS. MTT reduction assays showed that early autophagy inhibitors, 3-methyladenin (3-MA) or LY294002, enhanced the loss in cell viability induced by LPS exposure for 48 h. On the contrary, the inhibition of PLD1 and PLD2 prevented the loss in cell viability induced by LPS. In conclusion, our results show that even though LPS treatment promotes an inflammatory response in RPE cells, it also triggers the activation of the autophagic process which in turn may serve as a protective mechanism for the cells. In addition, we demonstrate that the PLD pathway modulates the autophagic process in RPE cells. Our findings contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases and open new avenues for potential therapeutic exploration.

Transcriptome-Wide Analysis of CXCR5 Deficient Retinal Pigment Epithelial (RPE) Cells Reveals Molecular Signatures of RPE Homeostasis

“…We have also observed the cytokines, chemokines, and prostaglandins such as IL13RA1, IL7, IL13RA2, CCL28, CCL2, PTGDR, PTGES3, PTGER2, PTGIS and PTGS2 (COX-2) to be up-regulated in CXCR5-deficient RPE which may suggest the involvement of RPE cells in pro-inflammation signaling and immune cells recruitment. Our previous ex-vivo experiments in aged CXCR5-deficient mice have indicated increased microglia migration to RPE cell layer sub-RPE infiltration with CD4 + cells, as well as increased expression of COX-2 in the mouse RPE-choroid complexes [39]. However, the exact cellular source of these genes has remained unclear.…”

A Comprehensive Proteomic and Phosphoproteomic Analysis of Retinal Pigment Epithelium Reveals Multiple Pathway Alterations in Response to the Inflammatory Stimuli

“…Hence, integrated understanding of multiple models was recommended [21]. LPS, an agonist of toll-like receptor 4 (TLR4) used in this study for inducing inflammatory pathology, has frequently been utilized in RPE studies as a priming or stimulating agent because LPS-triggered downstream signals and phenotypes have been reported as similar to those seen in RPE pathobiology [15][16][17][18][19][20], as mentioned in the introduction. Interestingly, our results identified an early and strong metabolic response after LPS challenge (Figure 1B,C), implying that subsequent metabolic overload of the mitochondria could occur, leading to various biological alterations.…”

“…Lipopolysaccharide (LPS) has been widely used to induce ocular inflammation and to recapitulate various retinal diseases such as uveitis, diabetic retinopathy (DR), and age-related macular degeneration (AMD) [11][12][13][14][15]. In the RPE, LPS is known to trigger pathological conditions such as inflammation, oxidative stress, and aging through altering multiple pathways including ERK1/2 activation, NF-κB nuclear translocation, and ROS generation [15,16]. Specifically, LPS has been reported as involved in the production of the proforms of NLRP3 inflammasome-dependent cytokines IL-1β and IL-18, whose maturation and secretion results in degeneration of the RPE [17].…”

Protective or Harmful: The Dual Roles of Autophagy in Diabetic Retinopathy

“…Bermúdez et al ( 89 ) studied the inflammatory response in DR, with ARPE-19 and D407 cultured in lipopolysaccharide (LPS, 10 or 25 μg/ml). They explored the autophagic flux and its regulation by the phospholipases D (PLD) pathway in cells exposed to LPS.…”

“…Moreover, 3-MA increased the loss in cell viability instigated by LPS. Therefore, although LPS treatment induced an inflammatory activation in RPE cells, it also promoted the activation of the autophagic process which in turn may elicit a protective effect on the cells via PLD pathway modulation ( 89 ).…”