YDJC is downregulated in inflamed intestinal mucosa and CD4+ T cells from IBD patients
To clarify the tissue-specific expression pattern of YDJC, we first utilized a publicly available dataset from the Genotype-Tissue Expression (GTEx) Project (https://www.gtexportal.org) and found that YDJC expression was relatively high in human Epstein-Barr virus (EBV)-transformed lymphocytes, as well as in the spleen, small intestine, and colon (Supplementary Fig. 1A). In mice, Ydjc was predominantly expressed in intestinal tissues (including the jejunum, duodenum, and ileum) and lymphoid tissues (spleen and MLN) (Supplementary Fig. 1B). Next, to explore the involvement of YDJC in the pathogenesis of IBD, we analyzed its expression in the intestinal mucosa of IBD patients. We observed that YDJC mRNA expression was lower in the inflamed mucosa of active IBD patients than in that of healthy controls (HCs) and IBD patients in remission, with no significant differences between HCs and IBD patients in remission (Fig. 1A). Furthermore, YDJC expression was significantly lower in inflamed mucosa than in uninflamed mucosa from the same IBD patients (Fig. 1B). Consistent with these findings, the microarray data from the GEO database (GSE73094) also revealed a decrease in YDJC expression in inflamed IBD colon tissues compared with HCs and uninflamed tissues (Fig. 1C). Additionally, immunohistochemistry (IHC) and immunofluorescence analyses further confirmed the downregulation of YDJC protein levels in the intestinal mucosa of IBD patients (Fig. 1D, E).
Next, we aimed to elucidate the relationship between YDJC expression and the clinical disease characteristics of IBD patients. Correlation analysis revealed that YDJC mRNA expression in the intestinal mucosa was inversely correlated with the Crohn's Disease Activity Index (CDAI) and the Simple Endoscopic Score for Crohn's Disease (SES-CD) in CD patients (Fig. 1F), as well as with the Mayo index and the Ulcerative Colitis Endoscopic Index of Severity (UCEIS) in UC patients (Fig. 1G). Furthermore, YDJC expression was inversely correlated with clinical indicators such as C-reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) (Fig. 1F, G). These results suggest a potential link between YDJC expression and the disease activity of IBD.
To identify the expression pattern across different immune cells through which YDJC influences the perpetuation of IBD, we analyzed YDJC expression across various immune cell types within the intestinal mucosa (Fig. 1H) and peripheral blood (PB) (Fig. 1I) of HCs, which presented relatively high levels of YDJC expression in CD4 T cells. Additionally, single-cell RNA sequencing (scRNA-seq) data from publicly available datasets (GSE134809, SCP259) revealed high levels of YDJC expression in T cells, B cells, and myeloid cells from the intestinal mucosa of IBD patients (Supplementary Fig. 1C). To investigate the immunoregulatory effects of YDJC expression on immune cells, we isolated CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and neutrophils from the peripheral blood of HCs and assessed YDJC expression following activation. Notably, YDJC expression was significantly downregulated in CD4⁺ and CD8⁺ T cells following in vitro activation, whereas no appreciable changes were observed in CD19⁺ B cells or neutrophils (Supplementary Fig. 1D).
Given the pivotal role of CD4 T cells in the pathogenesis of IBD, we focused our subsequent study on CD4 T cells. We noted a significant decrease in the expression of YDJC in CD4 T cells from IBD patients, as observed in our experiments and in publicly available microarray data (Fig. 1J, Supplementary Fig. 1E). scRNA-seq data further revealed significant and consistent downregulation of YDJC in CD4 T cells from the inflamed mucosa of IBD patients compared with those from the uninflamed mucosa and normal mucosa of HCs (Fig. 1K, L). Immunofluorescence analysis further validated these findings, revealing a significant reduction in YDJC expression in the CD4 T cells of IBD patients (Fig. 1M). Moreover, a significant decrease in YDJC expression was also observed in the colons and splenic CD4 T cells of dextran sulfate sodium (DSS)-treated wild-type (WT) mice compared with those of the control groups (Supplementary Fig. 1F, G). Analysis of microarray data from the intestinal mucosa revealed a significant negative correlation between the expression levels of YDJC and major proinflammatory cytokines (e.g., IFNG and TNF) involved in the pathogenesis of IBD (Supplementary Fig. 1H). Collectively, these findings underscore a significant association of YDJC expression with the pathogenesis of IBD.
