Dapansutrile (Dapan) is a newly developed anti-inflammatory molecule that supresses the production of NLRP3 inflammasome-dependent IL-1β. Its hepatoprotective effects against autoimmune hepatitis (AIH) have not yet been explored. Hence, this study was conducted to examine the possible protective effects of Dapan against concanavalin A (Con A)-induced hepatitis in mice.

Mice were randomly divided into five groups (n = 6): control, Con A (15 mg/kg), Dapan (60 mg/kg), Dapan (6 mg/kg) + Con A, and Dapan (60 mg/kg) + Con A. Mice were euthanised at the end of the study, and blood and hepatic tissues were collected.

Hepatic function testing using lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase levels, in addition to hepatic tissue histological examination, revealed that intraperitoneal administration of Dapan noticeably ameliorated Con A-induced hepatic enzyme impairment and histopathological disruption. Moreover, Dapan-treated mice had significantly lower malondialdehyde hepatic content and elevated reduced glutathione, superoxide dismutase, and total antioxidant capacity levels than non-treated mice in a dose-dependent manner. The Dapan-treated groups showed significantly lower levels of the inflammatory mediators, NLRP3, TNF-α, IL-6, and IL-1β, in addition to the immunomodulators CD8, CD4, INF-γ, and NFκB and inhibition of JNK and p38 MAPK levels compared to the Con A-treated group.

Our results showed that intraperitoneal administration of Dapan could be a therapeutic opportunity to inhibit the development of AIH via inhibition of inflammatory pathways.

Severe immune responses regulate the various clinical hepatic injuries, including autoimmune hepatitis and acute viral hepatitis. N6-methyladenosine (m6A) modification is a crucial regulator of immunity and inflammation. However, the precise role of YTHDF1 in T cell-mediated hepatitis remains incompletely characterized. To address this, we utilized Concanavalin A (ConA)-induced mouse liver damage as an experimental model for T cell-mediated hepatitis. Our findings found that hepatic YTHDF1 protein rapidly decreased during ConA-induced hepatitis, and YTHDF1-deficient (Ythdf1 -/- ) mice showed more susceptibility to ConA-induced liver injury, along with an intensified inflammatory storm accompanied by aggravated hepatic inflammatory response via ERK and NF-κB pathways. Interestingly, hepatic-specific over-expression or deletion of YTHDF1 exhibited redundancy in ConA-induced liver injury. Validation in bone marrow chimeric mice confirmed the necessity of YTHDF1 in hematopoietic cells for controlling the response to ConA-induced hepatitis. Additionally, our data revealed that YTHDF1 deletion in macrophages exacerbated the inflammatory response induced by lipopolysaccharide. In summary, our study uncovered that YTHDF1 deficiency exacerbates the immune response in ConA-induced hepatitis by modulating the expression of inflammatory mediators, highlighting the potential of YTHDF1 as a therapeutic target for clinical hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune disease mediated by abnormal autoimmune. The pathogenesis and pathological manifestation of immune-mediated liver injury, induced by concanavalin A (ConA) in mice, closely parallel those observed in human AIH. However, the sensitivity and stability of mice to ConA vary depending on the strain and sex of the mice. Therefore, this study aimed to compare the sensitivity and stability of Balb/c, C57BL/6J, and ICR mice to ConA-induced acute liver injury. In this study, the mice in ConA group were injected with ConA (15 mg/kg·bw) via tail vein. After 8 h, the blood, liver, and spleen were collected for subsequent analysis. The liver index of Balb/c mice was increased (P < 0.05). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels of male C57BL/6J mice in ConA-treated group were the highest among the three strains of mice, followed by female Balb/c mice (P < 0.05). After ConA challenge, ICR, Balb/c, and C57BL/6J mice (both male and female) appeared markedly inflammatory cell infiltration and hepatocyte necrosis. Furthermore, hemorrhagic necrosis is more severe in females than in males. Lastly, male C57BL/6J and female Balb/c mice had the lowest coefficient of variation in serum ALT, AST, and LDH activities, while female Balb/c mice had the minimum coefficient of variation of the liver index, suggesting that they have good stability to ConA. Altogether, our study found that Balb/c female and C57BL/6J male mice have high sensitivity and good stability to ConA challenge, which were suitable for mimicking the pathology of AIH in humans.

