Chunguang Ren

Background: Protein availability critically influences innate immune reactivity and the extent of immune-mediated tissue injury. A key unresolved question is how specific protein homeostasis networks within immune cells such as neutrophils can be modulated to mitigate the detrimental outcomes of acute inflammation. Methods: We established an acute lung injury (ALI) model in immune-specific ubiquitin-specific peptidase 4 (USP4) knockout chimeric (BM-Usp4 ^−/− ) and control (BM-WT) mice. Using cytometry by time-of-flight (CyTOF) mass cytometry, quantitative proteomics, immunoprecipitation-mass spectrometry and neutrophil functional assays, we elucidated the molecular mechanism of USP4 in a specific neutrophil migratory behaviour—spreading—as well as the therapeutic potential of USP4 inhibitors in acute respiratory distress syndrome (ARDS). Results: USP4 expression was upregulated in blood and pulmonary leucocytes from patients with ARDS and ALI mice (p=0.049, p=0.0086), but downregulated in mouse lung parenchyma (lipopolysaccharide vs control, p=0.0019). BM-Usp4 ^−/− mice exhibited attenuated lung injury and neutrophil infiltration (in lung tissue and bronchoalveolar lavage fluid, p−/− neutrophils showed a reduction in both the proportion and area of spreading (p+/+ neutrophils). Mechanistically, USP4 deubiquitinated and upregulated IQGAP1, enhancing CDC42 activity (p=0.0286) and F-actin formation (p<0.0001). Notably, USP4 and IQGAP1 expression correlated positively for patients with severe pneumonia (p=0.0438, R²=0.7898). Moreover, USP4 inhibitor Vialinin A in ALI mice decreased neutrophil infiltration, improved lung function and survival and alleviated lung injury progression. Conclusion: USP4 links protein homeostasis to neutrophil recruitment during inflammation. Targeting the USP4-IQGAP1 axis with Vialinin A represents a promising therapeutic strategy for patients with ARDS secondary to severe pneumonia.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract characterized by a complex interplay of genetic, environmental, and immunological factors. Neutrophils, as a key component of the innate immune system, play a critical role in IBD pathogenesis due to their dysregulated infiltration, impaired apoptosis, and resultant epithelial damage during the disease process. Conventional approaches aimed at suppressing neutrophil activity have achieved only modest clinical efficacy and highlight the need for strategies that recalibrate neutrophil responses without compromising their essential antimicrobial functions. In this study, we employed high-throughput chemical screening using neutrophil-specific transgenic zebrafish larvae to identify compounds capable of modulating neutrophil homeostasis, and subsequently evaluating their efficacy in a dextran sodium sulfate (DSS)-induced IBD model. We identified epothilone B (Epo B), a traditional chemotherapeutic drug, as a potential therapeutic candidate for IBD. Our findings demonstrate that Epo B protects against IBD by promoting intestinal epithelial recovery, alleviating inflammatory responses, and rebalancing the gut microbiota. Mechanistically, Epo B selectively stimulates neutrophil apoptosis by targeting and enhancing Caspase-3 cleavage, thereby inhibiting neutrophilic inflammation. This study underscores the utility of in vivo high-throughput drug screening in IBD and highlights Epo B as a promising candidate for drug repurposing in IBD treatment, offering a novel therapeutic strategy by targeting neutrophil apoptosis.

