Dynamic Transition of Regulatory T Cells to Cytotoxic Phenotype Amid Systemic Inflammation in Graves' Ophthalmopathy.

Graves' disease (GD) is an autoimmune condition that can progress to Graves' Ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, GD patients without GO, newly diagnosed GO patients, and treated GO patients. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated regulatory T cells (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yinyang1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results revealed the dynamic changes in immune landscape during GO progression and provided novel insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.

Carbon dioxide laser excision as a novel treatment for large xanthelasma palpebrarum: long-term efficacy and safety.

AIMS: This study evaluates the long-term efficacy and safety of carbon dioxide (CO2) laser excision as a novel treatment for large xanthelasma palpebrarum. METHODS: The study included 295 patients diagnosed with xanthelasma palpebrarum, categorised as 246 patients with grade I, 16 with grade II, 22 patients with grade III and 11 with grade IV lesions. All underwent CO2 laser excision and were followed up for a 12-month period. Treatment efficacy was assessed through clearance and recurrence rates, evaluated using digital photography and complications were recorded. RESULTS: The participant group consisted of 66 males and 229 females, with an average age of 41.7±11.6 years. Clearance rates exceeded 99% for grades I-III and were around 95% for grade IV lesions. The recurrence rate was 6.8%, with higher recurrence rates observed in lesions over 2 mm in height compared with those under 2 mm (p<0.001). The main complications over the 12-month follow-up were scarring (4.4%), hyperpigmentation (8.1%) and hypopigmentation (8.5%), with no severe complications reported. CONCLUSION: CO2 laser excision emerges as a precise, minimally invasive and effective modality for treating xanthelasma palpebrarum, marking a significant advancement in treatment modalities. Further research is required to reinforce these results and refine treatment protocols.

Experimental Study of Ultra-Pulsed CO 2 Fractional Laser Combined With Recombinant Human Epidermal Growth Factor Gel in the Treatment of Eyelid Keloid.

OBJECTIVE: Keloid (KD) and hypertrophic scars are prevalent and result from excessive growth of dermal tissue after skin damage. This review focused on the clinical application of the ultra-pulsed CO 2 fractional laser combined with recombinant human epidermal growth factor (rHEGF) gel in patients with eyelid KD. METHODS: Patients (N = 98) with KD who underwent surgery were randomly divided into a study group (ultra-pulsed CO 2 fractional laser combined with rHEGF gel therapy, N = 49) and a control group (ultra-pulsed CO 2 fractional laser therapy, N = 49). Besides, 5 cases dropped out of the study, including 2 cases in the study group and 3 cases in the control group. Finally, 47 cases of the study group and 46 cases of the study group were included in the analysis. The clinical baseline data such as sex, age, body mass index, scar area, etiology, Vancouver Scar Scale score, Patient and Observer Scar Assessment Scale score, four-item itch questionnaire score, serum interleukin-6 (IL-6), IL-10, and tumor necrosis factor-α level expression were recorded in the study group (N = 47) and the control group (N = 46). RESULTS: There was no significant difference in gender, age, body mass index, scar area, etiology, Vancouver Scar Scale score, Patient and Observer Scar Assessment Scale score, 4-item itch questionnaire score, IL-6, IL-10, and tumor necrosis factor-α levels between the patients treated with ultra-pulse CO 2 fractional laser + rHEGF gel and those only treated with ultra-pulse CO 2 fractional laser ( p > 0.05). Vancouver Scar Scale scores, Patient and Observer Scar Assessment Scale scores, and four-item itch questionnaire scores of patients with eyelid KD decreased to a greater extent than those treated with ultra-pulsed CO 2 fractional laser combined with rHEGF gel ( p <0.01). Compared with ultra-pulsed CO 2 fractional laser treatment, ultra-pulsed CO 2 fractional laser combined with rHEGF gel was more efficacious in treating patients with eyelid KD, with a lower incidence of adverse effects and a 1-year recurrence rate. CONCLUSIONS: Ultra-pulsed CO 2 fractional laser combined with rHEGF gel can significantly improve the scar status and scar itching in patients with eyelid KD, with an obvious therapeutic effect, a low incidence of adverse effects, a 1-year recurrence rate, and high safety, which is worthy of popularization and application.

