Itaconate Ameliorates Experimental Autoimmune Uveitis by Modulating Teff/Treg Cell Imbalance Via the DNAJA1/CDC45 Axis

Abstract

Purpose: The aim of this study was to elucidate the effect of itaconate (ITA) on experimental autoimmune uveitis (EAU), to explore its potential mechanism, and to identify potential therapeutic targets.

Methods: We established an animal model of EAU by constructing an immune map of mice treated with ITA and exploring the therapeutic mechanism of ITA by single-cell RNA sequencing and flow cytometry.

Results: ITA mitigated ocular inflammation associated with EAU and reversed the pathogenic differentiation linked to Th17 induction by EAU, along with the reactive oxygen species (ROS) and oxidative stress pathways. Subsequent to ITA intervention, the downregulated differentially expressed genes in the T-cell subset primarily centered around the heat shock protein (HSP) family. Activation of HSPs reversed the anti-inflammatory effects of ITA in EAU mice. ITA decreased ROS levels and HSP expression in CD4⁺ T cells, with DnaJ heat shock protein family (HSP40) member A1 (DNAJA1) exhibiting the most notable alterations among the HSPs. ITA suppressed the expression of DNAJA1/cell division cycle protein 45 (CDC45), thereby disrupting the pathogenic division cycle of CD4⁺ T cells and reducing their proliferation. Inhibiting DNAJA1 also held promise for modulating the Th17/Treg imbalance. Notably, ITA curtailed the expansion of CD4⁺ T cells in uveitis patients.

Conclusions: Our research delved into the potential therapeutic mechanisms underlying ITA therapy in EAU, offering fresh perspectives on its utility in the treatment of autoimmune conditions. DNAJA1 emerges as a promising candidate for targeted therapeutic interventions in uveitis.

Uveitis is an inflammatory ocular condition linked to aberrant immune system functionality that affects the iris, ciliary body, choroid, and adjacent ocular tissues.^1,2 Recurrent uveitis causes cumulative and irreversible intraocular tissue damage, increasing the risk of vision loss and imposing substantial medical and socioeconomic burdens.^3,4 The elusive pathogenesis of uveitis poses a therapeutic challenge due to the lack of specificity in treatment modalities.⁵ The broad immunomodulatory effects of corticosteroids and immunosuppressants, especially in high doses and long-term regimens, pose risks of adverse reactions.^6–8 Therefore, understanding uveitis pathogenesis, identifying novel therapeutic targets, precisely modulating these targets, and developing safe, effective, and target-specific treatments are crucial for improving patient outcomes. T cells, particularly CD4⁺ T cells, play a pivotal role in the initiation and progression of uveitis.⁹ Effector T (Teff) cells, including T helper 1 (Th1) cells and Th17 within the CD4⁺ Teff population, drive the pathological evolution of uveitis, whereas regulatory T (Treg) cells modulate Teff cell function.¹⁰ The balance between Teff and Treg cells is critical for immune homeostasis.^11,12 However, the precise mechanisms underlying the imbalance between Teff and Treg cells in uveitis pathogenesis remain unclear. Further research is necessary to develop interventions that restore the Teff/Treg cell equilibrium and ameliorate uveitis progression. 

Itaconate (ITA) is an intermediate metabolite of the mitochondrial tricarboxylic acid (TCA) cycle. When macrophages and myeloid cells are exposed to pathological stimuli such as lipopolysaccharide (LPS), ITA is formed in the TCA cycle through the oxidation and decarboxylation of citrate by aconitate decarboxylase 1 (ACOD1), encoded by the immunoresponsive gene 1 (Irg1).¹³ ITA was first discovered in 2011 to be abundantly produced in LPS-activated macrophages. Subsequent studies have shown its potent anti-inflammatory, antiviral, and antibacterial properties. For example, ITA inhibits the production of inflammatory cytokines in macrophages, such as IL-1β and IL-6, exerting anti-inflammatory effects.¹⁴ ITA also directly inhibits bacterial isocitrate lyase¹⁵ and acetyl-CoA carboxylase,¹⁶ leading to antibacterial effects, and it effectively suppresses viral replication and the host's excessive inflammatory response to human pathogenic viruses.¹⁷ Recent studies have shown that CD8⁺ T cells can uptake ITA, influencing their proliferation and activation and reducing the secretion of IFN-γ, granzymes, and perforin.¹⁸ ITA exhibits anti-inflammatory effects in preclinical models of sepsis, psoriasis, gout, ischemia/reperfusion injury, and pulmonary fibrosis, suggesting its potential for treating various immune/inflammatory diseases.¹⁹ However, the medicinal value of ITA in uveitis treatment, its mechanisms of action, and specific molecular pathways remain unexplored. 

Single-cell RNA sequencing (scRNA-seq), a powerful technology with high throughput and resolution, can reveal genetic and expression differences among specific cells, enabling in-depth exploration of cellular heterogeneity.²⁰ This study demonstrated that ITA ameliorated retinal inflammation in experimental autoimmune uveitis (EAU) mice, with heat shock protein (HSP) agonist co-intervention reversing this therapeutic effect. ITA reduced Th17 differentiation, IL-17 signaling, reactive oxygen species (ROS), protein folding, and oxidative stress-related pathways. ITA primarily reduced the expression of ROS and HSPs in CD4⁺ T cells, leading to decreased expression of interferon-gamma (IFN-γ) and IL-17, reduced proportions of Th1 and Th17 cells, and increased Treg proportions, thereby restoring Teff/Treg cell balance. Additionally, ITA influenced the expression of DnaJ heat shock protein family (HSP40) member A1 (DNAJA1) within the HSP family and cell division cycle protein 45 (CDC45), affecting the cell cycle of CD4⁺ T cells and reducing their proliferation and pathogenicity. DNAJA1, which can reverse Teff/Treg imbalance, may represent a potential therapeutic target for uveitis. 

Methods

Collection of Human Venous Blood Samples

Venous blood samples from five patients with Vogt–Koyanagi–Harada (VKH) disease were obtained for flow cytometry to evaluate the pharmacological effects of ITA. These samples were collected at the Zhongshan Ophthalmic Center of Sun Yat-Sen University. According to the revised diagnostic criteria, VKH diagnosis is based on clinical manifestations, continuous optical coherence tomography, and indocyanine green fluorescence fundus angiography results.²¹ All patients were newly diagnosed and clinically active and had not received any prior treatment. Individuals with diabetes, cancer, hypertension, or other systemic diseases were excluded. The study was conducted with the informed consent of all donors and approved by the Medical Ethics Committee of Guangzhou Zhongshan Ophthalmology Center (2020KYPJ124). 