The rising prevalence of autoimmune hepatitis (AIH) and its intricate pathogenesis has escalated it to a global health issue. This study centered on investigating the effects of allyl methyl disulfide (AMDS) against concanavalin A (ConA)-induced AIH in mice and elucidate the possible mechanisms. Histopathology and blood biochemistry were performed to assess the protective effects of AMDS on ConA-challenged liver injury in C57BL/6 male mice. Then, Immunohistochemistry, Immunofluorescence, RT-qPCR, ELISA and Western blot assays were performed to test changes in the M1 polarization of macrophage and NLRP3 inflammasome activation. Additionally, J774A.1 and AML12 cells were co-cultured to further investigate protective mechanism of AMDS against AIH. We found that AMDS pretreatment significantly alleviated the elevation of the levels of liver injury marker enzymes, and liver pathological changes triggered by ConA. Additionally, AMDS antagonized liver neutrophil infiltration, liver macrophage M1 polarization, and the increase in serum IL-6 and TNF-α levels induced by ConA. Furthermore, the changes in protein and mRNA levels of crucial molecules in the NF-κB and NLRP3 inflammasome pathways after ConA challenge were restored by AMDS. Additionally, AMDS significantly ameliorated the ConA-induced morphological alterations, the release of IL-6 and TNF-α, and the activation of NLRP3 inflammasome pathway in J774A.1 macrophages. Lastly, in a conditioned co-culture system of AML12 and J774A.1 cells, administration of AMDS at a concentration of 25 μM prominently inhibited the mRNA levels of Tnf and Nos2 in AML12 cells. Collectively, AMDS ameliorates ConA-induced AIH by alleviating hepatic neutrophil infiltration, inhibiting M1-type macrophage polarization, and antagonizing NLRP3 inflammasome activation.

Liver fibrosis is potentially a reversible form of liver disease that evolved from the early stage of liver scarring as a consequence of chronic liver injuries. Recurrent injuries in the liver without any appropriate medication cause the injuries to get intense and deeper, which gradually leads to the progression of irreversible cirrhosis or carcinoma. Unfortunately, there are no approved treatment strategies for reversing hepatic fibrosis, making it one of the significant risk factors for developing advanced liver disorders and liver disease-associated mortality. Consequently, the interpretation of the fundamental mechanisms, etiology, and pathogenesis is crucial for identifying the potential therapeutic target as well as evaluating novel anti-fibrotic therapy. However, despite innumerable research, the functional mechanism and disease characteristics are still obscure. To accelerate the understanding of underlying disease pathophysiology, molecular pathways and disease progression mechanism, it is crucial to mimic human liver disease through the formation of precise disease models. Although various in vitro and in vivo liver fibrotic models have emerged and developed already, a perfect clinical model replicating human liver diseases is yet to be established, which is one of the major challenges in discovering proper therapeutics. This review paper will shed light on pathophysiology, signaling pathways, preclinical models of liver fibrosis, and their limitations.

Transforming growth factor-β (Tgf-β) contributes to the development of liver diseases through its regulation of various cell types. While Tgf-β signaling to hepatic stellate cells (HSCs) and hepatocytes was shown to mediate hepatic damage, the effect of Tgf-β on other cells in liver is yet to be clearly defined. Herein we identified a regulatory function of macrophage Tgf-β signaling in liver injury. We found that transgenic mice expressing constitutively active Tgf-β receptor type I (TβRI CA ) under the control of Fsp1-Cre (TβRI CA /Fsp1-Cre mice) were less susceptible to concanavalin A (conA)-induced autoimmune hepatitis. Liver tissue examination showed a decrease of necrotic area in conA-treated TβRI CA /Fsp1-Cre liver compared to those of wild-type mice. Blood test revealed that serum aminotransferases were significantly reduced in conA-treated TβRI CA /Fsp1-Cre mice as compared to those of wild-type mice. Immunohistochemistry for CD3 and myeloperoxidase demonstrated that there was a decreased accumulation of T cells and neutrophils, respectively, whereas ELISA showed that IL-4, IL-5, IL-10, IL-12 and IFN-γ was increased in livers of conA-treated TβRI CA /Fsp1-Cre mice. Alternatively activated macrophage (M2) polarization was significantly elevated in livers of conA-treated TβRI CA /Fsp1-Cre mice as indicated by enhanced hepatic expression of CCR2 and CD206 as well as increased numbers of liver macrophages expressing M2 subtype marker, CD163. qPCR analysis indicated an increased expression of TβRI CA , Arg1, Ym1, CD206, Snail1, Foxo1 and IRF4 as well as a decreased expression of MHC class II and CD1d in liver macrophages that were isolated from TβRI CA /Fsp1-Cre mice. Moreover, flow cytometry analysis showed a lower number of NKT cells in livers of conA-treated TβRI CA /Fsp1-Cre mice when compared to those of wild-type mice. In conclusion, Fsp1-Cre-mediated expression of TβRI CA lead to a decreased conA-induced liver injury that was associated with enhanced M2 macrophage polarization and reduced NKT cell recruitment.

Extracellular vesicles (EVs) are generated in all cells. Systemic administration of allogenic EVs derived from epithelial and mesenchymal cells has been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cell-derived EVs can be modified to acquire the capacity to induce an immune response, we engineered 293T EVs to harbor the immunomodulatory molecules CD80, OX40L, and PD-L1. We demonstrated abundant levels of these proteins in the engineered cells and EVs. Functionally, the engineered EVs efficiently elicited positive and negative costimulation of human and murine T cells. In the setting of cancer and autoimmune hepatitis, the engineered EVs modulated T cell functions and altered disease progression. OX40L EVs also provided enhanced antitumor activity in combination with anti-CTLA-4 in melanoma-bearing mice. In addition, we added multiple immunomodulatory proteins in EVs (EVmIM), attempting to elicit an immune response in both lymphoid and myeloid compartments. The EVmIM containing CD80, 4-1BBL, CD40L, CD2, and CD32 engaged both T cells and antigen presenting cells (APCs) in melanoma tumors, demonstrating the capacity for EVmIM to elicit antitumor activity. Our work provides evidence that EVs can be engineered to induce specific immune responses with translational potential to modulate immune cell functions in pathological settings.