Deciphering the impact of aging on splenic endothelial cell heterogeneity and immunosenescence through single-cell RNA sequencing analysis.

BACKGROUND: Aging is associated with significant structural and functional changes in the spleen, leading to immunosenescence, yet the detailed effects on splenic vascular endothelial cells (ECs) and their immunomodulatory roles are not fully understood. In this study, a single-cell RNA (scRNA) atlas of EC transcriptomes from young and aged mouse spleens was constructed to reveal age-related molecular changes, including increased inflammation and reduced vascular development and also the potential interaction between splenic endothelial cells and immune cells. RESULTS: Ten clusters of splenic endothelial cells were identified. DEGs analysis across different EC clusters revealed the molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the loss in vascular development function of aged ECs. Notably, four EC clusters with immunological functions were identified, suggesting an Endothelial-to-Immune-like Cell Transition (EndICLT) potentially driven by aging. Pseudotime analysis of the Immunology4 cluster further indicated a possible aging-induced transitional state, potentially initiated by Ctss gene activation. Finally, the effects of aging on cell signaling communication between different EC clusters and immune cells were analyzed. CONCLUSIONS: This comprehensive atlas elucidates the complex interplay between ECs and immune cells in the aging spleen, offering new insights into endothelial heterogeneity, reprogramming, and the mechanisms of immunosenescence.

CD169+ classical monocyte as an important participant in Graves' ophthalmopathy through CXCL12-CXCR4 axis.

Patients with Graves' disease (GD) can develop Graves' ophthalmopathy (GO), but the underlying pathological mechanisms driving this development remain unclear. In our study, which included patients with GD and GO, we utilized single-cell RNA sequencing (scRNA-seq) and multiplatform analyses to investigate CD169+ classical monocytes, which secrete proinflammatory cytokines and are expanded through activated interferon signaling. We found that CD169+ clas_mono was clinically significant in predicting GO progression and prognosis, and differentiated into CD169+ macrophages that promote inflammation, adipogenesis, and fibrosis. Our murine model of early-stage GO showed that CD169+ classical monocytes accumulated in orbital tissue via the Cxcl12-Cxcr4 axis. Further studies are needed to investigate whether targeting circulating monocytes and the Cxcl12-Cxcr4 axis could alleviate GO progression.

Single-cell chromatin accessibility and transcriptomic characterization of Behcet's disease.

Behect's disease is a chronic vasculitis characterized by complex multi-organ immune aberrations. However, a comprehensive understanding of the gene-regulatory profile of peripheral autoimmunity and the diverse immune responses across distinct cell types in Behcet's disease (BD) is still lacking. Here, we present a multi-omic single-cell study of 424,817 cells in BD patients and non-BD individuals. This study maps chromatin accessibility and gene expression in the same biological samples, unraveling vast cellular heterogeneity. We identify widespread cell-type-specific, disease-associated active and pro-inflammatory immunity in both transcript and epigenomic aspects. Notably, integrative multi-omic analysis reveals putative TF regulators that might contribute to chromatin accessibility and gene expression in BD. Moreover, we predicted gene-regulatory networks within nominated TF activators, including AP-1, NF-kB, and ETS transcript factor families, which may regulate cellular interaction and govern inflammation. Our study illustrates the epigenetic and transcriptional landscape in BD peripheral blood and expands understanding of potential epigenomic immunopathology in this disease.

VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Single-cell analysis of immune cells on gingiva-derived mesenchymal stem cells in experimental autoimmune uveitis.

