Single­cell RNA sequencing reveals inflammatory retinal microglia in experimental autoimmune uveitis.

Autoimmune uveitis (AU) is a kind of immune-mediated disease resulting in irreversible ocular damage and even permanent vision loss. However, the precise mechanism underlying dynamic immune changes contributing to disease initiation and progression of AU remains unclear. Here, we induced an experimental AU (EAU) model with IRBP651-670 and found that day[D]14 was the inflammatory summit with remarking clinical and histopathological manifestations and the activation of retinal microglia exhibited a time-dependent pattern in the EAU course. We conducted single-cell RNA sequencing of retinal immune cells in EAU mice at four time points and found microglia constituting the largest proportion, especially on D14. A novel inflammatory subtype (Cd74high Ccl5high) of retinal microglia was identified at the disease peak that was closely associated with modulating immune responses. In vitro experiments indicated that inflammatory stimuli induced proinflammatory microglia with the upregulation of CD74 and CCL5, and CD74 overexpression in microglia elicited their proinflammatory phenotype via nuclear factor-kappa B signaling that could be attenuated by the treatment of neutralizing CCL5 antibody to a certain extent. In-vivo blockade of Cd74 and Ccl5 effectively alleviated retinal microglial activation and disease phenotype of EAU. Therefore, we propose targeting CD74 and CCL5 of retinal microglia as promising strategies for AU treatment.

A de novo missense mutation in MPP2 confers an increased risk of Vogt-Koyanagi-Harada disease as shown by trio-based whole-exome sequencing.

Vogt-Koyanagi-Harada (VKH) disease is a leading cause of blindness in young and middle-aged people. However, the etiology of VKH disease remains unclear. Here, we performed the first trio-based whole-exome sequencing study, which enrolled 25 VKH patients and 50 controls, followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations. A total of 15 de novo mutations in VKH patients were identified, with one of the most important being the membrane palmitoylated protein 2 (MPP2) p.K315N (MPP2-N315) mutation. The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions. Additionally, this mutation appears rare, being absent from the 1000 Genome Project and Genome Aggregation Database, and it is highly conserved in 10 species, including humans and mice. Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis (EAU). In vitro, we used clustered regularly interspaced short palindromic repeats (CRISPR‒Cas9) gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315. Levels of cytokines, such as IL-1ß, IL-17E, and vascular endothelial growth factor A, were increased, and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells. Mechanistically, the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315, as shown by LC‒MS/MS and Co-IP, and resulted in activation of the ERK3/IL-17E pathway. Overall, our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

Integrative Single-Cell Transcriptomics and Epigenomics Mapping of the Fetal Retina Developmental Dynamics.

The underlying mechanisms that determine gene expression and chromatin accessibility in retinogenesis are poorly understood. Herein, single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing are performed on human embryonic eye samples obtained 9-26 weeks after conception to explore the heterogeneity of retinal progenitor cells (RPCs) and neurogenic RPCs. The differentiation trajectory from RPCs to 7 major types of retinal cells are verified. Subsequently, diverse lineage-determining transcription factors are identified and their gene regulatory networks are refined at the transcriptomic and epigenomic levels. Treatment of retinospheres, with the inhibitor of RE1 silencing transcription factor, X5050, induces more neurogenesis with the regular arrangement, and a decrease in Müller glial cells. The signatures of major retinal cells and their correlation with pathogenic genes associated with multiple ocular diseases, including uveitis and age-related macular degeneration are also described. A framework for the integrated exploration of single-cell developmental dynamics of the human primary retina is provided.

SOX2-positive retinal stem cells are identified in adult human pars plicata by single-cell transcriptomic analyses.

