Pigment Dispersion Contributes to Ocular Immune Privilege in a DBA/2J Mouse Model of Pigmentary Glaucoma

Abstract

Purpose: To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma.

Methods: DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry.

Results: Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells.

Conclusions: Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Ocular immune privilege is a crucial aspect of the eye's defense against intraocular inflammation. This privilege plays a vital role in immune protection and safeguarding the eye from potential vision impairment caused by inflammatory responses.¹ This mechanism of immunosuppression and immunomodulation can be explained by the ocular immunosuppressive microenvironment and anterior chamber-associated immune deviation (ACAID). After soluble antigens are introduced into the anterior chamber, ocular antigen-presenting cells (APCs) recognize and process these antigens in response. These processed antigens are then circulated to the spleen, selectively activating regulatory T cells (Tregs) and inhibiting the production of antigen-specific delayed-type hypersensitive (DTH) and complement-fixing antibodies, a specific immune-regulatory process known as ACAID.² Previous studies have demonstrated the critical role of Tregs in this process. Specifically, Stein-Streilein et al.³ have identified two distinct types of Tregs involved in ACAID: CD4⁺ T cells, which act as “afferent regulators,” and CD8⁺ T cells, which function as “efferent regulators.” Both types of Tregs are essential for the induction and maintenance of ACAID, playing complementary roles in modulating the immune response. The transcription factor forkhead box P3 (Foxp3) is more specifically expressed on CD4⁺CD25⁺ Treg cells and is a key regulator in the maintenance of Treg cell function; thus, it is widely believed that CD4⁺CD25⁺Foxp3 regulatory T cells are Tregs.^4–6 Tregs are important for mediating immunosuppression and maintaining immune homeostasis.^7,8 ACAID-induced Treg cells are a pivotal aspect of the molecular mechanism responsible for maintaining immune privilege. 

Pigment dispersion syndrome (PDS) encompasses a range of clinical symptoms caused by loss and dispersion of iris pigment particles, which are deposited in the anterior segment of the eye with aqueous humor (AH) circulation.⁹ It can cause elevated intraocular pressure (IOP) and progress into glaucomatous optic neuropathy, eventually leading to the transformation of PDS into pigmentary glaucoma (PG).¹⁰ PG is a subtype of secondary glaucoma with a low incidence, mild clinical symptoms, and inconspicuous early symptoms. However, it significantly impacts vision and can be easily misdiagnosed as other types of open-angle glaucoma during clinical diagnosis.¹¹ Therefore, studying the pathogenesis of PDS/PG is clinically important for early diagnosis and treatment. The progression of PDS to PG involves a complex interplay of several etiologic factors. Some researchers have revealed that immune factors are involved in abnormal pigment dispersion, thus confirming the importance of immune factors in the progression of PDS/PG.^12–14 However, the immune factors that affect the onset and development of PDS/PG remain unknown. 

As a classical animal model of hereditary glaucoma, DAB/2J mice exhibit pathogenesis and clinical manifestations similar to those of human PDS/PG, providing a novel approach for investigating the complex underlying mechanisms.^15–17 The primary manifestation associated with aging is the continuous loss and dispersion of iris pigment particles throughout the anterior segment of the eye. As specific foreign bodies, pigment particles are released into the AH, which may alter the ocular immunosuppressive microenvironment and affect immune privilege. Our previous studies revealed that functional blebs still existed in 66.7% (12/18) of the PG eyes for 8 years after trabeculectomy.¹⁸ Functional blebs survived longer than that in patients with primary open-angle glaucoma of similar age but without pigment dispersion in the anterior chamber. This suggests that pigment dispersion in the anterior chamber improves the long-term survival of functional blebs. The survival time of the functional blebs after glaucoma surgery can indirectly reflect the immunosuppressive microenvironment of the anterior chamber and the normalization of immune privilege status. Given the important role of Tregs in maintaining ocular immune privilege, we hypothesized that pigment dispersion within the anterior chamber contributes to modulating the immune response through the production of Tregs. 

