Genetic landscape and autoimmunity of monocytes in developing Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada (VKH) disease is a systemic autoimmune disorder affecting multiple organs, including eyes, skin, and central nervous system. It is known that monocytes significantly contribute to the development of autoimmune disease. However, the subset heterogeneity with unique functions and signatures in human circulating monocytes and the identity of disease-specific monocytic populations remain largely unknown. Here, we employed an advanced single-cell RNA sequencing technology to systematically analyze 11,259 human circulating monocytes and genetically defined their subpopulations. We constructed a precise atlas of human blood monocytes, identified six subpopulations-including S100A12, HLA, CD16, proinflammatory, megakaryocyte-like, and NK-like monocyte subsets-and uncovered two previously unidentified subsets: HLA and megakaryocyte-like monocyte subsets. Relative to healthy individuals, cellular composition, gene expression signatures, and activation states were markedly alternated in VKH patients utilizing cell type-specific programs, especially the CD16 and proinflammatory monocyte subpopulations. Notably, we discovered a disease-relevant subgroup, proinflammatory monocytes, which showed a discriminative gene expression signature indicative of inflammation, antiviral activity, and pathologic activation, and converted into a pathologic activation state implicating the active inflammation during VKH disease. Additionally, we found the cell type-specific transcriptional signature of proinflammatory monocytes, ISG15, whose production might reflect the treatment response. Taken together, in this study, we present discoveries on accurate classification, molecular markers, and signaling pathways for VKH disease-associated monocytes. Therapeutically targeting this proinflammatory monocyte subpopulation would provide an attractive approach for treating VKH, as well as other autoimmune diseases.

A Cellular MicroRNA Facilitates Regulatory T Lymphocyte Development by Targeting the FOXP3 Promoter TATA-Box Motif.

The CD4+CD25+FOXP3+ regulatory T cells (Tregs) mediate immunological self-tolerance and suppress various immune responses. FOXP3 is a key transcriptional factor for the generation and development of Tregs. Its expression is regulated by various cytokines including TGF-ß, IL-2, and IL-10. It is important to further identify the regulatory factors for Tregs. Given that many microRNAs (miRNAs) could specifically interact with the core promoter region and specifically enhance the transcription of many target genes, we searched for any possible miRNA(s) targeting the core promoter region of the FOXP3 gene. We found that miR-4281, an miRNA specifically expressed in hominids, can potently and specifically upregulate FOXP3 expression by directly interacting with the TATA-box motif in the human FOXP3 promoter. Consequently, miR-4281 significantly accelerated the differentiation of human naive cells to induced Tregs (iTregs) that possess immune suppressor functions and weaken the development of graft-versus-host disease in a humanized mouse model. Interestingly, iTregs induced by the combination of TGF-ß, IL-2, and chemically synthesized miR-4281 were more stable and functional than those induced by TGF-ß and IL-2 alone. Moreover, we found that the IL-2/STAT5 signal transduction upregulates FOXP3 expression not only through the classical pathway, but also by enhancing the expression of the miR-4281 precursor gene (SNCB) and, correspondingly, miR-4281. This study reveals a novel mechanism regulating FOXP3 expression and human iTreg development and, therefore, offers a new therapeutic target to manipulate immunosuppressive system.

Quantitative Analysis of Retinal Microvascular Changes after Conbercept Therapy in Branch Retinal Vein Occlusion Using Optical Coherence Tomography Angiography.