Stem cell therapy is a promising strategy to rescue visual impairment caused by retinal degeneration. Previous studies have proposed controversial theories about whether in situ retinal stem cells (RSCs) are present in adult human eye tissue. Single-cell RNA sequencing (scRNA-seq) has emerged as one of the most powerful tools to reveal the heterogeneity of tissue cells. By using scRNA-seq, we explored the cell heterogeneity of different subregions of adult human eyes, including pars plicata, pars plana, retinal pigment epithelium (RPE), iris, and neural retina (NR). We identified one subpopulation expressing SRY-box transcription factor 2 (SOX2) as RSCs, which were present in the pars plicata of the adult human eye. Further analysis showed the identified subpopulation of RSCs expressed specific markers aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12). We, therefore, isolated this subpopulation using these two markers by flow sorting and found that the isolated RSCs could proliferate and differentiate into some retinal cell types, including photoreceptors, neurons, RPE cells, microglia, astrocytes, horizontal cells, bipolar cells, and ganglion cells; whereas, AQP1- TSPAN12- cells did not have this differentiation potential. In conclusion, our results showed that SOX2-positive RSCs are present in the pars plicata and may be valuable for treating human retinal diseases due to their proliferation and differentiation potential.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

Galectin-3 regulates microglial activation and promotes inflammation through TLR4/MyD88/NF-kB in experimental autoimmune uveitis.

Galectin-3, an attractive molecule of innate immunity, has been reported to be involved in the neuroinflammatory diseases. However, the role of Galectin-3 in autoimmune uveitis is still unclear. The purpose of this study was to investigate the effect and mechanism of Galectin-3 on microglial activation and inflammation of experimental autoimmune uveitis (EAU). We immunized female C57BL/6 J mice with IRBP651-670 to induce EAU and the specific inhibitor was intravitreally injected in EAU mice. Disease severity was evaluated by clinical and histopathological scores. Immunofluorescence, western blot, qRT-PCR analysis and immunoprecipitation were used to detect the functional phenotypes and mechanisms on microglia after Galectin-3 inhibition. Our results showed that the expression of Galectin-3 was conspicuously increased in microglia of EAU retinas. The specific inhibitor of Galectin-3, TD139 was found to ameliorate the clinical and histological manifestations of EAU mice. In addition, TD139 reduced the expression of proinflammatory factors in vivo and vitro, which are related to the severity of uveitis. In mechanism, TD139 down-regulated the expression of TLR4 and MyD88, and then inhibited the activation of NF-κB p65 in microglia. In conclusion, Galectin-3 may play important roles in a variety of immune related diseases including autoimmune uveitis. Additionally, the inhibition of Galectin-3 may attenuate the microglial activation and inflammatory response through TLR4/MyD88/NF-κB pathway, highlighting a potential therapeutic target of Galectin-3 for autoimmune uveitis.

[Prediction of Pathogenesis and Potential Therapeutic Targets for Asthma Based on Proteomics of Mouse Lung Tissue].

Objective To predict the mechanism and potential therapeutic targets of asthma based on proteomic analysis and network pharmacology.Methods The mouse model of asthma was established via intraperitoneal injection of 200 µl suspension containing 100 µg ovalbumin(OVA)and 2 mg aluminum hydroxide and intranasal administration with 5% OVA.Maxquant system was used to retrieve the protein and gene data.The analysis of variance and t test were performed to obtain differential proteins,and then clustering map and target set of differential proteins were established.The protein-protein interaction network of differential proteins was constructed.The pathogenesis of asthma was investigated via gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis.Results A total of 5063 genes were identified,from which 904 differentially expressed genes were selected with the thresholds of fold change(model/control)≥2 and P≤0.05 as well as thresholds of fold change(model/control)≤1/2 and P≤0.05.The 904 genes were classified into 3 clusters.The 904 differentially expressed genes included 595 up-regulated genes and 309 down-regulated genes in the model group compared with the control group.The pathogenesis of asthma was associated with regulatory metabolism,Fc gamma-R mediated phagocytosis,leukocyte transendothelial migration,tumor necrosis factor signaling pathway,Toll-like receptor signaling pathway,B cell receptor signaling pathway,phosphoinositol 3-kinase/protein kinase B signaling pathway,vascular smooth muscle contraction and cell adhesion signaling pathway.ITGB3,CYBB,SYK,VWF,ITGB2,MYD88,COMP,VEGFA,and FCGR2B were identified as the therapeutic targets for asthma.Meanwhile,the biological processes such as signal transduction,redox process,immune response,inflammatory response,cell adhesion,positive regulation of GTPase activity,apoptosis,and extracellular matrix formation were the main participants in asthma.Conclusion This study systematically revealed the pathogenesis,biological processes,and 9 potential therapeutic targets of asthma.

