Hepatic steatosis is a significant independent risk factor for liver surgery because of its vulnerability to ischemia-reperfusion (IR) injury. Invariant natural killer T (iNKT) cells contribute to IR injury in healthy liver. We previously reported that activated iNKT cells mitigate liver IR injury and facilitate liver repair by interacting with macrophages. This study aimed to assess the role of activated iNKT cells in IR injury in steatotic livers.

Male C57/BL6 mice were fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks before liver IR. iNKT cells were activated by intraperitoneal injection of α-galactosylceramide (α-GC) into HFD-fed mice. This study assessed liver injury, cytokine levels, and immune cell accumulation.

HFD-fed mice exhibited increased levels of liver injury, pro-inflammatory mediators, and macrophages compared to those of ND-fed mice. Administration of α-GC to HFD-fed mice enhanced liver IR injury that was associated with increased numbers of iNKT cells and pro-inflammatory macrophages compared with those in the vehicle-treated group. Additionally, liver repair was delayed in α-GC-treated HFD-fed mice, as demonstrated by the increased necrotic area and decreased proliferating cell nuclear antigen expression. This was accompanied by reduced levels of anti-inflammatory mediators and reparative macrophages. Pro-inflammatory cytokine levels were increased in activated hepatic iNKT cells co-cultured with macrophages isolated from HFD-fed mice.

The activation of hepatic iNKT cells aggravates steatotic liver IR injury by upregulating pro-inflammatory mediators and macrophages, while suppressing anti-inflammatory mediators and reparative macrophages.

Gasdermins (GSDMs) are an important family of proteins that have received extensive attention in tumor research in recent years. They directly induce tumor cell death by mediating pyroptosis and also regulate the recognition and clearance of tumor cells by the immune system by affecting the microenvironment. Therefore, it is of great significance to investigate the role of GSDMs in tumor development and tumor microenvironment. It can not only reveal new mechanisms of cancer development, but also provide theoretical basis for the development of novel anti-tumor therapeutic strategies. This literature review aims to systematically summarize the dual roles of GSDMs in tumor development and their interactions with the tumor microenvironment, and to focus on the importance of GSDM-mediated pyroptosis in anti-cancer therapy, with a view to providing guidance for future research directions.

The pathophysiology of diabetic kidney disease (DKD) is complex. Interfering with the processes of pyroptosis and fibrosis is an effective strategy for slowing DKD progression. Previous studies have revealed that nuclear receptor subfamily 4 group A member 1 (NR4A1) may serve as a novel pathogenic element in DKD; however, the specific mechanism by which it contributes to pyroptosis and fibrosis in DKD is unknown.

To investigate the role of NR4A1 in renal pyroptosis and fibrosis in DKD and possible molecular mechanisms.

Streptozotocin 60 mg/kg was injected intraperitoneally to establish a rat model of DKD. Typically, 45 mmol/L glucose [high glucose (HG)] was used to activate HK-2 cells to mimic the DKD model in vitro. HK-2 cells were transfected with NR4A1 siRNA to silence NR4A1.

NR4A1 was elevated in renal tissues of DKD rats and HG-stimulated HK-2 cells. Concurrently, NOD-like receptor protein 3 (NLRP3) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways were triggered, and pyroptosis and expression of fibrosis-linked elements was increased in vivo and in vitro. These alterations were significantly reversed via NR4A1 silencing.

Inhibition of NR4A1 mitigated pyroptosis and fibrosis via suppressing NLRP3 activation and the PI3K/AKT pathway in HG-activated HK-2 cells.

Osteoarthritis (OA) is a common degenerative joint disease. The occurrence of OA slowly destroys the soft tissue structure of the patient's joint. Severe cases could lead to disability. Current studies had shown that inhibition of chondrocytes pyroptosis could slow down the progression of OA. Our work aimed to explore the specific mechanisms and ways of regulating this process.

In this work, the level of N6-methyladenosine (m6A) in clinical tissues was detected by ribonucleic acid (RNA) m6A dot blot. qRT-PCR (quantitative real-time polymerase chain reaction) was used to detect the messenger RNA (mRNA) expression level of m6A modified enzyme in clinical tissues. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromid) and flow cytometry were used to detect the effect of sh-METTL3 (methyltransferase like 3) and NIMA-related kinase 7 (NEK7) transfection on chondrocytes pyroptosis in OA. Western blot was used to detect the protein expression levels of pyroptosis-related proteins. ELISA (enzyme-linked immunosorbent assay) was used to measure the protein concentration of inflammatory cytokines. The SRAMP online database was used to predict the m6A site of NEK7. HE staining was used to assess the progression of OA in mice.

