Collagen-induced arthritis (CIA) model in C57BL/6 (H-2b background) mice provides a valuable model for studying the pathogenesis of rheumatoid arthritis (RA). However, the immune mechanism underlying this model remains unclear. This study aims to comprehensively understand the dynamic multi-dimensional characteristics of CIA, especially the temporal changes in immune cell subsets, providing a basis for future research on immune-targeted therapy for RA. The CIA course was divided into onset, peak, and chronic stages based on arthritis scores. Joint pathology was assessed by histology; immune cell subsets in the spleen and lymph nodes were profiled by flow cytometry; and serum cytokines were measured by ELISA. The results revealed distinct stage-specific immune alterations during CIA progression. M1 polarization occurred at the onset stage. DCs were markedly activated at the peak stage, accompanied by elevated frequencies of Th1, Th17, and Tfh cells, while the Breg cell proportions declined. Moreover, DC activation and abundant Th1 and Tfh cells persisted into the chronic stage. Neutrophils remained elevated throughout disease progression to sustain inflammatory responses. Furthermore, upregulated TNF-α and decreased TGF-β1 represented the core cytokine alterations driving CIA pathogenesis. Collectively, this study provided in-depth insights into dynamic immune shifts during CIA progression and offered valuable clues for developing novel RA immunotherapies.

Wutou decoction (WTD) is a traditional remedy in Chinese medicine for rheumatoid arthritis (RA). However, its active ingredients are complex, and how it counteracts RA is still not clear. The objective of this research was to investigate the key components and targets of WTD action. RA-related genes were analyzed using the GSE55235 dataset. The active components of WTD were analyzed using online databases. Component-target-pathology (CTP) networks were built using the Cytoscape software. The functions and pathways of the key targets were analyzed using GO and KEGG. The component-target interaction was assessed by molecular docking. Moreover, the impact of WTD on RA progression and its mechanism were validated using cell and animal experiments. The results showed that 1361 genes were related to RA, and they were enriched in 2035 biological processes and 97 signaling pathways. The five herbs of WTD had 221 common targets, and each herb had numerous components. A total of 66297 interactions were found among 440 active ingredients and 2638 targets within the WTD in the CTP network. Next, 114 core functional component groups were selected, and the types and quantities of target-enriched signaling pathways they covered were greater than those of RA-related genes alone. In vivo, WTD attenuated RA-induced arthritis and bone destruction. In vitro, benzoic acid, quercetin, apigenin, and decanoic acid inhibited the inflammatory response of macrophages by inactivating the PI3K/ATK and MAPK pathways. In conclusion, WTD exerts a therapeutic effect on RA through multi-component and multi-target, providing novel methods to study the mechanism of WTD.

Managing lupus nephritis (LN) remains challenging due to relapses after immunosuppressive induction and toxicity from long-term glucocorticoid (GC) maintenance therapy. Dietary omega-3 fatty acids prevent LN onset in preclinical models, but their role in maintaining remission post-induction remains unstudied. The silica-accelerated LN (SALN) model using lupus-prone NZBWF1 mice was used to evaluate how docosahexaenoic acid (DHA), an omega-3 fatty acid, influenced LN remission durability after cyclophosphamide (CYC) induction, alone or with a moderate dose of prednisone (PDN). Mice received intranasal silica weekly from 8 to 11 weeks. After LN developed at 21 weeks, groups were injected weekly with CYC (human equivalent dose [HED] = 31 mg/day) or vehicle (VEH) for 8 weeks, during which CYC groups also received control, DHA (HED = 5 g/day), PDN (HED = 9 mg/day), or DHA+PDN diets. Disease activity was monitored via proteinuria, autoantibodies, and survival. Six weeks post-CYC, multiorgan histopathology and immunohistochemistry were assessed. VEH-treated mice developed severe LN with early death. CYC slowed disease temporarily in control- and PDN-fed mice; relapses occurred after cessation. DHA and DHA+PDN increased tissue omega-3 levels and prolonged remission. PDN and DHA monotherapies and co-therapy improved survival, but DHA+PDN was most effective at sustaining remission as reflected by reduced histopathologic markers of lupus severity in the kidney, spleen, lung, and brain. DHA+PDN optimally maintained LN remission after CYC, supporting omega-3 supplementation as a potential GC-sparing strategy to improve immunosuppressive therapy and prevent relapses.