Experimental Animals

C57BL/6J mice 6 to 8 weeks old (n = 5 per group) were purchased from the Medical Laboratory Animal Center in Guangzhou, China. The mice were raised under specific pathogen free conditions, with a room temperature of 21°C ± 1°C, relative humidity of 60% ± 5%, and a 12-hour light/dark cycle. All animal experiments were conducted in accordance with the guidelines and institutional policies for the use of animals in ophthalmology and visual research at the Zhongshan Ophthalmic Center of Sun Yat-Sen University, Guangzhou, China. The experimental plan was approved by the Animal Experiment Management Committee. 

Induction of EAU in Mouse Model

After anesthesia, each C57BL/6J mouse was subcutaneously injected with 200 µL of a mixture containing 2-mg/mL retinal antigen interphotoreceptor retinoid-binding protein 1–20 (IRBP1-20; GL Biochem, Shanghai, China) and 2.5-mg/mL Mycobacterium tuberculosis dissolved in complete Freund's adjuvant (BD Biosciences, Franklin Lakes, NJ, USA). Additionally, 0.25 µg of pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, USA) was injected intraperitoneally on the day of immunization and again 2 days later. 

On the 14th day post-immunization, fundus microscopy was performed on anesthetized mice to assess retinal infiltration and vasculitis. Clinical scores, ranging from 0 to 4, were evaluated using a blind method (Supplementary Table S1).²² The murine eyeballs were then washed with PBS, fixed in paraformaldehyde for 48 hours, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and sealed. Three slices were taken from each murine eyeball through the optic disc, resulting in a total of six slices per mouse. Section images were collected under a microscope, and pathological scores were evaluated blindly (Supplementary Table S1).²² Representative images were selected for display based on average values. 

Drug Intervention and Mouse Tissue Extraction

ITA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile PBS and the HSP agonist ML346 (MedChemExpress, Monmouth Junction, NJ, USA) in dimethylsulfoxide (DMSO, 0.1%; Sigma-Aldrich). Then, the ML346 with 20% sulfobutylether-β-cyclodextrin was diluted in a 90% saline solution (MedChemExpress). From days 0 to 14 of establishing the EAU mouse models, ITA treatment group mice were intraperitoneally injected with ITA (50 mg/kg) every other day. In the rescue experiment, EAU mouse models were divided into a Control group, ITA group, and ITA+ML346 group, with the latter two groups receiving ITA and ITA plus ML346 interventions, respectively. The ITA+ML346 group was intraperitoneally injected with ML346 (10 mg/kg) and ITA (50 mg/kg) every other day. The EAU mice (Control group) received an equal volume of PBS solution. On day 14 post-immunization, retinas, cervical draining lymph nodes (LN), and spleen (SP) tissues were extracted from the mice. Cells were isolated and analyzed by flow cytometry. 

For in vitro experiments, cells isolated from LN of the EAU mice were cultured with IRBP1-20 alone; IRBP1-20 plus ITA (6-mM); IRBP1-20 plus ITA (6-mM) plus ML346 (1-µM); or IRBP1-20 plus 116-9e, a DNAJA1 inhibitor (40-µM), at 37°C for 72 hours and analyzed by flow cytometry. 

Extraction of Mononuclear Cells From Peripheral Blood of Human Veins

Human heparinized venous blood samples were immediately processed and subjected to standard density gradient centrifugation using Ficoll–Hypaque solution (GE HealthCare, Chicago, IL, USA) to isolate peripheral blood mononuclear cells (PBMCs). 

Preparation of Single-Cell Suspensions

Mouse LN cells were harvested on day 14 post-immunization and combined into a single sample for single-cell library preparation. The LN tissue was ground, and the cells were mixed with RPMI-1640 medium (Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), penicillin/streptomycin (Thermo Fisher Scientific), 3-mg/mL collagenase IV (Sigma-Aldrich), and 40-mg/mL deoxyribonuclease I (DNase I; Sigma-Aldrich). The mixture was incubated at 37°C for 15 minutes. The digested cells were then collected and filtered through a 70-µm cell strainer. The resulting single-cell suspension contained 1 × 10⁷ cells/mL with a viability of ≥85%, as determined by the Countess II Automated Cell Counter (Thermo Fisher Scientific). 

Th17/Treg Cell Polarization

Naïve CD4⁺ T cells were isolated from the murine SP by magnetic cell sorting with the Naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, BC, Canada) with the purity of CD4⁺ T cells > 95%. They were stimulated for 3 days with anti-CD3 (0.25 µg/mL; BioLegend, San Diego, CA, USA) and anti-CD28 (0.5 µg/mL; BioLegend) antibodies. The Th17 cell differentiation condition utilized anti–IL-4 antibody (2 µg/mL; BioLegend), anti–IFN-γ antibody (2 µg/mL; BioLegend), IL-6 (30 ng/mL; BioLegend), and TGF-β (0.3 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany). The Treg cell differentiation condition utilized anti–IL-4 antibody (2 µg/mL; BioLegend), anti–IFN-γ antibody (2 µg/mL; BioLegend), IL-2 (20 ng/mL; BioLegend), and TGF-β (1 ng/mL; Miltenyi Biotec). 

Flow Cytometry and Cell Culture

Mouse LN, SP, and retinas were collected and homogenized, then mixed with the RPMI-1640 medium containing 2% FBS, penicillin/streptomycin, 3-mg/mL collagenase IV, and 40-mg/mL DNase I. They were then incubated at 37°C for 15 minutes. The digested cells were collected and filtered through a 70-µm strainer. For flow cytometry analysis, cells were stained with live/dead cell dyes (BioLegend) at 4°C for 10 minutes. Subsequently, surface antibodies against mouse CD4 (BioLegend) and human CD4 (BioLegend) were added and incubated for 15 minutes at 4°C. After fixation and membrane permeabilization, intracellular antibody staining was performed. Cells were incubated with phorbol 12-myristate 13-acetate (PMA; 1 µg/mL), Brefeldin A (BFA; 20 µg/mL), and ionomycin (10 µg/mL) at 37°C for 5 hours, followed by overnight incubation at 4°C with antibodies against murine/human IFN-γ, IL-17A, forkhead box P3 (FOXP3), and DNAJA1 (Abcam, Cambridge, UK), as well as CDC45 (Bioss Antibodies, Woburn, MA, USA). Flow cytometry analysis was conducted, and data were processed using FlowJo 10.8.1 software. 