Proinflammatory cytokines are known to contribute to the perpetuation and deterioration of IBD [16]. To investigate whether these cytokines can modulate YDJC expression, we isolated CD4 T cells from the PB of HCs and cultured them with various cytokines in vitro for 3 days. The results demonstrated that IL-1β significantly downregulated YDJC expression (Fig. 2A) and that its expression was markedly decreased in PB CD4 T cells under Th1-polarizing conditions but remained comparable in Th0, Th2, Th17, and Treg cells (Fig. 2B). To further corroborate these findings in mice, CD4⁺ T cells were isolated from the spleens of WT mice and activated with anti-CD3 and anti-CD28 mAbs in vitro. Consistently, Ydjc expression was significantly downregulated following T-cell activation (Supplementary Fig. 2A). Notably, stimulation with specific cytokines and inflammatory stimuli, including IL-12, IL-1β, and PMA, further suppressed Ydjc expression in CD4⁺ T cells (Supplementary Fig. 2B). These findings suggest that TCR and proinflammatory signaling lead to the downregulation of YDJC in CD4⁺ T cells, indicating a potential role of YDJC in modulating the T-cell immune response during IBD.
Next, we examined the influence of YDJC on CD4 T-cell differentiation. PB CD4 T cells from HCs and IBD patients were transfected with YDJC-expressing lentivirus (LV-YDJC) or a negative control (LV-NC) as indicated (Fig. 2C). YDJC expression was significantly greater in LV-YDJC-transfected CD4 T cells than in control T cells, indicating high transfection efficacy (Fig. 2D). We then assessed cytokine and transcription factor expression in YDJC-transfected CD4 T cells. As expected, there was a significant reduction in Th1-related cytokines (i.e., IFNG and TNF) and the transcription factor TBX21 in YDJC-transfected CD4 T cells, whereas other Th cell-related cytokines (e.g., IL4, IL17A, and IL10) and transcription factors (e.g., GATA3, RORC, and FOXP3) were unaffected (Fig. 2D). Flow cytometry further demonstrated that YDJC-transfected CD4 T cells exhibited a diminished capacity to differentiate into IFN-γ Th1 cells (Fig. 2E). Collectively, these data suggest that the overexpression of YDJC in CD4 T cells could attenuate the Th1-associated immune response in IBD patients, highlighting its potential role in modulating CD4 T-cell function and participating in the development of intestinal inflammation.
To better understand the role of YDJC in regulating CD4 T-cell function, we generated Ydjc mice via the CRISPR/Cas9 technique (Supplementary Fig. 3A), leveraging the high genetic homology between mice and humans (Supplementary Fig. 3B). The absence of YDJC in the colon tissue and CD4 T cells of Ydjc mice was confirmed through Western blotting and qRT‒PCR (Supplementary Fig. 3C-E). Ydjc mice presented no abnormalities in thymocyte development (Supplementary Fig. 3F), with comparable percentages of CD4 T cells in the spleen and mesenteric lymph nodes (MLNs). However, the frequency of CD4 T cells in lamina propria mononuclear cells (LPMCs) was greater in Ydjc mice than in their WT littermates, and no changes in B cells or CD8 T cells were detected (Supplementary Fig. 3G). These results suggest that YDJC may play a crucial role in regulating the intestinal immune balance, primarily the function of CD4 T cells.
