Effects of Cathepsin S Inhibition in the Age-Related Dry Eye Phenotype

Purpose Aged C57BL/6J (B6) mice have increased levels of cathepsin S, and aged cathepsin S (Ctss−/−) knockout mice are resistant to age-related dry eye. This study investigated the effects of cathepsin S inhibition on age-related dry eye disease. Methods Female B6 mice aged 15.5 to 17 months were randomized to receive a medicated diet formulated by mixing the RO5461111 cathepsin S inhibitor or a standard diet for at least 12 weeks. Cornea mechanosensitivity was measured with a Cochet–Bonnet esthesiometer. Ocular draining lymph nodes and lacrimal glands (LGs) were excised and prepared for histology or assayed by flow cytometry to quantify infiltrating immune cells. The inflammatory foci (>50 cells) were counted under a 10× microscope lens and quantified using the focus score. Goblet cell density was investigated in periodic acid–Schiff stained sections. Ctss−/− mice were compared to age-matched wild-type mice. Results Aged mice subjected to cathepsin S inhibition or Ctss−/− mice showed improved conjunctival goblet cell density and cornea mechanosensitivity. There was no change in total LG focus score in the diet or Ctss−/− mice, but there was a lower frequency of CD4+IFN-γ+ cell infiltration in the LGs. Furthermore, aged Ctss−/− LGs had an increase in T central memory, higher numbers of CD19+B220−, and fewer CD19+B220+ cells than wild-type LGs. Conclusions Our results indicate that therapies aimed at decreasing cathepsin S can ameliorate age-related dry eye disease with a highly beneficial impact on the ocular surface. Further studies are needed to investigate the role of cathepsin S during aging.

constantly in contact with the external environment.It is subject to various challenges that can lead to inflammation, infection, and other diseases.To maintain the health and function of the ocular surface, a sophisticated and finely regulated immune system is in place to detect and respond to any threats. 1,2Conversely, immune dysregulation is a core pathophysiologic mechanism in several ocular surface disorders.Although not a disease itself, aging also brings about dysregulatory changes in the ocular surface and is thus associated with an increased prevalence of dry eye and allergy. 3mong the numerous age-related changes described, aged ocular surface antigen-presenting cells (APCs) are more mature and better promote the differentiation of proinflammatory Th1 CD4 + T cells than their young counterparts. 4In line with this, we reported that aged mice display impaired ocular mucosal tolerance, 1 a key regulatory immune mechanism to keep inflammation at bay. 3,[5][6][7][8][9][10] APCs are master regulators of the immune response because they prime naive T cells into different Th subsets.Antigen presenta-tion involves the processing of antigen-derived peptides by the concerted action of lysosomal proteases.][13][14] Although most of these proteases have rather broad cleavage specificities, the system is not entirely redundant.Agerelated changes in antigen presentation-associated proteases are thought to contribute to the increase in autoimmunity observed in the elderly. 15athepsin S is a cysteine protease primarily expressed in the lysosomes of phagocytic cells, such as macrophages, APCs, and lacrimal gland acinar cells. 16,17One of its primary functions is degrading the MHC II-associated invariant chain (Ii).The Ii peptide acts as a placeholder within the antigenic peptide groove of the MHC II complex during its assembly in the endoplasmic reticulum to prevent the unwanted presentation of other endogenous peptides as potential antigens.9][20] In addition, a transgenic mouse with overexpression of cathepsin S displays spontaneous age-related systemic inflammation. 21Elevated cathepsin S activity in tears has been proposed as a potential biomarker for dry eye and Sjögren syndrome, as published data have shown increased activity in non-Sjögren syndrome dry eye patients compared to healthy controls. 22][25] On the other hand, increased levels of cathepsin S in plasma correlate with higher mortality in the elderly. 26,27he age-associated increase in inflammation might create a vicious cycle, as inflammatory cytokines like IFN-γ , TNF, and IL-1β are known to upregulate cathepsin S, 28,29 which in turn upregulates IL-1β and TNF (TNF is no longer named TNF-α 30 ).5][36] Because Sjögren syndrome and aged lacrimal glands share many similarities and activation of pathways related to immune activation, 10 and because our work showed that 1-year-old Ctss −/− mice have less severe dry eye phenotype than aged wild-type controls, 33 we hypothesized that a diet containing a cathepsin S inhibitor would also be beneficial during aging.The cathepsin S inhibitor RO5461111 was chosen since it is a selective inhibitor of cathepsin S. 34 The objective of this study was twofold: (1) to investigate the effects of RO5461111 on age-related dry eye phenotype and (2) to investigate the effects of pharmacologic and genetic deletion of cathepsin S on the immune infiltrates that accompany aging in the lacrimal gland.To this end, we subjected 15-to 17-month-old C57BL/6 mice to a 12or 28-week regimen with a cathepsin S inhibitor in the diet.We observed several positive effects on the ocular surface (improvement of goblet cell density and mechanical sensitivity).While the total immune infiltrates in the lacrimal gland did not change, we observed a decrease in Th1 cells infiltrating the lacrimal glands and a change in the B-cell landscape.These results were recapitulated in aged Ctss −/− mice.These results indicate that cathepsin S inhibition improves age-related dry eye without affecting the total immune infiltration in the lacrimal gland.Further studies are necessary to investigate if topical administration of a cathepsin S inhibitor would also benefit age-related dry eye.

