Liraglutide protects ß-cells in novel human islet spheroid models of type 1 diabetes.

To enable accurate, high-throughput and longer-term studies of the immunopathogenesis of type 1 diabetes (T1D), we established three in-vitro islet-immune injury models by culturing spheroids derived from primary human islets with proinflammatory cytokines, activated peripheral blood mononuclear cells or HLA-A2-restricted preproinsulin-specific cytotoxic T lymphocytes. In all models, ß-cell function declined as manifested by increased basal and decreased glucose-stimulated insulin release (GSIS), and decreased intracellular insulin content. Additional hallmarks of T1D progression such as loss of the first-phase insulin response (FFIR), increased proinsulin-to-insulin ratios, HLA-class I expression, and inflammatory cytokine release were also observed. Using these models, we show that liraglutide, a glucagon-like peptide 1 receptor agonist, prevented loss of GSIS under T1D-relevant stress, by preserving the FFIR and decreasing immune cell infiltration and cytokine secretion. Our results corroborate that liraglutide mediates an anti-inflammatory effect that aids in protecting ß-cells from the immune-mediated attack that leads to T1D.

The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation.

Several subsets of Foxp3(+) regulatory T cells (T(reg) cells) work in concert to maintain immune homeostasis. However, the molecular bases underlying the phenotypic and functional diversity of T(reg) cells remain obscure. We show that in response to interferon-gamma, Foxp3(+) T(reg) cells upregulated the T helper type 1 (T(H)1)-specifying transcription factor T-bet. T-bet promoted expression of the chemokine receptor CXCR3 on T(reg) cells, and T-bet(+) T(reg) cells accumulated at sites of T(H)1 cell-mediated inflammation. Furthermore, T-bet expression was required for the homeostasis and function of T(reg) cells during type 1 inflammation. Thus, in a subset of CD4(+) T cells, the activities of the transcription factors Foxp3 and T-bet are overlaid, which results in T(reg) cells with unique homeostatic and migratory properties optimized for the suppression of T(H)1 responses in vivo.

T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor ß2.

Foxp3(+) regulatory T (Treg) cells limit inflammatory responses and maintain immune homeostasis. Although comprised of several phenotypically and functionally distinct subsets, the differentiation of specialized Treg cell populations within the periphery is poorly characterized. We demonstrate that the development of T-bet(+) Treg cells that potently inhibit T helper 1 (Th1) cell responses was dependent on the transcription factor STAT1 and occurred directly in response to interferon-γ produced by effector T cells. Additionally, delayed induction of the IL-12Rß2 receptor component after STAT1 activation helped ensure that Treg cells do not readily complete STAT4-dependent Th1 cell development and lose their ability to suppress effector T cell proliferation. Thus, we define a pathway of abortive Th1 cell development that results in the specialization of peripheral Treg cells and demonstrate that impaired expression of a single cytokine receptor helps maintain Treg cell-suppressive function in the context of inflammatory Th1 cell responses.

Anti-IL-21 monoclonal antibody combined with liraglutide effectively reverses established hyperglycemia in mouse models of type 1 diabetes.

Immunotherapy for type 1 diabetes (T1D) has previously focused on suppressing the autoimmune response against pancreatic beta cells to preserve endogenous insulin production and regulate glucose levels. With increased attention toward combination therapy strategies, studies indicate the multifunctional cytokine interleukin-21 (IL-21) may be a suitable target as an immuno-modulatory arm, while glucagon-like peptide-1 receptor (GLP-1R) agonists may be appropriate as a beta cell protective arm in combination therapy for T1D. We report here that treatment with anti-IL-21 monoclonal antibody delays diabetes onset in the spontaneous non-obese diabetic (NOD) and NOD.scid adoptive transfer models, while its effect in reversing recent-onset hyperglycemia is limited. However, the combination of anti-IL-21 plus the GLP-1R agonist liraglutide is effective in reversing established disease compared to either monotherapy in both the NOD and rat insulin promotor-lymphocytic choriomeningitis virus glycoprotein (RIP-LCMV-GP) models of autoimmune diabetes. Enhanced efficacy is particularly evident in severely hyperglycemic mice, with return to normoglycemia remaining stable for the majority of mice even after therapy is withdrawn. Importantly, increased beta cell proliferation does not appear to be the predominant mechanism. In conclusion, combination therapy with anti-IL-21 and liraglutide is able to consistently reverse disease in mouse models of T1D. The observed effects rival the most effective experimental disease-modifying treatments tested in preclinical studies.