Autoimmune hepatitis (AIH) is an immune-mediated hepatic disease associated with intense complications. AIH is more common in females and needs effective drugs to treat. Guizhi Fuling Wan (GZFLW) is a traditional Chinese herbal formula for treating various gynecologic diseases. In this study, we aim to extend the new use of GZFLW for AIH.

The tandem MS-based analysis was used to identify secondary metabolites in GZFLW. Therapeutic effects of GZFLW were tested in a concanavalin A (Con A)-induced AIH model in mice. Ethnopharmacological mechanisms underlying the antiapoptotic, antioxidant, and immunomodulatory protective effects were determined.

Oral administration of GZFLW attenuates AIH in a Con A-induced hepatotoxic model in vivo. The tandem MS-based analysis identified 15 secondary metabolites in GZFLW. The Con A-induced AIH syndromes, including hepatic apoptosis, inflammation, reactive oxygen species accumulation, function failure, and mortality, were significantly alleviated by GZFLW in mice. Mechanistically, GZFLW restrained the caspase-dependent apoptosis, restored the antioxidant system, and decreased pro-inflammatory cytokine production in the livers of Con A-treated mice. Besides, GZFLW repressed the Con A-induced hepatic infiltration of inflammatory cells, splenic T cell activation, and splenomegaly in mice.

Our findings demonstrate the applicable potential of GZFLW in treating AIH. It prompts further investigation of GZFLW as a treatment option for AIH and possibly other hepatic diseases.

Hepatic fibrosis (HF) is an important pathological state in the progression of chronic liver disease to end-stage liver disease and is usually triggered by alcohol, nonalcoholic fatty liver, chronic hepatitis viruses, autoimmune hepatitis (AIH), or cholestatic liver disease. Research on novel therapies has become a hot topic due to the reversibility of HF. Research into the molecular mechanisms of the pathology of HF and potential drug screening relies on reliable and rational biological models, mainly including animals and cells. Hence, a number of modeling approaches have been attempted based on human dietary, pathological, and physiological factors in the development of HF. In this review, classical and novel methods of modeling HF in the last 10 years were collected from electronic databases, including Web of Science, PubMed, ScienceDirect, ResearchGate, Baidu Scholar, and CNKI. Animal models of HF are usually induced by chemical toxicants, special diets, pathogenic microorganisms, surgical operations, and gene editing. The advantages and limitations of hepatic stellate cells (HSCs), organoids, and 3D coculture-based HF modeling methods established in vitro were also proposed and summarized. This information provides a scientific basis for the discovery of the pathological mechanism and treatment of HF.

Turtle hepatocytes are a nonexcitable model for metabolic depression during low-temperature and/or anoxic overwintering conditions. Cytoskeletal structure and mitochondrial distribution are continuously modified in cells, and we hypothesized that metabolic depression would inhibit such processes as cell attachment and spreading and promote withdrawal of cell protrusions and peripheral mitochondria. After developing a methodology for culturing painted turtle hepatocytes, two-dimensional (2-D) area and maintenance of cell attachment after a media change were used as indicators of structural rearrangement and spreading/volume. These were measured after incubating cells at varying temperatures and with or without the inclusion of cyanide (chemical proxy for anoxia). Experiments were performed using cells from 22°C- or 5°C-acclimated turtles. Live-cell imaging was used to monitor the effect of cyanide exposure on the distribution of mitochondria. We also acclimated cultured cells from 22°C-acclimated turtles to 4°C in vitro and scored withdrawal of protrusions. Only cells isolated from 5°C-acclimated turtles and incubated at 4°C had reduced attachment to fibronectin substrate, but cyanide exposure had no effect. These cells also had a 30% smaller 2-D area than those from 22°C-acclimated turtles. There was no change in mitochondrial distribution during cyanide perfusion. Finally, 4°C acclimation in vitro resulted in the withdrawal of protrusions over 14 days. Taken together with the results from cells acclimated to low temperature in vivo, this suggests inhibition of structural rearrangement and protrusion stability by low temperature acclimation, but not cyanide exposure. Our cultured primary hepatocyte system will facilitate further study of the role of structural dynamics in reversible metabolic depression.NEW & NOTEWORTHY We have optimized a methodology for two-dimensional (2-D) culturing of primary western painted turtle hepatocytes and used this model to study the effects of cyanide and temperature on structural rearrangement, and the effect of cyanide on mitochondrial distribution. Our results suggest that low temperature acclimation, either in vivo or in vitro, inhibits cell protrusions and structural rearrangement. Acute cyanide exposure did not inhibit structural rearrangement or alter mitochondrial distribution.