Animals
The Institutional Animal Care and Use Committee at Baylor College of Medicine and Jackson Laboratories approved all animal experiments.In addition, all studies adhered to the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic and Vision Research and the NIH Guide for the Care and Use of Laboratory Animals. 37he experiments were performed at the Ocular Surface Center, Department of Ophthalmology, Baylor College of Medicine (Houston, TX, USA).
Mice were housed in specific pathogen-free facilities of Baylor College of Medicine and Jackson Laboratories.They were kept on diurnal cycles of 12 hours/light and 12 hours/dark with ad libitum access to food, water, and environmental enrichment.No intervention was made to the mice; therefore, our experiments did not induce pain, suffering, or distress.Criteria for early euthanasia included loss of 20% or more of body weight, extensive ulcerative dermatitis, or cornea opacification.Mice subjected to early euthanasia were not included in the study.
Because dry eye is more frequent in women, 38,39 and aged male mice do not develop corneal barrier disruption, 40 we used female mice.An effort was made to collect multiple tissues from each mouse.A final sample size per endpoint can be found in figure legends.

Diet Regimen
Three different cohorts of animals aged 15.5 to 17 months were randomized to receive either a medicated diet formulated by mixing the cathepsin S inhibitor (R05461111, 262.5 mg/kg chow; Roche, Switzerland) 19,34 or a standard diet (placebo).The medicated diet was commercially prepared by LabDiet (San Antonio, TX, USA).Mice were weighed biweekly.
Cohorts 1 and 2 received a medicated diet for 12 weeks (n = 15/group and n = 21), while cohort 3 received the diet for 28 weeks (n = 16/group).The age at euthanasia for cohorts 1/2 and 3 was ∼17 to 19 months and 19 to 22 months, respectively.All endpoints were tested at euthanasia, but corneal sensitivity measurements were performed in live animals 1 week before euthanasia.A final sample size per endpoint can be found in the figure legends.

Corneal Mechanical Sensitivity
Corneal sensitivity was measured under a surgical loupe with the Luneau Cochet-Bonnet instrument (Western Ophthalmics, Lynnwood, WA, USA).This instrument relies on increasing pressure as the filament shortens (range, 6-0.5 cm).This test was performed by two experimenters.While holding the animal (experimenter 1), a nylon filament was applied to the central cornea by the second experimenter.Experimenter 2 was blinded to the animal group/treatment.
As the initial test, the nylon thread was extended to 4. The step technique was used; if a specific length exhibited no response, the next lower step (meaning higher pressure) was tested until a positive response was obtained.A similar technique using a higher step (meaning lower pressure) was used if the initial length tested negative.A positive response was indicated by a clear stimulus-evoked blink and retraction of the eye into the ocular orbit.The central cornea was tested six times at each filament length.The response was considered negative when the monofilament touch elicited no blink.A positive response was considered when the animal blinked more than or equal to 50% of the times tested.