CD4⁺ T-Cell Adoptive Transfer Experiment

LN cells from EAU mice (day 14) were incubated with IRBP1-20 (20 µg/mL) or IRBP1-20+ITA (6 mM) at 37°C for 72 hours. CD4⁺ T cells were subsequently sorted, purified, and collected for injection into C57BL/6J mice (n = 5 per group) via the tail vein (2 × 10⁷ live cells per mouse). 

CFSE Proliferation Experiment

Mouse CD4⁺ T cells were sorted using a flow cytometer, labeled with 2.5-mM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; BD Biosciences) for 10 minutes with continuous agitation at 37°C, and then stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 beads (Thermo Fisher Scientific). Cells were incubated with ITA (3–9 mM), ML346 (0.1–5 µM), or 116-9e (20–80 µM) for 72 hours, and proliferation was assessed by flow cytometry. Human CD4⁺ T cells were sorted using a flow cytometer, labeled with 2.5-mM CFDA-SE for 10 minutes with continuous agitation at 37°C, and then stimulated with the Gibco CD3/CD28 immunomagnetic beads. Cells were incubated with ITA (3–9 mM) for 72 hours, and proliferation was assessed by flow cytometry. 

Single-Cell RNA-Seq Data Analysis

For scRNA-seq analysis, raw data were processed using the “cellranger count” function of CellRanger (10x Genomics, Pleasanton, CA, USA) and mapped to the mouse reference genome. The outputs of multiple samples were aggregated using “cellranger aggr” and analyzed to generate a quantitative expression matrix. Quality control ensured that mitochondrial transcript percentages were <15%, and identified genes per cell ranged from 200 to 4000. Data were log normalized using the “NormalizeData” function in Seurat, and batch effects were corrected with the Harmony algorithm. Major cell clusters were identified using the “FindClusters” function (res = 0.5), and data were visualized with the Uniform Manifold Approximation and Projection (UMAP) algorithm using the “RunUMAP” function. Marker genes for different clusters were identified using “FindAllMarkers,” and differentially expressed genes (DEGs) were determined using the “FindMarkers” function with Wilcoxon rank-sum tests and Bonferroni correction (|FC| > 0.25 and P_adj < 0.05). 

Pathway Enrichment Analysis and Protein–Protein Interaction Network Construction

DEGs underwent pathway enrichment analysis and protein–protein interaction network construction using the Metascape webtool (www.metascape.org), including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heatmaps highlighting five to 10 uveitis-associated pathways from the top 100 enriched GO pathways for different cell types were generated using the Comprehensive R Archive Network (CRAN) package pheatmap 1.0.12 and the ggplot2 package in R 3.2.1 (R Foundation for Statistical Computing, Vienna, Austria). 

Statistical Analysis

All experiments were conducted in triplicate, and data were analyzed using Prism 9.0.2 (GraphPad, Boston, MA, USA). Statistical significance was determined using unpaired two-tailed Student's t-test, one-way ANOVA, or two-way ANOVA as appropriate, with P ≤ 0.05 considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). 

Results

ITA Alleviated EAU Inflammation Clinically and Histologically

To evaluate the potential therapeutic efficacy of ITA in uveitis, we conducted preliminary experiments using ITA and EAU models. We induced EAU models in C57BL/6J mice by immunizing with IRBP1-20 and administered ITA (50 mg/kg) via intraperitoneal injection every 2 days from day 0 to day 14. Subsequently, we assessed the fundus signs and histopathological severity of both the ITA-untreated mice (Control group) and ITA-treated mice (ITA group), calculating clinical and pathological scores for the fundus. Comparative fundus photography analysis revealed significant retinal choroidopathy, cellular infiltration, and extensive retinal folding in the Control group, symptoms that were ameliorated in mice treated with ITA (Figs. 1A–1D). Their clinical and pathological scores were markedly lower than those of ITA-untreated EAU mice (Figs. 1A–1D), indicating the ability of ITA to alleviate inflammation caused by EAU in terms of clinical symptoms and histology. CD4⁺ T cells play a crucial role in the pathogenesis of uveitis, with increased activity of Teff cells, especially Th1 and Th17, contributing to the pathological progression of uveitis, whereas Treg cells exert an inhibitory effect on Teff cells.¹¹ To evaluate the effects of ITA on EAU, we labeled mouse CD4⁺ T cells using flow cytometry (Supplementary Fig. S4A). In the in vivo experiments, flow cytometry analysis revealed that the proportion of CD4⁺ T cells (Figs. 1E, 1F) in retina (Re), IL-17A+CD4+ T cells (Th17) (Figs. 1K–1N) and IFN-γ+CD4+ T cells (Th1) (Figs. 1G–1J) in LN cells, and SP cells of the ITA group was lower than that in Control group. The proportion of FOXP3+CD4+ T cells (Treg) was higher in ITA-treated mice compared to the Control group (Figs. 1S–1V). Additionally, decreased granulocyte–macrophage colony-stimulating factor (GM-CSF) secreted by Th17 post-ITA intervention indicated reduced Th17 pathogenicity (Figs. 1O–1R; Supplementary Fig. S4J). To investigate the potential therapeutic impact of ITA on persistent inflammation, we initiated ITA treatment on the 7th day of EAU induction in mice (Supplementary Figs. S4B–S4I). Our findings revealed that ITA maintained a stable and prolonged anti-inflammatory effect in EAU mice (Supplementary Figs. S4B–S4I). 

Figure 1.

