Background: Pathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood–retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli. Methods: RMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H₂O₂), or 25 mM d-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA. Results: Pathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation. Conclusions: Pathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING–TBK1 signaling pathway.

In this study, we aimed to investigate whether chronic retinal inflammation is involved in the pathogenesis of form-deprivation myopia (FDM) using tree shrews as an animal model. Twenty-one tree shrews were randomly divided into 7-day/14-day FDM (FDM7/FDM14) groups and their corresponding 7-day/14-day control groups. Refraction and axial length were measured. To determine the effects of form deprivation on inflammation, we used real-time polymerase chain reaction (PCR) and immunohistochemistry to assess the expression levels of several proinflammatory cytokines. At day 0, the eyes in the FDM and control groups were hyperopic. However, after 7 and 14 days of form deprivation, the refractive error of the eyes in the FDM7 and FDM14 groups shifted from +6.6 ± 0.3 diopters (D) to +4.0 ± 0.5 D and from +6.4 ± 0.3 D to +5.0 ± 0.3 D, respectively. The levels of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and nuclear factor κB were increased in the FDM eyes, compared with those in the control eyes. The increase in matrix metalloproteinase-2 expression was greater in the FDM eyes than in the contralateral and control eyes, whereas collagen type I expression was downregulated. In conclusion, chronic inflammation may play a crucial pathogenic role in form-deprivation myopia in tree shrews.

Myopia is a highly prevalent refractive disorder. We investigated the effect of diacerein on monocular form deprivation (MFD) in hamsters as a possible therapeutic intervention. Diacerein is an anthraquinone derivative drug whose active metabolite is rhein. Diacerein or atropine was applied to the MFD hamsters, and their refractive error and axial length were measured after 21 days. The refractive error (control: −0.91 ± 0.023, atropine: −0.3 ± 0.08, and diacerein: −0.27 ± 0.07 D) and axial length (control: 0.401 ± 0.017, atropine: 0.326 ± 0.017, and diacerein: 0.334 ± 0.016 mm) showed statistically significant differences between control, atropine‐treated, and diacerein‐treated MFD eyes. Furthermore, we determined the level of transforming growth factor‐beta‐ (TGF‐) β1, matrix metalloproteinase‐ (MMP‐) 2, type I collagen, interleukin‐ (IL‐) 6, IL‐8, and monocyte chemoattractant protein‐ (MCP‐) 1 in the retina. Atropine and diacerein suppressed levels of the myopia‐related TGF‐β1 and MMP‐2 while increasing type I collagen expression. They also inhibited the interleukin IL‐6, IL‐8, and MCP‐1 levels. Diacerein reduced the IL‐6, IL‐8, and MCP‐1 expression in ARPE‐19 cells. Furthermore, diacerein inhibited inflammation by attenuating the phosphorylation of protein kinase B (AKT) and nuclear factor kappa‐light‐chain‐enhancer of activated B (NF‐κB) pathway. This suggests that diacerein has a therapeutic effect on myopia and is a potential treatment option.

Background: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. Methods: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. Results: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-β mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-β were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. Conclusions: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Achromatopsia (ACHM) is a severe congenital autosomal recessive retinal disorder caused by loss of cone photoreceptors. Here, we aimed to determine the underlying genetic lesions and phenotypic correlations in two Chinese families with ACHM. Medical history and clinical evaluation were obtained from both families. Targeted exome sequencing (TES) was performed on 201 disease-causing genes of inherited retinal dystrophies to screen for ACHM causative mutations in the two probands. The compound heterozygous mutations in CNGA3 (c.1074G > A, p.W358X; c.1706G > A, p.R569H) were identified in the first proband, and a novel homozygous mutation (c.968C > A, p.A323D) was detected in the other pedigree. The proposed topological model of the CNGA3 polypeptide suggested that the missense mutations primarily affected the transmembrane helix 5 and the cGMP-binding domain, respectively. Crystal structure modeling of the cyclic nucleotide-gated cation channel α-3 (CNGA3) protein encoded by the CNGA3 gene revealed an abnormal combined structure generated by R569H. We firstly used the TES approach to identify genetic alterations in patients with ACHM. We uncovered three mutations in CNGA3, including one novel mutation. Our results not only expand the genotypic spectrum for CNGA3 mutations, but also demonstrate that the TES approach is a valuable tool for molecular diagnosis.