The level of m6A in clinical samples of OA patients was higher, and METTL3 was significantly higher expressed in clinical samples of OA patients. We provided evidence that low expression of METTL3 inhibited chondrocytes pyroptosis. In addition, Rescue experiments and in vivo experiments had shown that METTL3 in combination with NEK7 inhibited the progression of OA by promoting chondrocytes pyroptosis.

METTL3 regulates m6A modification of NEK7 and inhibits OA progression.

Glial cell-induced neuroinflammation in the spinal cord is the critical pathology underlying complete Freund's adjuvant (CFA)-induced inflammatory pain. Previously, we showed that spinal glial cells undergo ferroptosis after CFA injection, which may contribute to the development of neuroinflammation and inflammatory pain. However, the mechanism underlying the occurrence of ferroptosis during inflammatory pain remains unclear. The aim of this study was to investigate the molecular factors involved in the occurrence of ferroptosis during the development of inflammatory pain. Bulk and single-cell RNA sequencing were performed to identify the key genes involved in the ferroptosis of spinal astrocytes, microglia, and oligodendrocytes in rats. We identified nuclear receptor 4A1 (NR4A1) as a common ferroptosis-related gene present in all three types of glial cells. Western blotting and immunostaining revealed increased NR4A1 levels in the spinal glial cells of the CFA-treated rats. Moreover, intrathecal injection of DIM-C-pPhOH (an NR4A1 inhibitor) effectively alleviated mechanical and thermal hypersensitivity in the CFA-treated rats by attenuating ferroptosis and neuroinflammation in spinal glial cells. Proteomic analysis revealed that mitogen-activated protein kinase 3 (MAPK3) may be the target protein of NR4A1. In addition, the combined results of chromatin immunoprecipitation and dual-luciferase assays indicated that NR4A1 can bind to the promoter region and promote transcription of MAPK3, ultimately leading to lipid peroxidation. In conclusion, this study demonstrated that increased expression of NR4A1 promotes the progression of CFA-induced inflammatory pain by enhancing ferroptosis through the transcriptional activation of MAPK3 and subsequent lipid peroxidation. Furthermore, inhibition of NR4A1 was found to suppress ferroptosis and reduce the release of pro-inflammatory cytokines in the spinal cord of rats with inflammatory pain. Collectively, these findings outline a novel pathological mechanism and identify potential therapeutic targets for the treatment of inflammatory pain.

Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion.

We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion.

During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1 , encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes.

Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP.

Steatotic livers exhibit higher susceptibility to ischemia reperfusion (IR) injury, which increase the risk of primary graft non-function following liver transplantation. S100A9 is identified as a pivotal innate immune sensor that regulates the progression of liver diseases. However, its significance in steatotic liver IR injury remains under-investigated.

In mice model, we generated S100A9 knockout (S100A9 KO) mice to investigate the role of S100A9 in IR-stimulated steatotic livers. In vitro, primary bone marrow-derived macrophages were utilized to explore the effect of S100A9 in regulating macrophage polarization and inflammation.

S100A9 expression was markedly increased in steatotic livers of mice subjected to IR insult. S100A9 deletion significantly attenuated liver inflammatory injury, as evidenced by the diminished infiltration of both monocytes/macrophages and neutrophils (p < 0.05). The expression of proinflammatory factors was reduced (p < 0.05) at the same time. Additionally, S100A9-deficient livers demonstrated M1 polarization decrease and Toll-like receptor 4 (TLR4) suppression (p < 0.05). In vitro, genetic TLR4 inhibition led to nuclear factor kappa B (NF-κB) inactivation and subsequent M1 polarization decrease (p < 0.05) in macrophages treated with recombinant S100A9. Conclusion In this study, we highlight the pivotal role of TLR4/NF-κB as a critical mediator of S100A9 in inducing M1 macrophage polorization- dependent inflammation in steatotic livers IR injury.