Oral insulin (human, murine, or porcine) does not prevent diabetes in the non-obese diabetic mouse.

Studies have shown oral insulin prevents type 1 diabetes (T1D) in mouse models, however human trials were inconclusive. We tested the ability of different insulins to prevent T1D in non-obese diabetic mice. Mice received oral insulin or PBS twice weekly and disease was monitored. Contrary to previous studies, no insulin tested showed significant ability to prevent T1D, nor did testing of linked suppression in a delayed type hypersensitivity model have reproducible effect. To investigate delivery of antigen within the GI tract, blue dye was fed to mice. Dye traveled 5-8 cm from stomach to small intestine within 10s, suggesting orally administered antigen may not get digested in the stomach in mice. Insulin incubated with jejunum extracts was instantly digested. Thus, in humans large doses of insulin may be required to achieve tolerance as antigen may be more vulnerable to digestion in the stomach even before reaching the small intestine.

Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease.

CD4(+)Foxp3(+) regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4(+)CD103(hi) phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4(+)Foxp3(+) T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.

Type-1 immunity drives early lethality in scurfy mice.

Foxp3(+) regulatory T (Treg) cells modulate the functions of multiple immune cell types, and loss of Treg cells causes lethal, CD4(+) T-cell-dependent multiorgan autoimmune disease in both mice and humans. However, how different effector T-cell subets contribute to the severe autoimmunity observed in the absence of Treg cells remains controversial. We found that although expanded populations of Th1, Th2, and Th17 cells can be detected in scurfy (sf) mice, Th1 cells predominate. Moreover, using a genetic approach, we found that sf mice with deficiencies in type-1 immunity (sf × Ifngr1(-/-), sf × Tbx21(-/-), and sf × Ifngr1(-/-)/Tbx21(-/-)) have an extended lifespan that is associated with altered cytokine production and attenuated cutaneous and hepatic inflammation. By contrast, sf mice deficient in type-2 immune responses (sf × Stat6(-/-)) display a significantly reduced lifespan with increased hepatic inflammation, but decreased dermatitis. These data indicate that Th1 cells and their associated cytokines drive early immunopathology in Foxp3-deficient sf mice, highlighting the essential role of Treg cells in restraining Th1-cell-mediated autoimmunity.

A self-reactive TCR drives the development of Foxp3+ regulatory T cells that prevent autoimmune disease.

Although Foxp3(+) regulatory T cells (Tregs) are thought to express autoreactive TCRs, it is not clear how individual TCRs influence Treg development, phenotype, and function in vivo. We have generated TCR transgenic mice (termed SFZ70 mice) using Tcra and Tcrb genes cloned from an autoreactive CD4(+) T cell isolated from a Treg-deficient scurfy mouse. The SFZ70 TCR recognizes a cutaneous autoantigen and drives development of both conventional CD4(+) Foxp3(-) T cells (T(conv)) and Foxp3(+) Tregs. SFZ70 Tregs display an activated phenotype evidenced by robust proliferation and expression of skin-homing molecules such as CD103 and P-selectin ligand. Analysis of Foxp3-deficient SFZ70 mice demonstrates that Tregs inhibit T(conv) cell expression of tissue-homing receptors and their production of proinflammatory cytokines. In addition, Treg suppression of SFZ70 T(conv) cells can be overcome by nonspecific activation of APCs. These results provide new insights into the differentiation and function of tissue-specific Tregs in vivo and provide a tractable system for analyzing the molecular requirements of Treg-mediated tolerance toward a cutaneous autoantigen.

Cellular requirements for diabetes induction in DO11.10xRIPmOVA mice.

Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing ß-islet cells in the pancreas. The genetic and environmental mechanisms promoting the development of this disease remain poorly understood. We have explored the cellular requirements for T1D development in DO11.10xRIPmOVA (DORmO) mice, which carry a TCR transgene specific for an MHC class II-restricted epitope from OVA and express membrane-bound OVA in the pancreas under the control of the rat insulin promoter. We found that DORmO.RAG2(-/-) mice do not develop insulitis and are completely protected from diabetes, demonstrating that endogenous lymphocyte receptor rearrangement is required for disease development. Diabetes in DORmO mice is preceded by the development of OVA-specific autoantibodies and is delayed in B cell-deficient DORmO.JhD(-/-) mice, demonstrating that B cells contribute to disease progression. In addition, transfer of CD8(+) T cells from diabetic animals into DORmO.RAG2(-/-) mice promoted insulitis by OVA-specific CD4(+) T cells. Finally, although diabetes develops in DORmO mice in the presence of a significant population of Foxp3(+) OVA-specific regulatory T cells, boosting regulatory T cell numbers by injecting IL-2 immune complexes dampens autoantibody production and prevents development of insulitis and overt diabetes. These results help define the events leading to diabetes in DORmO mice and provide new insights into the cellular interactions required for disease development in an Ag-specific model of T1D.

Peripheral autoreactive CD8 T-cell frequencies are too variable to be a reliable predictor of disease progression of human type 1 diabetes.

OBJECTIVES: The detection of a peripheral immune cell signature that specifically reflects autoimmunity in type 1 diabetes would enable the prediction and staging of disease on an individual basis. However, defining such a signature is technically challenging. Reliable interpretation of immune cell-related biomarkers depends on their inherent variability and, to understand this variability, longitudinal analyses are required. METHODS: We performed a longitudinal observational study in which 40 individuals with elevated genetic risk of type 1 diabetes and persistent islet autoantibodies provided a blood sample every 4-6 weeks for > 1 year. RESULTS: Peripheral immune cell composition (T cells, NK cells and monocytes) was assessed using well-validated flow cytometry panels and demonstrated that, while non-antigen-specific immune cell subsets were stable over time, autoantigen-reactive T-cell frequencies were highly variable in and between individuals. Neither the frequency nor phenotype of non-antigen-specific subsets or autoreactive CD8+ T cells associated with clinical onset of T1D. CONCLUSION: The findings from the Type 1 Diabetes Longitudinal BIomarker Trial underscore the inherent challenge of evaluating changes in peripheral immune cell populations as surrogates of organ-specific disease activity. The variability of peripheral antigen-specific T cells precludes their use as a prognostic marker and clearly demonstrates that a reliable prognostic cell signature remains elusive.

Multicomponent Plasmid Protects Mice From Spontaneous Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease in which insulin-secreting ß-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613).

Multicomponent Plasmid Protects Mice From Spontaneous Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease in which insulin-secreting ß-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613).

Systematic Assessment of Immune Marker Variation in Type 1 Diabetes: A Prospective Longitudinal Study.

Immune analytes have been widely tested in efforts to understand the heterogeneity of disease progression, risk, and therapeutic responses in type 1 diabetes (T1D). The future clinical utility of such analytes as biomarkers depends on their technical and biological variability, as well as their correlation with clinical outcomes. To assess the variability of a panel of 91 immune analytes, we conducted a prospective study of adults with T1D (<3 years from diagnosis), at 9-10 visits over 1 year. Autoantibodies and frequencies of T-cell, natural killer cell, and myeloid subsets were evaluated; autoreactive T-cell frequencies and function were also measured. We calculated an intraclass correlation coefficient (ICC) for each marker, which is a relative measure of between- and within-subject variability. Of the 91 analytes tested, we identified 35 with high between- and low within-subject variability, indicating their potential ability to be used to stratify subjects. We also provide extensive data regarding technical variability for 64 of the 91 analytes. To pilot the concept that ICC can be used to identify analytes that reflect biological outcomes, the association between each immune analyte and C-peptide was also evaluated using partial least squares modeling. CD8 effector memory T-cell (CD8 EM) frequency exhibited a high ICC and a positive correlation with C-peptide, which was also seen in an independent dataset of recent-onset T1D subjects. More work is needed to better understand the mechanisms underlying this relationship. Here we find that there are a limited number of technically reproducible immune analytes that also have a high ICC. We propose the use of ICC to define within- and between-subject variability and measurement of technical variability for future biomarker identification studies. Employing such a method is critical for selection of analytes to be tested in the context of future clinical trials aiming to understand heterogeneity in disease progression and response to therapy.

