Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina.

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

Beneficial mechanisms of dimethyl fumarate in autoimmune uveitis: insights from single-cell RNA sequencing.

BACKGROUND: Dimethyl fumarate (DMF) is a fumaric acid ester that exhibits immunoregulatory and anti-inflammatory properties. However, the function of DMF in autoimmune uveitis (AU) is incompletely understood, and studies comprehensively exploring the impact of DMF on immune cells are still lacking. METHODS: To explore the function of DMF in uveitis and its underlying mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) on the cervical draining lymph node (CDLN) cells of normal, experimental autoimmune uveitis (EAU), and DMF-treated EAU mice. Additionally, we integrated scRNA-seq data of the retina and CDLNs to identify the potential impact of DMF on ocular immune cell infiltration. Flow cytometry was conducted to verify the potential target molecules of DMF. RESULTS: Our study showed that DMF treatment effectively ameliorated EAU symptoms. The proportional and transcriptional alterations in each immune cell type during EAU were reversed by DMF treatment. Bioinformatics analysis in our study indicated that the enhanced expression of Pim1 and Cxcr4 in EAU was reversed by DMF treatment. Further experiments demonstrated that DMF restored the balance between effector T (Teff) /regulatory T (Treg) cells through inhibiting the pathway of PIM1-protein kinase B (AKT)-Forkhead box O1 (FOXO1). By incorporating the scRNA-seq data of the retina from EAU mice into analysis, our study identified that T cells highly expressing Pim1 and Cxcr4 were enriched in the retina. DMF repressed the ocular infiltration of Teff cells, and this effect might depend on its inhibition of PIM1 and CXCR4 expression. Additionally, our study indicated that DMF might reduce the proportion of plasma cells by inhibiting PIM1 expression in B cells. CONCLUSIONS: DMF effectively attenuated EAU symptoms. During EAU, DMF reversed the Teff/Treg cell imbalance and suppressed the ocular infiltration of Teff cells by inhibiting PIM1 and CXCR4 expression. Thus, DMF may act as a new drug option for the treatment of AU.

Single-cell sequencing of the retina shows that LDHA regulates pathogenesis of autoimmune uveitis.

Autoimmune uveitis (AU) is a severe disorder causing poor vision and blindness. However, the cellular dynamics and pathogenic mechanisms underlying retinal injury in uveitis remain unclear. In this study, single-cell RNA sequencing of the retina and cervical draining lymph nodes in experimental autoimmune uveitis mice was conducted to identify the cellular spatiotemporal dynamics and upregulation of the glycolysis-related gene LDHA. Suppression of LDHA can rescue the imbalance of T effector (Teff) cells/T regulator (Treg) cells under inflammation via downregulation of the glycolysis-PI3K signaling circuit and inhibition of the migration of CXCR4+ Teff cells towards retinal tissue. Furthermore, LDHA and CXCR4 are upregulated in the peripheral blood mononuclear cells of Vogt-Koyanagi-Harada patients. The LDHA inhibitor suppresses CD4+ T cell proliferation in humans. Therefore, our data indicate that the autoimmune environment of uveitis regulates Teff cell accumulation in the retina via glycolysis-associated LDHA. Modulation of this target may provide a novel therapeutic strategy for treating AU.

Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophil extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. METHODS: We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. RESULTS: We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. CONCLUSION: In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.

An aging-related immune landscape in the hematopoietic immune system.