Liver ischemia-reperfusion (I/R) injury is a clinically relevant pathophysiological process that determines the effectiveness of life-saving liver transplantation, to which aberrant ROS accumulation plays a key role. In the present study we investigated the role of SUV39H1, a lysine methyltransferases, in this process focusing on regulatory mechanism and translational potential. We report that SUV39H1 expression was up-regulated in the liver tissues of mice subjected to ischemia-reperfusion and in hepatocytes exposed to hypoxia-reoxygenation (H/R) in a redox-sensitive manner. Mechanistically, coactivator associated arginine methyltransferases 1 (CARM1) mediated redox-sensitive Suv39h1 trans-activation by promoting histone H3R17 methylation. Consistently, pharmaceutical CARM1 inhibition attenuated liver I/R injury. In addition, global or hepatocyte conditional Suv39h1 KO mice were protected from liver I/R injury. RNA-seq revealed that aldehyde dehydrogenase 1 family 1a (Aldh1a1) as a novel target for SUV39H1. SUV39H1 directly bound to the Aldh1a1 promoter and repressed Aldh1a1 transcription in H/R-challenged hepatocytes. ALDH1A1 silencing abrogated the protective effects of SUV39H1 deficiency on H/R-inflicted injuries whereas ALDH1A1 over-expression mitigated liver I/R injury in mice. Importantly, administration of a small-molecule SUV39H1 inhibitor achieved similar hepatoprotective effects as SUV39H1 deletion. Finally, increased Suv39h1 expression and decreased Aldh1a1 expression were observed in liver I/R specimens in humans. In conclusion, our data uncover a regulatory role for SUV39H1 in liver I/R injury and serve as proof-of-concept that targeting the SUV39H1-ALDH1A1 axis might be considered as a reasonable approach for the intervention of liver I/R injury.

Caspase-6 is an important regulatory factor in innate immunity, inflammasome activation, and host defense, but its role in preeclampsia (PE) is unknown. This study aims to investigate the mechanism of Caspase-6 in the interaction between PE rats and macrophage-trophoblast cells, in order to provide a new theoretical basis for the treatment of PE.

Co-cultures of THP-1 cells and HTR8/SVneo cells were employed to investigate the HMGB1 signaling in macrophages (transfection with si-Caspase-6) and HTR8/SVneo cells. The PE rat model was constructed by using the reduced uterine perfusion pressure (RUPP) surgery to explore the therapeutic effects of bone marrow-derived macrophages (BMDM) transfected with si-Caspase-6 in PE rats. ELISA, Western blot, immunofluorescence, etc., were employed to characterize the expression of ferroptosis-related markers.

Caspase-6 expression was significantly increased in CD14+ macrophages in the placental tissue of PE rats. Overexpression of Caspase-6 in THP-1 cells induced ferroptosis of HTR8/SVneo cells, but this process was blocked by anti-HMGB1 neutralizing antibody. Knockdown of Caspase-6 in macrophages could alleviate ferroptosis of HTR8/SVneo cells and restore its basic characteristics. Knockdown of Caspase-6 in BMDM downregulated ferroptosis in placental tissue of PE rats through HMGB1, thereby improving the disease phenotype in rats.

Knocking down Caspase-6 in BMDM regulated the crosstalk between macrophages and HTR8/SVneo cells through HMGB1, inhibiting HTR8/SVneo cell ferroptosis, thereby improving adverse pregnancy outcomes of PE.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

Fatty livers are widely accepted as marginal donors for liver transplantation but are more susceptible to liver ischemia and reperfusion (IR) injury. Increased macrophage-related inflammation plays an important role in the aggravation of fatty liver IR injury. Here, we investigate the precise mechanism by which endoplasmic reticulum (ER) stress activates macrophage NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling by regulating mitochondrial calcium overload in fatty liver IR.

Control- and high-fat diet-fed mice were subjected to a partial liver IR model. The ER stress, mitochondrial calcium levels, and NLRP3 signaling pathway in macrophages were analyzed.

Liver steatosis exacerbated liver inflammation and IR injury and enhanced NLRP3 activation in macrophages. Myeloid NLRP3 deficiency attenuated intrahepatic inflammation and fatty liver injury following IR. Mechanistically, increased ER stress and mitochondrial calcium overload were observed in macrophages obtained from mouse fatty livers after IR. Suppression of ER stress by tauroursodeoxycholic acid effectively downregulated mitochondrial calcium accumulation and suppressed NLRP3 activation in macrophages, leading to decreased inflammatory IR injury in fatty livers. Moreover, Xestospongin-C-mediated inhibition of mitochondrial calcium influx decreased reactive oxygen species (ROS) expression in macrophages after IR. Scavenging of mitochondrial ROS by mito-TEMPO suppressed macrophage NLRP3 activation and IR injury in fatty livers, indicating that excessive mitochondrial ROS production was responsible for macrophage NLRP3 activation induced by mitochondrial calcium overload. Patients with fatty liver also exhibited upregulated activation of NLRP3 and the ER stress signaling pathway after IR.