An in vitro model of posterior capsular opacity: SPARC and TGF-beta2 minimize epithelial-to-mesenchymal transition in lens epithelium.

PURPOSE: This report presents a novel model for studies of extracellular matrix (ECM) in posterior capsular opacification (PCO) in vitro. Lens epithelial cells (LEC) were cultured with an intraocular lens (IOL) on a surface of type IV collagen in an evaluation of the importance of the ECM-cell interaction in formation of PCO. Abnormal migration, proliferation, and expression of proteins associated with the epithelial-to-mesenchymal transition (EMT) that characterizes PCO were observed in the presence and absence of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine), which regulates matrix-cell interactions. METHODS: The model for PCO in vitro consisted of an IOL placed on a membrane coated with collagen IV, a major constituent of the lens capsule. LECs from the lenses of wild-type (WT) and SPARC-null (SP-null) mice were cultured in the presence or absence of 10 ng/mL TGF-beta2 and 20 mug/mL recombinant human SPARC (rhSP) for up to 6 days. The migration of LECs was quantified. Labeling with BrdU and the measurement of DNA synthesis were assays for cell proliferation. Expression of the EMT markers, collagen type I, fibronectin, and alpha-smooth muscle actin were assessed using immunocytochemistry or Western immunoblots. RESULTS: LEC migration, proliferation, and the synthesis of EMT markers were enhanced in SP-null compared with WT LECs. TGF-beta2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-beta2 increased the production of collagen type I, fibronectin, and alpha-SMA. The responses of SP-null LECs were rescued by the addition of recombinant human (rh)SP. CONCLUSIONS: A simple IOL culture system was useful for the evaluation of the effects of SPARC and TGF-beta2 on PCO in vitro. The action of TGF-beta2 on LEC migration and proliferation is influenced by SPARC, a regulator of matrix-cell interactions. The results indicate a functional intersection between pathways activated by TGF-beta2 and SPARC in the formation of PCO.

Absence of SPARC in murine lens epithelium leads to increased deposition of laminin-1 in lens capsule.

PURPOSE: To investigate the role of SPARC in the regulation of the expression and deposition of extracellular matrix (ECM) proteins in the lens capsule. METHODS: Wild-type (SP+/+) and SPARC-null (SP-/-) mice of embryonic day (E)14 to 3 months of age were examined. Transcript levels of lens basement membrane (BM) components were analyzed by semiquantitative RT-PCR with mRNA from lens epithelia. Expression of ECM proteins in lens capsule and lens epithelium was analyzed by immunohistochemistry and Western blot analysis. Cell attachment was assessed by lens epithelial explant culture. Coimmunoprecipitation was performed to identify intracellular protein interactions. RESULTS: From postnatal day 5 to 3 months of age, SPARC-null lens capsules exhibited higher levels of laminin-1 deposition relative to their wild-type counterparts, as revealed by immunohistochemistry and immunoblot analysis. An uneven and aggregated distribution of laminin-1 protein was apparent in the anterior region of SPARC-null lens capsules. SPARC and laminin-1 were expressed abundantly in the endoplasmic reticulum (ER) of lens epithelial cells. Coimmunoprecipitation identified that SPARC associates with laminin-1 before laminin secretion. Furthermore, increased laminin-1 in lens capsule promoted the attachment of lens epithelial explants in culture. CONCLUSIONS: SPARC affects the secretion and deposition of laminin-1 protein in lens epithelial cells. Because abnormal deposition of laminin-1 in the lens BM could influence lens epithelial cell adhesion and fiber cell differentiation, the authors propose that SPARC is important to lens homeostasis through its regulation of lens BM matrix organization.

Foxp3+ regulatory T cells maintain immune homeostasis in the skin.