Recently, the number of patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and its more advanced condition, metabolic dysfunction-associated steatohepatitis (MASH), has been increasing. These patients are at a higher risk of cardiovascular events and thromboembolism. However, the direct impact of high-fat diet (HFD), a cause of MASLD, on liver coagulation function is not well understood. Previously, we demonstrated that a short-term, 4-day intake of a HFD exacerbates concanavalin A (Con A)-induced acute liver injury in mice by promoting coagulation and inflammation. This model demonstrates that the liver exposed to a short-term HFD is vulnerable even before disease onset. In this study, using this model, we elucidated the detailed mechanisms by which short-term HFD intake promotes coagulation, considering primary and secondary hemostasis.

C57BL/6 mice normally fed a normal diet (ND) were subjected to a HFD for 4 days. Liver tissue and blood samples were collected before and 4 and 24 h after Con A administration. Histological analysis, flow cytometry for platelet analysis, and blood coagulation tests related to secondary hemostasis were performed.

Even with short-term consumption of a HFD alone, platelet and fibrinogen levels increased in the peripheral blood and liver. Additionally, when Con A was administered to mice on a short-term HFD, an increase in P-selectin expression was observed in the liver, with no upregulation in peripheral blood platelets. Furthermore, in mice subjected to a short-term HFD and treated with Con A, prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed.

Consuming a HFD in short-term can enhance primary and secondary hemostasis, thereby increasing the risk of thrombosis. These conditions are presumed to be a risk factor that exacerbates Con A-induced liver injury. The findings provide insight into early intervention strategies for chronic liver diseases, such as MASLD and MASH.

Autoimmune hepatitis (AIH), an immune-mediated liver injury, plays an important role in the development and pathogenesis of several liver diseases. However, therapeutic alternatives for the treatment of AIH remain limited. Zingerone (ZIN) is a natural non-toxic phenolic compound extracted from ginger that possesses various pharmacological activities. Thus, this study aimed to investigate the effect of ZIN on AIH using a mouse model of acute liver injury induced by concanavalin A (Con A). To establish liver injury, C57BL/6J mice were intraperitoneally administered ZIN, followed by 20 mg/kg Con A after 3 h. Thereafter, the liver and serum were collected for analysis. The results revealed that ZIN pretreatment significantly suppressed the elevation of liver injury markers induced by Con A exposure and improved the survival of mice. Additionally, ZIN significantly ameliorated liver histopathological injury, hepatocyte apoptosis, and oxidative stress. Notably, ZIN inhibited hepatic M1 macrophage polarization and decreased the expression of M1 macrophage-associated pro-inflammatory genes and cytokines, including interleukin-1β (IL-1β), IL-12, IL-6, and tumor necrosis factor-α (TNF-α). Western blotting analysis indicated that ZIN inhibited the phosphorylation of extracellular receptor kin, c-Jun N-terminal kinase, and p65 in vitro. Taken together, these results suggest that ZIN exerts a protective effect in the Con A-induced acute liver injury model by inhibiting M1 macrophage polarization and suppressing NF-κB, mitogen-activated protein kinase, and interferon regulatory factor signaling pathways. This highlights the possibility of using ZIN as a safe drug for the treatment of liver injury and provides a novel therapeutic direction for clinical studies on liver diseases.

To explore the prophylactic and therapeutic effects of Alhagi maurorum ethanolic extract (AME) in concanavalin A (Con A)-induced hepatitis (CIH) as well as possible underlying mechanisms.

Polyphenols in AME were characterized using high performance liquid chromatography (HPLC). Swiss albino mice were divided into 4 groups. Normal group received intravenous phosphate-buffered saline (PBS); Con A group received 40 mg/kg intravenous Con A. Prophylaxis group administered 300 mg/(kg·d) AME orally for 5 days before Con A intervention. Treatment group received intravenous Con A then administered 300 mg/kg AME at 30 min and 3 h after Con A intervention. After 24 h of Con A injection, hepatic injury, oxidative stress, and inflammatory mediators were assessed. Histopathological examination and markers of apoptosis, inflammation, and CD4+ cell infiltration were also investigated.

HPLC analysis revealed that AME contains abundant polyphenols with pharmacological constituents, such as ellagic acid, gallic acid, ferulic acid, methylgallate, and naringenin. AME alleviated Con A-induced hepatic injury, as manifested by a significant reduction in alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase (P<0.01). Additionally, the antioxidant effect of AME was revealed by a significant reduction in oxidative stress markers (nitric oxide and malondialdehyde) and restored glutathione (P<0.01). The levels of proinflammatory cytokines (tumor necrosis factor-α, interferon-γ, and interleukin-6) and c-Jun N-terminal kinase (JNK) activity were reduced (P<0.01). Histopathological examination of liver tissue showed that AME significantly ameliorated necrotic and inflammatory lesions induced by Con A (P<0.01). Moreover, AME reduced the expression of nuclear factor kappa B, pro-apoptotic protein (Bax), caspase-3, and CD4+ T cell hepatic infiltration (P<0.01). The expression of anti-apoptotic protein Bcl-2 was increased (P<0.01).