Histology, Periodic Acid-Schiff Staining, and Quantification of Focus Score
Eyes and ocular adnexa were excised, fixed in 10% formalin, paraffin embedded, and cut into 5-μm sections using a microtome (Microm HM 340E; Thermo Fisher Scientific, Waltham, MA, USA).Eye sections cut from paraffinembedded globes were stained with periodic acid-Schiff (PAS) reagent.The goblet cell density was measured in the superior and inferior bulbar and tarsal conjunctiva using NIS-Elements software (AR, version 5.20.2;Nikon Melville, NY, USA) and expressed as the number of positive cells per millimeter. 41ymphocytic infiltration foci were counted in hematoxylin and eosin-stained lacrimal gland sections by standard light microscopy using a 10× objective (Eclipse E400; Nikon) by two masked observers.A minimum of 50 mononuclear cells was counted as one focus, and the total number of foci per gland was recorded.Slides were scanned to obtain digital images using PathScan Enabler V (Meyer Instruments, Houston, TX, USA) and were calibrated according to the manufacturer's instructions (2.54 μm/px) using NIS Elements software.The lacrimal glands' total area was measured using the "autodetect area" function of the Nikon Elements software or was manually circumscribed using the polyline function.Finally, focus scores were calculated by dividing the number of foci per mm 2 and quantifying the number of inflammatory cell foci per 4 mm 2 tissue area.

Flow Cytometry Analysis
Single-cell suspensions of the lacrimal glands, conjunctiva, and cervical lymph nodes were prepared as previously reported. 23Then, 1 × 10 6 cells from the single-cell suspensions of the lacrimal gland and cervical lymph nodes (n = 7 to 10/group) were plated and then stained with different panels.Cells were blocked with CD16/CD32 (BioLegend, San Diego, CA, USA), washed, incubated with live/dead cell discriminator (IR; Invitrogen-Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), and stained using different panels as below.
A BD LSRII Benchtop cytometer was used for data acquisition, and data were analyzed using BD Diva Software (BD Pharmingen, Franklin Lakes, NJ, USA) and FlowJo software (version 10.1; Tree Star, Inc., Ashland, OR, USA).Biological replicates were averaged.

Statistical Analysis
Based on pilot studies, the sample size was calculated with StatMate2 Software (GraphPad Software, San Diego, CA, USA) to have at least 90% power to detect differences with an α of 0.5.Statistical analyses were performed with Graph-Pad Prism software (version 9.2; GraphPad Software).Data were first evaluated for normality with the Kolmogorov-Smirnov normality test.Appropriate parametric (t-test) or nonparametric (Mann-Whitney) statistical tests were used to compare the two age groups.Whenever adequate, one-way or two-way ANOVA or Kruskal-Wallis followed by post hoc tests were used.The final sample per experiment is shown in the figure legends.

Protective Effects of Cathepsin S Inhibition on the Ocular Surface During Aging
We previously reported that aged mice have increased cathepsin S activity in tears and Ctss −/− mice have diminished age-related corneal barrier disruption and do not lose goblet cells with age. 33In this study, we subjected mice aged 15.5 to 17 months to a cathepsin S inhibition diet to investigate if cathepsin S blockade could be used to revert the age-related dry eye phenotype in mice. 10,44Mice show signs of dry eye disease as early as 12 months. 44ince our regimen was not meant to be preventive, we chose to evaluate aged mice that already have dry eye.Two different cohorts of mice were treated for 12 consecutive weeks, and the combined data from the two experiments are shown in Figures 1 and 2. Mice were weighed weekly, and no changes in body mass were observed after 12 weeks of diet (Supplementary Fig. S1).Aged mice on a cathepsin S inhibition diet had greater conjunctival goblet cell density and corneal mechanosensitivity than those receiving normal chow (Figs.1A-C).These results show that a cathepsin S inhibition diet ameliorates the age-associated decrease in conjunctival goblet cell density and corneal mechanosensitivity. 40,44,45These results support the notion that cathepsin S inhibition improves the aged ocular surface phenotype and agree with our publication showing that aged Ctss −/− mice are resistant to age-related dry eye disease. 33