This study aimed to investigate whether pigment particles affect the immune function of the anterior chamber by regulating Treg cells. Further research is anticipated to introduce novel clinical therapeutic strategies targeting this molecular process and may pave the way for a new field of glaucoma immunomodulation therapy. 

Materials and Methods

Animals and Husbandry

All animal experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and protocols were approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University, Beijing, China (Project ID: AEEI-2022-194). In this study, a total of 220 mice were employed, consisting of 130 DBA/2J mice and 90 C57BL/6J mice. Sixty DBA/2J mice were specifically designated for ACAID induction at 3, 6, and 9 months of age and were systematically divided into four groups: ACAID group (n = 15), positive control group (n = 15), negative control group (n = 15), and normal group (n = 15). The remaining mice were allocated for other experimental purposes. DBA/2J and C57BL/6J mice were provided by Beijing Huafukang Biology Technology Co., Ltd. (Beijing, China). All mice were housed in the Experimental Animal Center of Capital Medical University and maintained in a 12-hour dark/light cycle with standard chow and water available ad libitum. Before commencing the experiments, the mice were kept until they reached 3, 6, and 9 months of age. 

IOP Measurements

IOP was measured in mice anesthetized with inhaled isoflurane (4% isoflurane) using a noninvasive Tonolab tonometer (iCare Finland Oy, Vantaa, Finland). For each measurement, the system automatically recorded the average of five measurements as a single IOP value; three measurements were obtained for each eye and averaged. All measurements were performed by the same operator from 9:00 AM to 11:00 AM. 

Anterior Segment Examination

In DBA/2J mice, morphologic changes of the anterior segments, such as iris transillumination defects (a typical feature of iris diseases), were observed using slit-lamp microscopy (BX900, Haag-Streit AG, Switzerland) at 3, 6, and 9 months of age. All the photographs were captured under the same conditions. 

AH Collection and Analysis

The AH was collected using a 34-gauge needle connected to a 1-mL syringe based on the siphon principle at 3, 6, and 9 months of age. The protein concentration of AH was detected using 2 µL of supernatant with a BCA protein assay kit (Beyotime, Shanghai, China) at 4000 rpm for 4 minutes. The remaining supernatants were pooled (8–10 eyes/pool). In accordance with the methodology described by Yamaguchi et al.,¹⁹ immune cells of AH were detected by cytologic analysis using a hematoxylin and eosin staining kit (Servicebio), and specific cell types were identified using immunocytochemistry. Immunocytochemistry was performed with anti-CD10 mouse mAb (Servicebio) and recombinant anti-myeloperoxidase (MPO) rabbit mAb (Servicebio) and visualized by Cy3-conjugated goat anti-mouse secondary antibody (Servicebio) and Alexa Fluor 488 anti-rabbit secondary antibody (Servicebio), respectively. TGF-β2 protein levels in the AH were measured using an ELISA detection kit (R&D Systems, Biotechne, Minneapolis, MN, USA) by analyzing the same volume of sample. Notably, TGF-β2 in the AH requires a process of acid activation and neutralization to be activated into an immunoreactive form. 

Measurements of Protein and Cytokine Levels in Mouse Serum

Serum was collected from mice at the ages of 3, 6, and 9 months to analyze serum protein concentrations with the BCA protein assay kit (Elabscience Biotechnology, Wuhan, China) and specific cytokine concentrations with the ELISA assay kit, including interferon-γ (IFN-γ) (Elabscience Biotechnology), interleukin 10 (IL-10) (Elabscience Biotechnology), and transforming growth factor–β2 (TGF-β2) (R&D Systems, Biotechne). All BCA and ELISA kits were used strictly under the manufacturer's instructions. 

