Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels.

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 µM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease.

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into "epithelial" type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.

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

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 E2 (PGE2) 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 A1 (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.

Carrier free indomethacin microparticles for dry powder inhalation.

The present studies were designed to evaluate inhalatory microparticles carrying indomethacin (IN) for potential local (specific and non-specific bronchial inflammatory asthma responses) and systemic treatments (joint inflammation, rheumatoid arthritis and osteoarthritis pain) by optimizing microparticle properties, characterizing their lung deposition, drug release, evaluating cytotoxicity and also pharmacological effect in vitro. The acidic groups of IN were complexed with the cationic groups of the polyelectrolyte polylysine in order to increase the drug water compatibility. The polylysine/indomethacin ratio was fixed and the pH was adjusted in different formulations. Microparticles were obtained by spray drying using a relatively high atomization air flowrate (742 L/min) and a high-performance cyclone in order to optimize the production of microparticles with adequate attributes for inhalatory delivery. The produced microparticles exhibited high process yield and IN loading, volumetric mean diameters smaller than 5 µm and narrow particle size distributions. According to demonstrated aerosolization performance, the powders were suitable for inhalatory indomethacin local and systemic treatments. Emitted fraction was higher than 90%, the MMAD was around 3 µm and the GSD lower than 3. The respirable fraction for particles with aerodynamic diameters smaller than 5 µm was around 29% while for particles with aerodynamic diameters smaller than 3 µm the value was around 17%. The addition of lactose as carrier worsened the aerodynamic performance of the microparticles. The developed powdered systems got wet and dissolved quickly and presented higher release rates respect to pure IN in simulated lung physiological conditions. Furthermore, the assays performed in RAW 264.7 cell line showed that the microparticles exhibited the same anti-inflammatory capability as the pure drug. The developed particles did not affect the RAW 264.7 cell viability. In conclusion, a promising powder formulation for DPIs has been developed to treat, locally and systemically, inflammatory diseases.

Soluble factors from Lactobacillus reuteri CRL1098 have anti-inflammatory effects in acute lung injury induced by lipopolysaccharide in mice.

We have previously demonstrated that Lactobacillus reuteri CRL1098 soluble factors were able to reduce TNF-α production by human peripheral blood mononuclear cells. The aims of this study were to determine whether L. reuteri CRL1098 soluble factors were able to modulate in vitro the inflammatory response triggered by LPS in murine macrophages, to gain insight into the molecular mechanisms involved in the immunoregulatory effect, and to evaluate in vivo its capacity to exert anti-inflammatory actions in acute lung injury induced by LPS in mice. In vitro assays demonstrated that L. reuteri CRL1098 soluble factors significantly reduced the production of pro-inflammatory mediators (NO, COX-2, and Hsp70) and pro-inflammatory cytokines (TNF-α, and IL-6) caused by the stimulation of macrophages with LPS. NF-kB and PI3K inhibition by L. reuteri CRL1098 soluble factors contributed to these inhibitory effects. Inhibition of PI3K/Akt pathway and the diminished expression of CD14 could be involved in the immunoregulatory effect. In addition, our in vivo data proved that the LPS-induced secretion of the pro-inflammatory cytokines, inflammatory cells recruitment to the airways and inflammatory lung tissue damage were reduced in L. reuteri CRL1098 soluble factors treated mice, providing a new way to reduce excessive pulmonary inflammation.