 

ITA alleviated EAU inflammation clinically and histologically. (A, B) Representative fundus images (A) and clinical scores (B) of eyes from EAU mice (Control group) and ITA-treated mice (ITA group) after immunization at day 14 (n = 5 per group). White arrows mark inflammatory exudation and vascular cuffing. (C, D) The H&E staining images (C) and pathological scores (D) of eyes from EAU mice and ITA-treated mice after immunization at day 14. Black arrows mark infiltration of inflammatory cells and retinal folding. Scale bars: 20 µm. (E–V) Proportions of CD4⁺ T cells in retina (Re) (E, F); CD4+IFN-γ+ cells (Th1) in cervical draining LN cells (G, H) and in SP cells (I, J); CD4+IL-17A+ cells (Th17) in LN cells (K, L) and in SP cells (M, N); CD4+IL-17A+GM-CSF+ cells in LN cells (O, P) and in SP cells (Q, R); and CD4+Foxp3+ cells (Treg) in LN cells (S, T) and in SP cells (U, V) were measured by flow cytometry after immunization at day 14 in EAU mice and ITA-treated mice. Data are shown as mean ± SEM. Significance was evaluated using unpaired two-tailed Student's t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001.

ITA Modulated Th17 and Treg Cell Differentiation and Pathogenicity

ITA can effectively inhibit the proliferation response of CD4⁺ T cells with a half maximal inhibitory concentration (IC₅₀) of 6 mM (Figs. 2A–2F), so we chose this concentration for in vitro culture in cell experiments. We isolated LN cells from EAU mice and incubated them with or without ITA in the presence of IRBP1-20 for 72 hours. After antibody staining and flow cytometry analysis, we found that ITA intervention reduced the proportion of Th1 (Figs. 2G, 2H) and Th17 (Figs. 2I, 2J) cells in LN cells of EAU mice while increasing the proportion of Treg cells (Figs. 2M, 2N). Similarly, the pathogenic ability of Th17 cells to secrete GM-CSF was impaired by ITA (Figs. 2K, 2L). ITA inhibited Th17 cell differentiation and enhanced Treg cell differentiation (Figs. 2O–2R). To further validate the therapeutic effect of ITA on EAU, we conducted adoptive transfer experiments. At day 14 after immune induction of EAU, we isolated CD4⁺ T cells from EAU mice and cultured them with IRBP1-20 or IRBP1-20+ITA for 72 hours. Subsequently, we transferred CD4⁺ T cells treated with or without ITA via tail vein injection into normal C57BL/6J mice. It was observed that ITA treatment significantly attenuated the EAU-inducing function of IRBP1-20 specific CD4⁺ T cells, confirming that ITA exerts a therapeutic effect on EAU by reducing the pathogenicity of CD4⁺ T cells (Figs. 2S–2V). 

Figure 2.

 

ITA modulated Th17 and Treg cell differentiation and pathogenicity. (A, B) The proliferation rates of CD4⁺ T cells treated by escalating doses of ITA (0, 3, 6, and 9 mM) for 72 hours were measured by flow cytometry. (C–N) LN cells from EAU mice were cultured with IRBP1-20 or IRBP1-20+ITA for 72 hours. The proportions of CD3⁺ (C, D); CD4⁺ (E, F); CD4⁺IFN-γ⁺ (G, H); CD4⁺IL-17A⁺ (I, J); and CD4⁺Foxp3⁺ (M, N) cells on the gate of CD4⁺ cells and CD4+IL-17A+GM-CSF+ (K, L) on the gate of CD4+IL-17A+ cells were measured by flow cytometry. (O–R) Representative flow plots and cumulative data regarding the differentiation of murine naïve CD4⁺ T cells activated under Th17 and Treg conditions in the presence or absence of ITA (6 mM) after 3-day cultures. (S, T) Representative fundus images and clinical scores of eyes from mice injected with IRBP1-20–specific CD4⁺ T cells cultured with IRBP1-20 (ITA-AT) or IRBP1-20+ITA (ITA+AT). White arrows mark inflammatory exudation. (U, V) The H&E staining plots and pathological scores of eyes from mice injected with IRBP1-20–specific CD4⁺ T cells cultured with IRBP1-20 or IRBP1-20+ITA. Black arrows mark retinal folding and inflammatory cell infiltration. Scale bars: 20 µm. Data are shown as mean ± SEM (n = 5 per group). Significance was evaluated using unpaired two-tailed Student’s t-test. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001.

EAU-Related Gene Expression Alteration Rescued by ITA Therapy

We performed scRNA-seq analysis on single-cell suspensions of LN cells from healthy control mice (HC group), EAU mice (Control group), and ITA-treated EAU mice (ITA group) (Fig. 3A). Based on their typical lineage markers, these cells were classified into eight cell types: T cells (Cd3e+), B cells (Cd79a+), natural killer cells (NK, Ncr1+), macrophages (Macro, C1qc+), monocytes (Mono, Ccr2+Ifitm3+), neutrophils (Neu, Lcn2+Csf3r+), conventional dendritic cells (cDCs, Fscn1+), and plasmacytoid dendritic cells (pDC, Siglech+) (Fig. 3B, Supplementary Fig. S1A). We compared DEGs between the Control and HC groups (referred to as EAU-DEGs) and DEGs between the ITA and Control groups (referred to as ITA-DEGs). From volcano plots, it was evident that DEGs related to transcriptional activation and migration (Fos/Fosb, S100a, and Bhlhe40) were upregulated in the Control group but downregulated after ITA intervention (Fig. 3D). It was observed that ITA significantly altered the proportions of various cell subtypes, with the most affected being T cells and B cells (Fig. 3E). In order to further explore the specificity of inflammation characteristics and ITA treatment, we focused on the upregulated EAU-DEGs and downregulated ITA-DEGs. GO analysis revealed that upregulated EAU-DEGs were enriched in pathways related to antigen presentation, lymphocyte activation and proliferation, Th17 cell differentiation, and IL-17 signaling, whereas downregulated ITA-DEGs were abundant in pathways related to Th17 cell differentiation, IL-17 signaling, ROS, protein folding, oxidative stress, the TCA cycle, and respiratory electron transport (Figs. 3F, 3G). This suggests that inflammation induced by EAU stimulates the upregulation of immune functions, particularly associated with significant activation of Th17 cells. Intervention with ITA negatively regulates Th17 cells, suppresses IL-17 secretion, and is closely related to changes in ROS and oxidative stress functions. This finding provides guidance for ITA regulation in disease management. 

Figure 3.