AME has hepatoprotective and ameliorative effects in CIH mice. These beneficial effects are likely due to the anti-inflammatory, antioxidant, and anti-apoptotic effects of the clinically important polyphenolic content. AME could be a novel and promising hepatoprotective agent for managing immune-mediated hepatitis.

The complex interaction between the immune system and autoinflammatory disorders highlights the centrality of autoimmune mechanisms in the pathogenesis of autoinflammatory diseases. With the exploration of PSTPIP2, it has been discovered to play an inhibitory role in immune diseases, suggesting its potential utility in the research and treatment of rheumatic diseases. This review outlines the mechanisms of PSTPIP2 in chronic multifocal osteomyelitis (CMO), rheumatoid arthritis (RA), synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome, liver diseases, renal diseases, pressure ulcer sepsis and diabetic obesity. The mechanisms include inhibiting the IL-1β inflammatory responses, NF-κB, ERK phosphorylation etc., promoting Erβ, and modulating the polarization of macrophage to prevent the inflammatory diseases. This review summarized current findings and offered perspectives on future research directions, laying a foundation for applying of PSTPIP2 in inflammatory diseases.

Galectin-1 is implicated in several pro-tumourigenic mechanisms and is considered immune-suppressive. The pharmacological inhibition of galectin-1 may be beneficial in cancers in which galectin-1 is overexpressed and driving cancer progression. This study aimed to further characterise the immunosuppressive cytokines influenced by galectin-1 in in vitro immune cell cultures and an in vivo inflammatory model using a recently discovered selective inhibitor of galectin-1, GB1908. To enable a translational approach and link mouse and human pharmacology, anti-CD3/anti-CD28 stimulated T cells cultured from human whole blood and mouse spleens were compared. For in vivo studies of T cell-mediated inflammation, the concanavalin-A (Con-A) mouse model was used to induce a T lymphocyte-driven acute liver injury phenotype. The inhibition of galectin-1 with GB1908 reduced IL-17A, IFNγ and TNFα in a concentration-dependent manner in both mouse and human T cells in vitro. The immunosuppressive cytokines measured in Con-A-treated mice were all upregulated compared to naïve mice. Subsequently, mice treated with GB1908 demonstrated a significant reduction in IL-17A, IFNγ, IL-6 and TNFα compared to vehicle-treated mice. In conclusion, galectin-1 induced the production of several important immune-suppressive cytokines from T cells in vitro and in vivo. This result suggests that, in the context of cancer therapy, a selective galectin-1 could be a viable approach as a monotherapy, or in combination with chemotherapeutic agents and/or checkpoint inhibitors, to enhance the numbers and activity of cytotoxic T cells in the tumour microenvironment of high galectin-1 expressing cancers.

Rhoifolin (ROF) exhibits a diverse range of biological activities, encompassing anticancer, hepatoprotective, antidiabetic, antirheumatic, and antiviral properties. However, the specific protective effects and possible mechanisms of the compound against T-cell-mediated autoimmune hepatitis have not been previously elucidated. In the present study, adult male mice were administered Con A (20 mg/kg, intravenously) for 8 h. In the treated groups, mice were pretreated with ROF daily (20 mg/kg and 40 mg/kg, orally) for 7 days before Con A intoxication. The results showed that ROF significantly decreased serum biochemical indices (ALT, AST, ALP, and LDH) and regulated related oxidative stress indicators (MDA, SOD, and GSH), reduced hepatic necrosis areas and immune cells infiltration, inhibited the release of various inflammatory factors (TNF-α, IFN-γ, IL-2, and IL-17), and improved hepatic tissue apoptosis, thereby alleviating hepatic damage induced by Con A. Additionally, we have also confirmed that ROF efficiently inhibited Th1/Th17 cells polarization via modulation of the JAK2/JAK3/STAT1/STAT3 signaling pathways both in vivo and in vitro. Moreover, the molecular mechanism examination also demonstrated that ROF regulated apoptotic cascade signaling through IL-6/JAK2/STAT1/STAT3 controlling BNIP3 activity in primary hepatocytes. These effects were in good agreement with the bioinformatics analysis of ROF treatment for AIH. In conclusion, our findings provide new insights into the potential use of ROF for AIH therapy, which may result from the specific regulation of the T cell subtype polarization and the apoptosis of liver cells via modulation of the JAKs/STATs signaling pathways.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is spreading worldwide, largely due to unhealthy lifestyles that contribute to the rise in diabetes, metabolic syndrome, and obesity. In this situation, the progression of injury to metabolic steatohepatitis can evolve to cirrhosis and, eventually, to hepatocellular carcinoma (HCC). It is well known that serine protease enzymes with different functions in cellular homeostasis act as signaling molecules that regulate liver inflammation by activating the protease-activated receptors (PARs) family members, expressed on the cellular plasma membrane. Among them, PAR2 plays a central role in the activation of signaling pathways in response to changes in the extracellular microenvironment. Experimental data have provided evidence that PAR2 is involved not only in inflammatory response but also in insulin resistance, lipid metabolism, and cancer. The major aims of this narrative review are addressed to assess PAR2 involvement in inflammation, metabolism, and liver disease progression and to explore possible therapeutic strategies, based on PAR2 inhibition, in order to prevent its biological effects in the context of MAFLD and cancer.

