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Crystallin βb2 (crybb2) is upregulated in regenerating retinas and in various pathological conditions of the retina, including uveoretinitis. However, the role of crybb2 in this disease is largely unknown. Therefore, we used recombinant crybb2 (rcrybb2) as intravitreal treatment of B10.RIII mice prior to immunization with human interphotoreceptor retinoid-binding protein peptide 161–180 (hIRBPp161-180) in complete Freund’s adjuvant (CFA) and concomitant injection of pertussis toxin (PTX) to induce experimental autoimmune uveoretinitis (EAU). In naïve mice, more beta III-tubulin (TUBB3) + and RNA-binding protein with multiple splicing (RBPMS) + cells were found in the ganglion cell layer of the retina than in EAU eyes, suggesting a loss of retinal ganglion cells (RGC) during the development of EAU. At the same time, the number of glial fibrillary acidic protein (GFAP) + cells increased in EAU eyes. RGCs were better protected in EAU eyes treated with rcrybb2, while the number of GFAP+ cells decreased. However, in retinal flatmounts, both retinal ganglion cells and retinal endothelial cells stained positive for TUBB3, indicating that TUBB3 is present in naïve B10.RIII mouse eyes not exclusive to RGCs. A significant decline in the number of RBPMS-positive retinal ganglion cells was observed in retinal flatmounts from EAU retinas in comparison to naïve retinas or EAU retinas with intravitreal rcrybb2 treatment. Whereas no significant decrease in TUBB3 levels was detected using Western blot and RT-qPCR, GFAP level, as a marker for astrocytes, increased in EAU mice compared to naïve mice. Level of Bax and Bcl2 in the retina was altered by treatment, suggesting better cell survival and inhibition of apoptosis. Furthermore, our histologic observations of the eyes showed no change in the incidence and severity of EAU, nor was the immune response affected by intravitreal rcrybb2 treatment. Taken together, these results suggest that intravitreal injection of rcrybb2 reduces retinal RGC death during the course of EAU, independent of local or systemic autoimmune responses. In the future, treating posterior uveitis with rcrybb2 to protect RGCs may offer a promising novel therapeutic strategy.

Patients with chronic anterior uveitis are at particularly high risk of developing secondary glaucoma when corticosteroids [e.g., dexamethasone (Dex)] are used or when inflammatory activity has regressed. Macrophage migration into the eye increases when secondary glaucoma develops and may play an important role in the development of secondary glaucoma. Our aim was to evaluate in vitro if increased hydrostatic pressure and corticosteroids could induce changes in macrophages phenotype. By using a pressure chamber cell culture system, we assessed the effect of increased hydrostatic pressure (HP), inflammation, and immunosuppression (Dex) on the M1/M2 phenotype of macrophages. Bone marrow-derived macrophages (BMDMs) were stimulated with medium, lipopolysaccharide (LPS, 100 ng/ml), Dex (200 ng/ml), or LPS + Dex and incubated with different HP (0, 20, or 60 mmHg) for 2 or 7 days. The numbers of CD86+/CD206− (M1 phenotype), CD86–/CD206+ (M2 phenotype), CD86+/CD206+ (intermediate phenotype), F4/80+/TNF-α+, and F4/80+/IL-10+ macrophages were determined by flow cytometry. TNF-α and IL-10 levels in cell culture supernatants were quantified by ELISA. TNF-α, IL-10, fibronectin, and collagen IV expression in BMDMs were detected by immunofluorescence microscopy. Higher HP polarizes macrophages primarily to an M1 phenotype (LPS, 60 vs. 0 mmHg, d2: p = 0.0034) with less extra cellular matrix (ECM) production and secondary to an M2 phenotype (medium, 60 vs. 0 mmHg, d7: p = 0.0089) (medium, 60 vs. 20 mmHg, d7: p = 0.0433) with enhanced ECM production. Dex induces an M2 phenotype (Dex, medium vs. Dex, d2: p < 0.0001; d7: p < 0.0001) with more ECM production. Higher HP further increased M2 polarization of Dex-treated macrophages (Dex, 60 vs. 0 mmHg, d2: p = 0.0417; d7: p = 0.0454). These changes in the M1/M2 phenotype by high HP or Dex treatment may play a role in the pathogenesis of secondary uveitic glaucoma- or glucocorticoid (GC)-induced glaucoma.

Microglia represent the primary resident immune cells of the central nervous system (CNS) and modulate local immune responses. Depending on their physiological functions, microglia can be classified into pro- (M1) and anti-inflammatory (M2) phenotype. Interleukin (IL)-10 is an important modulator of neuronal homeostasis, with anti-inflammatory and neuroprotective functions, and can be released by microglia. Here, we investigated how IL-10 deficiency affected the M1/2 polarization of primary microglia upon lipopolysaccharide (LPS) stimulation in vitro. Microglia phenotypes were analyzed via flow cytometry. Cytokine and chemokine secretion were examined by ELISA and bead-based multiplex LEGENDplexTM. Our results showed that genetic depletion of IL-10 led to elevated M1 like phenotype (CD86+ CD206−) under pro-inflammatory conditions associated with increased frequency of IL-6+, TNF-α+ cells and enhanced release of several pro-inflammatory chemokines. Absence of IL-10 led to an attenuated M2 like phenotype (CD86− CD206+) and a reduced secretion of TGF-β1 upon LPS stimulation. In conclusion, IL-10 deficiency may promote the polarization of microglia into M1-prone phenotype under pro-inflammatory conditions.