BACKGROUND: Aging is a holistic change that has a major impact on the immune system, and immunosenescence contributes to the overall progression of aging. The bone marrow is the most important hematopoietic immune organ, while the spleen, as the most important extramedullary hematopoietic immune organ, maintains homeostasis of the human hematopoietic immune system (HIS) in cooperation with the bone marrow. However, the overall changes in the HIS during aging have not been described. Here, we describe a hematopoietic immune map of the spleen and bone marrow of young and old mice using single-cell sequencing and flow cytometry techniques. RESULTS: We observed extensive, complex changes in the HIS during aging. Compared with young mice, the immune cells of aged mice showed a marked tendency toward myeloid differentiation, with the neutrophil population accounting for a significant proportion of this response. In this change, hypoxia-inducible factor 1-alpha (Hif1α) was significantly overexpressed, and this enhanced the immune efficacy and inflammatory response of neutrophils. Our research revealed that during the aging process, hematopoietic stem cells undergo significant changes in function and composition, and their polymorphism and differentiation abilities are downregulated. Moreover, we found that the highly responsive CD62L + HSCs were obviously downregulated in aging, suggesting that they may play an important role in the aging process. CONCLUSIONS: Overall, aging extensively alters the cellular composition and function of the HIS. These findings could potentially give high-dimensional insights and enable more accurate functional and developmental analyses as well as immune monitoring in HIS aging.

Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis.

Sex and aging influence the human immune system, resulting in disparate responses to infection, autoimmunity, and cancer. However, the impact of sex and aging on the immune system is not yet fully elucidated. Using small conditional RNA sequencing, we found that females had a lower percentage of natural killer (NK) cells and a higher percentage of plasma cells in peripheral blood compared with males. Bioinformatics revealed that young females exhibited an overrepresentation of pathways that relate to T and B cell activation. Moreover, cell-cell communication analysis revealed evidence of increased activity of the BAFF/APRIL systems in females. Notably, aging increased the percentage of monocytes and reduced the percentage of naïve T cells in the blood and the number of differentially expressed genes between the sexes. Aged males expressed higher levels of inflammatory genes. Collectively, the results suggest that females have more plasma cells in the circulation and a stronger BAFF/APRIL system, which is consistent with a stronger adaptive immune response. In contrast, males have a higher percentage of NK cells in blood and a higher expression of certain proinflammatory genes. Overall, this work expands our knowledge of sex differences in the immune system in humans.

Intravitreal dexamethasone implants facilitate the management of refractory Behçet's uveitis with vasculitis.

To investigate the efficacy and safety of dexamethasone (DEX) implant, Ozurdex ®, as an adjunctive treatment for refractory Behçet's uveitis (BU), a total of 61 patients (80 eyes) were included in this cross-sectional study and divided into the non-DEX and DEX groups. After >12 months of treatment, the improvement in the fluorescein angiography score and vitritis score was significantly higher in the DEX group than in the non-DEX group. Although the posterior capsule opacification score was exacerbated, the rate of low-dose systemic glucocorticoid was higher and the relapse times were fewer in the DEX group. Therefore, Ozurdex® is an effective and safe option for patients with BU that are refractory to systemic immunosuppressant treatments by controlling vasculitis, stabilizing vitreous inflammation, preventing recurrence, and reducing daily glucocorticoid doses.

Empagliflozin targets Mfn1 and Opa1 to attenuate microglia-mediated neuroinflammation in retinal ischemia and reperfusion injury.

BACKGROUND: Neuroinflammation and mitochondrial dysfunction play crucial roles in retinal ischemia and reperfusion (IR) injury. Recent studies have identified mitochondrial function as a promising target for immunomodulation. Empagliflozin (EMPA), an anti-diabetic drug, has exhibited great potential as both an anti-inflammatory agent and a protector of mitochondrial health. This study aimed to assess the therapeutic efficacy of EMPA in retinal IR injury. METHODS: To evaluate the protective effects of EMPA, the drug was injected into the vitreous body of mice post-retinal IR. Single-cell RNA sequencing (scRNA-seq) analysis was conducted to uncover the underlying mechanisms, and the results were further validated through in vivo and in vitro experiments. RESULTS: EMPA effectively protected retinal ganglion cells (RGCs) from IR injury by attenuating local retinal inflammation. The scRNA-seq analysis revealed that EMPA downregulated the nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) signaling pathway and restored mitochondrial dynamics by upregulating the expression of mitochondrial fusion-related genes, Mitofusin 1 (Mfn1) and optic atrophy 1 (Opa1). These findings were further corroborated by Western blotting. In vitro experiments provided additional insights, demonstrating that EMPA suppressed lipopolysaccharide (LPS)-induced cell inflammation and NLRP3 inflammasome activation. Moreover, EMPA enhanced mitochondrial fusion, neutralized mitochondrial reactive oxygen species (mtROS), and restored mitochondrial membrane potential (MMP) in BV2 microglia. Notably, genetic ablation of Mfn1 or Opa1 abolished the anti-inflammatory effects of EMPA. CONCLUSIONS: Our findings highlight the positive contribution of Mfn1 and Opa1 to the anti-inflammatory therapeutic effect of EMPA. By restoring mitochondrial dynamics, EMPA effectively mitigates microglia-mediated neuroinflammation and prevents RGC loss in retinal IR injury.

