Internalized E. coli particles but not carboxylated beads are delivered to the lysosomes and increase lysosomal mass in TM cells.

<p>(A) Confluent cultures of TM cells were subjected for three days to phagocytic challenge to either FITC-labeled E.coli (left panel) or Fluoresbrite® blue carboxylate microspheres (right panel), and then incubated with LTR (100 nM) for one hour at 37 ^oC. Co-localization of E. coli or carboxylated beads with LTR was observed under confocal microscopy. Images are representative of three independent experiments. (B) Representative electron microscopy images of TM cells phagocytically challenged for three days to either carboxylate microspheres (left panel) or pigment particles (right panel). <i>B</i> = beads in isolated phagosomes; <i>P</i> = pigment in isolated phagosomes; * = phagolysosome containing engulfed particle. (C) Lysosomal mass quantified by flow cytometry in the FL3 channel of TM cells phagocytically challenged to 1 x10⁶ particles/mL of either FITC-labeled E. coli, Fluoresbrite® blue carboxylate microspheres or autologous iris pigment. Data is represented as percentage of control. Values are means ± SD. *p<0.05; **p<0.001 (t-test, n=3).</p

Phagocytic activity in TM cells.

<p>Representative brightfield (A–D) and fluorescence microscopy (E, F) images of control TM cells (A) or TM cells phagocytically challenged for ten days to 1 x10⁶ particles/mL of (B, E) FITC-labeled E. coli; (C, F) Fluoresbrite® Blue carboxylate microspheres, 1 μM diameter; and (D) autologous iris pigment. (G) Flow cytometry quantification of internalized pHRodo-labeled E.coli particles overtime.</p

Cathepsin B Is Up-Regulated and Mediates Extracellular Matrix Degradation in Trabecular Meshwork Cells Following Phagocytic Challenge

<div><p>Cells in the trabecular meshwork (TM), a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment). Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB). Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.</p> </div

Phagocytosis Increases Cell Surface Expression and Secretion of CTSB in TM Cells.

<p>(A) Protein levels of CTSB evaluated by western-blot analysis in conditioned media (15 µL) from TM cells phagocytically challenged for ten days to either FITC-labeled E. coli, collagen I-coated beads, carboxylate microspheres, or iris pigment. The levels of the non-related protein, Chi3L1, were used as loading control. (B) Protein levels of CTSB analyzed by western blot in clarified aqueous humor samples (15 µL) collected from porcine cadaver eyes. (C) Constitutive Porcine TM cells were transfected with 2 µg of the plasmid GFP-CTSB. Two days after transfection cells were fixed, and GFP fluorescence analyzed under confocal microscopy. White arrows indicate the presence of GFP signal at the periphery of the cell. (D) Cathepsin B activity as visualized by in vivo confocal microscopy in porcine TM cells incubated for one hour with 10 μL of MR-(RR)₂. CTSB activity at the periphery of the cell is indicated with white arrows. (E) Protein levels of CTSB evaluated by western-blot analysis in purified cell surface fraction and washing effluent (20 µL) from TM cells phagocytically challenged for ten days FITC-labeled E. coli. The levels of the non-specific band at 60 kDa served as loading control. Western-blots and confocal images are representative from three different experiments.</p

Phagocytosis Promotes CTSB-mediated ECM Remodeling in TM Cells.

<p>(A) Confluent cultures of porcine TM cells grown in 96-well plate were phagocytically challenged to E. coli in the presence of vehicle or DQ-gelatin (10 μg/mL), with or without Ca074Me (40 μM). Fluorescence peptides released by the enzymatic cleavage of the substrates were measured in a microplate reader at the indicated times (Em: 495 nm; Exc: 515 nm). All values were corrected for background fluorescence. Values are mean ± SD. * compare E. coli-exposed cultures versus control; ^# compare Ca074M-treated cultures versus non-treated, ^{*, #} p<0.05, ^{**, ##} p<0.01, ^{***, ###} p<0.001 (t-test, n=3). (B) Confluent cultures of TM cells were subjected to phagocytic challenge to either E.coli or collagen I-coated beads for ten days. To avoid proteases contained in the serum to interfere with the assays, cells were shifted to serum-free media at day nine after phagocytic challenged. Serum-free cell culture supernatant samples (25 μl) were subjected to gelatin, casein, and plasminogen/casein gel zymography. Areas of proteolytic activity appeared as clear bands. Casein and plasminogen/casein color pictures have been reversed to improve sensititivity using ImageJ.</p

Phagocytosis of collagen I-coated beads also upregulates CTSB expression and activity in TM cells.