Preparation and Anterior Chamber Injection of Pigment Particles

Pigment particles were prepared as described previously.^20,21 In brief, fresh C57BL/6J mouse eyeballs were disinfected for 2 minutes in 5% povidone–iodine solution. The irises were then isolated on ice and placed into 10 mL PBS. The samples were frozen at –80°C for 2 hours and thawed at room temperature. After two freeze–thaw cycles, pigments were dislodged by pipetting up and down 100 times with a 3-mL Pasteur pipette. The samples were then filtered through a 40-µm cell strainer and centrifuged at 3000 rpm for 15 minutes, and the supernatant was discarded. The pigment particles were resuspended in 10 mL PBS and centrifuged again to remove the supernatant. Finally, the particles were resuspended in 2 mL PBS and stored at 4°C as stock solution. The concentration of pigment particles was measured using a hemocytometer. The suspension was diluted 1000-fold and observed under a microscope at 600× magnification to calculate the concentration. 

The concentration of pigment particles was adjusted to 1 × 10⁷ pigment particles per milliliter. Referring to the methods of ACAID induction²² and microbead-induced glaucoma,^23–25 we injected 2 µL of 1 × 10⁷ pigment particles per milliliter into the anterior chamber of DBA/2J mice at 3, 6, and 9 months of age. 

Induction of ACAID

ACAID was induced by microinjection of ovalbumin (OVA) antigen (Sigma-Aldrich, St. Louis, MO, USA) into the anterior chamber of DBA/2J mice at 3, 6, and 9 months of age. The mice were divided into four groups: ACAID group, positive control group, negative control group, and normal group. After the mice were anesthetized with an intraperitoneal injection of pentobarbital, 2 µL OVA solution (50 mg/mL) was injected into the anterior chamber of the right eye. On day 7, 100 µL OVA solution (2.5 mg/mL), completely emulsified in complete Freund's adjuvant (1:1; Sigma-Aldrich), was subcutaneously injected into the neck of the mice for immunization. The negative control group received only anterior chamber injections of PBS. The positive control group received only subcutaneous immunization. The normal group did not receive any intervention. 

DTH Assay

On day 7 after subcutaneous immunization, digimatic micrometers (Mitutoyo, Kanagawa, Japan) with a measurement resolution of 0.001 mm were used to measure the thickness of both ear pinnae in each group of mice. Then, 20 µL OVA solution (10 mg/mL) and 20 µL PBS solution were injected subcutaneously into the right and left ear pinnae, respectively. The thickness of both ear pinnae was measured again with a micrometer 24 hours after injection, and the formula used for calculating ear swelling is as follows: specific ear swelling = (24-hour measurement – 0-hour measurement) of the right ear – (24-hour measurement – 0-hour measurement) of the left ear. Ear swelling responses were used to evaluate the DTH. 

Flow Cytometry Analysis

The spleen was harvested after anesthesia, ground, filtered, and prepared into a single-cell suspensions, and erythrocytes were removed. A mixture of cells (1 × 10⁶) was stained with Fixable Viability Dye eFluor 780 (eBioscience Invitrogen, Carlsbad, CA, USA), incubated at 4°C, and protected from light for 30 minutes to assess cell viability. The cells were surface-stained with anti-mouse CD3 (eFluor 450; eBioscience Invitrogen), anti-mouse CD4 (FITC; eBioscience Invitrogen), and anti-mouse CD25 (APC; eBioscience Invitrogen); fixed and permeabilized with a Foxp3 Staining Set (eBioscience Invitrogen); and intracellularly stained with anti-mouse Foxp3 (PE; eBioscience Invitrogen). The cells were washed thrice with flow staining buffer. Cell detection and analysis were performed using a BD LSR Fortessa (BD Biosciences). 

Statistical Analysis

Prism 9.0 (GraphPad Software, San Diego, CA, USA) was used to graph and analyze the data. Statistical significance was evaluated using ANOVA. All experiments were performed at least three times to ensure practicability. All data are expressed as the mean  ±  SD. P values < 0.05 were considered statistically significant. 