Progesterone attenuates Th17-cell pathogenicity in autoimmune uveitis via Id2/Pim1 axis.

BACKGROUND: Autoimmune uveitis (AU) is the most common ophthalmic autoimmune disease (AD) and is characterized by a complex etiology, high morbidity, and high rate of blindness. AU remission has been observed in pregnant female patients. However, the effects of progesterone (PRG), a critical hormone for reproduction, on the treatment of AU and the regulatory mechanisms remain unclear. METHODS: To this end, we established experimental autoimmune uveitis (EAU) animal models and constructed a high-dimensional immune atlas of EAU-model mice undergoing PRG treatment to explore the underlying therapeutic mechanisms of PRG using single-cell RNA sequencing. RESULTS: We found that PRG ameliorated retinal lesions and inflammatory infiltration in EAU-model mice. Further single-cell analysis indicated that PRG reversed the EAU-induced expression of inflammatory genes (AP-1 family, S100a family, and Cxcr4) and pathological processes related to inflammatory cell migration, activation, and differentiation. Notably, PRG was found to regulate the Th17/Treg imbalance by increasing the reduced regulatory functional mediators of Tregs and diminishing the overactivation of pathological Th17 cells. Moreover, the Id2/Pim1 axis, IL-23/Th17/GM-CSF signaling, and enhanced Th17 pathogenicity during EAU were reversed by PRG treatment, resulting in the alleviation of EAU inflammation and treatment of AD. CONCLUSIONS: Our study provides a comprehensive single-cell map of the immunomodulatory effects of PRG therapy on EAU and elaborates on the possible therapeutic mechanisms, providing novel insights into its application for treating autoimmune diseases.

N,N-Dimethyl-3ß-hydroxycholenamide attenuates neuronal death and retinal inflammation in retinal ischemia/reperfusion injury by inhibiting Ninjurin 1.

BACKGROUND: Retinal ischemia-reperfusion (RIR) injury refers to an obstruction in the retinal blood supply followed by reperfusion. Although the molecular mechanism underlying the ischemic pathological cascade is not fully understood, neuroinflammation plays a crucial part in the mortality of retinal ganglion cells. METHODS: Single-cell RNA sequencing (scRNA-seq), molecular docking, and transfection assay were used to explore the effectiveness and pathogenesis of N,N-dimethyl-3ß-hydroxycholenamide (DMHCA)-treated mice with RIR injury and DMHCA-treated microglia after oxygen and glucose deprivation/reoxygenation (OGD/R). RESULTS: DMHCA could suppress inflammatory gene expression and attenuate neuronal lesions, restoring the retinal structure in vivo. Using scRNA-seq on the retina of DMHCA-treated mice, we provided novel insights into RIR immunity and demonstrated nerve injury-induced protein 1 (Ninjurin1/Ninj 1) as a promising treatment target for RIR. Moreover, the expression of Ninj1, which was increased in RIR injury and OGD/R-treated microglia, was downregulated in the DMHCA-treated group. DMHCA suppressed the activation of the nuclear factor kappa B (NF-κB) pathways induced by OGD/R, which was undermined by the NF-κB pathway agonist betulinic acid. Overexpressed Ninj1 reversed the anti-inflammatory and anti-apoptotic function of DMHCA. Molecular docking indicated that for Ninj1, DMHCA had a low binding energy of - 6.6 kcal/mol, suggesting highly stable binding. CONCLUSION: Ninj1 may play a pivotal role in microglia-mediated inflammation, while DMHCA could be a potential treatment strategy against RIR injury.