Acute myeloid leukemia (AML) cells rely heavily on mitochondrial oxidative phosphorylation (OXPHOS) for energy, making mitochondrial function a promising therapeutic target. TCF12, a transcription factor belonging to the basic helix-loop-helix (bHLH) family, has been implicated in various cancers and is highly expressed in AML, where it may contribute to disease progression. However, the effects of TCF12 on AML progression and the underlying mechanisms remain unclear. This study aimed to elucidate the role of TCF12 in promoting AML cell survival and uncover the underlying mechanism. We found that TCF12 was highly expressed in AML and was associated with poor patient prognosis. Knockdown of TCF12 significantly inhibited AML cell growth, suppressed glycolysis, increased ROS accumulation, and induced mitochondrial dysfunction. Additionally, TCF12 was shown to promote EZH2 transcription, whereas its knockdown increased TXNIP expression, thereby inhibiting AML progression. In summary, TCF12 promotes AML progression by regulating EZH2-mediated suppression of TXNIP, thereby enhancing glycolysis, maintaining mitochondrial function, regulating ROS levels, and promoting cell survival, which underscores its potential as a therapeutic target.

Emerging evidence links reactive oxygen species modulator 1 (ROMO1), a key mitochondrial ROS regulator, to rheumatoid arthritis (RA) pathogenesis. However, its exact mechanism remains elusive given the conflicting evidence about its specific function. We used a four-level integrative framework combining multi-omics data and literature‑supported mechanistic inference. At the genetic level, Mendelian randomization (MR) was performed to explore potential causal relationships between ROMO1, IL2RA, HLA-DR, MIF, and RA risk, followed by differential expression analysis and machine learning-based feature selection to identify key mROS genes. The temporal expression dynamics of ROMO1 were assessed in RA progression. At the cellular and tissue levels, we integrated single-cell RNA sequencing and spatial transcriptomics to map cell-type-specific expression and synovial localization of ROMO1-related immune cells and pathways. Finally, our multi-omics findings were contextualized with literature-supported mechanistic inference. (1) MR results were consistent with a potential protective effect of ROMO1 on RA (OR = 0.52) and its potential regulation of risk factors IL2RA (OR = 0.46) and HLA-DR (OR = 0.40). Conversely, IL2RA (OR = 1.42), HLA-DR (OR = 1.88), and MIF (OR = 1.17) were positively associated with RA risk. Additionally, ROMO1 was identified as a top candidate diagnostic predictor with stage-specific dynamics: downregulated in the early but upregulated in the late/remission stages. (2) Single-cell RNA sequencing showed ROMO1's cell-specific expression in CD14+ HLA-DR+ CD74+ monocytes and CD4+ IL2RA+ T cells. Cell communication analysis further suggested that these cells may participate in MIF pathway regulation. Spatial transcriptomics subsequently identified that ROMO1-related cells localized to synovial pathological regions, with MIF pathway changes correlated with RA progression. (3) Finally, literature-supported mechanistic inference suggests that ROMO1 may modulate mROS levels to promote anti-inflammatory M2 macrophage polarization, which could theoretically contribute to reduced systemic inflammation and the alleviation of multi-organ decline in RA. This integrated multi-omics investigation, supported by literature-based mechanistic inference, suggests ROMO1 as a stage-dependent biomarker candidate and potential immune regulator in RA.