Given the elevated IFN-γ expression in the colonic tissues of DSS-treated Ydjc mice, together with its established role as a key effector cytokine of the Th1-mediated immune response, we hypothesized that the exacerbated mucosal inflammation was at least partly dependent on Th1 responses driven by IFN-γ. To directly assess the role of IFN-γ in facilitating mucosal inflammation, we administered a neutralizing anti-IFN-γ mAb during DSS treatment. Notably, blockade of IFN-γ significantly alleviated colitis severity in Ydjc mice, as evidenced by reduced weight loss, restored colon length and histopathological scores, and decreased infiltration of CD4⁺ T cells in colon tissues (Supplementary Fig. 6A-F). Collectively, these findings indicate that IFN-γ plays a critical role in promoting the proinflammatory response in the gut mucosa of Ydjc-deficient mice during colitis.
To further clarify the role of Ydjc deficiency in CD4⁺ T cells in the increase in susceptibility to colitis in mice, we established a chronic colitis model via adoptive transfer of CD25CD45RB CD4 T cells from either WT or Ydjc mice into syngeneic Rag1 mice (Fig. 5A, Supplementary Fig. 7). Rag1 mice reconstituted with YdjcCD45RBCD4 T cells presented more severe colitis, as evidenced by greater weight loss, shortened colons, and enlarged spleens, along with higher DAI and histopathological scores (Fig. 5B-G). IHC staining revealed increased accumulation of CD4 T cells and MPO neutrophils in the colons of Rag1 mice transplanted with YdjcCD4 T cells (Fig. 5H). Flow cytometric analysis revealed increased proportions of IFN-γ and TNF-αCD4 T cells in Rag1 recipients reconstituted with YdjcCD45RBCD4 T cells (Fig. 5I, J). Consistently, the mRNA levels of Tbx21, Ifng, and Tnf were elevated in the colonic tissues of these mice, whereas no significant changes were observed in the mRNA levels of Rorc, Il17a, Gata3, Il4, Foxp3, and Il10 (Fig. 5K). Taken together, these results suggest that Ydjc deficiency in CD4 T cells exacerbates intestinal mucosal inflammation by promoting a Th1 cell-driven proinflammatory immune response.
To assess whether Ydjc deficiency in nonhematopoietic compartments is involved in colitis severity, we generated bone marrow chimeric mice by transplanting WT bone marrow cells into lethally irradiated WT and Ydjc recipients (WT → WT and WT→Ydjc). Compared with control recipients, Ydjc recipients presented significantly greater weight loss and higher DAI scores, accompanied by more pronounced colonic shortening, increased histopathological damage, and impaired epithelial barrier integrity, as indicated by diminished E-cadherin staining in the colonic mucosa (Supplementary Fig. 8A-G). These results indicate that Ydjc deficiency in nonhematopoietic cells also contributes to the exacerbation of intestinal inflammation.
To elucidate the mechanism by which YDJC regulates CD4 T-cell function and protects against colitis in mice, we isolated splenic CD4 T cells from Ydjc and WT mice for RNA-seq and proteomics analyses (Fig. 6A). We identified 902 DEGs and 264 DEPs (Supplementary Fig. 9A, C). GO enrichment analysis of the DEGs revealed that the upregulated genes were involved primarily in immune-related processes and lipid metabolism, including sterol biosynthesis, cholesterol biosynthesis, steroid biosynthesis, and fatty acid biosynthesis, most of which are involved in cholesterol biosynthesis (Supplementary Fig. 9B). Enrichment analysis of upregulated DEPs in YdjcCD4 T cells also revealed a dysregulated cholesterol biosynthesis pathway (Supplementary Fig. 9D). Integrative transcriptomic and proteomic analyses revealed enrichment of pathways related to plasma membrane organization, T-cell proliferation, and the cholesterol biosynthetic process (Fig. 6B). GSEA and a heatmap revealed upregulated cholesterol biosynthesis-related genes (e.g., Hmgcs1, Hmgcr, and Cyp51) (Fig. 6C, D). qRT‒PCR further validated the upregulation of key enzymes (Hmgcs1, Hmgcr, and Cyp51) in the cholesterol biosynthesis pathway in CD4 T cells from the spleens and MLNs of Ydjc mice (Supplementary Fig. 9E, F). In addition, heatmap analysis revealed marked upregulation of genes associated with cell chemotaxis and leukocyte migration, including Cxcl9, Cxcl10, Ccr3, Ccl22 and S1pr1, indicating the enhanced migratory potential of YdjcCD4⁺ T cells (Supplementary Fig. 9G).