Gingiva-derived mesenchymal stem cells (GMSCs) have shown astonishing efficacy in the treatment of various autoimmune diseases. However, the mechanisms underlying these immunosuppressive properties remain poorly understood. Here, we generated a lymph node single-cell transcriptomic atlas of GMSC-treated experimental autoimmune uveitis mice. GMSC exerted profound rescue effects on T cells, B cells, dendritic cells, and monocytes. GMSCs rescued the proportion of T helper 17 (Th17) cells and increased the proportion of regulatory T cells. In addition to globally altered transcriptional factors (Fosb and Jund), we observed cell type-dependent gene regulation (e.g., Il17a and Rac1 in Th17 cells), highlighting the GMSCs' cell type-dependent immunomodulatory capacity. GMSCs strongly influenced the phenotypes of Th17 cells, suppressing the formation of the highly inflammatory CCR6-CCR2+ phenotype and enhancing the production of interleukin (IL) -10 in the CCR6+CCR2+ phenotype. Integration of the glucocorticoid-treated transcriptome suggests a more specific immunosuppressive effect of GMSCs on lymphocytes.

PDGF-D-induced immunoproteasome activation and cell-cell interactions.

Platelet-derived growth factor-D (PDGF-D) is abundantly expressed in ocular diseases. Yet, it remains unknown whether and how PDGF-D affects ocular cells or cell-cell interactions in the eye. In this study, using single-cell RNA sequencing (scRNA-seq) and a mouse model of PDGF-D overexpression in retinal pigment epithelial (RPE) cells, we found that PDGF-D overexpression markedly upregulated the key immunoproteasome genes, leading to increased antigen processing/presentation capacity of RPE cells. Also, more than 6.5-fold ligand-receptor pairs were found in the PDGF-D overexpressing RPE-choroid tissues, suggesting markedly increased cell-cell interactions. Moreover, in the PDGF-D-overexpressing tissues, a unique cell population with a transcriptomic profile of both stromal cells and antigen-presenting RPE cells was detected, suggesting PDGF-D-induced epithelial-mesenchymal transition of RPE cells. Importantly, administration of ONX-0914, an immunoproteasome inhibitor, suppressed choroidal neovascularization (CNV) in a mouse CNV model in vivo. Together, we show that overexpression of PDGF-D increased pro-angiogenic immunoproteasome activities, and inhibiting immunoproteasome pathway may have therapeutic value for the treatment of neovascular diseases.

PDGFD switches on stem cell endothelial commitment.

The critical factors regulating stem cell endothelial commitment and renewal remain not well understood. Here, using loss- and gain-of-function assays together with bioinformatic analysis and multiple model systems, we show that PDGFD is an essential factor that switches on endothelial commitment of embryonic stem cells (ESCs). PDGFD genetic deletion or knockdown inhibits ESC differentiation into EC lineage and increases ESC self-renewal, and PDGFD overexpression activates ESC differentiation towards ECs. RNA sequencing reveals a critical requirement of PDGFD for the expression of vascular-differentiation related genes in ESCs. Importantly, PDGFD genetic deletion or knockdown increases ESC self-renewal and decreases blood vessel densities in both embryonic and neonatal mice and in teratomas. Mechanistically, we reveal that PDGFD fulfills this function via the MAPK/ERK pathway. Our findings provide new insight of PDGFD as a novel regulator of ESC fate determination, and suggest therapeutic implications of modulating PDGFD activity in stem cell therapy.

Insights gained from Single-Cell analysis of immune cells on Cyclosporine A treatment in autoimmune uveitis.