 

EAU-related gene expression alteration was rescued by ITA therapy. (A) Schematic of the experimental design for the scRNA-seq analysis. LN cells were harvested from healthy control (HC), ITA-treated (ITA), and ITA-untreated (Control) EAU mice and were subject to scRNA-seq. The HC and Control groups each contained two samples and the ITA group contained one sample. Each sample included three mice. (B) UMAP plot of LN cells from all groups. (C, D) Volcano plots showing upregulated and downregulated DEGs of LN cells in the EAU/HC (C) and the ITA/EAU (D) groups. Orange and green dots indicate upregulated and downregulated DEGs, respectively. (E) Rose diagrams showing the numbers of upregulated and downregulated DEGs of LN cells in the EAU/HC and ITA/EAU groups. (F, G) GO analysis of upregulated EAU/HC DEGs (F) and downregulated ITA/EAU DEGs (G) of LN cells. The p value was calculated using the Benjamini–Hochberg procedure.

Similar situations were also reflected in the subpopulations related to myeloid cells (MCs) (Supplementary Figs. S5A–S5I). ITA intervention modulated MCs by downregulating genes linked to innate immunity, antigen processing and presentation, response to biotic stimulus, oxidative phosphorylation, and TNF signaling (Supplementary Figs. S5A–S5C). It also affected interferon signaling and lymphocyte activation in cDCs and electron transport chain and cellular respiration in macrophages, suggesting a broad anti-inflammatory effect and potential antioxidant properties of ITA (Supplementary Figs. S5D–S5I). 

HSPs as Core Targets for ITA Treatment of EAU

We compared the downregulated ITA-DEGs with the upregulated EAU-DEGs, as well as the upregulated ITA-DEGs with the downregulated EAU-DEGs, referred to as down-rescued DEGs and up-rescued DEGs, respectively (Supplementary Figs. S2A, S2B). We also studied the biological effects of these rescued DEGs through GO and pathway enrichment analysis. Our results indicate that, in addition to exerting their effects by inhibiting Th17-related functions, the 73 downregulated rescue differential genes were mainly enriched in pathways related to protein folding and cellular response to ROS. On the other hand, the 87 upregulated rescue differential genes were significantly associated with the negative regulation of programmed cell death 1 (PD-1) and lymphocyte function, suggesting that our ITA treatment intervened in the oxidative stress function of cells (Supplementary Figs. S2C, S2D). Using UpSet plots, we demonstrated specific down-rescued DEGs in cell subtypes to further identify the potential immunosuppressive mechanism of ITA against EAU (Supplementary Figs. S2E, S2F). With regard to T cells and B cells, multiple DEGs associated with the HSP family were observed to be downregulated by ITA, in addition to genes associated with inflammation (Supplementary Fig. S2G). In T cells and B cells, GO analysis of immune cells from different cell types also revealed downregulated rescue DEGs enrichment in pathways associated with protein folding, Th17 cell differentiation, and IL-17 signaling (Supplementary Fig. S2H). 

Due to the significant role of T cells in the pathogenesis of uveitis, we further classified T cells into eight subgroups based on classical gene markers: naive CD4⁺ (NCD4) T cells (Cd4+Ccr7+), naïve CD8+ (NCD8) T cells (Cd8a+Igfbp4+), cytotoxic T cells (CTLs, Ccl5+Ctla2a+), T follicular helper (Tfh) cells (CD4+Cxcr5+Bcl6+), Treg cells (CD4+Foxp3+Il2ra+Mki67–), Th17 cells (CD4+Il17a+Cxcr3–), Th1 cells (CD4+Cxcr3+Bcl6–Mki67–), and proliferative T cells (Stmn1+Mki67+) (Figs. 4A, 4B; Supplementary Fig. S1B). Further analysis indicates that in the T-cell subgroups, Th17, Th1, and proliferative T cells dominated in EAU, whereas ITA intervention reversed the cell proportions, thus restoring immune balance (Figs. 4C, 4D). We generated a multiomics volcano plot, focusing on the characteristic changes of upregulated EAU-DEGs and downregulated ITA-DEGs in different T-cell subgroups (Fig. 4E). It can be observed that, in addition to genes associated with inflammation activation being upregulated in various T-cell subclusters in EAU and downregulated after ITA treatment, this trend was also widely distributed in genes related to the HSP family, mitochondrial electron transport chain, and ubiquitination. This finding further indicates that ITA intervention extensively alters the HSP-related mitochondrial redox mechanism in T cells (Fig. 4E). In the rescued DEGs and rescue ratio, we can also observe the importance of naïve T cells, especially NCD4 (Figs. 4F, 4G). In the GO and KEGG analysis of T cells, the rescued DEGs were enriched in pathways related to ROS, T-cell activation, and Th17 cell differentiation (Fig. 4H). Our analysis highlights the important role of heat shock response (HSR)-associated oxidative stress pathways in EAU inflammation and ITA treatment. 

Figure 4.

 

HSPs as core targets for ITA treatment of EAU. (A) UMAP plot of T-cell subsets from all mice groups. (B) Heatmap showing scaled expression of discriminative gene sets for T-cell subsets. (C) Line charts contrasted the T-cell subcluster proportions among three groups derived from the scRNA-seq data. (D) Bar chart shows the relative proportion of each T-cell subset in the three groups. (E) Multiomics volcano plot showing upregulated EAU-DEGs and downregulated ITA-DEGs in different T-cell subgroups. Orange and green dots indicate upregulated EAU-DEGs and downregulated ITA-DEGs, respectively. (F) The rose diagram shows the number of down-rescued and up-rescued DEGs in the T-cell subtypes. (G) The pod map shows the proportion of rescued DEGs to EAU-DEGs in T-cell subsets. (H) GO and KEGG analysis of down-rescued DEGs in T cells after ITA intervention. The p value was calculated using the Benjamini–Hochberg procedure.