Single-Cell Analysis Reveals the Heterogeneity of Monocyte-Derived and Peripheral Type-2 Conventional Dendritic Cells.

Dendritic cells (DCs) are critical for pathogen recognition and Ag processing/presentation. Human monocyte-derived DCs (moDCs) have been extensively used in experimental studies and DC-based immunotherapy approaches. However, the extent of human moDC and peripheral DCs heterogeneity and their interrelationship remain elusive. In this study, we performed single-cell RNA sequencing of human moDCs and blood DCs. We identified seven subtypes within moDCs: five corresponded to type 2 conventional DCs (cDC2s), and the other two were CLEC10A+CD127+ cells with no resemblance to any peripheral DC subpopulations characterized to date. Moreover, we defined five similar subtypes in human cDC2s, revealed the potential differentiation trajectory among them, and unveiled the transcriptomic differences between moDCs and cDC2s. We further studied the transcriptomic changes of each moDC subtype during maturation, demonstrating SLAMF7 and IL15RA as maturation markers and CLEC10A and SIGLEC10 as markers for immature DCs. These findings will enable more accurate functional/developmental analyses of human cDC2s and moDCs.

The Calcium Channel Inhibitor Nimodipine Shapes the Uveitogenic T Cells and Protects Mice from Experimental Autoimmune Uveitis through the p38-MAPK Signaling Pathway.

Autoimmune uveitis (AU) is a sight-threatening ocular inflammatory disorder, characterized by massive retinal vascular leakage and inflamed lesions with infiltration of the uveitogenic T cells in the retina and disorders of the T cell-related immune response in the system. Stimulation of TCRs can trigger calcium release and influx via Ca2+ channels and then transmit signals from the surface to the nucleus, which are important for energy metabolism, proliferation, activation, and differentiation. Inhibition of Ca2+ influx by pharmacological modulation of Ca2+ channels may suppress T cell function, representing a novel anti-inflammatory strategy in the treatment of AU. This study investigated the effects of the l-type voltage-gated calcium channel blocker nimodipine in experimental AU (EAU). Nimodipine was found to not only decrease the clinical and histopathological inflammation score of EAU (C57BL/6J mice) but also dwindle the infiltration of uveitogenic CD4+ T cells into the retina. Moreover, nimodipine decreased the effector T cells and increased the regulatory T cells in the immune system. In vitro, nimodipine reduced the effector T cell differentiation of the IRBP1-20-specific CD4+ T cells of EAU mice and LPS-stimulated PBMCs of uveitis patients. Meanwhile, nimodipine suppressed the energy metabolism, proliferation, activation, and Th1 cell differentiation of T cells. Further studies on RNA sequencing and molecular mechanisms have established that nimodipine alleviates EAU by regulating T cells response through the p38-MAPK pathway signaling. Taken together, our data reveal a novel therapeutic potential of the l-type Ca2+ channels antagonist nimodipine in AU by regulating the balance of T cell subsets.

Design, methodology, and preliminary results of the non-human primates eye study.

PURPOSE: To describe the normative profile of ophthalmic parameters in a healthy cynomolgus monkey colony, and to identify the characteristic of the spontaneous ocular disease non-human primates (NHP) models. METHODS: The NHP eye study was a cross-sectional on-site ocular examination with about 1,000 macaques held in Guangdong Province, southeastern China. The NHPs (Macaca fascicularis, cynomolgus) in this study included middle-aged individuals with a high prevalence of the ocular disease. The NHP eye study (NHPES) performed the information including systematic data and ocular data. Ocular examination included measurement of intraocular pressure (IOP), anterior segment- optical coherence tomography (OCT), slit-lamp examination, fundus photography, autorefraction, electroretinography, etc. Ocular diseases included measurement of refractive error, anisometropia, cataract, pterygium, etc. RESULTS: A total of 1148 subjects were included and completed the ocular examination. The average age was 16.4 ± 4.93 years. Compared to the male participants, the females in the NHPES had shorter axial length and the mean Average retinal nerve fiber layer (RNFL) thickness (except for the nasal quadrants). The mean IOP, anterior chamber depth, lens thickness, axial length, central corneal thickness, choroid thickness and other parameters were similar in each group. CONCLUSION: The NHPES is a unique and high-quality study, this is the first large macaque monkey cohort study focusing on ocular assessment along with comprehensive evaluation. Results from the NHPES will provide important information about the normal range of ophthalmic measurements in NHP.