After deciphering the upregulation of cholesterol metabolism-related genes in YdjcCD4 T cells, we subsequently performed untargeted metabolomic analyses to investigate the changes in the levels of cellular metabolites in these cells. As expected, the majority of differentially abundant metabolites belonged to the lipid superclass (54.93%) (Fig. 6E), with a predominance of glycerophospholipids (51.28%) and sphingolipids (15.38%) (Supplementary Fig. 10A). Elevated levels of glycerophospholipids, sphingolipids, and steroids, which are crucial for maintaining cellular structural integrity and pivotal for cell proliferation and signal transduction, were detected in YdjcCD4 T cells (Supplementary Fig. 10B) [17]. By integrating transcriptomic and metabolic data, we identified cholesterol biosynthesis as the predominant pathway, highlighting the important role of cholesterol metabolism in YDJC-mediated regulation of CD4 T-cell function (Fig. 6F).
To further confirm these findings, we assessed cholesterol levels in splenic CD4 T cells from WT and Ydjc mice via confocal microscopy. Filipin staining revealed a significant increase in both membrane and intracellular cholesterol levels in YdjcCD4 T cells. Additionally, LipidTox and BODIPY C12 staining confirmed elevated levels of neutral lipids, particularly cholesterol esters, in lipid droplets (LDs) (Fig. 6G). These observations were further validated quantitatively by flow cytometry and biochemical analyses, which revealed increased cholesterol in YdjcCD4 T cells (Fig. 6H, Supplementary Fig. 10C). We also detected increased total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) in the serum of Ydjc mice, with no significant changes in triglyceride (TG) or low-density lipoprotein cholesterol (LDL-C) (Supplementary Fig. 10D). Given that the liver is the primary site of cholesterol biosynthesis, we next evaluated the expression of SREBP2 and enzymes involved in cholesterol biosynthesis in the liver. Notably, no significant differences in the expression of Srebf2 or its downstream targets in liver tissue were detected between WT and Ydjc mice (Supplementary Fig. 10E). Consistently, the lipid content in the liver was not altered by Ydjc deficiency, as evidenced by H&E and Oil Red O staining, as well as biochemical analyses (Supplementary Fig. 10F-H). Moreover, Ydjc expression in the liver was relatively low compared with that of other deacetylases (e.g., Sirt1 and Hdac1) (Supplementary Fig. 10I). Collectively, these results indicate that YDJC selectively orchestrates cholesterol biosynthesis in CD4⁺ T cells without affecting cholesterol metabolism in the liver.
To investigate whether YDJC regulates CD4 T-cell function through cholesterol biosynthesis in vitro, we used simvastatin, an HMGCR inhibitor and rate-limiting enzyme of cholesterol biosynthesis, to block cholesterol production (Supplementary Fig. 11). Similarly, simvastatin effectively suppressed the proliferation (Fig. 6I, K) and Th1 cell differentiation of YdjcCD4 T cells (Fig. 6J, L) but did not alter their activation status (Supplementary Fig. 12A-C). These results underscore the pivotal role of the cholesterol biosynthesis pathway in modulating Ydjc-mediated CD4 T-cell function.
Next, we assessed the therapeutic potential of simvastatin in DSS-induced colitis. The mice received oral simvastatin once daily during colitis induction (Fig. 6M). Notably, simvastatin-treated Ydjc mice presented alleviated colitis, characterized by less weight loss, modest colon shortening, and a lower DAI (Fig. 6N-R). Additionally, simvastatin treatment also reduced the infiltration of CD4 T cells and MPO neutrophils in the colon of Ydjc mice (Supplementary Fig. 13), especially IFN-γ and TNF-α CD4 T cells (Fig. 6S). Moreover, simvastatin treatment also alleviated colitis in WT mice, which is consistent with previous studies demonstrating its protective effects [18]. Therefore, these data suggest a potential therapeutic role of simvastatin in protecting against intestinal mucosal inflammation in both Ydjc and WT mice during colitis.