PURPOSE: To quantitatively evaluate microvascular changes in eyes with macular oedema due to branch retinal vein occlusion (BRVO) using optical coherence tomography angiography (OCTA) before and after intravitreal conbercept injection and the correlation of such changes with best-corrected visual acuity (BCVA) and retinal thickness. METHODS: Twenty-eight eyes of 28 patients treated with a single intravitreal injection of conbercept for macular oedema due to BRVO were included in this study. The automatically measured values of the vessel density in the superficial (SCP) and deep retinal capillary plexus (DCP), the foveal avascular zone (FAZ) area, the FAZ perimeter, the vessel density within a 300 µm wide ring surrounding the FAZ (FD-300), the acircularity index (AI), the choriocapillaris flow area and the retinal thickness were obtained via OCTA before and at 1 month after initial injection and compared with those of age- and sex- matched healthy subjects. RESULTS: In BRVO eyes, the vascular density in the SCP and DCP, the FD-300 and the flow area of choriocapillaris were significantly lower than those in healthy eyes, while the AI and the retinal thickness were significantly increased. After treatment, the retinal thickness in eyes with BRVO was significantly decreased in all quadrants, and the mean BCVA dramatically increased from 20/162 to 20/78 (p = 0.0017). The mean flow area of choriocapillaris significantly improved after treatment. Moreover, negative correlations between the logMAR BCVA and the whole vascular density in the SCP and DCP as well as the flow area of choriocapillaris were observed. CONCLUSION: OCTA enables non-invasive, layer-specific and quantitative assessment of microvascular changes in eyes with BRVO before and after treatment, and it can be used as a valuable imaging tool for the evaluation of the follow-up in BRVO patients.

TLR4-MyD88 pathway promotes the imbalanced activation of NLRP3/NLRP6 via caspase-8 stimulation after alkali burn injury.

Alkali burn (AB) is one of the most serious ocular traumas in the world, characterized by extreme ocular surface disorders, critical secondary dry eye and irreversible vision loss. The exact mechanisms involved are unknown. Innate immunity, including the involvement of Toll-like receptors (TLRs) and NOD-like receptors (NLRs), is believed to participate in the pathogenesis of the epithelia, but the exact mechanisms by which TLRs transduce signals to NLRs and downstream molecules to initiate innate immunity remain poorly defined. In this present study, we used murine models of AB and AB concomitant desiccating stress (DS) to investigate the potential functions and mechanisms of TLR4 in regulating NLRP3 and NLRP6 during AB injury and secondary dry eye. We demonstrated that AB injury induced activation of the TLR4-MyD88 pathway, leading to imbalanced NLRP3 and NLRP6 via the activation of caspase-8 signaling. DS worsened ocular surface disorders post-AB injury by magnifying this phenomenon. Caspase-8 signaling promoted NLRP3 upregulation via the nuclear factor (NF)-κB pathway, while NLRP6 suppressed NF-κB activation. Our findings also revealed that TLR4-MyD88 knockout can alleviate AB-induced or DS-worsened ocular surface disorders, shedding light on the potential therapeutic strategies in the future for AB injury. Taken together, our findings demonstrate that AB promotes the TLR4-MyD88-caspase-8 axis to cause imbalanced NLRP3/NLRP6, and DS exacerbates ocular surface damage via magnifying this imbalance.

A novel function and mechanism of ischemia-induced retinal astrocyte-derived exosomes for RGC apoptosis of ischemic retinopathy.

Retinal ischemia is a common clinical event leading to retinal ganglion cell (RGC) death, resulting in irreversible vision loss. In the retina, glia-neuron communication is crucial for multiple functions and homeostasis. Extracellular vesicles, notably exosomes, play a critical role. The functions and mechanisms of retinal astrocyte-secreted exosomes remain unclear. Here, we isolated astrocyte-derived exosomes under hypoxia or normoxia and explored their role in an in vivo retinal ischemia-reperfusion (RIR) model. We found that hypoxia triggered astrocytes to produce a significantly increased number of exosomes, which could be internalized by RGCs in vivo or in vitro. Also, in the RIR model, the hypoxia-induced exosomes ameliorated the RIR injury and suppressed the RGC apoptosis. Furthermore, microRNA sequencing of retinal astrocyte-secreted exosomes revealed different patterns of exosomal miRNAs under hypoxia, particularly enriched with miR-329-5p. We verified that miR-329-5p was specifically bound to mitogen-activated protein kinase 8 mRNA, and subsequent JNK-pathway molecules were downregulated. We anticipated that the miR-329-5p/JNK pathway is a key to suppressing RGC apoptosis and preventing RIR injury. Such findings provided insights into the therapeutic potential of hypoxia-induced astrocyte-secreted exosomes and the miR-329-5p for treating retina ischemic diseases.