Discovery of Clinical Manifestations in Spontaneous Glaucoma Suspect Nonhuman Primates.

INTRODUCTION: The primary aim of this study was to assess the longitudinal changes in retinal nerve fiber layer (RNFL) thickness and relative ONH parameters using spectral-domain optical coherence tomography (SD-OCT) in the living eyes of nonhuman primates with spontaneous glaucoma. METHODS: Totally 9 macaque subjects underwent a standard fundus photo and OCT examination, including 3 adult monkeys with stable elevated intraocular pressure (IOP) as the glaucoma suspect group and 6 normal IOP monkeys as the control group. Each eye of primates with IOP measurement was repeated three times. OCT measurements recorded RNFL, Bruch's membrane opening (BMO) minimum rim width (MRW). The follow-up scan of glaucomatous subjects was undergone in 10 months. RESULTS: The mean ages of glaucoma and healthy subjects are 19.33 ± 0.33 and 20.5 ± 1.46 years. Elevated IOP was achieved in three nonhuman primate eyes with an average increase of 10 mm Hg over the study period. Elevated IOP was associated with decreased RNFL thickness in all the regions and decreased RNFL phase retardation in the superior and inferior regions. When averaged over the entire retinal area, only the whole RNFL and temporal regions showed a significant decrease. The mean IPL thicknesses are 87.17 ± 2.15 µm in glaucomatous and 93.33 ± 1.51 µm in healthy eyes (p = 0.03). Lamina cribrosa parameters are measured from the OCT images and showed significant differences between glaucoma primates and normal primates. CONCLUSIONS: Of the measured parameters, decreased RNFL and MRW were correlated with glaucomatous damage. Natural glaucoma primate can be a natural glaucoma model which is closer to glaucoma in humans.

Single-cell RNA sequencing reveals a landscape and targeted treatment of ferroptosis in retinal ischemia/reperfusion injury.

BACKGROUND: The aim of this study was to establish a complete retinal cell atlas of ischemia-reperfusion injury by single-cell RNA sequencing, and to explore the underlying mechanism of retinal ischemia-reperfusion injury in mice. METHODS: Single-cell RNA sequencing was used to evaluate changes in the mouse retinal ischemia reperfusion model. In vivo and in vitro experiments were performed to verify the protective effect of inhibiting ferroptosis in retinal ischemia-reperfusion injury. RESULTS: After ischemia-reperfusion injury, retinal cells were significantly reduced, accompanied by the activation of myeloid and a large amount of blood-derived immune cell infiltration. The IFNG, MAPK and NFKB signaling pathways in retinal neuronal cells, together with the TNF signaling pathway in myeloid give rise to a strong inflammatory response in the I/R state. Besides, the expression of genes implicating iron metabolism, oxidative stress and multiple programed cell death pathways have changed in cell subtypes described above. Especially the ferroptosis-related genes and blocking this process could apparently alleviate the inflammatory immune responses and enhance retinal ganglion cells survival. CONCLUSIONS: We established a comprehensive landscape of mouse retinal ischemia-reperfusion injury at the single-cell level, revealing the important role of ferroptosis during this injury, and targeted inhibition of ferroptosis can effectively protect retinal structure and function.

Melatonin, an endogenous hormone, modulates Th17 cells via the reactive-oxygen species/TXNIP/HIF-1α axis to alleviate autoimmune uveitis.