Characterized by renal inflammation and structural damage, lupus nephritis (LN) is a severe and often debilitating complication of systemic lupus erythematosus. The dynamic regulation of RNA processing, stability, and translation by N6-methyladenosine (m6A) modification has been implicated in the pathophysiology of LN. As an m6A reader, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) plays a crucial role in the pathogenesis of various kidney diseases, yet its precise function in LN remains unclear. IGF2BP2 expression was measured in kidney specimens from LN patients and MRL/LPR mice. MRL/lpr mice were administered IGF2BP2 short hairpin RNA adeno-associated viral vectors, followed by evaluation of proteinuria, renal function, and histology. In vitro experiments employed lipopolysaccharide (LPS) stimulation of human kidney tubular epithelial cells (HK-2) to simulate LN inflammatory responses and further investigated the mechanism of IGF2BP2. Molecular interactions were validated via RNA immunoprecipitation and luciferase reporter assays. IGF2BP2 expression is significantly upregulated in LN and positively correlated with proinflammatory factor levels. Knockdown of IGF2BP2 significantly improved renal injury and alleviated renal inflammatory responses in MRL/LPR mice, as well as reduced LPS-induced inflammatory responses in HK-2 cells. Mechanistically, under LN pathological conditions, IGF2BP2 recognized m6A modifications on signal transducer and activator of transcription 1 (STAT1) mRNA and enhanced its stability, leading to the activation of proinflammatory signaling, exacerbation of renal inflammation, and ultimately renal injury. The m6A reader IGF2BP2 promotes proinflammatory signaling in LN by recognizing m6A-modified STAT1 mRNA. Targeting the IGF2BP2-STAT1 axis may represent a potential therapeutic strategy to ameliorate renal inflammatory responses in LN.

Tc17 cells and Tc17.1 cells contribute synovial inflammation during the development of rheumatoid arthritis (RA). However, the modulatory mechanisms of synovial Tc17 cells and Tc17.1 cells remain elusive. In the current study, we performed transcriptome profiling analysis on sorted live synovial Tc17 cells and Tc17.1 cells from a mouse model of collagen-induced arthritis (CIA). We found an array of differentially expressed genes (DEGs), which might be involved in the metabolism, cytokine signaling, and other biological aspects in synovial Tc17 and Tc17.1 cells. Importantly, we identified zinc finger protein 24 (ZNF24), a transcription factor, among the DEGs. ZNF24 knockdown impaired Tc17 differentiation and function in vitro, as evidenced by lower expression of IL-17A, RORγt, GM-CSF, IL-22, perforin, and granzyme B. ZNF24 knockdown resulted in similar effects on Tc17.1 differentiation and function in vitro. Furthermore, ZNF24 knockdown weakened the effects of Tc17 cells and Tc17.1 cells on inducing pro-inflammatory mediator expression in synovial fibroblasts. Adoptive transfer of Tc17.1 cells significantly exacerbated CIA development, whereas ZNF24-knockdown ameliorated the detrimental effect of Tc17.1 cells. Synovial fibroblasts up-regulated ZNF24 expression in Tc17 and Tc17.1 cells in a direct contact manner. The expression pattern and effect of ZNF24 were also observed in patients with RA and human Tc17 cells and Tc17.1 cells. Taken together, our study suggests that ZNF24 is a positive regulatory factor for Tc17 and Tc17.1 cells to exert pro-inflammatory effects in RA. We thus unveiled a novel mechanism by which the functions of synovial Tc17 cells and Tc17.1 cells are modulated.