Results

Longitudinal Changes in IOP

Changes in the IOP of 3-, 6-, and 9-month-old DBA/2J and age-matched C57BL/6J mice were observed longitudinally. As shown in Figure 1, the IOP began to rise as iris pigment particles spread in the anterior chamber, and the difference in IOP among the groups before and after pigment dispersion was statistically significant in DBA2J. In contrast, no significant changes occurred in C57BL/6J mice. 

Figure 1.

 

Changes in IOP in 3-, 6-, and 9-month-old DBA/2J mice and C57BL/6J mice. Data are expressed as the mean ± SD (n = 10 /group). ****P < 0.0001.

Anterior Eye Morphology

To assess pigment dispersion in the anterior chamber of DBA/2J mice, we observed changes in the anterior segment of the eyes of 3-, 6-, and 9-month-old DBA/2J mice. The iris texture of 3-month-old mice was clear and no obvious abnormalities were observed under a slit-lamp microscope. At 6 months of age, the iris texture was blurry, the iris stroma began to show atrophy, and the iris pigment particles began to disperse. At 9 months of age, the anterior chamber depth was increased, corneal neovascularization was visible, the dispersion of iris pigment particles was more pronounced, and the iris stroma was extensively atrophied with substantial iris transillumination defects. Representative images are shown in Figure 2. The C57BL/6J mice showed no changes (data not shown). 

Figure 2.

 

Slit-lamp photography of changes in the morphology of the anterior eye with age in DBA/2J mice. (A–F) Changes in the anterior segment abnormalities. (G–I) The degree of iris transillumination defects (magnification, 25×).

Inflammation Occurs in the Eye After Pigment Dispersion

Normally, the protein levels in the AH of C57BL/6J mice are extremely low. Protein levels in the AH of 3-month-old DBA/2J mice resembled those of C57BL/6J mice. In contrast, AH protein levels in DBA/2J mice began to increase at 6 months of age and were highest at 9 months of age (Fig. 3A). Protein levels in the serum of C57BL/6J mice had weak changes with age. In contrast, serum protein levels in DBA/2J mice began to increase at 6 months of age and were highest at 9 months of age (Fig. 3B). We found that the number of AH immune cells significantly increased after pigment dispersion at 9 months of age, compared with DBA/2J mice at 3 and 6 months of age. In contrast, no immune cells were found in the AH of C57BL/6J mice (Fig. 3C). We also found that the immune cells in the AH of DBA/2J mice were positive for CD10 and MPO, indicating that they are neutrophils (Fig. 3C). These findings indicate that the blood–aqueous barrier was compromised. 

Figure 3.

 

Pigment dispersion causes intraocular inflammation. (A, B) Protein concentrations in the AH and serum were assessed using bicinchoninic acid. (C) Quantification of immune cells in the AH by hematoxylin and eosin. Immune cells were positively stained for CD10, MPO, and DAPI. Scale bar: 50 µm. (D–F) Concentrations of IL-10, IFN-γ, and TGF-β2 in mice serum were examined via an ELISA kit. Data are expressed as the mean ± SD of three independent experiments (n = 4/group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To further evaluate the effect of pigment dispersion on intraocular inflammation, we assayed the expression of inflammatory cytokines (IL-10, IFN-γ) in serum. The results displayed a high level of IL-10 (Fig. 3D) and a low level of IFN-γ (Fig. 3E) after pigment dispersion. The level of TGF-β2 in serum was decreased in DBA/2J mice as pigment dispersed (Fig. 3F). In summary, inflammation was present in the DBA/2J mice after pigment dispersion. 

Pigment Dispersion Affects the Immunosuppressive Microenvironment of AH

Pigment dispersion can induce changes in the immunosuppressive microenvironment of AH. The ocular microenvironment is enriched with immunomodulatory molecules. Previous research has shown that elevated TGF-β2 levels, a major immunomodulatory factor, are crucial for the activity of immune cells within AH.^26,27 At 3 months of age, TGF-β2 levels in the AH of DBA/2J mice did not significantly differ compared to C57BL/6J mice. However, at 6 months of age, TGF-β2 levels were elevated in the AH of DBA/2J mice. Moreover, TGF-β2 levels were significantly higher at 9 months of age. These findings indicate that TGF-β2 in the AH of DBA/2J mice gradually increased after pigment dispersion, with statistically significant differences observed in all pairwise comparisons (Fig. 4). 