Effects of Cathepsin S Inhibition in Immune Infiltration in the Aged Lacrimal Glands
Cathepsin S inhibition in autoimmune mice decreases immune infiltration in their salivary and lacrimal glands. 18,46s cathepsin S inhibition could also target aging-induced changes in the lacrimal gland, we quantified immune infiltration.Histopathology evaluation of aged (17-to 19-monthold) lacrimal glands 12 weeks posttreatment showed no difference in focus score/4 mm 2 (a measurement of total lacrimal gland infiltration) (Figs.2A, 2B).
We repeated the experiment in the third cohort of mice for 28 weeks to rule out that treatment with a cathepsin S inhibitor diet for 12 weeks was insufficient to see an effect in the lacrimal gland infiltrates.In this experiment, 15.5-to 17-month-old mice received normal chow or a cathepsin S inhibition diet for 28 weeks.Mice were weighed weekly.Mice receiving a cathepsin S inhibition diet for 28 consecutive weeks gained 20% more body mass than mice receiving normal chow (Supplementary Fig. S1C).We repeated the corneal esthesiometry and conjunctival goblet cell density evaluation.Both measurements showed a significant improvement in the treated animals compared to the placebo diet (Figs.3A, 3B), confirming our results with the 12-week treatment regimen.Next, we investigated focus scores in lacrimal gland histologic sections.Like the 12-week treatment regimen, a longer cathepsin S inhibition diet did not improve the total lacrimal gland infiltration.
These results indicate that short-and long-term treatment with cathepsin S inhibitor benefits the cornea and conjunctiva by preserving cornea sensitivity and conjunctival goblet cell density, two cardinal signs of aging in the eye.However, a cathepsin S inhibitor diet does not significantly affect ectopic lymphoid structures in the aged lacrimal gland.

Aged Ctss −/− Mice Are Resistant to the Age-Related Dry Eye Phenotype
Since diet-based inhibition might not completely abrogate cathepsin S activity due to pharmacologic limitations, we validated the previously described findings in aged Ctss −/− mice.As previously shown, aged Ctss −/− mice did not display age-related goblet cell loss (Fig. 4A). 33Moreover, aged Ctss −/− mice did not display age-related corneal sensitivity loss (Fig. 4B) but had comparable total lacrimal gland infiltration to aged wild-type mice (Figs.4C, 4D).We then characterized these immune infiltrates with flow cytometry based on several markers.While both strains have an agerelated increase in Th1, Th17, and Treg cells (Fig. 4E) in their lacrimal glands, the frequency of Th1 and Th17 is smaller in the 24-month-old Ctss −/− mice (Fig. 4E), in agreement with our previous publication showing that aged lacrimal glands have infiltrating T effectors, Tregs, and B cells. 10,43Similar results were present in the ocular draining cervical lymph nodes (Fig. 4E).These results confirm that cathepsin S is critical for ocular health since it preserves corneal nerve sensitivity and goblet cell density.It is also critical for preventing Th1 and Th17 infiltration in aged lacrimal glands.