Our findings suggest that ER stress promotes mitochondrial calcium overload to activate ROS/NLRP3 signaling pathways within macrophages during IR-stimulated inflammatory responses associated with fatty livers.

Doxorubicin (DOX) is a widely used antitumor drug, but its clinical applicability is hampered by the unfortunate side effect of DOX-induced cardiotoxicity (DIC). In our current study, we retrieved three high-throughput sequencing datasets related to DIC from the Gene Expression Omnibus (GEO) datasets. We conducted differential analysis using R (DESeq2) to pinpoint differentially expressed genes (DEGs, and identified 11 genes that were consistently altered in both the control and DOX-treated groups. Notably, our Random Forest analysis of these three GEO datasets highlighted the significance of nuclear receptor subfamily 4 group A member 1 (NR4A1) in the context of DIC. The DOX-induced mouse model and cell model were used for the in vivo and in vitro studies to reveal the role of NR4A1 in DIC. We found that silencing NR4A1 by adeno-associated virus serotype 9 (AAV9) contained shRNA in vivo alleviated the DOX-induced cardiac dysfunction, cardiomyocyte injury and fibrosis. Mechanistically, we found NR4A1 silencing was able to inhibit DOX-induced the cleavage of NLRP3, IL-1β and GSDMD in vivo. Further in vitro studies have shown that inhibition of NR4A1 suppressed DOX-induced cytotoxicity and oxidative stress through the same molecular mechanism. We prove that NR4A1 plays a critical role in DOX-induced cardiotoxicity by inducing pyroptosis via activation of the NLRP3 inflammasome, and it might be a promising therapeutic target for DIC.

Liver ischemia-reperfusion injury (IRI) remains a common issue and with the increasing incidence of Nonalcoholic fatty liver disease (NAFLD), which are more sensitive to IRI, it is crucial to explore the possible strategy to alleviate the steatotic liver IRI. Several modes of cell death are involved in hepatocytes and immune cells during hepatic IRI, and the effects of different cell death inhibitors including apoptosis, necroptosis, pyroptosis, and ferroptosis in steatotic liver IRI have not been investigated. We established 70% IRI model on steatotic liver in mice. Apoptosis, necroptosis, pyroptosis and ferroptosis inhibitors were used to evaluate their effects on liver injury, inflammatory response, and immune cell infiltration. Immunofluorescence and immunohistochemical results demonstrated that there were apoptosis, necroptosis, pyroptosis, and ferroptosis in the progression of IRI in steatotic liver. All four types of cell death inhibitors showed protective effects, but ferroptosis inhibitor Fer-1 and pyroptosis inhibitor VX765 exerted better protective effects compared the apoptosis inhibitor Z-VAD and necroptosis inhibitor Nec-1. Further, we found that pyroptosis occurred mainly in macrophages and ferroptosis occured primarily in hepatocytes during steatotic liver IRI. Ferroptosis in heaptocytes and pyroptosis in macrophages are two major cell death types involved in steatotic liver IRI and inhibiting these cell death exerted good protective effects.

Macrophages play a critical role in innate immunity, with approximately 90% of the total macrophage population in the human body residing in the liver. This population encompasses both resident and infiltrating macrophages. Recent studies highlight the pivotal role of liver macrophages in various aspects such as liver inflammation, regeneration, and immune regulation. A novel pro-inflammatory programmed cell death, pyroptosis, initially identified in macrophages, has garnered substantial attention since its discovery. Studies investigating pyroptosis and inflammation progression have particularly centered around macrophages. In liver diseases, pyroptosis plays an important role in driving the inflammatory response, facilitating the fibrotic process, and promoting tumor progression. Notably, the role of macrophage pyroptosis cannot be understated. This review primarily focuses on the role of macrophage pyroptosis in liver diseases. Additionally, it underscores the therapeutic potential inherent in targeting macrophage pyroptosis.

Caspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury.

Human liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury.

In human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages.

Our findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury. Video Abstract.