BACKGROUND: Melatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis. METHODS: EAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1-20 (IRBP1-20). Melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were graded to evaluate the disease progression. T lymphocytes accumulation and the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR, respectively. T helper 1 (Th1), T helper 17 (Th17), and regulatory T (Treg) cells were detected via flow cytometry for both in vivo and in vitro experiments. Reactive-oxygen species (ROS) from CD4 + T cells was tested via flow cytometry. The expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α) proteins were quantified via western blot. RESULTS: Melatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells by suppressing their transcription factors and potentiated Treg cells. In vitro studies corroborated that melatonin restrained the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU. CONCLUSIONS: Melatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

A pilot study of the use of dynamic analysis of cell-free DNA from aqueous humor and vitreous fluid for the diagnosis and treatment monitoring of vitreoretinal lymphomas.

The diagnosis of vitreoretinal lymphoma (VRL), a rare subtype of primary central nervous system lymphoma, is challenging. We aimed to investigate the mutational landscape of VRL by sequencing circulating tumor DNA (ctDNA) from aqueous humor (AH) and/or vitreous fluid (VF), as well as applying ctDNA sequencing to diagnosis and treatment monitoring. Baseline AH and/or VF specimens from 15 VRL patients underwent comprehensive genomic profiling using targeted next-generation sequencing. The molecular profiles of paired baseline AH and VF specimens were highly concordant, with comparable allele frequencies. However, the genetic alterations detected in cerebrospinal fluid ctDNA only partially overlapped with those from simultaneously collected AH/VF samples, with much lower allele frequencies. Serial post-treatment AH or VF samples were available for five patients and their changes in ctDNA allele frequency displayed a similar trend as the changes in interleukin-10 levels; an indicator of response to treatment. A cohort of 23 patients with primary central nervous system lymphoma was included as a comparison group for the genetic landscape and evaluations of the efficacy of ibrutinib. More MYD88 mutations, but fewer IRF4 mutations and CDKN2A/B copy number losses were observed in the baseline samples of primary central nervous system lymphoma than VRL patients. The objective response rate to ibrutinib treatment was much higher for patients with primary central nervous system lymphoma (64.7%, 11/17) than for those with VRL (14.3%, 1/7). In summary, we provide valuable clinical evidence that AH is a good source of tumor genomic information and can substitute VF. Moreover, molecular profiling of AH has clinical utility for the diagnosis of VRL and treatment monitoring.

Interleukin-35 suppresses pyroptosis and protects against neuronal death in retinal ischaemia/reperfusion injury.

Retinal ischaemia-reperfusion (I/R) is a pathological process in many eye disorders. Neuroinflammation and cell pyroptosis have been recognized as important in the pathogenesis of tissue damage in retinal I/R. Interleukin (IL)-35 is a novel heterodimeric cytokine that exhibits anti-inflammatory activity in various autoimmune diseases, but its role in retinal I/R and the underlying molecular mechanisms remain unexplored. This research investigated the effect of IL-35 on retinal I/R and the inhibition of pyroptosis and neuronal death. In our study, a murine retinal I/R model was used to explore the neuroprotective effect of recombinant IL-35 protein in vivo. Primary murine microglial cells in pyroptosis and retinal ganglion cells (RGCs) in oxygen and glucose deprivation/reoxygenation (OGD/R) models were employed to assess the antipyroptotic and antiapoptotic effects of IL-35 in vitro. The data showed that IL-35 decreases retinal damage, RGC death, and inner plexiform layer (IPL) thinning in mice with retinal I/R injury, with significant attenuation of pyroptosis in the retina. The study also demonstrated the anti-pyroptotic action of IL-35 in primary microglia stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Furthermore, primary RGC apoptosis induced by OGD/R was directly suppressed by IL-35, and IL-35-mediated neuroprotection was abrogated when miR-21 was blocked. In conclusion, our results suggest the mechanisms of RGC apoptosis and a new therapeutic target, IL-35, that exerts a robust neuroprotective effect against retinal I/R.

TET-dependent GDF7 hypomethylation impairs aqueous humor outflow and serves as a potential therapeutic target in glaucoma.