Low Expression of YTH Domain-Containing 1 Promotes Microglial M1 Polarization by Reducing the Stability of Sirtuin 1 mRNA.

The N6-methyladenosine (m6A) modification is the most abundant posttranscriptional mRNA modification in mammalian cells and is dynamically modulated by a series of "writers," "erasers," and "readers." Studies have shown that m6A affects RNA metabolism in terms of RNA processing, nuclear export, translation, and decay. However, the role of the m6A modification in retinal microglial activation remains unclear. Here, we analyzed the single-cell RNA sequencing data of retinal cells from mice with uveitis and found that the m6A-binding protein YTH domain-containing 1 (YTHDC1) was significantly downregulated in retinal microglia in the context of uveitis. Further studies showed that YTHDC1 deficiency resulted in M1 microglial polarization, an increased inflammatory response and the promotion of microglial migration. Mechanistically, YTHDC1 maintained sirtuin 1 (SIRT1) mRNA stability, which reduced signal transducer and activator of transcription 3 (STAT3) phosphorylation, thus inhibiting microglial M1 polarization. Collectively, our data show that YTHDC1 is critical for microglial inflammatory response regulation and can serve as a target for the development of therapeutics for autogenic immune diseases.

A Single-Cell Transcriptome Atlas of the Human Retinal Pigment Epithelium.

Human retinal pigment epithelium cells are arranged in a monolayer that plays an important supporting role in the retina. Although the heterogeneity of specific retinal cells has been well studied, the diversity of hRPE cells has not been reported. Here, we performed a single-cell RNA sequencing on 9,302 hRPE cells from three donors and profiled a transcriptome atlas. Our results identified two subpopulations that exhibit substantial differences in gene expression patterns and functions. One of the clusters specifically expressed ID3, a macular retinal pigment epithelium marker. The other cluster highly expressed CRYAB, a peripheral RPE marker. Our results also showed that the genes associated with oxidative stress and endoplasmic reticulum stress were more enriched in the macular RPE. The genes related to light perception, oxidative stress and lipid metabolism were more enriched in the peripheral RPE. Additionally, we provided a map of disease-related genes in the hRPE and highlighted the importance of the macular RPE and peripheral RPE clusters P4 and P6 as potential therapeutic targets for retinal diseases. Our study provides a transcriptional landscape for the human retinal pigment epithelium that is critical to understanding retinal biology and disease.

Aminooxy-Acetic Acid Inhibits Experimental Autoimmune Uveitis by Modulating the Balance between Effector and Regulatory Lymphocyte Subsets.

PURPOSE: A small molecular compound, aminooxy-acetic acid (AOA), has been shown to modulate experimental autoimmune encephalomyelitis (EAE). The current study was designed to investigate whether AOA has a similar effect on the development of experimental autoimmune uveitis (EAU) and to further explore underlying mechanisms of this drug. METHODS: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). AOA (500µg or 750µg) or vehicle was administered by intraperitoneal injection from day 10 to 14 after EAU induction. The severity was assessed by clinical and histological scores. The integrity of the blood retinal barrier was detected with Evans Blue. Frequencies of splenic Th1, Th17 and Foxp3+ Treg cells were examined by flow cytometry. The production of cytokines was tested by ELISA. The mRNA expression of IL-17, IFN-γ and IL-10 was detected by RT-PCR. The expression of p-Stat1 and NF-κB was detected by Western Blotting. RESULTS: AOA was found to markedly inhibit the severity of EAU, as determined by clinical and histopathological examinations. AOA can relieve the leakage of blood retinal barrier (BRB). Functional studies found a decreased frequency of Th1 and Th17 cells and an increased frequency of Treg cells in EAU mice as compared with controls. Further studies showed that AOA not only downregulated the production of the proinflammatory cytokines including IFN-γ and IL-17 but also upregulated the expression of an anti-inflammatory cytokine such as IL-10, which might be caused by inhibiting the expressions of p-Stat1 and NF-κB. CONCLUSION: This study shows that AOA inhibits the severity and development of EAU by modulating the balance between regulatory and pathogenic lymphocyte subsets.