Mesenchymal stem cells (MSCs) exhibit a potent immunomodulatory capacity and have been applied to treat diseases such as graft versus host disease and severe autoimmune diseases. However, the mechanism underlying their immunosuppressive effect is not yet completely understood. Here, we investigated the role of the CD73/adenosine pathway in immune modulation by MSCs using a mouse model of experimental autoimmune uveitis (EAU). Moreover, we examined the in vitro modulatory effect of MSCs mediated through the CD73/adenosine pathway in human and mouse T cells. We found that the severity of EAU was significantly attenuated by MSCs; however, most therapeutic effects of MSCs were lost by pretreatment with a CD73 inhibitor. The inhibitory mechanism of MSCs might be contributed by CD73 on MSCs that cooperated with CD39 and CD73 on activated T cells to produce adenosine, resulting in inhibition of T-cell proliferation. Furthermore, MSCs increased the expression of CD73 on CD4 ⁺ T cells, and transforming growth factor-β1 (TGF-β1) was the only tested cytokine that contributed to upregulation of CD73. Hence, our study demonstrates that the CD73/adenosine pathway involves the immunomodulatory function of MSCs in autoimmune responses.

Background: Many studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Krüppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis. Purpose: In this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway. Methods: KLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs subcloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA` probe. Results: Ectopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio. Conclusion: The ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.

Hepatocyte growth factor (HGF) induced the proliferation of lens epithelial cells (LECs) and may be a major cause of posterior capsule opacification (PCO), which is the most frequent postoperative complication of cataract surgery. To date, several agents that can block LECs proliferation have been studied, but none have been used in clinic. Recently, accumulating evidence has suggested rapamycin, the inhibitor of mTOR (mammalian target of Rapamycin), was associated with the induction of apoptosis in LECs. The purpose of our study was to investigate the potential effects of rapamycin on HGF-induced LECs and the underlying mechanisms by which rapamycin exerted its actions. Using cell proliferation, cell viability and flow cytometric apoptosis assays, we found that rapamycin potently not only suppressed proliferation but also induced the apoptosis of LECs in a dose-dependent manner under HGF administration. Further investigation of the underlying mechanism using siRNA transfection revealed that rapamycin could promote apoptosis of LECs via inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK and JAK2/STAT3 signaling molecules. Moreover, the forced expression of AKT, ERK and STAT3 could induce a significant suppression of apoptosis in these cells after treatment of rapamycin. Together, these findings suggested that rapamycin-induced apoptosis in HGF-stimulated LECs is accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3 pathways, which supports its use to inhibit PCO in preclinical studies and provides theoretical foundation for future possible practice.

Microglial activation and subsequent pathological neuroinflammation contribute to diabetic retinopathy (DR). However, the underlying mechanisms of microgliosis, and means to effectively suppress pathological microgliosis, remain incompletely understood. Peroxisome proliferator-activated receptor alpha (PPARα) is a transcription factor that regulates lipid metabolism. The present study aimed to determine if PPARα affects pathological microgliosis in DR. In global Pparα mice, retinal microglia exhibited decreased structural complexity and enlarged cell bodies, suggesting microglial activation. Microglia-specific conditional Pparα^−/− (PCKO) mice showed decreased retinal thickness as revealed by optical coherence tomography. Under streptozotocin (STZ)-induced diabetes, diabetic PCKO mice exhibited decreased electroretinography response, while diabetes-induced retinal dysfunction was alleviated in diabetic microglia-specific Pparα-transgenic (PCTG) mice. Additionally, diabetes-induced retinal pericyte loss was exacerbated in diabetic PCKO mice and alleviated in diabetic PCTG mice. In cultured microglial cells with the diabetic stressor 4-HNE, metabolic flux analysis demonstrated that Pparα ablation caused a metabolic shift from oxidative phosphorylation to glycolysis. Pparα deficiency also increased microglial STING and TNF-α expression. Taken together, these findings revealed a critical role for PPARα in pathological microgliosis, neurodegeneration, and vascular damage in DR, providing insight into the underlying molecular mechanisms of microgliosis in this context and suggesting microglial PPARα as a potential therapeutic target.

Background: The epithelial–mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis. Methods: We performed fundus fluorescein angiography, optical coherence tomography and hematoxylin–eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT–related markers, pERK1/2, pGSK3β and β-catenin. Results: SB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3β and the expression of β-catenin. Conclusion: SB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3β/β-catenin pathways. Graphical SB431542 inhibits EMT in RPE cells under high glucose conditions.