ITA Attenuated EAU and Th17 Cell Differentiation Through the ROS/HSP Axis

We generated a Venn diagram to explore the changes of downregulated rescued-DEGs among the eight T-cell subgroups and found that the HSP family was significantly downregulated in most naïve T-cell subgroups, especially in NCD4 (Fig. 5A). Protein–protein interactions (PPI) can reveal a complex network of functional relationships between biological molecules. By applying the molecular complex detection (MCODE) method, potential protein complexes can be calculated from the PPI network, and it labels entity groups interacting in specific modes.²³ Therefore, we conducted PPI analysis on T-cell subgroups, and the results indicated that the most enriched MCODE in T-cell subgroups was most relevant to protein complexes formed by the HSP family (Fig. 5B). These results suggest the key role of ROS and HSPs in the development of uveitis and the negative regulation by ITA. HSPs may serve as a key target for reversing inflammation and treating uveitis. Therefore, we first used flow cytometry to analyze the expression of ROS in CD4⁺ T cells of EAU after ITA intervention. Our results show that ITA significantly reduced the expression of ROS in CD4⁺ T cells of EAU in both in vivo and in vitro experiments (Figs. 5C, 5D). To further investigate the role of HSP in the pathogenesis of uveitis, we established EAU models as the Control group (n = 5), ITA-treated EAU mice (50 mg/kg) every two days as the ITA group (n = 5), and ITA (50 mg/kg) and HSP agonist ML346 (10 mg/kg) intervention as the ITA+ML346 group (n = 5). After ML346 intervention, we observed no significant improvement in retinal vascular leakage and retinal folding in fundus photographs of mice (Figs. 5E, 5F), and histological sections also showed tissue damage (Figs. 5G, 5H); meanwhile, flow cytometry analysis showed that ITA intervention reduced the expression of IFN-γ (Figs. 5I–5L) and IL-17A (Figs. 5M–5P) compared to the Control group and increased the expression of FOXP3 (Figs. 5Q–5T). Furthermore, when cells were disposed by ML346 on the basis of ITA intervention, we found a re-emergence of Teff/Treg cell imbalance. These findings indicate that the HSP agonist reversed the therapeutic effect of ITA on EAU mice, leading to a Teff/Treg cell imbalance in EAU mice. ITA alleviates EAU symptoms by inhibiting the expression of the ROS/HSP axis, thus reducing the expression of Teff cell inflammatory factors to reverse immune imbalance, indicating that HSPs may offer potential for the treatment of uveitis. 

Figure 5.

 

ITA attenuated EAU and Th17 cell differentiation through the ROS/HSP axis. (A) The Venn diagram shows the number of down-rescued DEGs among the eight T-cell subgroups. (B) PPI analysis of T-cell subsets by using MCODE. (C) Flow cytometry plots of ROS expression in CD4⁺ T cells from LN and SP cells of EAU mice with or without ITA treatment in experiment in vivo. (D) Flow cytometry plots of the expression of ROS in IRBP1-20–specific CD4⁺ T cells with or without ITA intervention. (E, F) Representative fundus images and clinical scores of eyes from EAU mice, ITA-treated mice, and mice treated with ITA+ML346 after immunization at day 14 (n = 5 per group). White arrows mark inflammatory exudation and vascular cuffing. (G, H) Day 14 H&E staining plots and pathological scores of eyes from EAU mice, ITA-treated mice, and mice treated with ITA+ML346 after immunization. Black arrows mark infiltration of inflammatory cells and retinal folding. Scale bars: 20 µm. (I–T) In the in vivo experiments among EAU mice (Control), ITA-treated mice (ITA), and mice treated with ITA+ML346, flow cytometry measured the proportions of Th1 cells in LN cells (I, J) and SP cells (K, L), Th17 cells in LN cells (M, N) and SP cells (O, P), and Treg cells in LN cells (Q, R) and SP cells (S, T). Data are shown as mean ± SEM. Significance was evaluated using one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001.

DNAJA1 As the Most Core Factor for ITA Intervention

The dosage for the ML346 in vitro experiments was screened through proliferation experiments (Supplementary Figs. S3A, S3B). In vitro cell experiments showed that ML346 significantly interfered with the therapeutic effect of ITA (Figs. 6A–6F). This observation further underscored the key role of HSPs in ITA treatment of EAU. However, HSPs are a large family. In order to further explore the heterogeneity of HSP-related EAU inflammation, we used PPI analysis to identify the top five key HSPs after ITA intervention in T cells—namely, DNAJA1, HSPA1A (HSP70), HSPE1 (HSP10), HSP90, and HSPH1 (HSP105) (Fig. 5B). The flow cytometry results showed that ITA intervention significantly inhibited the expression of DNAJA1 in CD4⁺ T cells specifically induced by IRBP1-20 (Figs. 6G, 6H), whereas the expression of the other HSPs did not show significant changes (Supplementary Figs. S3C–S3J). 

Figure 6.

 

DNAJA1 as the most core factor to ITA intervention. (A–F) LN cells from EAU mice were cultured under the intervention of IRBP1-20, IRBP1-20+ITA, or IRBP1-20+ITA+ML-346 for 72 hours. Proportions of Th17 (A, B), Th1 (C, D), and Treg (E, F) cells were measured using flow cytometry. (G, H) The expression of DNAJA1 was measured by flow cytometry in LN cells of EAU mice cultured with IRBP1-20 with or without ITA for 72 hours. (I) Heatmap shows the average expression of DNAJA1 of different T-cell subclusters among the healthy control (HC), EAU control (Control), and ITA-treated (ITA) groups. Data are shown as mean ± SEM. Significance was evaluated using one-way ANOVA. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Therefore, we analyzed the differences, at the average expression levels of DNAJA1, in T-cell subgroups in the single-cell data among the HC, Control, and ITA groups (Fig. 6I). We found that the expression of DNAJA1 was lower in all subclusters in the HC group, significantly increased in the Control group, and decreased in the ITA group, showing a significant reversal of ITA intervention (Fig. 6I). Especially in the Th17 and Th1 cell subgroups, the trend of increased expression of DNAJA1 in EAU and its reversal under the ITA intervention were particularly significant (Fig. 6I). This suggests that DNAJA1 may play a dominant role in the pathogenesis of uveitis. 