Diffuse alveolar hemorrhage (DAH) is a lethal, female-biased complication of systemic lupus erythematosus (SLE). While adaptive immunity is a known driver, the role of innate lymphoid cells (ILCs) in DAH and the contribution of sex hormones to this process remain unclear. To characterize pulmonary ILC remodeling during DAH and evaluate whether androgen signaling modulates natural killer (NK) cell homeostasis and disease severity. A pristane-induced DAH model was established in C57BL/6J and Rag2 [-/-] mice. Pulmonary ILCs were profiled by single-cell transcriptomics and flow cytometry. Baseline sex differences were compared between healthy humans and mice. In vivo interventions included male castration, female testosterone propionate (TP) administration, and antibody-mediated NK cell depletion. DAH was accompanied by a marked reduction in pulmonary NK cell frequency in both C57BL/6J and Rag2 [-/-] mice, indicating an innate feature independent of adaptive lymphocytes. DAH-associated NK cells displayed an activated but constrained state, showing increased expression of effector-associated genes Prf1 and Ifng, together with the inhibitory receptor gene Lag3. At baseline, healthy human females and female mice exhibited lower NK cell frequencies than males. While male castration did not significantly alter DAH severity, TP administration in females restored pulmonary NK cell dominance and significantly attenuated disease severity. NK cell depletion prior to challenge led to a trend toward more severe hemorrhage (p = 0.084). Pulmonary NK cells are reduced in a lupus-associated DAH model and may participate in disease development. Androgen treatment restores pulmonary NK cell predominance and attenuates DAH severity, supporting further investigation of strategies aimed at restoring or preserving pulmonary NK cell homeostasis and function as potential therapeutic approaches for DAH.

The course of systemic lupus erythematosus (SLE) is highly heterogeneous, with clinical manifestations varying considerably among patients and multiple immune pathways implicated in disease progression. Regardless of this variability, lupus nephritis (LN) is a frequent organ manifestation of SLE and often progresses to renal failure and dialysis dependence. Despite advances in immunosuppressive therapy, there remains a lack of targeted and individualized treatment options to improve outcomes for patients with LN. Immune dysregulation in SLE is reflected by increased interferon (IFN) activity and signaling, accompanied by the upregulation of canonical IFN‑stimulated genes (ISGs). The resulting IFN signature is widely used as a readout of IFN pathway activation, and numerous studies have evaluated this signature as a biomarker of SLE and LN activity. The specific contributions of type I, II, and III IFNs to this signature remain incompletely defined, although research over the past years has focused primarily on type I IFNs as key drivers. Notably, in the context of LN, renal cells themselves exhibit an IFN signature, underscoring their active contribution to disease progression. To define their suitability as therapeutic targets, the role of IFN activity in LN pathogenesis requires further clarification. Experimental models support distinct LN‑promoting roles of type I, II, and III IFNs, suggesting that targeting upstream triggers of IFN production, in addition to directly inhibiting IFN signaling, may represent promising therapeutic strategies. The recent approval of the type I IFN receptor (IFNαR)-blocking antibody anifrolumab has substantiated the relevance of type I IFN signaling in SLE overall, although its specific benefit in LN remains to be fully established. Based on the studies summarized in this narrative review, increased IFN activity or an IFN signature is detectable in a substantial proportion of patients with SLE and may carry prognostic information but appears insufficient to reliably predict disease flares. In LN in particular, blood-derived IFN activity or signatures alone may not be adequate, and the evaluation of renal tissue or urine-derived cells warrants further investigation. IFN signaling across all three IFN types contributes to LN progression by activating and dysregulating renal resident cells; therefore, targeting pathways that induce IFN expression, alongside direct inhibition of IFN signaling, may offer additional therapeutic opportunities.

Interleukin 2 (IL-2) is a cytokine essential for the development and function of regulatory T cells, which are key players in maintaining immune tolerance by suppressing autoreactive lymphocytes. Patients with systemic lupus erythematosus (SLE) often exhibit reduced IL-2 production, resulting in impaired Treg function and a consequent breakdown of immune tolerance. Hematological abnormalities, mainly leukopenia and lymphopenia, are prominent features of SLE. This study aimed to explore the relationship between serum IL-2 concentrations and the altered blood cell populations observed in patients with SLE. We recruited 235 patients with SLE. Complete blood counts, including red blood cells, white blood cells, and platelets, were obtained, and IL-2 levels were quantified using the ultrasensitive single-molecule array (Simoa) technique. Multivariate linear and logistic regression analyses were performed to evaluate the associations between IL-2 concentrations and hematological parameters. Multivariate linear regression revealed significant inverse associations between the serum IL-2 concentration and the hemoglobin level, hematocrit, mean corpuscular hemoglobin concentration, and total leukocyte, neutrophil, lymphocyte, and basophil counts. Similarly, after adjustment for covariates, serum IL-2 was associated with increased odds of anemia, leukopenia, and lymphopenia but not neutropenia or thrombocytopenia. In conclusion, circulating IL-2 levels are independently associated with specific hematological abnormalities, including anemia, leukopenia, and lymphopenia, in patients with SLE. These findings highlight the potential role of IL-2 and regulatory T cell dysfunction in the hematologic manifestations of SLE.

