Rheumatoid arthritis (RA) is a chronic autoimmune disease causing joint inflammation, dysfunction, and deformity, along with systemic inflammatory manifestations. Inhibitor of differentiation-2 (ID2) is a transcription factor containing a helix-loop-helix (HLH) structure. Studies suggest that ID2 regulates innate and adaptive immunity and inhibits the differentiation of osteoclasts. However, the effects and underlying molecular mechanisms of ID2 on rheumatoid arthritis (RA) remain unclear. In the present study, we found that exogenous supplementation of human recombinant ID2 (hID2) protein significantly reduced paw swelling and arthritis index scores in adjuvant-induced arthritis (AIA) rats, and improved ankle joint pathology. Analysis of pro-inflammatory factor levels in peripheral blood mononuclear cells and synovial tissues indicated that hID2 attenuated inflammatory responses in AIA rats. Furthermore, RNA sequencing demonstrated that hID2 down-regulated the JAK-STAT pathway, and the phosphorylation of its key molecule, Signal Transducer and Activator of Transcription 3 (STAT3), was inhibited in synovial tissues. Additionally, the expression of chemokine-related genes was noticeably down-regulated in synovial tissues, though further investigation is needed to understand the underlying mechanisms. Overall, these findings suggest that hID2 effectively attenuated the inflammatory response and joint destruction in AIA rats, highlighting the potential of hID2 as a therapeutic agent for the treatment of RA.

This research investigates the role of inhibitor of differentiation 2 (Id2) in the synthesis of pro-inflammatory cytokines, specifically interferon-γ (IFN-γ) and interleukin-17 (IL-17), by various subsets of T cells, and its pathogenic role in rheumatoid arthritis (RA).

Flow cytometry was employed to assess T-cell activation and Id2 expression in 72 RA patients and 23 healthy controls. In vitro, peripheral blood mononuclear cells were treated with either an Id2 inhibitor or a T-cell co-stimulation inhibitor. An in vivo collagen-induced arthritis (CIA) model was established using T-cell-specific Id2 knockout mice. Additionally, follow-up observations were conducted among treated RA patients.

T-cell activation levels in RA synovial fluid were significantly elevated. A positive correlation was found between increased IFN-γ and Id2 expression. In vitro, antagonising Id2 reduced IFN-γ production after T-cell activation. T-cell-specific Id2 knockout mice exhibited a diminished occurrence and severity of CIA, along with a significant decrease in IFN-γ expression. Clinical monitoring indicated that Id2-induced circulating T-cell IFN-γ expression significantly decreased following treatment with the T-cell activation inhibitor abatacept.

The data suggest that high Id2 expression is a critical regulator of pro-inflammatory cytokine upregulation, particularly IFN-γ, by hyperactivated T cells in RA, potentially exacerbating the disease. Inhibiting Id2 expression or function may offer new therapeutic approaches for RA joint inflammation.

Pro-inflammatory cytokines are significantly upregulated in the synovial fluid T cells in rheumatoid arthritis patients. The expression of pro-inflammatory cytokine interferon-γ (IFN-γ) positively correlates with the high expression of inhibitor of differentiation 2 (Id2). The inhibition or ablation of Id2 can effectively suppress IFN-γ production and the onset and progression of arthritis.