ITA Inhibited the Proliferation of Teff Cells in EAU by Suppressing the Cell Cycle Via the DNAJA1/CDC45 Axis

DNAJA1 is a member of the DNAJ/HSP40 family of proteins, playing a critical role in protein translation, folding, unfolding, translocation, and degradation.²⁴ Studies have shown that DNAJA1 can promote the cell cycle by stabilizing CDC45, thereby promoting proliferation of colorectal cancer cells, tumor growth, and metastasis.²⁵ Our findings indicate that ITA intervention can suppress the expression of DNAJA in Teff cells of EAU, reducing the proportion of Teff cells. Therefore, we hypothesize that ITA inhibits the cell cycle of Teff cells by suppressing DNAJA1/CDC45, thus reducing the proportion of pathogenic CD4⁺ T cells and thereby alleviating the inflammatory immune state. To validate this hypothesis, we isolated LN cells from EAU mice with ITA and/or DNAJA1 inhibitor 116-9e in the presence of IRBP1-20 for 72 hours. Because Teff cells mainly consist of Th17 and Th1 cells, we used flow cytometry to analyze the expression of DNAJA1 and CDC45 in Th17 and Th1 cells. The results showed that IRBP-specific induction led to a significant increase in the expression of DNAJA1 (Figs. 7A–7D) and CDC45 (Figs. 7E–7H) in Th17 and Th1 cells, which were significantly reduced under ITA treatment (Figs. 7A–7H; Supplementary Fig. S3K). We further used the DNAJA1 inhibitor 116-9e to investigate whether intervening with DNAJA1 had a statistically significant impact on Teff cells and the expression of CDC45. Proliferation experiments revealed that 116-9e effectively inhibited the proliferation response of CD4⁺ T cells at an IC₅₀ of 40 µM (Figs. 7I, 7J). Therefore, we chose this concentration for in vitro culture (Figs. 7I, 7J). Under the intervention of 116-9e, the expression of IFN-γ (Figs. 7K, 7L) and IL-17A (Figs. 7M, 7N) in CD4⁺ T cells decreased significantly, and the proportion of Treg cells increased (Figs. 7O, 7P). In Th17 and Th1 cells, the expression of CDC45 also decreased significantly under the ministration of 116-9e (Figs. 7Q–7T), which indicates that inhibiting DNAJA1 indeed suppressed the expression of CDC45 in Teff cells. We then investigated the effects of ITA and DNAJA1 on CDC45 and their impact on the cell cycle of CD4⁺ T cells. Flow cytometry results confirmed our expectations, showing that both ITA and 116-9e interventions led to cell cycle arrest in the S phase of CD4⁺ T cells, with a noteworthy increase in the proportion of cells in the G0/G1 phase (Figs. 7U, 7V). Above all, our experiments indicate that ITA treatment can reduce the expression of DNAJA1 and CDC45 in Teff cells, and DNAJA1 inhibitors can inhibit the expression of CDC45 in Teff cells. Interventions with ITA or DNAJA1 inhibitors both lead to CD4⁺ T-cell cycle arrest. This clearly demonstrates that ITA blocks the Teff cell cycle and reduces its pathogenicity by inhibiting the DNAJA1/CDC45 pathway, thereby achieving the therapeutic goal of treating EAU. 

Figure 7.

 

ITA inhibited the proliferation of Teff cells in EAU by suppressing the cell cycle via the DNAJA1/CDC45 axis. (A–D) The percentages of DNAJA1 gated on Th1 (A, B) and Th17 (C, D) cells. (E–H) The proportion of CDC45 gated on Th1 cells (E, F) and Th17 (G, H) cells. (I–J) Proliferation rate of CD4⁺ T cells treated by escalating doses of DNAJA1 inhibitor 116-9e (20, 40, 80 µM). (K–P) The expression of IFN-γ (K, L), IL-17A (M, N), and Foxp3 (O, P) in CD4⁺ T cells with or without the administration of 116-9e. (Q–T) The percentages of CDC45 gated on Th1 (Q, R) and Th17 (S, T) cells before and after 116-9e intervention. (U, V) Percentages of IRBP1-20–specific CD4⁺ T cells in G0/G1, S, and G2/M phase with ITA or 116-9e intervention. Data are shown as mean ± SEM. Significance was evaluated using one-way ANOVA. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Restraint of Activated CD4⁺ T-Cell Function in VKH Patients by ITA

The above results are limited to animal models of EAU and mouse cell experiments. To further validate the role of ITA in human uveitis, we conducted in vitro experiments using cells from patients with uveitis. VKH is one of the most prevalent forms of uveitis in Asia, distinguished by bilateral ocular inflammation and often accompanied with manifestations in the nervous system (meninges), hearing, and skin.²¹ We isolated PBMCs from venous blood samples of VKH patients during the clinical attack period. ITA effectively inhibited the proliferation response of CD4⁺ T cells in PBMCs at an IC₅₀ of 6 mM (Figs. 8A, 8B), so we chose this dose for subsequent experiments. PBMCs were cultured for 72 hours under CD3/CD28 stimulation with or without ITA intervention and were analyzed using flow cytometry. The results showed that ITA effectively reduced the proportion of Th1 cells (Figs. 8C, 8D) and Th17 cells (Figs. 8E, 8F) induced by CD3/CD28 while increasing the proportion of Treg cells (Figs. 8G, 8H). This finding indicates that ITA can also play an important role in regulating the Teff/Treg cell balance in PBMCs of VKH patients. Meanwhile, we also used cell cycle dye to label CD4⁺ T cells and applied flow cytometry to analyze the impact of ITA on the cell cycle of PBMCs. We found that ITA can block the division cycle of CD4⁺ T cells, leading to an increase in the proportion of cells staying in the G₀/G₁ phase (Figs. 8I, 8J). This also indicates that ITA has the same inhibitory effect on the target molecules in the pathway and cell cycle of Teff cells, providing further validation of the function of the DNAJA1/CDC45 pathway in uveitis patients. 

Figure 8.

 

ITA restraint of activated CD4⁺ T-cell function in VKH patients. (A, B) Proliferation rate of CD4⁺ T cells from PBMCs of VKH patients treated with escalating doses of ITA (3, 6, 9 mM) for 72 hours. Proliferation rates were measured by flow cytometry. (C–H) PBMCs from VKH patients were cultured with CD3/CD28 with or without ITA for 72 hours. The proportions of CD4⁺ T cells expressing IFN-γ (C, D), IL-17A (E, F), and Foxp3⁺ (G, H) were measured by flow cytometry. (I, J) Percentages of CD4⁺ T cells from VKH patients' PBMCs in G0/G1, S and G2/M phase under the intervention of CD3/28 without or with ITA. Data are shown as mean ± SEM. Significance was determined using two-way ANOVA. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

This study, based on scRNA-seq data, revealed the impact of ITA on the immune characteristics of EAU at the gene and cellular levels, providing valuable insights into the effective anti-inflammatory action of ITA. ITA decreased ocular inflammation in EAU mice by reducing the differentiation of pathogenic Th17 cells, ROS production, and HSR-related oxidative stress pathways. The inhibition of DNAJA1 is expected to regulate the Th17/Treg cell imbalance. This analysis also shed light on the molecular mechanisms of uveitis and identified potential therapeutic targets, offering ideas for improving uveitis treatment strategies. 