Background Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract characterized by complex interplay of genetic, environmental, and immunological factors. Neutrophils, as a key component of the innate immune system, play a critical role in IBD pathogenesis due to their dysregulated infiltration, impaired apoptosis, and resultant epithelial damage during the disease process.However, effective intervention strategies by modulating neutrophils to mitigate the severity of IBD have yet to be established. Methods High-throughput chemical screening was conducted utilizing neutrophil-specific transgenic zebrafish embryos to identify compounds capable of modulating neutrophil homeostasis. The identified compounds were subsequently evaluated in a dextran sodium sulfate (DSS)-induced zebrafish model of IBD by assessing intestinal damage, leukocyte infiltration, the expression of inflammatory cytokines, and alterations in intestinal bacterial microbiota. The effects of Epothilone B (Epo B) on neutrophil apoptosis were assessed via flow cytometry and whole-mount terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays, both in vivo and in vitro. Potential targets of Epo B in the context of IBD were identified through network pharmacology and molecular docking analyses, followed by validation at the protein level via Western blotting. Results Chemical screening identified four compounds, including Epo B, that influence neutrophil homeostasis. Epo B protects against IBD in zebrafish by promoting intestinal epithelial recovery, alleviating inflammatory responses, and rebalancing gut microbiota. Mechanistically, Epo B selectively stimulates neutrophil apoptosis by targeting and enhancing Caspase-3 cleavage, thereby inhibiting neutrophilic inflammation. Conclusion This study underscores the utility of zebrafish in drug screening for IBD and highlights Epo B as a promising candidate for repurposing in IBD treatment, offering a novel therapeutic strategy by targeting neutrophil apoptosis.

Purpose Acute respiratory distress syndrome (ARDS)/Acute lung injury (ALI), characterized by severe hypoxemia and pulmonary edema, involves mitochondrial dysfunction. Cytidine/uridine monophosphate kinase 2 (Cmpk2), a mitochondrial metabolic enzyme, modulates inflammation and senescence, yet its role in ARDS remains unclear. We investigated Cmpk2’s function in Pseudomonas aeruginosa (P. aeruginosa)-induced ALI using Cmpk2 global knockout (KO) mice. Methods Cmpk2 was identified through mitochondrial gene expression analysis of ARDS datasets (GEO). Murine ALI was induced by intratracheal P. aeruginosa injection. Lung pathology (hematoxylin and eosin staining), leukocyte recruitment (flow cytometry), and cytokines (ELISA) were assessed. GO/KEGG analyses were conducted to identify Cmpk2-associated biological processes and pathways. The expression of Cmpk2 in leukocyte populations was analyzed using single-cell RNA sequencing (scRNA-seq) data from ARDS patient samples. Mouse neutrophils’ phagocytosis of P. aeruginosa was quantified by flow cytometry. Zebrafish embryos were infected with P. aeruginosa and Staphylococcus aureus for bacterial burden and survival assays. Results Cmpk2 expression was significantly upregulated in ARDS. Cmpk2 KO exacerbated P. aeruginosa-induced ALI in mice, as evidenced by increased pathological damage and permeability, elevated proinflammatory cytokines and enhanced neutrophil infiltration. GO/KEGG analyses linked Cmpk2 to innate immunity. scRNA-seq analysis revealed an enriched expression of Cmpk2 in neutrophils. Cmpk2 deficiency impaired neutrophil phagocytosis and reduced host survival during bacterial infection, accomplished by decreased STING expression. The differences in phagocytosis between the wild-type and Cmpk2 KO mouse neutrophils/zebrafish embryos were eliminated by STING inhibitor C176. Conclusion Cmpk2 protects against pneumonia by attenuating neutrophil recruitment and enhancing bacterial phagocytosis via STNG-dependent mechanisms.

Coat protein complex I (COPI) vesicles mediate the retrograde transfer of cargo between Golgi cisternae and from the Golgi to the endoplasmic reticulum (ER). However, their roles in the cell cycle and proliferation are unclear. This study shows that TANGO6 associates with COPI vesicles via two transmembrane domains. The TANGO6 N- and C-terminal cytoplasmic fragments capture RNA polymerase II subunit B (RPB) 2 in the cis-Golgi during the G1 phase. COPI-docked TANGO6 carries RPB2 to the ER and then to the nucleus. Functional disruption of TANGO6 hinders the nuclear entry of RPB2, which accumulates in the cytoplasm, causing cell cycle arrest in the G1 phase. The conditional depletion or overexpression of TANGO6 in mouse hematopoietic stem cells results in compromised or expanded hematopoiesis. Our study results demonstrate that COPI vesicle-associated TANGO6 plays a role in the regulation of cell cycle progression by directing the nuclear transfer of RPB2, making it a potential target for promoting or arresting cell expansion.