Cathepsin S Controls the T-Cell Landscape in Aged Lacrimal Glands
Since we observed that aged Ctss −/− mice had diminished Th1 and Th17 infiltration into the lacrimal glands, we performed additional flow cytometry studies investigating the frequency of naive, T central memory, and T effector memory cells in the lacrimal glands using CD44 and CD62L antibodies (Fig. 5A).The lacrimal glands of both aged Ctss −/− and B6 mice had a decrease in the proportion of effector memory T cells (CD44 + CD62L − ) and an increase in the proportion of central memory and naive T cells (Fig. 5B, only significant in the aged Ctss −/− mice).Interestingly, the proportion of central memory (CD44 + CD62L + ) and naive (CD44 − CD62L + cells) T cells was greater in aged Ctss −/− than in aged B6 mice (Fig. 5B).We also investigated T follicular helper (Tfh) cell frequency using CXCR5, PD-1, and BCL-6 antibodies (Figs.6A, 6B).In the Tfh compartment, there was an equivalent age-related increase in CD4 + CXCR5 + PD-1 + cells in both strains.Young B6 lacrimal glands had greater frequency and mean fluorescence intensity of the Tfh-specific transcription factor BCL-6 than young Ctss −/− lacrimal glands (Figs.6C-E).These results indicate that cathepsin S controls the pool of the different CD4 + T-cell compartments within the ectopic lymphoid structures that develop in the lacrimal glands during aging.

B-Cell Infiltration Differs in the Aged Ctss −/− Mice Compared to Wild-Type Mice
Most immune cells within aged lacrimal glands are B cells. 10 B-cell expansion within these ectopic lymphoid structures relies on T-cell-derived signals elicited by B-cell-driven antigenic presentation.Cathepsin S also participates in germinal center development. 14Thus, we quantified the impact of Ctss deletion on age-associated lacrimal gland B-cell infiltrates using CD19 and B220 staining and flow cytometry analysis (Figs. 7A, 7B).We observed an increase in CD19 + B220 − cells in both aged B6 and Ctss −/− mice, but the increase was higher in Ctss −/− than in B6 mice.On the other hand, mature CD19 + B220 + cells also increased in both strains but was higher in the B6 lacrimal glands (Fig. 7B).We also further characterized the B220 + popula-tion into marginal zone-like cells (IgM + CD23 − ) or follicularlike B cells (IgM + CD23 + ) after gating on B220 + CD93 − cells (Figs. 7C, 7D).We recently showed that aged lacrimal glands have an increase in marginal zone-like B cells. 10 There was an age-related increase in marginal zone-like and follicularlike B cells in both strains but no difference between the aged B6 and Ctss −/− mice.Altogether, these results indicate cathepsin S represses the CD19 + B220 − cell compartment size while promoting the expansion of CD19 + B220 + cells.