Microglia are the most important immune cells in the central nervous system (CNS), which can defend against external pathogens and stimuli. Dysregulation of microglia releases excessive proinflammatory cytokines and leads to neuroinflammation, which is fundamental to the pathophysiology of multiple neurological diseases. However, the molecular mechanisms underlying the regulation of proinflammatory cytokines in microglia are still not well-understood. Here, we identified that inhibitor of DNA binding protein 2 (Id2) was a negative regulator of tumor necrosis factor-α (TNFα) in cultured microglia. Knockdown of Id2 significantly increased the expression of TNFα in microglia, while overexpression of Id2 inhibited TNFα expression. Furthermore, by interacting with the p65 subunit of nuclear factor kappa-B (NF-κB), Id2 suppressed the transcription activation of NF-κB and inhibited TNFα expression. Interestingly, in lipopolysaccharides (LPS)-treated microglia, Id2 increased and underwent a cytoplasmic relocation. Immunoprecipitation and immunostaining results showed that by binding to the LIM domain of Id2, a scaffold protein PDZ and LIM 5 (PDLIM5) involved in the Id2 cytoplasmic relocation, which inactivated Id2 and resulted in higher TNFα expression in LPS-treated microglia. Collectively, our data delineate a novel effect of Id2 on TNFα regulation in microglia, which may shed a light on the proinflammatory cytokines regulating in microglia associated neuroimmune disorders.

The human Inhibitor of Differentiation-2 (hID2) protein is a promising candidate for the treatment of colitis. However, its relatively low molecular weight limits its clinical application. To extend the therapeutic half-life, an albumin-binding domain (ABD), known for its high affinity for human serum albumin (HSA), was fused to hID2, resulting in a recombinant ABD-hID2. The anti-colitis bioactivity of ABD-hID2 than that of hID2 was evaluated in this study.

Western blotting, size-exclusion high-performance chromatography, HSA binding assay, and pharmacokinetic studies were used to characterise ABD-hID2, which was induced by dextran sulfate sodium salt (DSS), Citrobacter rodentium (CR), and ABD-hID2 and hID2. The Disease Activity Index, histological pathologies, inflammatory response, Alcian blue or tuft cell staining, and tight junction proteins were determined. Alterations in the intestinal microbiota after ABD-hID2 treatment were analysed via 16S rRNA gene sequencing.

Compared with hID2, ABD-hID2 exhibited a decreased dimer complex, bound to HSA with high affinity, and demonstrated an extended blood retention time in vivo. Consequently, ABD-hID2 exhibited increased therapeutic efficacy in both DSS- and CR-induced colitis mouse models, as evidenced by the alleviation of colitis symptoms, preservation of goblet and tuft cell functions, restoration of the intestinal mucus barrier, and suppression of abnormal immune-inflammatory responses. Additionally, the modulation of the gut microbiota may play a role in the protective effects of ABD-hID2 in mice with CR-induced ulcerative colitis.

ABD-hID2 enhances the bioactivity of hID2 and has the potential for further development as a treatment for colitis.

The innate immune system (IIS) is an ancient and essential defense mechanism that protects animals against a wide range of pathogens and diseases. Although extensively studied in mammals, our understanding of the IIS in other taxa remains limited. The zebrafish (Danio rerio) serves as a promising model organism for investigating IIS-related processes, yet the immunogenetics of fish are not fully elucidated. To address this gap, we conducted a meta-analysis of single-cell RNA sequencing (scRNA-seq) datasets from zebrafish kidney marrow, encompassing approximately 250,000 immune cells. Our analysis confirms the presence of key genetic pathways in zebrafish innate immune cells that are similar to those identified in mammals. Zebrafish macrophages specifically express genes encoding cathepsins, major histocompatibility complex class II proteins, integral membrane proteins, and the V-ATPase complex and demonstrate the enrichment of oxidative phosphorylation ferroptosis processes. Neutrophils are characterized by the significant expression of genes encoding actins, cytoskeleton organizing proteins, the Arp2/3 complex, and glycolysis enzymes and have demonstrated their involvement in GnRH and CLR signaling pathways, adherents, and tight junctions. Both macrophages and neutrophils highly express genes of NOD-like receptors, phagosomes, and lysosome pathways and genes involved in apoptosis. Our findings reinforce the idea about the existence of a wide spectrum of immune cell phenotypes in fish since we found only a small number of cells with clear pro- or anti-inflammatory signatures.