Immune-mediated liver injury is a common characteristic of various liver diseases, including autoimmune and viral hepatitis. Here, we investigated the role of DEAD-box helicase 3, X-linked (DDX3X) in immune-mediated liver injury. Liver injury was induced in C57BL/6J mice via concanavalin A (Con A). DDX3X hepatocyte-specific knockout (DDX3XΔHep) mice and control (DDX3Xfl/fl) mice were utilized to investigate the role of DDX3X in liver injury. Primary hepatocytes were treated with tunicamycin (TM) to induce ER stress in vitro. The expression of DDX3X in patients with various liver diseases was evaluated. Hepatic DDX3X expression increased, and DDX3X translocated from the cytoplasm to the nucleus during Con A-induced liver injury. DDX3X deficiency ameliorated mouse liver injury and reduced ER stress in liver tissue. The inhibition of ER stress with 4-PBA significantly attenuated liver injury while decreasing DDX3X levels in liver tissue. However, the upregulation of hepatic DDX3X expression reversed Con A-induced liver injury and negated the protective effect of 4-PBA. Mechanistically, the nuclear translocation of DDX3X promoted ER stress-induced apoptosis through the transcriptional induction of CHOP. Moreover, DDX3X was elevated and translocated into the nucleus in patients with HBV-LF and AIH. Additionally, serum DDX3X levels markedly increased in patients with HBV-LF, and a consistent decrease in DDX3X was associated with a good prognosis. The cytoplasmic-to-nuclear translocation of DDX3X promotes ER stress-induced apoptosis, which is an obligatory step that drives hepatic necrosis and tissue damage. Notably, DDX3X is a potential therapeutic target for immune-mediated liver injury.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors.

Astaxanthin (ASX) is an oxygen-containing non-vitamin A carotenoid pigment. However, the role of ASX in autoimmune hepatitis (AIH) remains unclear. In this study, a mouse model of AIH is established induced by concanavalin A (ConA). Mass cytometry and single-cell RNA sequencing (scRNA-seq) are used to analyze the potential role of ASX in regulating the immune microenvironment of AIH. ASX treatment effectively alleviated liver damage induced by ConA and downregulated pro-inflammatory cytokines production in mice. Mass cytometry and scRNA-seq analyses revealed a significant increase in the number of CD8+ T cells following ASX treatment. Functional markers of CD8+ T cells, such as CD69, MHC II, and PD-1, are significantly downregulated. Additionally, specific CD8+ T cell subclusters (subclusters 4, 13, 24, and 27) are identified, each displaying distinct changes in marker gene expression after ASX treatment. This finding suggests a modulation of CD8+ T cell function by ASX. Finally, the key transcription factors for four subclusters of CD8+ T cells are predicted and constructed a cell-to-cell communication network based on receptor-ligand interactions probability. In conclusion, ASX holds the potential to ameliorate liver damage by regulating the number and function of CD8+ T cells.

Acute liver injury is a life-threatening disease. Although immune responses are involved in the development and exacerbation of acute liver injury, the cellular and molecular mechanisms are not fully understood. Intravenous administration of the plant lectin concanavalin A (ConA) is widely used as a model of acute liver injury. ConA triggers T cell activation and cytokine production by crosslinking glycoproteins, including the T cell receptor, leading to the infiltration of myeloid cells into the liver and the subsequent amplification of inflammation in the liver. Thus, the pathogenesis of ConA-induced acute liver injury is considered a model of immune-mediated acute liver injury or autoimmune hepatitis in humans. However, the severity of the liver injury and the analyses of immune cells and non-hematopoietic cells in the liver following ConA injection are significantly influenced by the experimental conditions. This article outlines protocols for ConA-induced acute liver injury in mice and evaluation methods for liver injury, immune cells, and non-hematopoietic cells in the liver. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Induction of acute liver injury by ConA injection Basic Protocol 2: Evaluation of inflammatory cytokines in mouse plasma Basic Protocol 3: Preparation of liver sections and histological analysis of liver injury Basic Protocol 4: Preparation of liver immune cells Basic Protocol 5: Preparation of hepatocytes, endothelial cells, and hepatic stellate cells Basic Protocol 6: Flow cytometry of immune and non-hematopoietic liver cells Basic Protocol 7: Flow cytometric sorting of endothelial cells and hepatic stellate cells Basic Protocol 8: Quantitative reverse transcription polymerase chain reaction.