Mitophagy Protects the Retina Against Anti-Vascular Endothelial Growth Factor Therapy-Driven Hypoxia via Hypoxia-Inducible Factor-1α Signaling.

Anti-VEGF drugs are first-line treatments for retinal neovascular diseases, but these anti-angiogenic agents may also aggravate retinal damage by inducing hypoxia. Mitophagy can protect against hypoxia by maintaining mitochondrial quality, thereby sustaining metabolic homeostasis and reducing reactive oxygen species (ROS) generation. Here we report that the anti-VEGF agent bevacizumab upregulated the hypoxic cell marker HIF-1α in photoreceptors, Müller cells, and vascular endothelial cells of oxygen-induced retinopathy (OIR) model mice, as well as in hypoxic cultured 661W photoreceptors, MIO-MI Müller cells, and human vascular endothelial cells. Bevacizumab also increased expression of mitophagy-related proteins, and mitophagosome formation both in vivo and in vitro, but did not influence cellular ROS production or apoptosis rate. The HIF-1α inhibitor LW6 blocked mitophagy, augmented ROS production, and triggered apoptosis. Induction of HIF-1α and mitophagy were associated with upregulation of BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1), and overexpression of these proteins in culture reversed the effects of HIF-1α inhibition. These findings suggest that bevacizumab does induce retinal hypoxia, but that concomitant activation of the HIF-1α-BNIP3/FUNDC1 signaling pathway also induces mitophagy, which can mitigate the deleterious effects by reducing oxidative stress secondary. Promoting HIF-1α-BNIP3/FUNDC1-mediated mitophagy may enhance the safety of anti-VEGF therapy for retinal neovascular diseases and indicate new explanation and possible new target of the anti-VEGF therapy with suboptimal effect.

Distinguishing Microvasculature Features of Vogt-Koyanagi-Harada in Patients in Acute and Convalescent Phases Using Optical Coherence Tomography Angiography.

Purpose: To evaluate retinal and choroidal microvascular features of VKH patients in acute and convalescent phases after treatment using OCTA.Methods: A prospective, observational study was conducted in patients with initial VKH at the acute stage (n = 15) and healthy participants (n = 15) served as controls. After 3-month systemic corticosteroid treatment, patients' vascular parameters were recorded by OCTA before and after treatment and compared with results observed in healthy participants.Results: Our findings first uncovered that there are two types of abnormalities in the choriocapillary layer of patients with VKH in the acute stage: one is characterized as multiple dark spots of choriocapillary flow void and the other involves highly reflective areas surrounded by light spots with an increased flow area. During the convalescent stage, all eyes showed multifocal dark spots in the choriocapillary layer, leading to a reduced choroidal flow area.Conclusions: OCTA provides a better display of the microvascular appearance of the choroid to noninvasively evaluate choriocapillaris abnormalities in VKH disease.

Vascular Abnormalities in Peripapillary and Macular Regions of Behcet's Uveitis Patients Evaluated by Optical Coherence Tomography Angiography.

Purpose: To investigate the involvement of peripapillary zone vascular abnormalities in Behcet's uveitis (BU) and associated visual dysfunction. We evaluated the retinal and choroidal microvascular features in both macular and peripapillary areas of BU patients to identify vascular abnormalities contributing to reduced best-corrected visual acuity (BCVA) using optical coherence tomography angiography (OCTA). Methods: A prospective, observational study was conducted in 24 eyes of 13 patients with BU and 24 eyes of 15 healthy participants as controls. They received a standard eye examination and were recorded by OCTA measurements of macular and peripapillary areas. The vascular densities of superficial capillary plexus (SCP) and deep capillary plexus (DCP), choroidal flow area, radial peripapillary capillary network (RPCN) density, foveal avascular zone (FAZ) area and perimeter, full retinal thickness (FRT), and peripapillary retinal nerve fiber layer thickness (pRNFLT) were measured.Correlations among microvascular, structural, and functional changes were assessed. Results: Our findings uncovered that the vascular density was significantly reduced in the peripapillary zone of BU eyes compared to healthy eyes, especially in the inferior subfield of the RPCN. The vascular densities of SCP and DCP quadrants within the macular zone had no significant difference between BU and control groups except for DCP density of the nasal parafoveal quadrant. Both FAZ area and perimeter were greater but without statistical significance in the BU group. Compared to healthy eyes, the choriocapillaris flow area was smaller while the FRT and pRNFLT were greater in the BU group. Notably, there was a significant correlation between the reduction in RPCN vascular density and decreased BCVA in BU patients. Conclusion: Based on OCTA, vascular changes associated with BU are more prominent in the peripapillary zone than those in the macular zone. The vascular density of the RPCN could serve as a sensitive indicator to monitoring BU pathogenic progression and treatment response using a non-invasively method of OCTA.

