Clusterin seals the ocular surface barrier in mouse dry eye

Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye

Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye

Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye

Topical CLU ameliorates pre-existing ocular surface barrier disruption caused by desiccating stress.

<p><b>(Left).</b> The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. <b>(Left)</b> After the indicated time period, barrier disruption was confirmed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm) in a subset of mice. Values are expressed as the mean ± SD. *p<0.0001 (n = 4). <b>(Right)</b> The same desiccating stress (DS) protocol was continued for another 5 days while eyes with desiccating stress were treated topically with 1 uL of recombinant human CLU (rhCLU) formulated in PBS at 2 ug/mL, or with PBS control, 4 times/day. The fluorescein uptake test was then performed on these remaining mice. Values are expressed as the mean ± SD. *p<0.0001(n = 4).</p

Topical CLU protects the ocular surface barrier against proteolytic damage due to desiccating stress.

<p><b>(A)</b> The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. <b>(B)</b> The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. <b>(C)</b> Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)</p

Topical CLU protects the ocular surface barrier against functional disruption by desiccating stress.

<p>The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. <b>(A)</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). <b>(B)</b> The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). <b>(C)</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)</p

Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 <i>in vitro</i>.

<p><b>(A)</b> The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. <b>(B)</b> A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. <b>(C)</b> LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose</p

Topical CLU protects the ocular surface barrier via an all-or-none mechanism.

<p>The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. <b>(A) Dose response experiment.</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with <b>(Left)</b> recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), <b>(Middle)</b> recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or <b>(Right)</b> recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. <b>(B) Experiment comparing CLU with BSA.</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4)<b>. (C) Stress reduction experiment.</b> The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)</p

Causal association between endogenous CLU concentration in tears and ocular surface barrier vulnerability.

<p><b>(A)</b> Tears were collected from mice housed under normal ambient conditions or after application of the standard desiccating stress (DS) protocol for 5-days, and ELISA was used to measure CLU concentration (*P = 5x10^-8 n = 6, student’s t-test). <b>(B)</b> Representative transmission electron microscopy comparing images of anterior cornea from wild type C57BL6/J mice (A and C) and mice with homozygous CLU^-/- knockout on the C57BL6/J background (B and D). In low power (4000x) magnifications (A and B), five layers of epithelial cells divided into squamous, wing, and basal cell regions are visualized along with an intact basement membrane and Bowman's layer in both types of animals. Higher power images (C and D, 20,000x) of similar regions to those boxed in panels A and B show numerous surface microplicae (fat arrows) in both genotypes. Desmosomes (thin arrows) are similar in both frequency and structure. Higher power images (not shown) demonstrate intact adherens junctions in both genotypes. <b>(C)</b> Tears from wild type or heterozygous CLU^+/- knockout mice kept at ambient conditions were collected and ELISA was used to measure CLU concentration (p = 2.1x10^-5; n = 7, student’s t-test). <b>(D)</b> Wild type mice or heterozygous CLU^+/- knockout mice were subjected to the standard desiccating stress protocol, but without scopolamine injection for four weeks and then ocular surface barrier integrity was measured by fluorescein uptake (**p<0.0001, n = 4).</p