Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

T-Cell Repertoire Analysis in the Conjunctiva of Murine Dry Eye Model.

Purpose: Dry eye is closely related to the activation and proliferation of immune cells, especially T cells. However, the determination of the preferential T-cell clonotypes is technically challenging. This study aimed to investigate the characterization of T-cell receptor (TCR) repertoire in the conjunctiva during dry eye. Methods: A desiccating stress animal model was established using C57/BL6 mice (8-10 weeks, female). After 7 days of stress stimulation, the slit-lamp image and Oregon-green-dextran staining were used to evaluate the ocular surface injury. Periodic acid-Schiff staining was used to measure the number of goblet cells. Flow cytometry was used to detect the activation and proliferation of T cells in the conjunctiva and cervical lymph nodes. Next-generation sequencing was used to detect the αß TCR repertoire of the conjunctiva. Results: The αß TCR diversity increased significantly in the dry eye group, including the higher CDR3 amino acid length, marked gene usage on TCR V and J gene segments, extensive V(D)J recombination, and distinct CDR3 aa motifs. More important, several T-cell clonotypes were uniquely identified in dry eye. Furthermore, these perturbed rearrangements were reversed after glucocorticoid administration. Conclusions: A comprehensive analysis of the αß TCR repertoire in the conjunctiva of the dry eye mouse model was performed. Data in this study contributed significantly to the research on dry eye pathogenesis by demonstrating the TCR gene distribution and disease-specific TCR signatures. This study further provided some potential predictive T-cell biomarkers for future studies.

Studies on bacterial cellulose/poly(vinyl alcohol) hydrogel composites as tissue-engineered corneal stroma.

Corneal transplantation is currently the major solution in the treatment of severe corneal diseases. However, it is restricted due to the limited number of corneal donors. A tissue-engineered cornea is a potential substitute which could help overcome this limitation. This research envisages the development of a novel tissue-engineered corneal stroma consisting of bacterial cellulose (BC)/poly(vinyl alcohol) (PVA) hydrogel composites for reconstructing the cornea. It was found that the properties of BC/PVA were better suited for use as a corneal stroma material than the BC hydrogel. The human corneal stromal cells (hCSCs) were used to evaluate the cytotoxicity of the materials, wherein BC/PVA displayed excellent biocompatibility with these cells. Furthermore, in the in vivo studies, the BC/PVA was transplanted intrastromally in rabbits. After four weeks, the cornea remained almost transparent, and without obvious inflammation, sensitization or neovascularization, as confirmed by the clinical and histological examinations. Our results demonstrate that BC/PVA was well-tolerated in the rabbit cornea, and may be a potential substitute for corneal stroma.

Oxidative Stress Suppresses Cellular Autophagy in Corneal Epithelium.

Purpose: Oxidative stress is a major pathogenesis of certain ocular surface diseases. This study investigated the association of oxidative stress and cellular autophagy in corneal epithelium. Methods: We applied hydrogen peroxide (H2O2) to induce oxidative damage to cultured human corneal epithelial (HCE) cells and rat corneas. Cell viability, Western blotting of caspase 8, and TUNEL staining were conducted to measure the cellular injury. The production of reactive oxygen species (ROS) was measured and the levels of the following marker and key factors of ROS were also measured to detect oxidative stress: 3-nitrotyrosine, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), superoxide dismutase, catalase, and glutathione S-transferase P. The following key factors of autophagy were measured: LC3, beclin 1, Atg 12, and P62. We also applied an agonist of autophagy, rapamycin, in the experiment. Results: Cellular injury and oxidant damage were induced after exposure to H2O2 in HCE cells and rat corneas, such as increases of cell death and production of ROS; upregulation of a ROS generation enzyme, NOX4; and downregulation of degradation factors of ROS, superoxide dismutase, catalase, and glutathione S-transferase P. However, the process of cellular autophagy was suppressed by the measurements of LC3, beclin 1, Atg 12, and P62. Furthermore, application of rapamycin antagonized the cellular and oxidant injury induced by H2O2 but increased the level of autophagy in HCE cells. Conclusions: The oxidative stress of corneal epithelium is associated with the inhibition of cellular autophagy.

SERPINA3K Ameliorates the Corneal Oxidative Injury Induced by 4-Hydroxynonenal.

