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.

Kurarinone regulates Th17/Treg balance and ameliorates autoimmune uveitis via Rac1 inhibition.

INTRODUCTION: Autoimmune uveitis (AU) is a severe intraocular autoimmune disorder with a chronic disease course and a high rate of blindness. Kurarinone (KU), a major component of the traditional Chinese medicine Sophorae Flavescentis Radix, possesses a wide spectrum of activities and has been used to treat several inflammation-related diseases. OBJECTIVE: We aimed to investigate the effects of KU on AU and its modulatory mechanisms. METHODS: We used an experimental autoimmune uveitis (EAU) animal model and characterized the comprehensive immune landscape of KU-treated EAU mice using single-cell RNA sequencing (scRNA-seq). The retina and lymph nodes were analyzed. The siRNAs and selective inhibitors were used to study the signaling pathway. The effect of KU on peripheral blood mononuclear cells (PBMCs) from uveitis patients was also examined. RESULTS: We found that KU relieved chorioretinal lesions and immune cell infiltration in EAU model mice. Subsequent single-cell analysis revealed that KU downregulated the EAU-upregulated expression of inflammatory and autoimmune-related genes and suppressed pathways associated with immune cell differentiation, activation, and migration in a cell-specific manner. KU was implicated in restoring T helper 17 (Th17)/regulatory T (Treg) cell balance by alleviating inflammatory injury and elevating the expression of modulatory mediators in Tregs, while simultaneously ameliorating excessive inflammation by Th17 cells. Furthermore, Rac1 and the Id2/Pim1 axis potentiated the pathogenicity of Th17 cells during EAU, which was inhibited by KU treatment, contributing to the amelioration of EAU-induced inflammation and treatment of AU. In addition, KU suppressed inflammatory cytokine production in activated human PBMCs by inhibiting Rac1. Integration of the glucocorticoid-treated transcriptome suggests that KU has immunomodulatory effects on lymphocytes. CONCLUSION: Our study constructed a high-resolution atlas of the immunoregulatory effects of KU treatment on EAU and identified its potential therapeutic mechanisms, which hold great promise in treating autoimmune disorders.

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.

Therapeutic Effects of Upadacitinib on Experimental Autoimmune Uveitis: Insights From Single-Cell Analysis.

Purpose: This study aimed to elucidate the impact of upadacitinib, a Janus kinase 1 (JAK1)-specific inhibitor, on experimental autoimmune uveitis (EAU) and explore its underlying mechanisms. Methods: We utilized single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to investigate the JAK/signal transducer and activator of transcription (STAT) pathway in peripheral blood mononuclear cells (PBMCs) of 12 patients with Vogt-Koyanagi-Harada (VKH) disease and cervical draining lymph node (CDLN) cells of EAU. After treating EAU with upadacitinib, we analyzed immune cell gene expression and cell-cell communication by integrating scRNA data. Additionally, we applied flow cytometry and western blot to analyze the CDLN cells. Results: The JAK/STAT pathway was found to be upregulated in patients with VKH disease and EAU. Upadacitinib effectively alleviated EAU symptoms, reduced JAK1 protein expression, and suppressed pathogenic CD4 T cell (CD4TC) proliferation and pathogenicity while promoting Treg proliferation. The inhibition of pathogenic CD4TCs by upadacitinib was observed in both flow cytometry and scRNA data. Additionally, upadacitinib was found to rescue the interferon-stimulated gene 15 (ISG15)+ CD4TCs and CD8 T and B cell ratios and reduce expression of inflammatory-related genes. Upadacitinib demonstrated the ability to inhibit abnormally activated cell-cell communication, particularly the CXCR4-mediated migration pathway, which has been implicated in EAU pathogenesis. CXCR4 inhibitors showed promising therapeutic effects in EAU. Conclusions: Our findings indicate that the JAK1-mediated signaling pathway is significantly upregulated in uveitis, and upadacitinib exhibits therapeutic efficacy against EAU. Furthermore, targeting the CXCR4-mediated migration pathway could be a promising therapeutic strategy.