DISCUSSION
Cathepsin S levels increase in tears and lacrimal glands with aging.As augmented activity of this protease is linked with autoimmunity, its inhibition represents a potential therapeutic target for age-associated dry eye.Here we show that oral delivery of the cathepsin S inhibitor RO5461111 through specially formulated chow effectively reduces the impact of age-associated dry eye on the ocular surface of mice.These positive ocular effects are accompanied by changes in immune infiltration in the ocular draining lymph nodes and lacrimal glands.Aged Ctss −/− mice exhibit a comparable phenotype, confirming that these findings result from the on-target effects of the cathepsin S inhibitor.Altogether, our results demonstrate the feasibility of oral administration of a cathepsin S inhibitor and its potential usefulness in ameliorating age-associated dry eye while shedding light on the underlying therapeutic mechanisms.
The combined interpretation of the data on the T-and B-cell compartments in the lacrimal glands and ocular draining lymph nodes allows us to put forth a mechanism of action in the context of age-associated dry eye.Aging impacts T-cell homeostasis through different pathways, resulting in loss of tolerance and increased autoimmunity. 1,3,47Since cathepsin S activity favors antigenic presentation of self-derived peptides, 14 we believe that prolonged diet-based cathepsin S blockade interferes with the aberrant development of autoreactive T cells during aging.We have previously shown that the aging process is accompanied by the appearance of ectopic lymphoid structures with appreciable germinal centers in the lacrimal gland. 10In line with this, we report herein that diet-based cathepsin S inhibition modifies the age-associated immune infiltration of the lacrimal gland in terms of quality but not in quantity.
In Sjögren syndrome, a disease with lacrimal and salivary gland infiltrates, the initial events in ectopic lymphoid tissue development (i.e., lymphoid neogenesis) are related to local tissue inflammation, which ensues in response to intrinsic lacrimal gland dysfunction and acinar inflammation. 48Recently, a study showed that tissue-specific knockout of the autophagy pathway in acinar and ductal cells of salivary glands is sufficient to induce gland dysfunction before immune infiltration. 49Comparable studies on age-related lacrimal gland deficiency are lacking, but consistent with our hypothesis that loss of cell homeostasis such as in inflammation, Tnf −/− mice experience less age-related infiltration and decreased number of ectopic lymphoid structures. 50,51ntrasting with the initial events of lymphoid neogenesis, its progression requires antigen presentation, which in this context probably involves self-antigens.This process ensures adequate T follicular and B-cell cooperation and B-cell expansion.We propose that diet-based cathepsin S inhibition prevents autoantigen presentation in this scenario, as it has been demonstrated for young autoimmune-prone mice. 18,46In line with this, we observed fewer activated macrophages (professional antigen-presenting cells), fewer proinflammatory Th1 CD4 + T cells, and more regulatory CD4 + T cells, in agreement with our previous publications that these cells are important players in age-related dry eye. 43,52Aged cathepsin S-deficient mice replicated these findings, confirming that they were due to the specific blockade of cathepsin S activity by the inhibitor diet.Supporting our hypothesis, aged Ctss −/− mice had a reduced proportion of effector memory T cells and a higher proportion of naive and central memory T cells within the lacrimal gland infiltrates.Moreover, these mice harbored fewer T follicular cells in the lacrimal glands as assessed by the expression of the signature transcription factor Bcl-6.These cells are required to drive T-cell-dependent B-cell responses. 53,54Like the antigen-presenting cells that activate and induce T follicular cells, B cells also rely on cathepsin S for MHC II processing and thus obtain cooperative signaling from cognate antigen-specific T follicular cells. 55Therefore, cathepsin S is also essential for the expansion and proliferation of the corresponding B cells in physiologic humoral immune responses, 13 but cathepsin S inhibition does not alter T-cellindependent responses.Our findings on the landscape of the lacrimal gland infiltrates of aged Ctss −/− mice are consistent with this framework: we observed an increased proportion of CD19 + B220 − B cells, which do not rely on T cells for antibody production, and a reduced proportion of CD19 + B220 + B cells, which depend on T follicular cells for expansion. 56hus, the changes in both T-and B-cell compartments within the lacrimal gland ectopic lymphoid tissue of aged cathepsin S-deficient mice are likely linked by the reduction in local autoantigen presentation.However, because the antigen in dry eye remains elusive, further studies are necessary to investigate this.
By contrast, the beneficial effects of the cathepsin S inhibitor-containing diet on the ocular surface of aged mice are probably due to the blockade of extracellular cathepsin S activity.We have shown that aged C57BL/6J mice have increased cathepsin S activity levels in tears and lacrimal gland lysates. 33The source of increased cathepsin S levels in aged tears is most likely dysregulated exocytosis of endolysosomal vesicles in the lacrimal gland acinar cells, as it has been demonstrated to occur in a murine Sjögren syndrome model. 57Cathepsin S exocytosis is also differentially modulated by sympathetic and parasympathetic autonomic stimuli. 58Intriguingly, the increase in sympathetic activity that occurs with aging 59 is associated with increased cathepsin S secretion through an alternative pathway in lacrimal gland acinar cells. 58Regarding its pathogenic action, cathepsin S disrupts intercellular tight junctions in cultured corneal epithelial cells, and topical ocular administration to Ctss −/− mice breaks down the corneal epithelial barrier, thus replicating the phenotype. 33In addition, cathepsin S elicits extracellular matrix remodeling in other tissues 60,61 and can degrade the lubricating proteoglycans of the ocular surface. 62Contrasting the knowledge of its pathogenic effects on the corneal epithelium, the impact of cathepsin S on corneal nerves and conjunctival goblet cells is less understood.IFN-γ deficiency protects mice from agerelated goblet cell loss. 40As both corneal nerves and goblet cells are negatively affected by Th1 CD4 + T cells, 41,[63][64][65][66] it is tempting to speculate that cathepsin S inhibition during aging protects corneal nerves and goblet cells by reducing Th1 immune responses on the ocular surface.Supporting this idea, we observed fewer IFN-γ -producing CD4 + T cells in the lacrimal glands and a comparable trend in the eyedraining lymph nodes.However, more work is warranted to prove or discard this hypothetical mechanism of action.
Our research provides preclinical evidence that an oral approach to cathepsin S inhibition could be therapeutic in age-related dry eye.Comparable strategies have shown preclinical efficacy in Sjögren syndrome 19,67 using a different small molecule (RO5459072).By contrast, a recent phase II study that evaluated a 12-week-long daily oral intake of RO5459072 cathepsin S inhibitor in patients with Sjögren syndrome did not detect a clinical benefit. 68Nonetheless, these findings do not necessarily rule out the therapeutical potential of cathepsin S blockade in this disease context for two reasons.First, assessing improvement in Sjögren syndrome is challenging as it relies mainly on subjective scoring that compounds into a disease activity index. 69herefore, the study documented a high individual variation. 68Second, the optimal duration for a treatment to show improvement in a chronic condition such as Sjögren syndrome is not known beforehand, especially with a therapeutic intervention that targets long-standing pathogenic processes (i.e., autoimmune responses).Both concerns also apply to age-related dry eye.In this regard, herein we show that 12-week and 28-week-long treatments with a cathepsin S inhibitor improved age-related dry eye parameters, a strength of our findings.Thus, dietary blockade of cathepsin S represents a potential therapeutic intervention in ageassociated dry eye.Further studies are necessary to investigate if topical eye drops would be as equally beneficial as dietary intake.