The advancement of hepatocellular carcinoma (HCC) is associated with metabolic reprogramming and immune escape. Compound kushen injection (CKI) is a cancer therapeutic agent with a long history. O-GlcNAcylation is reported to regulate tumor progression. We aimed to investigate how CKI affects HCC cell glycolysis and immune evasion and then clarify the underlying O-GlcNAcylation regulatory mechanism. HCC cell lines and subcutaneous transplantation mice were treated with CKI, and glycolysis and immune evasion were analyzed. The O-GlcNAcylation was analyzed using immunoprecipitation and western blotting. The results showed that CKI inhibited the viability, glycolysis, and immune escape of HCC cells, as well as hindered tumor growth, glycolysis, and immune evasion in vivo. Induction of glycolysis caused by hypoxia reduced the effect of CKI on immune evasion. Besides, CKI reduced the O-GlcNAc levels and OGT transcription activity. Overexpression of OGT counteracted the inhibition of immune evasion caused by CKI. Additionally, OGT catalyzed the O-GlcNAcylation of PD-L1 and enhanced its stability. In conclusion, CKI suppresses immune evasion of HCC cells in association with glycolysis suppression, which is related to the reduction of PGT-mediated PD-L1 O-GlcNAcylation. These findings suggest that CKI possesses an anti-HCC effect, and targeting PD-L1 O-GlcNAcylation may improve its therapeutic efficacy on HCC.

Increasing evidence shows that CD8[+] T cells are the pathogenic mediators of tissue injury in systemic lupus erythematosus (SLE), sustaining the chronic inflammation through the accumulation of long-lived cytotoxic memory populations. However, the transcriptional mechanisms that prevent the aberrant differentiation of pathogenic CD8[+] T cells remain poorly understood. Here, the transcription factor E4BP4 (NFIL3) was identified as a critical restraint of cytotoxic effector-memory CD8[+] T cells in lupus. E4BP4 expression was reduced in CD8[+] T cells from SLE patients and inversely correlated with disease activity. Using a lupus-like disease model, we found that E4BP4 deficiency accelerated disease progression, resulting in heightened autoantibody production, immune complex deposition, and renal pathology. This phenotype was associated with the systemic accumulation of cytotoxic effector-memory CD8[+] T cells. Depletion of CD8[+] T cells significantly ameliorated the disease phenotype, confirming the functional contribution of CD8[+] T cells to lupus-like immunopathology. Competitive adoptive transfer experiments revealed that E4BP4 functions cell-intrinsically to limit the cytotoxicity and proliferation of CD8[+] T cells in autoimmunity. Beyond autoimmunity, E4BP4 deficiency also resulted in an exuberant CD8[+] effector-memory T cell response to Listeria monocytogenes infection, indicating a broader role for E4BP4 in limiting CD8[+] T cell effector-memory responses. Collectively, these findings establish E4BP4 as a transcriptional checkpoint that restricts pathogenic CD8[+] effector-memory T cell responses to maintain immune homeostasis in autoimmunity and infection.