Background: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common respiratory disease characterized by persistent hypoxemia and an uncontrolled inflammatory response. Valsartan, an angiotensin II type 1 receptor antagonist, is clinically used to treat hypertension and has anti-inflammatory and antioxidant effects on gefitinib-induced pneumonia in rats. However, the potential therapeutic effects of valsartan on lipopolysaccharide (LPS)-induced ALI remain unclear. This study investigated the protective role of valsartan in LPS-induced ALI and its underlying mechanisms.Methods: LPS-treated BEAS-2B cells and ALI mouse model were established. BEAS-2B cells were treated with LPS (10 μg/mL) for 24h, with or without valsartan (20, 40, and 80 µM). For ALI mouse models, LPS (5 mg/kg) was administered through intratracheal injection to treat the mice for 24h, and valsartan (10 or 30 mg/kg) was injected intraperitoneally twice 2 h before and 12 h after the LPS injection. Pulmonary functional parameters were examined by an EMKA pulmonary system. Hematoxylin and eosin staining, flow cytometry, CCK-8 assay, qRT-PCR, ELISA, immunofluorescence, Western blotting, and related commercial kits were used to assess the pathological damage to the lungs, neutrophil recruitment in the lung tissue and bronchoalveolar lavage fluid (BALF), cell viability, inflammation, oxidative activity, and mucus production, respectively. Potential mechanisms were further explored using network pharmacology and Western blotting.Results: Valsartan rescued LPS-reduced cell viability of BEAS-2B cells, improved the pulmonary function, ameliorated pathological lung injury in mice with ALI, ameliorated LPS-induced neutrophil recruitment in BALF and lung tissue of mice, attenuated oxidative stress by increasing the level of SOD and decreasing that of MDA and GSSG, inhibited LPS-induced MUC5AC overproduction, decreased the LPS-induced increase in expression of pro-inflammatory cytokines/chemokines including TNF-α, IL-6, IL-1β, CXCL-1 and CXCL-2, and restored the expression of anti-inflammatory IL-10. Mechanistic studies showed that valsartan inhibits LPS-induced phosphorylation of nuclear factor-kappa B (NF-κΒ) and mitogen-activated protein kinases (MAPKs) including P38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in both LPS-treated cells and the mouse model of ALI.Conclusion: Valsartan protects against LPS-induced ALI by attenuating oxidative stress, reducing MUC5AC production, and attenuating the inflammatory response that may involve MAPK and NF-κΒ pathways.

Tissue injury-induced neutrophil recruitment is a prerequisite for the initiation and amplification of inflammatory responses. Although multiple proteases and enzymes involved in post-translational modification (PTM) of proteins regulate leukocyte recruitment, an unbiased functional screen of enzymes regulating inflammatory leukocyte recruitment has yet to be undertaken. Here, using a zebrafish tail fin amputation (TFA) model to screen a chemical library consisting of 295 compounds that target proteases and PTM enzymes, we identified multiple histone deacetylase (HDAC) inhibitors that modulate inflammatory neutrophil recruitment. AR-42, a pan-HDAC inhibitor, was shown to inhibit neutrophil recruitment in three different zebrafish sterile tissue injury models: a TFA model, a copper-induced neuromast damage and mechanical otic vesicle injury (MOVI) model, and a sterile murine peritonitis model. RNA sequencing analysis of AR-42-treated fish embryos revealed downregulation of neutrophil-associated cytokines/chemokines, and exogenous supplementation with recombinant human IL-1β and CXCL8 partially restored the defective neutrophil recruitment in AR-42-treated MOVI model fish embryos. We thus demonstrate that AR-42 non-cell-autonomously modulates neutrophil recruitment by suppressing transcriptional expression of cytokines/chemokines, thereby identifying AR-42 as a promising anti-inflammatory drug for treating sterile tissue injury-associated diseases.

Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter in vitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.