Figure 4.

 

Concentrations of TGF-β2 in the AH were determined using an ELISA assay. Data are expressed as the mean ± SD of three independent experiments (n = 5/group). *P < 0.05, **P < 0.01, and ****P < 0.0001.

Pigment Particles Influence Splenic Treg Cell Expression

It is reported that Treg cells are involved in stabilizing the immunosuppressive microenvironment within the ocular immune privilege mechanism and suppressing the DTH response induced by ocular antigens.²⁸ We investigated the expression of Treg cells in splenic T lymphocytes before and after pigment dispersion in DBA/2J mice and in age-matched C57BL/6J mice. In all age groups, Tregs were significantly more abundant in DBA/2J than in C57BL/6J mice (Figs. 5A–C). Furthermore, our results showed that Treg cells increased with pigment dispersion in DBA/2J mice, with the most pronounced increase observed at 9 months of age (Fig. 5D). 

Figure 5.

 

Expression of Treg cells on murine splenic T lymphocytes was analyzed via flow cytometry. (A–C) Expression levels of Treg cells in both groups of mice at 3, 6, and 9 months old. (D) Expression levels of Treg cells gradually increased with age in DBA/2J mice. Data are expressed as the mean ± SD of three independent experiments (n = 5/group). *P < 0.05, ***P < 0.001, and ****P < 0.0001.

To further validate the impact of pigment particles on splenic Treg cell expression, 3-, 6-, and 9-month-old DBA/2J mice received intracameral injections of pigment particles for 2 weeks, followed by flow cytometry to assess the expression of regulatory T cells. The results demonstrate that in all age groups receiving intracameral injections of pigment particles, the number of Treg cells was increased compared to the control group of the same age (Fig. 6). 

Figure 6.

 

Expression of Treg cells on murine splenic T lymphocytes was analyzed via flow cytometry. (A–C) Expression levels of Treg cells in the anterior chamber 2 weeks postinjection of pigment particles in DBA/2J mice at 3, 6, and 9 months old. Data are expressed as the mean ± SD of three independent experiments (n = 4/group). *P < 0.05, ***P < 0.001, and ****P < 0.0001.

Pigment Dispersion Enhances the Capacity of ACAID Induction

ACAID is a major manifestation of ocular immune privilege, and when the ability to induce ACAID is lost, ocular immune privilege is usually damaged. We investigated whether pigment dispersion supported ACAID induction in DBA/2J mice. ACAID was induced in DBA/2J mice at 3, 6, and 9 months of age. The ACAID-inducing capacity was assessed by evaluating DTH responses and Treg cell expression in each group. At both 3 and 6 months of age, ear swelling in the ACAID group was increased significantly compared to the normal and negative groups. However, compared to the positive control group, there was no significant difference in ear swelling in the ACAID group (Figs. 7A, 7C). At the same time, Treg cells were not highly expressed in the ACAID group (Figs. 7B, 7D). This indicated that the DTH response was not suppressed and the ability to induce ACAID is lost. There was no significant difference in ear swelling in the ACAID group compared to the normal and negative group at 9 months of age; however, the ear swelling in the ACAID group was decreased significantly compared to the positive control group (Fig. 7E). At the same time, Treg cells were significantly elevated in the ACAID group (Fig. 7F). This indicated that OVA injected into the anterior chamber of 9-month-old DBA/2J mice successfully inhibited the DTH response and elevated Treg cell expression. These results suggest that pigment dispersion enhances the capacity for ACAID induction. 

Figure 7.