Myasthenia gravis (MG) is a complex autoimmune neuromuscular disorder, and the role of lipid metabolism dysregulation in MG pathogenesis remains unclear. This study aimed to investigate the molecular mechanisms of lipid metabolism regulation in myasthenia gravis development by integrating single-cell and bulk transcriptomic data. This study analyzed bulk RNA sequencing data from the GSE85452 dataset (13 MG patients and 12 healthy controls) and single-cell RNA sequencing data from the GSE227835 dataset (10 MG patients and 10 healthy controls). Differential expression analysis was performed, and weighted gene co-expression network analysis (WGCNA) was conducted to identify disease-related modules. Key genes were screened through the intersection of differentially expressed genes (DEGs), WGCNA hub genes, and lipid metabolism-related genes. Functional enrichment analysis, protein‒protein interaction (PPI) network construction, immune infiltration analysis, and regulatory network analysis were performed. Single-cell analysis was used to characterize cellular heterogeneity and intercellular communication features. A nomogram prediction model was constructed and internally validated using leave-one-out cross-validation (LOOCV). Potential therapeutic compounds were identified through drug prediction and molecular docking analysis. Furthermore, key genes were validated by RT-qPCR in an independent cohort of 5 MG patients and 5 healthy controls. A total of 823 DEGs and 13 co-expression modules were identified, of which 4 modules were significantly associated with MG. Twenty-one candidate genes were screened, and 2 key genes (IRS2 and ALDH2) were ultimately determined. IRS2 was significantly downregulated while ALDH2 was significantly upregulated in MG patients. The nomogram model based on key genes demonstrated excellent predictive performance (AUC = 0.897). Immune infiltration analysis showed increased regulatory T cells (Tregs) and decreased CD4+ memory activated T cells in MG patients. Single-cell analysis identified 10 major cell types. Cell‒cell communication analysis revealed dense interactions among CD4+ T cells, B cells, and CD14+ monocytes. Drug prediction identified metformin and cyclophosphamide as potential therapeutic candidates, and molecular docking confirmed favorable binding affinities. This integrative study generated the hypothesis that lipid metabolism dysregulation, potentially mediated by IRS2 and ALDH2, may contribute to immune dysfunction in MG pathogenesis. These findings provide preliminary insights into the molecular mechanisms underlying MG development and suggest potential diagnostic biomarkers and therapeutic targets. However, these results are hypothesis-generating, and clinical translation is contingent upon rigorous mechanistic validation through functional experiments, animal models, and larger multicenter clinical cohorts.

While interferon regulatory factor 1 (IRF1) has been implicated in reactive oxygen species (ROS)-dependent neutrophil extracellular trap (NET) release during acute lung injury, its regulatory role in mitochondrial dysfunction-driven NETosis specific to pediatric pneumonia remains unexplored. Neutrophils were isolated from the bronchoalveolar lavage fluid (BALF) of pediatric pneumonia patients and the bone marrow of mice. IRF1 expression was quantified via qRT-PCR and Western blotting. Mitochondrial ROS (mtROS) and total ROS were measured by flow cytometry. Mitochondrial dysfunction was assessed by ATP quantification. NETosis was evaluated through immunofluorescence staining and ELISA quantification of neutrophil elastase (NE), myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3) levels. For in vivo studies, pneumonia-related lung injury was induced by intratracheal LPS instillation in mice, with pathological severity graded by H&E staining, pulmonary edema quantified via the wet/dry weight ratio, and inflammation assessed by the BALF protein concentration. Mechanistically, the IRF1/MMP9 interaction was predicted by bioinformatics (STRING database) and validated by co-immunoprecipitation (Co-IP) and immunofluorescence staining, while MMP9 overexpression was achieved via lentivirus transduction to delineate pathway regulation. IRF1 was significantly upregulated in BALF neutrophils from patients and correlated with elevated ROS production and mitochondrial dysfunction, as well as NETs release. IRF1 knockdown attenuated ROS-driven NETosis in vitro. Matrix metalloproteinase 9 (MMP9) was predicted to interact with IRF1, and MMP9 overexpression effectively reversed the beneficial effects of IRF1 deficiency on ROS release, mitochondrial dysfunction, neutrophil apoptosis, and NETosis. Consistently, in mouse models, MMP9 overexpression abolished the protective effects of IRF1 deficiency, exacerbating acute lung injury and restoring NETs levels in BALF.