Purpose: We previously demonstrated that SERPINA3K has anti-inflammatory, antiangiogenic, and antioxidant effects in corneas. Here we further investigated the effects of SERPINA3K on the corneal oxidant injury setting recently developed and induced by 4-hydroxynonenal (4-HNE). Methods: We applied the 4-HNE-induced corneal oxidant stress in cultured human corneal epithelial (HCE) cells in vitro and to the cornea of rats in vivo. The following experiments were conducted: cell counting kit 8 assay to detect cell viability; quantitative real-time PCR assay; Western blotting and immunofluorescent staining to measure gene expressions or protein levels of key reactive oxygen species (ROS)-associated factors (3-nitrotyrosine [3-NT]; nicotinamide adenine dinucleotide phosphate [NADPH]-oxidase 4 [NOX4]; superoxide dismutase [SOD]); catalase and nuclear factor [erythroid-derived 2]-like 2 [NRF2]); as well as main factors of the Wnt/ß-catenin signaling pathway (p-LRP6, ß-catenin and transcription factor 4 [TCF4]); histologic staining; and TUNEL staining to examine sections of rat corneas. Results: We found that SERPINA3K concentration dependently protected cell viability, decreased levels of ROS marker 3-NT, suppressed NOX4, and upregulated SOD and catalase. Furthermore, SERPINA3K inhibited the activation of the ROS pathway NRF2 and its downstream factors, NAD(P)H dehydrogenase (quinone) 1 (NQO1) and heme oxygenase 1 (HO1), and also suppressed the activation of the Wnt signaling pathway p-LRP6, ß-catenin, and TCF4 in HCE cells treated with 4-HNE. Meanwhile, SERPINA3K ameliorated the oxidant injury of rat corneas induced by 4-HNE and downregulated ROS systems and the Wnt/ß-catenin pathway. Conclusions: Our findings show that SERPINA3K protected the oxidant damage induced by 4-HNE in the cornea and its underlying mechanism was through suppression of the ROS system and inhibition of the activated Wnt/ß-catenin signaling pathway.

Down-Regulation of MicroRNA-184 Is Associated With Corneal Neovascularization.

PURPOSE: Although microRNA-184 (miR-184) is abundantly expressed in the corneas, the role of miR-184 in corneal neovascularization remains unknown. Here we investigated the association between miR-184 expression and corneal neovascularization. METHODS: Quantitative real-time PCR assay was conducted to detect the expression of miR-184 and its potential target genes in the corneal epithelium of rats with corneal suture-induced neovascularization. MicroRNA-184 was also applied topically to the suture rats. Mimic and inhibitor of miR-184 were transfected into the cultured human umbilical vein endothelial cells (HUVECs), human corneal epithelial (HCE) cells, and simian choroidal endothelial cells (RF/6A). The following experiments were performed to evaluate the effects of miR-184 in these transfected cells: cell proliferation by cell viability assay, cell migration by a scratch wound test, VEGF-induced tube formation, and VEGF and ß-catenin levels by Western blot analysis. RESULTS: The expression of miR-184 was significantly reduced, whereas the gene expression of frizzled-4, a receptor of the Wnt pathway, was up-regulated in the corneal epithelium of corneal suture rats. The corneal neovascularization induced by suture was ameliorated by topical administration of miR-184. In the cells transfected with mimic and inhibitor of miR-184, miR-184 significantly suppressed the cell proliferation and cell migration of HUVECs, miR-184 down-regulated VEGF, and ß-catenin expression in HUVECs and HCE cells. Furthermore, miR-184 inhibited the tube formation of RF/6A cells. CONCLUSIONS: Down-regulation of miR-184 is associated with up-regulation of VEGF and Wnt/ß-catenin expression as well as corneal neovascularization, indicating that miR-184 negatively regulates corneal neovascularization.

Male Men1 heterozygous mice exhibit fasting hyperglycemia in the early stage of MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome characterized by multiple tumors in the parathyroid glands, endocrine pancreas and anterior pituitary. Recent clinical studies have revealed a strong association between MEN1 syndrome and the risk of developing diabetes mellitus; however, the underlying mechanisms remain unknown. In this study, heterozygous Men1 knockout (Men1(+/-)) mice were used as MEN1 models to investigate MEN1-associated glucose metabolic phenotypes and mechanisms. Heterozygous deficiency of Men1 in 12-month-old male mice induced fasting hyperglycemia, along with increased serum insulin levels. However, male Men1(+/-) mice did not show insulin resistance, as evidenced by Akt activation in hepatic tissues and an insulin tolerance test. Increased glucose levels following pyruvate challenge and expression of key gluconeogenic genes suggested increased hepatic glucose output in the male Men1(+/-) mice. This effect could be partly due to higher basal serum glucagon levels, which resulted from pancreatic islet cell proliferation induced by heterozygous loss of Men1 Taken together, our results indicate that fasted male Men1(+/-) mice, in the early stage of development of MEN1, display glucose metabolic disorders. These disorders are caused not by direct induction of insulin resistance, but via increased glucagon secretion and the consequent stimulation of hepatic glucose production.

ROS-mediated Different Homeostasis of Murine Corneal Epithelial Progenitor Cell Line under Oxidative Stress.

The role of ROS in stem cell biology has not been fully illustrated and understood. Here we compared the different responses and investigated the mechanism underlying oxidative stress induced by hydrogen peroxide (H2O2) between murine corneal epithelial progenitor cell line (TKE2) and mature murine corneal epithelial cells (MCE). TKE2 showed a different homeostasis and strong resistance to H2O2. TKE2 reduced the production of ROS, inhibited ROS generation enzyme NADPH oxidase 4 (NOX4), and increased dual specificity phosphatase 6 (DUSP6). Furthermore, TKE2 activated nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway, regulated miR-125B1 and miR-29B1, and elevated levels of antioxidants glutathione S-transferase P (GSTP) and superoxide dismutases (SOD). The association with ROS of the cells was also verified by RNA interference approach and pharmacological antagonization. In addition, TKE2 enhanced the autophagy after exposure to H2O2. The novel evidence suggests that TKE2 cells have different homeostasis and strong antioxidant properties against oxidative stress via the regulation of ROS formation and pathway.