 

Pigment dispersion enhances the capacity of ACAID induction. In 3-, 6-, and 9-month-old DBA/2J mice, OVA antigen was injected into the anterior chamber of the right eye. On day 7, 100 µL OVA solution was thoroughly emulsified with an equal volume of complete Freund's adjuvant and subcutaneously immunized into the neck of mice. On day 14, the DTH assay was performed after the ear challenge. ACAID mice received all the above interventions. The positive control group received only subcutaneous immunizations and the ear challenge. The negative control group received anterior chamber injections of PBS and performed the ear challenge. The normal group underwent the ear challenge alone. (A, B) At 3 months of age, the results of ACAID induction and Treg cell expression levels in DBA/2J mice are shown. (C, D) At 6 months of age, the results of ACAID induction and Treg cell expression levels in DBA/2J mice are shown. (E, F) At 9 months of age, DBA/2J mice can suppress DTH and induce ACAID. Flow cytometry results showed considerable changes in the expression of Treg cells. Data are presented as the mean ± SD of three independent experiments (n = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for experimental groups versus positive controls. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 for experimental groups versus normal and negative controls.

Discussion

In this study, we explored the impact of pigment particles, as a specific foreign body in the AH, on ocular immune privilege. We confirmed that pigment dispersion causes alterations in the immunosuppressive microenvironment of AH, enhances splenic Treg cell expression, and restores ACAID, further suggesting that pigment dispersion promotes immune privilege. 

DBA/2J mice have been observed to develop ocular anterior segment abnormalities as they age, including corneal calcification, iris stromal atrophy, peripheral anterior synechiae, and iris transillumination defects. Of notable significance is the occurrence of varying degrees of pigment dispersion.¹⁵ We chose 3, 6, and 9 months as the main time points to observe the effect of varying degrees of pigment dispersion on ocular immune privilege in DBA2J mice according to our previous studies.²⁹ In our present study, we observed elevated levels of AH protein and serum protein, accompanied by an increase in the number of immune cells in the anterior chamber. These findings indicate the disruption of the blood–aqueous barrier and the occurrence of inflammation in eyes affected by pigment dispersion. We also observed that the serum level of the anti-inflammatory cytokine IL-10 was increased while that of the proinflammatory cytokine IFN-γ was decreased, further indicating that DBA/2J mice spontaneously experienced intraocular inflammation after pigment dispersion. Additionally, ocular immune privilege can induce apoptosis and promote the release of anti-inflammatory cytokines,³⁰ and changes in these inflammatory factors result from immune privilege–mediated antigen-specific immunomodulation. 

The ocular microenvironment within the AH consists of immunosuppressive factors that suppress the induction of inflammation and maintain immune privilege by modulating innate and adaptive immunity.²⁶ Under physiologic conditions, pigment particles do not exist in the AH. Nevertheless, patients with PDS/PG experience the prolonged dispersion of numerous pigment particles in the AH, leading to the deposition of these particles in the anterior eye segment. Our results suggest that this causes changes in the immunosuppressive microenvironment, which, in turn, affects immune privilege. The majority of TGF-β2 is present in the eye in a latent form, necessitating conversion to its active form, which plays a crucial role in conferring immunosuppressive activity. Nishida and Taylor³¹ found that AH can maintain a normal immunosuppressed ocular microenvironment by inducing Treg cell expression through the production of the immunoregulatory factor TGF-β2. After pigment dispersion, latent TGF-β2 was activated, TGF-β2 levels were elevated in the AH, and Treg activity was promoted, thereby increasing splenic Treg cell expression. This was similar to the process underlying the increased production of active TGF-β in the AH after injecting OVA into the anterior chamber.³¹ Interestingly, we also found that DBA/2J mice had higher levels of Treg cells than C57BL/6J mice prior to pigment dispersion. This differential expression may have been caused by gene mutations in the DBA/2J mice. 