Cutaneous immune responses must be tightly controlled to prevent unwanted inflammation in response to innocuous antigens, while maintaining the ability to combat skin-tropic pathogens. Foxp3(+) regulatory T (T reg) cells are potent immune regulators and are found at high frequency in both human and mouse skin. Although T reg cells migrate to the skin and can dampen immune responses during experimentally induced inflammation or infection, the importance of cutaneous T reg cells for maintaining normal immune homeostasis in the skin has not been addressed. To selectively block T reg cell function in the skin, we restored the T reg cell compartment in Foxp3-deficient scurfy mice with cells whose ability to migrate to the skin was impaired because of targeted mutation of alpha-1,3-fucosyltransferase VII (Fut7). Although Fut7-deficient T reg cells were present at normal frequency and could function in all other tissues examined, these animals rapidly developed severe cutaneous inflammation. Thus, skin-resident T reg cell are essential for maintaining normal immune homeostasis at this site.

Caveolin-1 is up-regulated in transdifferentiated lens epithelial cells but minimal in normal human and murine lenses.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types. Caveolae play a role in lipid transport, endocytosis, signal transduction, and cell transformation. Expression of caveolin-1, the principal component of caveolae and a regulator of caveolae-dependent signaling and endocytosis, was investigated in lens epithelial cells and lens fiber cells in wild-type (wt) and SPARC-null mice and normal human donors in vivo and in vitro. RT-PCR, immunofluorescence and immunoblot analyses of human and murine ocular tissues revealed that caveolin-1 was expressed in the corneal epithelium, corneal endothelial cells, and blood vessels of iris, ciliary body and retina, but minimal in the normal lens epithelia or fiber cells of murine and human lens. In contrast, caveolin-1 was significantly up-regulated in mesenchymal-transdifferentiated lens epithelia in SPARC-null cataract lenses. In addition, lens epithelial cells from primary culture or from cultures of immortalized lens epithelial cell lines expressed significant amounts of caveolin-1. The lens epithelial cells expressed epidermal growth factor (EGF) receptor and were responsive to EGF-mediated cell proliferation, but they did not show EGF-dependent caveolin-1 tyrosine phosphorylation. Caveolin-1 might have a role in the process of epithelial-mesenchymal transdifferentiation (EMT) in the lens, the most common cause of vision loss in human secondary cataracts.

Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells.

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.

Differential responses of human lens epithelial cells to intraocular lenses in vitro: hydrophobic acrylic versus PMMA or silicone discs.

BACKGROUND: The purpose of this study was to determine the influence of different materials of intraocular lenses (IOLs) on human lens epithelial cell behavior, including adhesion, migration, proliferation, apoptosis, and epithelial-mesenchymal transdifferentiation (EMT) in vitro. METHODS: Human lens epithelial cells (SRA 01/04) were grown on hydrophobic acrylic (Acrysof), polymethylmethacrylate (PMMA), and silicone IOLs. Cellular adhesion, migration, proliferation, and apoptotic assays were performed to assess cell behavior. The expression of EMT markers (fibronectin and type I collagen) produced by cells on IOLs was determined by immunoblotting and immunocytochemistry. RESULTS: Human lens epithelial cells exhibited preferred adhesion and reduced apoptosis when cultured on acrylic IOLs, in comparison to PMMA and silicone IOLs. Cells grown on acrylic lenses formed a confluent epithelial monolayer. Migration of lens epithelial cells under the acrylic lens was substantially blocked in an in vitro assay. In contrast, cells grown on PMMA and silicone lenses displayed a spindle-shaped, myofibroblast-like morphology, increased apoptosis, reduced adhesion, and enhanced production of EMT proteins such as fibronectin and type I collagen. The migration of lens epithelial cells under PMMA and silicone IOLs was substantial in the in vitro assay. CONCLUSION: This report demonstrates that hydrophobic acrylic lenses are more capsular biocompatible than PMMA and silicone lenses. The in vitro assays are reliable measurements for evaluating the responses of human lens epithelial cells to different IOL materials, and could advance our understanding of the preferential capsular opacification conferred by different IOL materials.