The Regulatory NOD-Like Receptor NLRC5 Promotes Ganglion Cell Death in Ischemic Retinopathy by Inducing Microglial Pyroptosis.

Retinal ischemia is a common pathological event that can result in retinal ganglion cell (RGC) death and irreversible vision loss. The pathogenic mechanisms linking retinal ischemia to RGC loss and visual deficits are uncertain, which has greatly hampered the development of effective treatments. It is increasingly recognized that pyroptosis of microglia contributes to the indirect inflammatory death of RGCs. In this study, we report a regulatory NOD-like receptor, NOD-, LRR- and CARD-containing 5 (NLRC5), as a key regulator on microglial pyroptosis and the retinal ischemia process. Through an in-depth analysis of our recently published transcriptome data, we found that NLRC5 was significantly up-regulated in retina during ischemia-reperfusion injury, which were further confirmed by subsequent detection of mRNA and protein level. We further found that NLRC5 was upregulated in retinal microglia during ischemia, while NLRC5 knockdown significantly ameliorated retinal ischemic damage and RGC death. Mechanistically, we revealed that knockdown of NLRC5 markedly suppressed gasdermin D (GSDMD) cleavage and activation of interleukin-1ß (IL-1ß) and caspase-3, indicating that NLRC5 promotes both microglial pyroptosis and apoptosis. Notably, we found that NLRC5 directly bound to NLRP3 and NLRC4 in inflammasomes to cooperatively drive microglial pyroptosis and apoptosis mediating retinal ischemic damage. Overall, these findings reveal a previously unidentified key contribution of NLRC5 signaling to microglial pyroptosis under ischemia or hypoxia conditions. This NLRC5-dependent pathway may be a novel therapeutic target for treatment of ischemic retinopathy.

Microvasculature Features of Vogt-Koyanagi-Harada Disease Revealed by Widefield Swept-Source Optical Coherence Tomography Angiography.

Background: Vogt-Koyanagi-Harada (VKH) disease is a multisystem autoimmune disorder which could induce bilateral panuveitis involving the posterior pole and peripheral fundus. Optical coherence tomography angiography (OCTA) provides several advantages over traditional fluorescence angiography for revealing pathological abnormalities of the retinal vasculature. Until recently, however, the OCTA field of view (FOV) was limited to 6 × 6 mm2 scans. Purpose: This study examined retinal vasculature and choriocapillaris abnormalities across multiple regions of the retina (15 × 9 mm2 wide field, macular, peripapillary regions) among acute and convalescent VKH patients using a novel widefield swept-source OCTA (WSS-OCTA) device and assessed correlations between imaging features and best-corrected visual acuity (BCVA). Methods: Twenty eyes of 13 VHK disease patients in the acute phase, 30 eyes of 17 patients in the convalescent phase, and 30 eyes of 15 healthy controls (HCs) were included in this study. Vascular length density (VLD) in superficial and deep vascular plexuses (SVP, DVP), vascular perfusion density (VPD) in SVP, DVP, and choriocapillaris (CC), and flow voids (FV) in CC were measured across multiple retinal regions via WSS-OCTA (PLEX Elite 9000, Carl Zeiss Meditec Inc., USA) using the 15 × 9 mm2 scan pattern centered on the fovea and quantified by ImageJ. Results: Compared to HCs, acute phase VKH patients exhibited significantly reduced SVP-VLD, SVP-VPD, and CC-VPD across multiple retinal regions (all p < 0.01). Notably, the FV area was more extensive in VKH patients, especially those in the acute phase (p < 0.01). These changes were reversed in the convalescent phase. Stepwise multiple linear regression analysis demonstrated that macular DVP-VLD and macular CC-VPD were the best predictive factors for BCVA in the acute and convalescent VKH groups. Conclusion: The wider field of SS-OCAT provides more comprehensive and detailed images of the microvasculature abnormalities characterizing VKH disease. The quantifiable and layer-specific information from OCTA allows for the identification of sensitive and specific imaging markers for prognosis and treatment guidance, highlighting WSS-OCTA as a promising modality for the clinical management of VKH disease.