Ulcerative colitis (UC) is a chronic inflammatory disease, the incidence of which is increasing worldwide. However, the etiology and pathogenesis of UC remains unclear. The n-butanol extract of Pulsatilla decoction (BEPD), a traditional Chinese medicine, has been shown to be effective in treating UC. This study aimed to explore the molecular mechanism underlying the effects of BEPD on UC, in particular its effects on neutrophil extracellular trap (NET) formation by neutrophils. High-performance liquid chromatography was used to determine the principal compounds of BEPD. UC was induced in mice using dextran sodium sulfate, and mice were treated with 20, 40, or 80 mg/kg BEPD daily for seven days. Colonic inflammation was determined by assessing the disease activity index, histopathology, colonic mucosal damage index, colonic mucosal permeability, and pro- and anti-inflammatory cytokine levels. The infiltration and activation status of neutrophils in the colon were determined by analyzing the levels of chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL2, reactive oxygen species, Ly6G, and numerous NET proteins. The findings suggest that BEPD improved the disease activity index, histopathology, and colonic mucosal damage index scores of mice with UC, and restored colonic mucosal permeability compared with untreated mice. The expression levels of the pro-inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α in colon tissues were significantly decreased, while the expression levels of anti-inflammatory cytokines in colon tissues were significantly increased, exceeding those of control mice. In addition, BEPD reduced the expression of the neutrophil chemokines CXCL1 and CXCL2 in the colon tissue of mice with UC, reduced neutrophil infiltration, reduced reactive oxygen species levels, and significantly reduced the expression of NET proteins. BEPD also significantly reduced NET formation. The results of this study suggest that BEPD exerts therapeutic effects in a murine model of UC by inhibiting neutrophil infiltration and activation in the colon, as well as by inhibiting the expression of key proteins involved in NET formation and reducing NET formation, thereby alleviating local tissue damage and disease manifestations.

Ulcerative colitis (UC) is characterized by chronic non-recessive inflammation of the intestinal mucosa involving both innate and adaptive immune responses. Currently, new targeted therapies are urgently needed for UC, and neutrophil extracellular traps (NETs) are new therapeutic options. NETs are DNA-based networks released from neutrophils into the extracellular space after stimulation, in which a variety of granule proteins, proteolytic enzymes, antibacterial peptides, histones, and other network structures are embedded. With the deepening of the studies on NETs, their regulatory role in the development of autoimmune and autoinflammatory diseases has received extensive attention in recent years. Increasing evidence indicates that excess NETs exacerbate the inflammatory response in UC, disrupting the structure and function of the intestinal mucosal barrier and increasing the risk of thrombosis. Although NETs are usually assigned a deleterious role in promoting the pathological process of UC, they also appear to have a protective role in some models. Despite such progress, comprehensive reviews describing the therapeutic promise of NETs in UC remain limited. In this review, we discuss the latest evidence for the formation and degradation of NETs, focusing on their double-edged role in UC. Finally, the potential implications of NETs as therapeutic targets for UC will be discussed. This review aims to provide novel insights into the pathogenesis and therapeutic options for UC.

Ulcerative colitis (UC) is a complex immune-mediated chronic inflammatory bowel disease. It is mainly characterized by diffuse inflammation of the colonic and rectal mucosa with barrier function impairment. Identifying new biomarkers for the development of more effective UC therapies remains a pressing task for current research. Ferroptosis is a newly identified form of regulated cell death characterized by iron-dependent lipid peroxidation. As research deepens, ferroptosis has been demonstrated to be involved in the pathological processes of numerous diseases. A growing body of evidence suggests that the pathogenesis of UC is associated with ferroptosis, and the regulation of ferroptosis provides new opportunities for UC treatment. However, the specific mechanisms by which ferroptosis participates in the development of UC remain to be more fully and thoroughly investigated. Therefore, in this review, we focus on the research advances in the mechanism of ferroptosis in recent years and describe the potential role of ferroptosis in the pathogenesis of UC. In addition, we explore the underlying role of the crosslinked pathway between ferroptosis and other mechanisms such as macrophages, neutrophils, autophagy, endoplasmic reticulum stress, and gut microbiota in UC. Finally, we also summarize the potential compounds that may act as ferroptosis inhibitors in UC in the future.