Liver-associated diseases affect millions of individuals worldwide. In developed countries, the incidence of viral hepatitis is reducing due to advancements in disease prevention, diagnosis, and treatment. However, with improvements in living standards, the prevalence of metabolic liver diseases, such as non-alcoholic fatty liver disease and alcohol-related liver disease, is expected to increase; notably, this rise in the prevalence of metabolic liver disease can lead to the development of more severe liver diseases, including liver failure, cirrhosis, and liver cancer. The growing demand for natural alternative therapies for chronic diseases has highlighted the importance of studying the pharmacology of bioactive compounds in plants. One such compound is oleanolic acid (OA), a pentacyclic triterpenoid known for its antioxidant, anti-inflammatory, anti-ulcer, antibacterial, antiviral, antihypertensive, anti-obesity, anticancer, anti-diabetic, cardioprotective, hepatoprotective, and anti-neurodegenerative properties. Recent studies have demonstrated that OA treatment can reduce the risk of pathological liver damage, ultimately alleviating liver dysregulation and restoring overall liver function. This review aims to explore the latest research on the biological effects of OA and its derivatives. Notably, it explores the mechanisms of action of these compounds in both in vitro and in vivo research models and, ultimately, highlights OA as a promising candidate for alternative therapies in the treatment and management of chronic liver disease.

Acute liver failure (ALF) is a life-threatening disorder that progresses from self-limiting acute liver injury (ALI). Microcirculatory disturbance characterized by sinusoidal hypercoagulation and subsequent massive hypoxic hepatocyte damage have been proposed to be the mechanism by which ALI deteriorates to ALF; however, the precise molecular pathway of the sinusoidal hypercoagulation remains unknown. Here, we analyzed ALI patients and mice models to uncover the pathogenesis of ALI with microcirculatory disturbance.

We conducted a single-center retrospective study for ALI and blood samples and liver tissues were analyzed to evaluate the microcirculatory disturbance in ALI patients (n = 120). Single-cell RNA sequencing analysis (scRNA-seq) was applied to the liver from the concanavalin A (Con A)‑induced mouse model of ALI. Interferon-gamma (IFNγ) and tumor necrosis factor-alpha knockout mice, and primary human liver sinusoidal endothelial cells (LSECs) were used to assess the mechanism of microcirculatory disturbance.

The serum IFNγ concentrations were significantly higher in ALI patients with microcirculatory disturbance than in patients without microcirculatory disturbance, and the IFNγ was upregulated in the Con A mouse model which presented microcirculatory disturbance. Hepatic IFNγ expression was increased as early as 1 hour after Con A treatment prior to sinusoidal hypercoagulation and hypoxic liver damage. scRNA-seq revealed that IFNγ was upregulated in innate lymphoid cells and stimulated hepatic vascular endothelial cells at the early stage of liver injury. In IFNγ knockout mice treated with Con A, the sinusoidal hypercoagulation and liver damage were remarkably attenuated, concomitant with the complete inhibition of CD40 and tissue factor (TF) upregulation in vascular endothelial cells. By ligand-receptor analysis, CD40-CD40 ligand interaction was identified in vascular endothelial cells. In human LSECs, IFNγ upregulated CD40 expression and TF was further induced by increased CD40-CD40 ligand interaction. Consistent with these findings, hepatic CD40 expression was significantly elevated in human ALI patients with microcirculatory disturbance.

We identified the critical role of the IFNγ-CD40 axis as the molecular mechanism of microcirculatory disturbance in ALI. This finding may provide novel insights into the pathogenesis of ALI and potentially contribute to the emergence of new therapeutic strategies for ALI patients.

Macrophages, as important immune cells of the organism, are involved in maintaining intrahepatic microenvironmental homeostasis and can undergo rapid phenotypic changes in the injured or recovering liver. In recent years, the crucial role of macrophage-programmed cell death in the development and regression of liver diseases has become a research hotspot. Moreover, macrophage-targeted therapeutic strategies are emerging in both preclinical and clinical studies. Given the macrophages' vital role in complex organismal environments, there is tremendous academic interest in developing novel therapeutic strategies that target these cells. This review provides an overview of the characteristics and interactions between macrophage polarization, programmed cell death, related biomarkers, and macrophage-targeted therapies. It aims to deepen the understanding of macrophage immunomodulation and molecular mechanisms and to provide a basis for the treatment of macrophage-associated liver diseases.

Type 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing β-cells. As insulin facilitates glucose absorption into cells and tissues, β-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt β-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation's effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue.

We utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in β-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and β-cell survival.

We discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in β-cells offers protection against β-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in β-cells, offering a promising strategy for T1D.

This study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Previous data indicated that nicotine could modulate the immune regulatory potential of mesenchymal stem cells (MSCs). Currently, we intend to assess the effects of a conditioned medium of nicotine-pulsed mesenchymal stem cells in the experimental model of autoimmune hepatitis (AIH). Bone marrow-derived MSCs pulsed with 0,.1,.5, or 1 μM nicotine until the cells reached 90% confluency. Correspondent to in vitro results, the least effective concentration of nicotine that led to an anti-inflammatory environment by the MSC-conditioned medium was 0.5 μM. The murine model of AIH induced by Intravenous injection Concanavalin A (ConA). Mice were allocated to pretreatment (Concomitant treatment with ConA administration) or treatment groups and received un-pulsed MSC-conditioned medium (CM) or conditioned medium of nicotine (0.5 µM)-pulsed MSCs (CMN). The levels of ALT, AST, MPO, TNF-α, IFN-γ, and IL-6 were the highest in the ConA group than in the other groups. Pretreatment or treatment with the CMN caused a significant reduction in hepatic enzymes and inflammatory cytokines compared to pretreatment or treatment with CM. Both CM or CMN significantly decreased the numbers of activated TCD4+ and TCD8+ in the blood. More importantly, pre-treatment or treatment with CMN caused a better improvement in the histopathological appearance than pre-treatment or treatment with CM. The results of this study show that CMN rapidly controls the AIH mouse model, and therefore it may be considered as a new therapeutic approach for the treatment of AIH patients.