FIGURE 1 .
FIGURE 1. Cathepsin S inhibition diet for 12 weeks improves goblet cell density and cornea mechanosensitivity in 17-to 19-month-old mice.(A) Representative images of conjunctival sections stained with PAS (purple magenta) showing increased goblet cell density with the cathepsin S inhibition diet.Insets are a higher magnification of the area on the left.Scale bar: 100 μm.(B) Cumulative data of conjunctival goblet cell density after treatment.Mann-Whitney U test; each dot represents one animal, n = 5-10/group.(C) Cornea sensitivity was assessed using the Cochet-Bonnet aesthesiometer.Mann-Whitney U test, each dot represents one animal, n = 5-10/group.

FIGURE 3 .
FIGURE 3. Cathepsin S inhibition diet for 28 weeks improves goblet cell density and cornea mechanosensitivity in 19-to 22-month-old mice.(A) Cumulative data of conjunctival goblet cell density.Mann-Whitney U test; each dot represents one animal, n = 4-8/group.(B) Cornea sensitivity assessed using the Cochet-Bonnet aesthesiometer.Mann-Whitney U test; each dot represents one animal, n = 16-17/group.(C) Total lacrimal gland infiltration measured as focus score.Mann-Whitney U test.Each dot represents one animal, n = 4-8/group.

FIGURE 7 .
FIGURE 7. Cathepsin S controls the influx of subtypes of B cells into the aged lacrimal glands.(A, B) Representative dot plots of lacrimal gland suspensions from young and aged lacrimal glands from C57BL/6 and Ctss −/− mice stained with CD19 and B220 antibodies (A) and cumulative data (B).Cells were gated on CD45 + live cells.Mean ± SD; each dot represents one lacrimal gland from one animal.Kruskal-Wallis with Dunn's multiple comparison test, n = 8-10/group.(C, D) Representative dot plots of lacrimal gland suspensions from young and aged lacrimal glands from C57BL/6 and Ctss −/− mice stained with IgM and CD23 antibodies (C) and cumulative data (D).Cells were gated on CD45 + B220 + CD93 − cells.Mean ± SD; each dot represents one lacrimal gland from one animal.Kruskal-Wallis with Dunn's multiple comparison test, n = 4/group.2-3M = 2-3 months; 24M+ = 24 months or older.Asterisks indicate intrastrain comparison of aged versus young lacrimal glands.*P < 0.05.****P < 0.0001.Fully written P values show interstrain comparisons.