Cyclosporine A (CsA) is a widely known immunosuppressive agent that is clinically important in autoimmune diseases owing to its selective suppression of T lymphocytes. Although it has long been recognized to inhibit T cell responses by blocking calcineurin, the potential targets and specific downstream mechanisms remain elusive. Herein, we built a comprehensive single-cell transcriptomic landscape of immune cells in the blank, untreated experimental autoimmune uveitis (EAU), and CsA-treated EAU mice. CsA reversed EAU-associated changes in cell type composition, genomic expression, cell trajectory, and cell-cell communication. We found that CsA reverses the proportion change of disease-related immune cells; regulates several crucial pathogenic factors (eg. IL1r1, CD48, and Bhlhe40) in T helper 17 cells (Th17), the transcription factor Bhlhe40 was also rescued in T helper 1 cells (Th1); and may differentiate Tregs into a state of enhanced immunosuppression. In addition, we revealed the rescued impact of CsA on all immune cell types, especially on plasma B cells differentiation and immunoglobulin secretion. Furthermore, comparisons with glucocorticoids showed that CsA might have a more premium rescue effect involved in attenuating the pathogenicity of autoreactive T cells. Our work provides a comprehensive single-cell transcriptional atlas of immune cells under CsA therapy, providing advanced insights into the mechanisms underlying CsA and a reference for developing new therapeutic strategies for autoimmune diseases.

Corrigendum: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization.

[This corrects the article DOI: 10.3389/fcell.2021.686886.].

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.

Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization.

Platelet-derived growth factor-D (PDGF-D) is highly expressed in immune cells. However, the potential role of PDGF-D in immune system remains thus far unclear. Here, we reveal a novel function of PDGF-D in activating both classical and alternative complement pathways that markedly increase chemokine and cytokine responses to promote macrophage polarization. Pharmacological targeting of the complement C3a receptor using SB290157 alleviated PDGF-D-induced neuroinflammation by blocking macrophage polarization and inhibited pathological choroidal neovascularization. Our study thus suggests that therapeutic strategies targeting both PDGF-D and the complement system may open up new possibilities for the treatment of neovascular diseases.

Mitogen-Inducible Gene 6 Inhibits Angiogenesis by Binding to SHC1 and Suppressing Its Phosphorylation.

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.

Inhibition of LPA1 Signaling Impedes Conversion of Human Tenon's Fibroblasts into Myofibroblasts Via Suppressing TGF-ß/Smad2/3 Signaling.

Purpose: Lysophosphatidic acid (LPA) is a growth factor-like phospholipid that has been recognized as a profibrotic mediator in numerous tissues, yet, whether it plays a role in subconjunctival fibrosis remains to be investigated. Therefore, this study was designed to examine the effect of LPA1-3 signaling inhibitor, Ki16425 on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts. Methods: Primary cultured HTFs were incubated with transforming growth factor-ß1 (TGF-ß1) alone or combined with Ki16425, the cell proliferation and migration were measured by Cell Counting Kit-8 and the scratch wound assay, respectively. HTFs contractility was evaluated with 3-dimensional (3D) Collagen Contraction assay. The mRNA and protein levels of α-smooth muscle actin (α-SMA), Snail and the phosphorylation levels of Smad2/3, p38MAPK, and ERK1/2 were determined by real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunofluorescence staining. Results: Ki16425 significantly prevent the proliferation and migration of Tenon's fibroblasts (HTFs) in a dose-dependent manner. Furthermore, Ki16425 blocked HTFs myofibroblast differentiation via downregulation of mRNA and protein expression of α-SMA. 3D collagen gel contraction assay demonstrated that Ki16425 effectively inhibits myofibroblast contraction induced by TGF-ß1. Mechanistically, we revealed that Ki16425 reduces Smad2/3 but not p38MAPK or ERK1/2 phosphorylation by TGF-ß1. By using an LPA1-specific inhibitor, AM095, we confirmed that LPA1 signaling but not LPA2 or LPA3 is involved in TGF-ß1 induced HTFs activation. Conclusions: Our results show that inhibition of LPA1 signaling presents potent antifibrotic effect in HTFs, which may serve as a promising intervention strategy for preventing subconjunctival fibrosis caused by glaucoma filtration surgery.

Novel multi-targeted inhibitors suppress ocular neovascularization by regulating unique gene sets.