NLRP12- and NLRC4-mediated corneal epithelial pyroptosis is driven by GSDMD cleavage accompanied by IL-33 processing in dry eye.

PURPOSE: Dry eye disease (DED) is a common and multifactor-induced autoimmune ocular surface disease. Environmental factors, such as desiccating stress (DS) and hyperosmolarity, affect the corneal epithelium to induce ocular surface inflammation in DED. We aimed to explore the potential mechanisms by which innate immunity and pyroptosis are initiated in the mucosal epithelium in response to environmental stress. METHODS: Experimental dry eye was established in C57BL/6 J mice and genetic mice on the background of C57BL/6 J mice by subcutaneous injection of scopolamine and exposure to a desiccating environment. SDHCEC cell line was subjected to hyperosmolarity stress (450 mOsM). The phenol red thread tear test and corneal epithelial defects evaluation were used as assessments of severity of DED. RNA-sequencing, quantitative real-time PCR, western blotting and immunofluorescence staining were performed in this study. RESULTS: Loss-of-function studies indicated that genetic deletion of GSDMD alleviates DS-induced corneal epithelium defects, and GSDMD is needed for IL-33 processing. We further found that NLRP12 collaborates with NLRC4 inflammasome to initiate GSDMD-dependent pyroptosis, which requires TLR4-induced caspase-8 (CASP8) activation in the mucosal corneal epithelium in response to DS. CONCLUSIONS: These findings provide compelling evidence that GSDMD-dependent pyroptosis plays a pivotal role in DED. A novel mechanism involving NLRP12 and NLRC4 inflammasomes-induced GSDMD-dependent pyroptosis, accompanied by IL-33 processing is responsible for ocular surface epithelial defects in response to environmental stress. GSDMD is required for IL-33 processing and the subsequent amplification of inflammatory cascades. These findings reveal novel therapeutic targets for treating DED.

NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma.

BACKGROUND: Acute glaucoma, characterized by a sudden elevation in intraocular pressure (IOP) and retinal ganglion cells (RGCs) death, is a major cause of irreversible blindness worldwide that lacks approved effective therapies, validated treatment targets and clear molecular mechanisms. We sought to explore the potential molecular mechanisms underlying the causal link between high IOP and glaucomatous RGCs death. METHODS: A murine retinal ischemia/ reperfusion (RIR) model and an in vitro oxygen and glucose deprivation/reoxygenation (OGDR) model were used to investigate the pathogenic mechanisms of acute glaucoma. RESULTS: Our findings reveal a novel mechanism of microglia-induced pyroptosis-mediated RGCs death associated with glaucomatous vision loss. Genetic deletion of gasdermin D (GSDMD), the effector of pyroptosis, markedly ameliorated the RGCs death and retinal tissue damage in acute glaucoma. Moreover, GSDMD cleavage of microglial cells was dependent on caspase-8 (CASP8)-hypoxia-inducible factor-1α (HIF-1α) signaling. Mechanistically, the newly identified nucleotide-binding leucine-rich repeat-containing receptor (NLR) family pyrin domain-containing 12 (NLRP12) collaborated with NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) downstream of the CASP8-HIF-1α axis, to elicit pyroptotic processes and interleukin-1ß (IL-1ß) maturation through caspase-1 activation, facilitating pyroptosis and neuroinflammation in acute glaucoma. Interestingly, processing of IL-1ß in turn magnified the CASP8-HIF-1α-NLRP12/NLRP3/NLRC4-pyroptosis circuit to accelerate inflammatory cascades. CONCLUSIONS: These data not only indicate that the collaborative effects of NLRP12, NLRP3 and NLRC4 on pyroptosis are responsible for RGCs death, but also shed novel mechanistic insights into microglial pyroptosis, paving novel therapeutic avenues for the treatment of glaucoma-induced irreversible vision loss through simultaneously targeting of pyroptosis.