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein that regulates the activity of FoF1-ATP synthase. Mice lacking ATPIF1 throughout their bodies (Atpif1-/-) exhibit a reduction in the number of neutrophils. However, it remains unclear whether the inactivation of ATPIF1 impairs the antibacterial function of mice, this study aimed to evaluate it using a mouse peritonitis model. Mice were intraperitoneally injected with E. coli to induce peritonitis, and after 24 h, the colonies of E. coli were counted in agarose plates containing mice peritoneal lavage fluids (PLF) or extract from the liver. Neutrophils were analyzed for glucose metabolism in glycolysis following LPS stimulation. Reactive oxygen species (ROS) and lactic acid (LA) levels in neutrophils were measured using flow cytometry and Seahorse analysis, respectively. N-Acetylcysteine (NAC) and 2-Deoxy-d-glucose (2-DG) were employed to assess the role of ROS and LA in neutrophil bactericidal activity. RNA-seq analysis was conducted in neutrophils to investigate potential mechanisms. In ATPIF1-/- neutrophils, bactericidal activity was enhanced, accompanied by increased levels of ROS and LA compared to wildtype neutrophils. The augmented bactericidal activity of ATPIF1-/- neutrophils was reversed by pretreatment with NAC or 2-DG. RNA-seq analysis revealed downregulation of multiple genes involved in glutathione metabolism, pyruvate oxidation, and heme synthesis, along with increased expression of inflammatory and apoptotic genes. This study suggests that the inactivation of the Atpif1 gene enhances glucose metabolism in neutrophils, resulting in increased bactericidal activity mediated by elevated levels of ROS and LA. Inhibiting ATPIF1 may be a potential approach to enhance antibacterial immunity.

Lacticaseibacillus paracasei strain PS23 (PS23) exhibits some probiotic properties. In this study, a genomic analysis of PS23 revealed no genes related to virulence or antibiotic resistance. Moreover, ornithine decarboxylase activity was not detected in vitro. In addition, PS23 was sensitive to the tested antibiotics. Genotoxicity tests for PS23 including the Ames test and chromosomal aberrations in vitro using Chinese hamster ovary cells and micronuclei in immature erythrocytes of ICR mice were all negative. Moreover, following a 28-day study involving repeated oral dose toxicity tests (40, 400, and 4000 mg/kg equal 1.28 × 10¹⁰, 1.28 × 10¹¹, and 1.28 × 10¹² CFU/kg body weight, respectively) using an ICR mouse model, no adverse effects were observed from any doses. In addition, supplementation with live or heat-killed PS23 ameliorates DSS-induced colonic inflammation in mice. Our findings suggest that PS23 is safe and has anti-inflammatory effects and may therefore have therapeutic implications.

Gut microbiota imbalances play an important role in inflammatory bowel disease (IBD), but no single pathogenic microorganism critical to IBD that is specific to the IBD terminal ileum mucosa or can invade intestinal epithelial cells has been found. Invasive Escherichia coli (E. coli) adhesion to macrophages is considered to be closely related to the pathogenesis of inflammatory bowel disease. Further study of the specific biological characteristics of adherent invasive E. coli (AIEC) may contribute to a further understanding of IBD pathogenesis. This review explores the relationship between AIEC and the intestinal immune system, discusses the prevalence and relevance of AIEC in Crohn's disease and ulcerative colitis patients, and describes the relationship between AIEC and the disease site, activity, and postoperative recurrence. Finally, we highlight potential therapeutic strategies to attenuate AIEC colonization in the intestinal mucosa, including the use of phage therapy, antibiotics, and anti-adhesion molecules. These strategies may open up new avenues for the prevention and treatment of IBD in the future.