Glaucoma is the leading cause of irreversible vision loss, affecting more than 70 million individuals worldwide. Circulatory disturbances of aqueous humor (AH) have long been central pathological contributors to glaucomatous lesions. Thus, targeting the AH outflow is a promising approach to treat glaucoma. However, the epigenetic mechanisms initiating AH outflow disorders and the targeted treatments remain to be developed. Studying glaucoma patients, we identified GDF7 (growth differentiation factor 7) hypomethylation as a crucial event in the onset of AH outflow disorders. Regarding the underlying mechanism, the hypomethylated GDF7 promoter was responsible for the increased GDF7 production and secretion in primary open-angle glaucoma (POAG). Excessive GDF7 protein promoted trabecular meshwork (TM) fibrosis through bone morphogenetic protein receptor type 2 (BMPR2)/Smad signaling and upregulated pro-fibrotic genes, α-smooth muscle actin (α-SMA) and fibronectin (FN). GDF7 protein expression formed a positive feedback loop in glaucomatous TM (GTM). This positive feedback loop was dependent on the activated TET (ten-eleven translocation) enzyme, which kept the GDF7 promoter region hypomethylated. The phenotypic transition in TM fortified the AH outflow resistance, thus elevating the intraocular pressure (IOP) and attenuating the nerve fiber layer. This methylation-dependent mechanism is also confirmed by a machine-learning model in silico with a specificity of 84.38% and a sensitivity of 89.38%. In rhesus monkeys, we developed GDF7 neutralization therapy to inhibit TM fibrosis and consequent AH outflow resistance that contributes to glaucoma. The neutralization therapy achieved high-efficiency control of the IOP (from 21.3 ± 0.3 to 17.6 ± 0.2 mmHg), a three-fold improvement in the outflow facility (from 0.1 to 0.3 µL/min · mmHg), and protection of nerve fibers. This study provides new insights into the epigenetic mechanism of glaucoma and proposes an innovative GDF7 neutralization therapy as a promising intervention.

First observation of intraocular extranodal natural killer/T-cell lymphoma secondary to a retroperitoneal tumour: a case report and comparative review.

BACKGROUND: Vitreoretinal lymphomas are difficult to diagnose due to their insidious onset and inaccessible focal points. Natural killer/T-cell derived malignancies are rare as intraocular lymphomas and usually have a rapid progression and a poor prognosis. Therefore, it is essential to make a definite diagnosis, especially differentially with B-cell-derived lymphomas, which account for most cases of vitreoretinal lymphomas. CASE PRESENTATION: This case report describes a 55-year-old female reporting a 10-month history of painless decline in her vision of the right eye. Optical coherence tomography of the patient revealed hyperreflective nodules and irregular humps in the retinal pigment epithelium layer. The right vitreous was aspirated for diagnostic assessment, revealing an interleukin-10 level of 39.4 pg/mL and an interleukin-10/interleukin-6 ratio of 1.05. The right vitreous humor was positive for Epstein-Barr virus DNA. Upon a systemic examination, a high metabolic nodule was found in the retroperitoneal area and proven to be positive for Epstein-Barr virus-encoded mRNA, CD2, CD3ε, TIA-1, and Ki-67. Considering the homology of the two lesions, the patient was diagnosed with metastatic vitreoretinal lymphoma secondary to retroperitoneal extranodal natural killer/T-cell derived lymphoma. The patient received systemic chemotherapy and regular intravitreal injections of methotrexate. Her visual acuity of the right eye had improved from 20/125 to 20/32 at the latest follow-up. No new lesions were found. CONCLUSIONS: A definitive diagnosis of vitreoretinal lymphoma is challenging. On some occasions in which pathological evidence is missing, the available examination results and clinical observations must be comprehensively considered. This study herein summarized pertinent pieces of literature and reports and reviewed available practicable methods to make a definitive diagnosis of intraocular extranodal natural killer/T-cell lymphoma, which was particularly distinct from the common diffuse large B-cell lymphomas.