Quantitative Analysis of Anterior Chamber Inflammation Using the Novel CASIA2 Optical Coherence Tomography.

PURPOSE: We evaluated the clinical utility of a novel anterior segment optical coherence tomography (AS-OCT) device, CASIA2, to evaluate parameters indicative of anterior chamber (AC) inflammation severity in uveitis, including AC cell number, flare, and keratic precipitates (KPs). DESIGN: Prospective evaluation of a diagnostic device. METHODS: Uveitis eyes were classified into active and inactive groups. The number of hyperreflective dots representing AC cells and optical density ratio (aqueous-to-air relative intensity [ARI] index) for flare qualification were calculated from AS-OCT images. In addition, a program was designed to quantify the posterior corneal surface smoothness (PCSS) of each image for KPs evaluation. The maximum, minimum, and average PCSS values were calculated from 128 images per eye and compared among active uveitis, inactive uveitis, and control eyes. Correlations between Standardization of Uveitis Nomenclature grade and both hyperreflective dot number and ARI index were evaluated. Receiver operating characteristic (ROC) curves were constructed to test the values of these indicators for uveitis diagnosis. RESULTS: AC hyperreflective dot count, ARI index, and maximum and average PCSS values were all significantly higher in the active uveitis group than in the inactive and control groups. Hyperreflective dot count and ARI index were associated with Standardization of Uveitis Nomenclature cell and flare grade. According to ROC curve analysis, maximum PCSS was the best indicator for the diagnosis of uveitis involving the anterior segment, meanwhile the hyperreflective dot number was the best to identify active AC inflammation from the inactive. CONCLUSIONS: Quantification of AC cell number, flare, and KPs using the CASIA2 device is a promising strategy for the objective assessment of AC inflammation.

Microvascular changes after conbercept therapy in central retinal vein occlusion analyzed by optical coherence tomography angiography.

AIM: To investigate microvascular changes in eyes with central retinal vein occlusion (CRVO) complicated by macular edema before and after intravitreal conbercept injection and evaluate correlations between these changes and best-corrected visual acuity (BCVA) and retinal thickness. METHODS: Twenty-eight eyes of 28 patients with macular edema caused by CRVO were included in this retrospective study. All patients received a single intravitreal conbercept injection to treat macular edema. BCVA and the results of optical coherence tomography angiography (OCTA) automatic measurements of the vessel density in the superficial (SCP) and deep retinal capillary plexus (DCP), the foveal avascular zone (FAZ) area, the FAZ perimeter (PERIM), the vessel density within a 300-µm wide ring surrounding the FAZ (FD-300), the acircularity index (AI), the choriocapillaris flow area, and retinal thickness were recorded before and at one month after treatment and compared with the results observed in age- and sex-matched healthy subjects. RESULTS: The vessel density in the SCP and DCP, the FD-300, and the flow area of the choriocapillaris were all significantly lower in CRVO eyes than in healthy eyes, while the AI and retinal thickness were significantly higher (all P<0.05). After treatment, retinal thickness was significantly decreased, and the mean BCVA had markedly improved from 20/167 to 20/65 (P=0.0092). The flow area of the choriocapillaris was also significantly improved, which may result from the reduction of shadowing effect caused by the attenuation of macular edema. However, there were no significant changes in SCP and DCP vessel density after treatment. The flow area of the choriocapillaris at baseline was negatively correlated with retinal thickness. CONCLUSION: OCTA enables the non-invasive, layer-specific and quantitative assessment of microvascular changes both before and after treatment, and can therefore be used as a valuable imaging tool for the evaluation of the follow-up in CRVO patients.