<p>Confluent cultures of TM cells were phagocytically challenged for 2, 5, and 10 days to FluoSpheres® collagen I-labeled microspheres (1.0 µm, 1 x10⁶ particles/mL). (A) Protein levels of CTSB and LAMP1 as evaluated by western-blot analysis in whole cell lysates (10 μg) using a specific antibody. β-Tubulin levels were used as loading control. Western-blots images are representative from three different experiments. (B) Proteolytic CTSB activity normalized by total protein content using the fluorogenic substrates z-RR-AMC (20 μM). Data is represented as percentage of control. Values are mean ± SD. ***p<0.001 (t-test, n=3). (C) Confluent cultures of TM cells were subjected for three days to phagocytic challenge to FluoSpheres® collagen I-labeled microspheres and then incubated with LTR (100 nM) for one hour at 37 ^oC. Co-localization of collagen I-coated beads (green color) with LTR (red color) was observed under confocal microscopy. Images are representative of three independent experiments. (D) Representative electron microscopy images of TM cells phagocytically challenged for three days to FluoSpheres® collagen I-labeled microspheres. CB: beads contained in isolated phagosomes; asterisks (*): beads contained in mature autophagolysosomes.</p

DQ-Gelatin is degraded intracellularly within lysosomes in association with CTSB activity.

<p>(A) Porcine TM cells were plated onto Lab-Tek II chambers coated with 20 µg/mL of DQ-gelatin. Two days later, cells were incubated for one hour with LTR (100 nM, red fluorescence, left panel) or MR-(RR)₂ (red fluorescence, right panel). Green signal indicates fluorescence peptides released by proteolytic degradation of the quenched DQ-gelatin. Co-localization of DQ-degradation products with lysosomes (left panel) or CTSB activity (right panel) is shown as orange/yellow signal. Asterisks (*) indicate the areas where DQ-gelatin is extracellularly degraded. Note that DQ-gelatin degradation products found in the extracellular space do not co-localize with either LTR or MR-(RR)₂. (B) In vivo CTSB activity in TM cells grown onto onto Lab-Tek II chambers coated with 20 µg/mL DQ-gelatin for one day and exposed for 24 hours to Ca074Me (40 µM). The fluorescent peptides released from the MR-(RR)₂ proteolytic cleavage (red fluorescence) and from the degradation of DQ-gelatin (green fluorescence) in the presence or absence of Ca074Me were visualized in vivo by confocal microscopy. The fluorescence intensities from five different images were quantified using ImageJ (C, D). Values are mean ± SD. *, p<0.05, ***p<0.001 (t-test, n=5).</p

Quantitative real-time PCR confirmation of selected genes with differential expression in phagocytically challenged human TM cells under physiological (black bars) and oxidative stress (stripped bars) conditions.

<p>The expression levels were calculated using the formula 2^−ΔCt, where ΔCt = Ct_gene−Ct _{average housekeeping}. β-Actin, GAPDH, and HPRT1 served as internal standard for normalization. Values represent mean ± SD. (*) compares phagocytically challenged versus control cultures; (^#) compares oxidatively stressed versus cultures grown under physiological conditions. *, ^# p<0.05, **, ^## p<0.005, ***, ^### p<0.0005 (t-test, n = 3).</p

Genes Significantly Downregulated (>1.5 fold, p<0.05) in HTM Cells Phagocytically Challenged Under Physiological Condition.

<p>Genes Significantly Downregulated (>1.5 fold, p<0.05) in HTM Cells Phagocytically Challenged Under Physiological Condition.</p

Collagenolytic activity of porcine TM cells phagocytically challenged to E.coli or pigment in the presence of the self-quenched fluorescent substrates DQ-Collagen I (A) or DQ-Collagen IV (B).

<p>Values represent mean ± SD. **p<0.005, ***p<0.0005 (t-test, n = 3). (C) Collagenolytic and caseinolytic activities in the culture media of porcine TM cells phagocytically challenged to E.coli or pigment evaluated by in-gel collagen I or casein zymography. Lytic activity is shown as clear bands. Zymograms are representative from three independent experiments.</p