The TCA cycle is a central pathway required for aerobic cellular energy metabolism. In addition to regulating energy and substance metabolism, its intermediate metabolites play a role in controlling chromatin modification, DNA methylation, hypoxia response, and signaling molecules of immune function.²⁶ ITA is one of the intermediate metabolites of the TCA cycle, with direct antimicrobial and antiviral effects and strong immunomodulatory properties. Our research demonstrated that ITA could reduce retinal inflammation and pathological scores in EAU mice. Furthermore, ITA inhibited Th17 cell differentiation and enhanced Treg cell differentiation. The transfer of IRBP-specific pathogenic CD4⁺ T cells to normal mice after ITA intervention did not induce disease. The scRNA-seq analysis revealed that ITA downregulated the expression of proinflammatory genes and reduced the enrichment and differentiation of Th17-related pathways and IL-17 signaling pathways. In myeloid cell subsets, our data indicated that ITA extensively downregulated the expression of inflammatory cytokines associated with leukocyte migration and adhesion and suppressed the enrichment of proinflammatory signaling pathways, including TNF and IFN. Particularly within the cDC and macrophage subsets, ITA inhibited the expression of key enzymes such as p38 mitogen-activated protein kinases (MAPKs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA), thereby modulating the expression of intracellular metabolic molecules and exerting anti-inflammatory effects, an observation that is congruent with the findings of prior studies.^19,27 Our cell experiments showed that ITA regulated the Teff/Treg cell imbalance by inhibiting Th17 cell differentiation and increasing the Treg cell differentiation rate. This regulation is central to the pathogenesis of uveitis, and this finding suggests the therapeutic potential of ITA in treating autoimmune diseases such as uveitis. 

HSPs constitute a large family of proteins that are key regulators of protein homeostasis in eukaryotic cells under physiological and stress conditions such as heat shock and ischemia.²⁸ Due to the involvement of hundreds of protein substrates, the HSP family participates in many cellular processes beyond maintaining proper protein folding,²⁹ including DNA repair and cell development, as well as various diseases such as autoimmune and neurodegenerative diseases.³⁰ The heat shock response is one of the important pathways in oxidative stress. Under stress conditions, there is an increase in ROS alertness, accompanied by mitochondrial and tissue damage, misfolded proteins, and high expression of HSPs. In our study, scRNA-seq analysis showed that ITA reduced gene enrichment in pathways related to ROS, protein folding, and oxidative stress. The MCODE among T-cell subgroups is most correlated with the protein complexes formed by the HSP family. Intervening with ITA in EAU mice could alleviate retinal inflammation, and co-treatment with the HSP agonist could lead to sustained inflammation, reversing the therapeutic effects of ITA. Flow cytometry demonstrated that ITA reduced the expression levels of ROS and HSPs in CD4⁺ T cells, with the most significant change occurring in DNAJA1 protein in HSPs. The DNAJA1 proteins belong to the DNAJ/HSP40 protein family³¹ and have been implicated in various human diseases, such as neurodegenerative diseases.³² However, the regulatory mechanisms of DNAJ/HSP40 protein activity are poorly understood, so further investigation is needed. Our study validated the suggestion that ITA intervention reduces the expression of DNAJA1 and CDC45 in Teff cells and the intervention of DNAJA1 inhibitor 116-9e decreases the protein levels of CDC45. Additionally, ITA could block the cell cycle of pathogenic CD4⁺ T cells, thus suggesting that ITA inhibits the proliferation of pathogenic CD4⁺ T cells by suppressing the expression of DNAJA1/CDC45 in Teff. 

The disruption of redox homeostasis results from an imbalance between the production of ROS and the corresponding antioxidant defenses. Antioxidation, such as scavenging ROS, has become a new approach for treating diseases and reshaping cellular homeostasis. However, due to the complexity of the biological redox mechanisms, our understanding of oxidative stress and its regulatory mechanisms remains limited. The theoretical and practical application of supplementing oxidative scavengers to prevent or reverse pathological changes related to oxidative stress is not always successful, and there is still some controversy. Therefore, our study validating the inhibition of ROS and the HSR pathway by ITA cannot be considered comprehensive, as it lacks direct evidence of interactions, and most experiments are based on animal models. The cell experiments on uveitis patients focused on VKH syndrome, which is common among Asians. Given the complex etiology, diverse types, and significant heterogeneity of uveitis, our findings may not be universally applicable to all forms of the disease. 

Conclusions

Our study provides a comprehensive overview of the impact of ITA treatment on immune cells and presents new discoveries regarding the specific mechanisms of ITA therapy for EAU. With regard to the pathogenesis mechanisms of uveitis, we confirmed a role of the HSR, an oxidative stress pathway, in immune inflammation. ITA significantly alleviated uveitis inflammation by inhibiting ROS expression and the differentiation of Th17 cells, enhancing the differentiation of Treg cells. However, stimulating HSP expression reversed the therapeutic effects of ITA, leading to unresolved inflammation. Further research has identified the role of DNAJA1, a key molecule within the extensive HSP family, which may affect the proliferation of pathogenic CD4⁺ T cells by influencing CDC45 and thus rescuing the imbalance of Teff/Treg cells and alleviating uveitis. DNAJA1 may be a potential key pathogenic factor in the onset and progression of uveitis. Our findings expand our understanding of the regulatory mechanisms of ITA and the pathophysiology of uveitis, with DNAJA1 potentially emerging as a promising therapeutic target, and it has opened up new avenues for the treatment of uveitis. 

Acknowledgments

Supported by grants from the National Outstanding Youth Science Fund Project of China (82122016); National Natural Science Foundation of China (823B2019, 82401239); The Science and Technology Foundation of Health Commission of Guizhou Province (gzwjkj2017-1-043); Guizhou Provincial Science and Technology Project (Qiankehe Fundamental Research-ZK [2023] General 396), and The Science and Technology Commission of Shanghai (17DZ2260100). 

Disclosure: Q. Jiang, None; Z. Li, None; Y. Huang, None; Z. Huang, None; J. Chen, None; X. Liu, None; C. Zhang, None; C. Gu, None; T. Wang, None; H. Li, None; Y. Li, None; W. Su, None