Neovascular diseases, such as many cancers and ocular disorders, are life threatening and devastating. Although anti-vascular endothelial growth factor A (VEGF-A) therapy is available, many patients are not responsive and drug resistance can develop. To try to overcome these problems, combination therapy targeting VEGF-A and platelet-derived growth factor B (PDGF-B) was tested. However, one obvious drawback was that the other VEGF and PDGF family members were not inhibited and therefore could compensate. Indeed, this was, at least to some extent, demonstrated by the disappointing outcomes. To this end, we designed novel multi-targeted inhibitors that can block most of the VEGF and PDGF family members simultaneously by making a fusion protein containing the ligand-binding domains of vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRß), which can therefore act as a decoy blocker for most of the VEGF and PDGF family members. Indeed, in cultured cells, the novel inhibitors suppressed the migration and proliferation of both vascular endothelial cells and smooth muscle cells, and abolished VEGFR2 and PDGFRß activation. Importantly, in a choroidal neovascularization model in vivo, the novel inhibitor inhibited ocular neovascularization more efficiently than the mono-inhibitors against VEGFR or PDGFR alone respectively. Mechanistically, a genome-wide microarray analysis unveiled that the novel inhibitor regulated unique sets of genes that were not regulated by the mono-inhibitors, further demonstrating the functional uniqueness and superiority of the novel inhibitor. Together, we show that the multi-targeted inhibitors that can block VEGFR1, VEGFR2 and PDGFRß simultaneously suppress pathological angiogenesis more efficiently than monotherapy, and may therefore have promising therapeutic value for the treatment of neovascular diseases.

Novel function of VEGF-B as an antioxidant and therapeutic implications.

Oxidative stress, due to insufficiency of antioxidants or over-production of oxidants, can lead to severe cell and tissue damage. Oxidative stress occurs constantly and has been shown to be involved in innumerable diseases, such as degenerative, cardiovascular, neurological, and metabolic disorders, cancer, and aging, thus highlighting the vital need of antioxidant defense mechanisms. Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago, and is abundantly expressed in most types of cells and tissues. VEGF-B remained functionally mysterious for many years and later on has been shown to be minimally angiogenic. Recently, VEGF-B is reported to be a potent antioxidant by boosting the expression of key antioxidant enzymes. Thus, one major role of VEGF-B lies in safeguarding tissues and cells from oxidative stress-induced damage. VEGF-B may therefore have promising therapeutic utilities in treating oxidative stress-related diseases. In this review, we discuss the current knowledge on the newly discovered antioxidant function of VEGF-B and the related molecular mechanisms, particularly, in relationship to some oxidative stress-related diseases, such as retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, glaucoma, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

MEK inhibition prevents TGF­ß1­induced myofibroblast transdifferentiation in human tenon fibroblasts.

Subconjunctival fibrosis represents the primary cause of postoperative failure of trabeculectomy, and at present there is a lack of effective intervention strategies. The present study aimed to investigate the effect of the mitogen­activated protein kinase kinase (MEK) inhibitor U0126 on human tenon fibroblast (HTF) myofibrosis transdifferentiation, and to illuminate the underlying molecular mechanisms involved. It was demonstrated that U0126 significantly inhibited the proliferation, migration and collagen contraction of HTFs stimulated with TGF­ß1. In addition, U0126 largely attenuated the TGF­ß1­induced conversion of HTFs into myofibroblasts, as indicated by a downregulation of the mRNA and protein expression of α­smooth muscle actin and zinc finger protein SNAI1, and by ameliorating the 3D­collagen contraction response. Mechanistically, U0126 suppressed the TGF­ß1­stimulated phosphorylation of mothers against decapentaplegic homolog 2/3, P38 mitogen­activated protein kinase and extracellular signal­regulated kinase 1/2, indicating that U0126 may inhibit HTF activation through the canonical and non­canonical signaling pathways of TGF­ß1. Therefore, U0126 exhibits a potent anti­fibrotic effect among HTFs, and the inhibition of MEK signaling may serve as an alternative intervention strategy for the treatment of trabeculectomy­associated fibrosis.