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.

Sleep loss potentiates Th17-cell pathogenicity and promotes autoimmune uveitis.

BACKGROUND: Sleep loss (SL) is a health issue associated with the higher risk of autoimmune and inflammatory disorders. However, the connection between SL, the immune system, and autoimmune diseases remains unknown. METHODS: We conducted mass cytometry, single-cell RNA sequencing, and flow cytometry to analyze how SL influences immune system and autoimmune disease development. Peripheral blood mononuclear cells from six healthy subjects before and after SL were collected and analyzed by mass cytometry experiments and subsequent bioinformatic analysis to identify the effects of SL on human immune system. Sleep deprivation and experimental autoimmune uveitis (EAU) mice model were constructed, and scRNA-seq data from mice cervical draining lymph nodes were generated to explore how SL influences EAU development and related autoimmune responses. RESULTS: We found compositional and functional changes in human and mouse immune cells after SL, especially in effector CD4+ T and myeloid cells. SL upregulated serum GM-CSF levels in healthy individuals and in patients with SL-induced recurrent uveitis. Experiments in mice undergoing SL or EAU demonstrated that SL could aggravate autoimmune disorders by inducing pathological immune cell activation, upregulating inflammatory pathways, and promoting intercellular communication. Furthermore, we found that SL promoted Th17 differentiation, pathogenicity, and myeloid cells activation through the IL-23Th17GM-CSF feedback mechanism, thus promoting EAU development. Lastly, an anti-GM-CSF treatment rescued SL-induced EAU aggravation and pathological immune response. CONCLUSIONS: SL promoted Th17 cells pathogenicity and autoimmune uveitis development, especially through the interaction between Th17 and myeloid cells involving GM-CSF signaling, providing possible therapeutic targets for the SL-related pathological disorders.

Insights gained from single-cell analysis of immune cells in tofacitinib treatment of Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada disease (VKH) is an important refractory uveitis mediated by pathological T cells (TCs). Tofacitinib (TOFA) is a JAK- targeted therapy for several autoimmune diseases. However, the specific pathogenesis and targeted therapeutics for VKH remain largely unknown. Based on single-cell RNA sequencing and mass cytometry, we present what we believe is the first multimodal, high-dimensional analysis to generate a comprehensive human immune atlas regarding subset composition, gene signatures, enriched pathways, and intercellular interactions of VKH patients undergoing TOFA therapy. Patients with VKH are characterized by TCs' polarization from naive to effector and memory subsets, together with accrued monocytes and upregulated cytokines and JAK/STAT signaling pathways. In vitro, TOFA reversed Th17/Treg imbalance and inhibited IL-2-induced STAT1/3 phosphorylation. TOFA alleviated VKH symptoms by restoring pathological TCs' polarization and functional marker expression and downregulating cytokine signaling and lymphocyte function. Remarkably, inflammation-related responses and intercellular interactions decreased after TOFA treatment, particularly in monocytes. Notably, we identified 2 inflammation- and JAK-associated monocyte subpopulations that were strongly implicated in VKH pathogenesis and mechanisms involved in TOFA treatment. Here, we provide a potentially novel JAK-targeted therapy for VKH and elaborate on the possible therapeutic mechanisms of TOFA, expanding our knowledge of VKH pathological patterns.

Functional analysis of human circulating immune cells based on high-dimensional mass cytometry.

With the advantages of high resolution and high dimension, mass cytometry is implemented to analyze the blood complex immune system in clinical settings. However, long-term clinical sample collection may cause batch effects that mask true biological results. Here, we present a validated and streamlined mass cytometry workflow that features fixed staining for clinical use and optimized barcode staining patterns. The reagents and approaches used in this workflow can help reduce batch effects, thereby extending the application range and advantages of mass cytometry.