Investigating the mechanism of ACAID has been a key area of interest for scientists, holding significant scientific value and providing guidance for clinical anti-inflammatory and immunotherapy. In corneal transplantation, studies have shown that ACAID contributes significantly to the survival of corneal allografts. The role of ACAID in corneal immune privilege is of great significance.^32–34 In general, antigens entering the anterior chamber of the eye do not cause DTH, thus protecting the eye against damage from inflammation due to the modulating effect of ACAID.³ Pigment particles dispersed in the AH can alter the immunosuppressive microenvironment and enhance the ACAID-inducing capacity. At 3 and 6 months of age, DTH was not suppressed in DBA/2J mice, and the capacity to support ACAID was lost. Our experimental results were consistent with those of the previous studies.^12,35 However, at 9 months of age, DBA/2J mice showed suppressed DTH and ACAID was induced. Treg cell numbers were also elevated in the ACAID group. To augment ACAID induction, we hypothesized that maintenance of an immunosuppressive microenvironment through the activation of latent TGF-β2 after pigment dispersion enhances Treg cell expression and reestablishes ACAID. 

It has been reported that active mechanisms of immunomodulation and immunosuppression exist in the ocular microenvironment. Immunosuppressive factors in the AH can suppress DTH and induce regulatory immunity in their own unique way.²⁷ Evidence indicates that TGF-β2 in the AH can confer the properties of conventional APC-induced ACAID and downregulate DTH, which has a promotive effect on ACAID.^2,36 Immune tolerance of ACAID promotes regulation of the immune response through the production of Tregs.¹ Given that antigen-specific Treg cells are direct mediators of the immune deviation profile of ACAID and have potent immunosuppressive properties, Foxp3⁺ Treg cells are considered key regulators of ocular immune privilege.¹ Therefore, pigment particle dispersion in the anterior chamber may contribute to immune response regulation by inducing more Tregs to reestablish ACAID. These findings suggest that the eye has developed new mechanisms to maintain immune privilege despite intraocular inflammation. 

While our study primarily focuses on the expression of Foxp3 in CD4⁺ Tregs in the spleen of ACAID-induced mice, it is crucial to acknowledge the significant role of CD8⁺ Tregs in this model. Stein-Streilein et al.³ have shown that CD8⁺ Tregs act as “efferent regulators,” suppressing effector T-cell responses during the execution phase of ACAID. Future studies should include detailed investigations into the expression and functional characteristics of CD8⁺ Tregs in ACAID. Understanding the interactions between CD4⁺ and CD8⁺ Tregs will allow for a more comprehensive understanding of the mechanisms by which pigment dispersion affects ocular immune privilege. 

In addition, we found that serum TGF-β2 levels were decreased. There is evidence that antigen-specific cell signaling in the peripheral blood is associated with ACAID induction, which selectively induces a state of systemic immunosuppression.³⁷ Pigment particles within the anterior chamber may act as foreign antigens, triggering the expression of TGF-β2 in the AH. This could generate a specific antigenic signal that travels through the blood circulation to the spleen, where most of the blood-borne signals may be selectively depleted, resulting in a decrease in serum TGF-β2. This process is similar to that of ACAID regulation. Further studies are necessary to investigate whether pigment particles can act as specific antigenic substances to induce ACAID when pigment particles are introduced into the anterior chamber. 

However, a limitation of our study is that it does not assess the effect of the degree of pigment dispersion, as well as the morphology, size, and number of pigment particles on ocular immune privilege. These factors will be the topic of our next study. 

In summary, our findings further support existing data showing that anterior chamber pigment dispersion promotes immune privilege and that Treg cells play an important regulatory role in pigment dispersion, thereby affecting ACAID. Theoretically, the ACAID-inducing capacity may be restored earlier and immune privilege reestablished if adoptive transfer of Treg cells is performed prior to pigment dispersion in DBA/2J mice. This will reduce inflammatory damage to the eye, on the one hand, and help arrest the progression of PDS/PG disease, on the other. Our study provides a novel approach for glaucoma treatment that targets immunomodulation. 

Acknowledgments

The authors thank Fengyang Lei for his constructive suggestions in the process of our research. 

Supported by grants from the National Natural Science Foundation of China (81970795). 

Disclosure: Q. Li, None; L. Pu, None; S. Cheng, None; S. Tang, None; J. Zhang, None; G. Qing, None