Cellular cholesterol metabolism is regulated primarily by sterol regulatory element-binding protein 2 (SREBP2) and liver X receptor (LXR) [19]. SREBP2 drives cholesterol biosynthesis and uptake, whereas LXR facilitates cholesterol efflux and inhibits cholesterol uptake [20]. We then investigated whether these genes modulate cholesterol metabolism in YdjcCD4 T cells. We found that SREBP2-regulated genes (e.g., Hmgcs1, Cyp51, Hmgcr, and Srebf2) were significantly upregulated in YdjcCD4 T cells, whereas LXR-regulated genes (e.g., Abca1 and Abcg1) were not altered (Fig. 7A). Upstream regulatory analysis of cholesterol biosynthesis-related DEGs revealed that SREBF2, encoding SREBP2, was the most prominent driver (Fig. 7B). Consistently, the upregulation of SREBP2 in YdjcCD4 T cells was also confirmed by qRT‒PCR and flow cytometry (Fig. 7C, D).
To explore whether Ydjc deficiency promotes cholesterol biosynthesis in CD4 T cells through the upregulation of SREBP2, we used fatostatin, an SREBP2 inhibitor, to modulate CD4 T-cell function. As expected, fatostatin effectively suppressed the increased proliferation and Th1 differentiation of YdjcCD4 T cells (Fig. 7E, F). These results indicate that Ydjc deficiency regulates CD4 T-cell function via the upregulation of SREBP2-mediated cholesterol biosynthesis.
We then assessed the therapeutic potential of fatostatin in preventing colitis development by administering fatostatin intraperitoneally to Ydjc mice once daily during colitis induction (Supplementary Fig. 14A). Compared with untreated Ydjc mice, fatostatin-treated Ydjc mice presented alleviated colitis, as evidenced by less weight loss, milder colon shortening, and lower histopathological scores (Supplementary Fig. 14B-F). Furthermore, the infiltration of CD4 T cells and MPO neutrophils was reduced (Supplementary Fig. 14G), especially the percentages of IFN-γ and TNF-α CD4 T cells in the colonic tissues of fatostatin-treated Ydjc mice (Supplementary Fig. 14H). Moreover, the levels of Tbx21, Ifng, and Tnf mRNA expression were also significantly reduced in the colonic mucosa (Supplementary Fig. 14I), indicating a therapeutic benefit of fatostatin in the treatment of colitis in Ydjc mice.
To further explore the therapeutic potential of modulating SREBP2 expression, we assessed the impact of AAV-sh-Srebf2 on colitis development in vivo in Ydjc mice. Following intraperitoneal injection of either AAV-sh-NC or AAV-sh-Srebf2, colitis was induced with DSS after a 14-day interval (Supplementary Fig. 15A). We found that Srebf2 expression was significantly lower in the colon tissues of AAV-sh-Srebf2-treated mice than in those of control mice (Supplementary Fig. 15B). Notably, compared with control mice, AAV-sh-Srebf2-treated mice presented attenuated colitis symptoms, including reduced body weight loss, less colon shortening, and lower histopathological scores (Supplementary Fig. 15C-G). Decreased infiltration of CD4 T cells and MPO neutrophils was also observed in the colon tissues of the AAV-sh-Srebf2-treated mice following DSS administration (Supplementary Fig. 15H). Additionally, the mRNA levels of Tbx21, Tnf, and Ifng were significantly diminished in the colon tissues of AAV-sh-Srebf2-treated mice (Supplementary Fig. 15I). Collectively, these findings suggest that targeting SREBP2 could alleviate DSS-induced colitis in Ydjc mice by suppressing Th1 cell immune responses.
To directly assess whether blockade of SREBP2 could ameliorate intestinal mucosal inflammation in a YdjcCD4⁻ T-cell-driven colitis model, we intravenously injected AAV-sh-NC or AAV-sh-Srebf2 into Ydjc mice. Two weeks later, naive CD4⁺ T cells from the spleens of Ydjc mice were intravenously transferred into Rag1 recipients to induce chronic colitis. Importantly, Rag1 mice receiving Srebf2-deficient YdjcCD4⁺ T cells developed substantially milder colitis than control mice did, as evidenced by reduced body weight loss, increased colon length, decreased CD4⁺ T-cell infiltration, and diminished Th1 responses in the colon (Supplementary Fig. 16A-G). Collectively, these findings underscore the critical role of SREBP2 in the initiation of intestinal mucosal inflammation in Ydjc mice.
Given the critical role of cholesterol metabolism in CD4 T-cell proliferation and differentiation, we next investigated SREBP2 expression in CD4⁺ T cells from IBD patients. We utilized two publicly available scRNA-seq datasets (scp259 and scp1884) and revealed elevated expression of SREBF2 in CD4⁺ T cells from the inflamed mucosa of IBD patients (Supplementary Fig. 17A, B). Consistently, our scRNA-seq data also revealed an upregulation of SREBF2 and its downstream target (HMGCS1, HMGCR) in the mucosal CD4⁺ T cells of IBD patients (Fig. 7G). Immunofluorescence staining further confirmed the increased expression of SREBP2 protein in intestinal mucosal CD4⁺ T cells (Fig. 7H), indicating that SREBP2-mediated cholesterol biosynthesis is increased in CD4 T cells from IBD patients.
The SREBP family, comprising SREBP1a, SREBP1c, and SREBP2, plays a critical role in regulating lipid metabolism. Among them, SREBP2 primarily controls genes related to cholesterol metabolism. SREBP2 is initially synthesized as an inactive precursor (pre-SREBP2) and is cleaved by site-1 protease (S1P) and site-2 protease (S2P) under low-cellular cholesterol conditions. The N-terminal transcriptionally mature fragment of SREBP2 (m-SREBP2) is liberated and translocates into the nucleus, where it binds sterol regulatory elements (SREs) to regulate the expression of genes involved in cholesterol metabolism [21]. The expression of SREBP2 is governed through multiple regulatory mechanisms, including transcriptional control, transport and cleavage processes, and posttranslational modifications such as acetylation, which intricately modulate SREBP2 stability and nuclear localization [22]. SIRT1, an NAD-dependent deacetylase, directly deacetylates SREBP2, resulting in reduced protein stability and consequent inhibition of cholesterol biosynthesis gene transcription [23].
Given the deacetylase activity of YDJC and its link to SREBP2-mediated cholesterol biosynthesis, we hypothesized that YDJC directly deacetylates SREBP2, thereby regulating its downstream target genes. To test this hypothesis, we transfected 293 T cells with LV-NC or LV-YDJC and found that the overexpression of YDJC significantly reduced the mRNA expression of SREBP2 and its target genes (e.g., HMGCS1 and HMGCR) (Fig. 7I). Additionally, YDJC overexpression decreased m-SREBP2 levels without affecting pre-SREBP2 levels (Fig. 7J). As SREBP2 regulates its own transcription, reduced expression of m-SREBP2, especially in the context of YDJC-mediated deacetylation, may account for decreased SREBP2 transcription.
We then conducted protein‒protein docking simulations to substantiate our hypothesis and identified a high-affinity binding mode between YDJC and SREBP2 (Fig. 7K), suggesting a direct interaction. We subsequently performed coimmunoprecipitation (co-IP) assays in 293 T cells transfected with LV-YDJC, further confirming this interaction (Fig. 7L). We determined the acetylation levels of SREBP2 in LV-YDJC- and LV-NC-transfected 293 T cells and found that overexpression of YDJC substantially reduced SREBP2 acetylation (Fig. 7M). In line with these findings, YdjcCD4 T cells presented increased SREBP2 acetylation (Fig. 7N). Taken together, these data indicate that YDJC directly interacts with and deacetylates SREBP2, modulating cholesterol biosynthesis in CD4 T cells.