The protease activated receptor 2 (Par2) plays a pivotal role in various damage models, influencing injury, proliferation, inflammation, and regeneration. Despite extensive studies, its binary roles- EITHER aggravating injury or promoting recovery-make a conclusive translational decision on its modulation strategy elusive. Analyzing two liver regeneration models, autoimmune hepatitis and direct hepatic damage, we discovered Par2's outcome depends on the injury's nature. In immune-mediated injury, Par2 exacerbates damage, while in direct tissue injury, it promotes regeneration. Subsequently, we evaluated the clinical significance of this finding by investigating Par2's expression in the context of autoimmune diabetes. We found that the absence of Par2 in all lymphocytes provided full protection against the autoimmune destruction of insulin-producing β-cells in mice, whereas the introduction of a β-cell-specific Par2 null mutation accelerated the onset of autoimmune diabetes. This pattern led us to hypothesize whether these observations are universal. A comprehensive review of recent Par2 publications across tissues and systems confirms the claim drafted above: Par2's initial activation in the immune system aggravates inflammation, hindering recovery, whereas its primary activation in the damaged tissue fosters regeneration. As a membrane-anchored receptor, Par2 emerges as an attractive drug target. Our findings highlight a crucial translational modulation strategy in regenerative medicine based on injury type.

FICZ (6-formylindolo[3,2-b]carbazole) is a potent aryl hydrocarbon receptor agonist that has a poorly understood function in the regulation of inflammation. In this study, we investigated the effect of aryl hydrocarbon receptor activation by FICZ in a murine model of autoimmune hepatitis induced by concanavalin A. High-throughput sequencing techniques such as single-cell RNA sequencing and assay for transposase accessible chromatin sequencing were used to explore the mechanisms through which FICZ induces its effects. FICZ treatment attenuated concanavalin A-induced hepatitis, evidenced by decreased T-cell infiltration, decreased circulating alanine transaminase levels, and suppression of proinflammatory cytokines. Concanavalin A revealed an increase in natural killer T cells, T cells, and mature B cells upon concanavalin A injection while FICZ treatment reversed the presence of these subsets. Surprisingly, concanavalin A depleted a subset of CD55+ B cells, while FICZ partially protected this subset. The immune cells showed significant dysregulation in the gene expression profiles, including diverse expression of migratory markers such as CCL4, CCL5, and CXCL2 and critical regulatory markers such as Junb. Assay for transposase accessible chromatin sequencing showed more accessible chromatin in the CD3e promoter in the concanavalin A-only group as compared to the naive and concanavalin A-exposed, FICZ-treated group. While there was overall more accessible chromatin of the Adgre1 (F4/80) promoter in the FICZ-treated group, we observed less open chromatin in the Itgam (CD11b) promoter in Kupffer cells, supporting the ability of FICZ to reduce the infiltration of proinflammatory cytokine producing CD11b+ Kupffer cells. Taken together, these data demonstrate that aryl hydrocarbon receptor activation by FICZ suppresses liver injury through the limitation of CD3+ T-cell activation and CD11b+ Kupffer cell infiltration.

Four years after its outbreak, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global challenge for human health. At its surface, SARS-CoV-2 features numerous extensively glycosylated spike proteins. This glycan coat supports virion docking and entry into host cells and at the same time renders the virus less susceptible to neutralizing antibodies. Given the high genetic plasticity of SARS-CoV-2 and the rapid emergence of immune escape variants, targeting the glycan shield by carbohydrate-binding agents emerges as a promising strategy. However, the potential of carbohydrate-targeting reagents as viral inhibitors remains underexplored. Here, we tested seven plant-derived carbohydrate-binding proteins, called lectins, and one crude plant extract for their antiviral activity against SARS-CoV-2 in two types of human lung cells: A549 cells ectopically expressing the ACE2 receptor and Calu-3 cells. We identified three lectins and an Allium porrum (leek) extract inhibiting SARS-CoV-2 infection in both cell systems with selectivity indices (SI) ranging between >2 and >299. Amongst these, the lectin Concanavalin A (Con A) exerted the most potent and broad activity against a panel of SARS-CoV-2 variants. We used multiplex super-resolution microscopy to address lectin interactions with SARS-CoV-2 and its host cells. Notably, we discovered that Con A not only binds to SARS-CoV-2 virions and their host cells, but also causes SARS-CoV-2 aggregation. Thus, Con A exerts a dual mode-of-action comprising both, antiviral and virucidal, mechanisms. These results establish Con A and other plant lectins as candidates for COVID-19 prevention and basis for further drug development.

Acute hepatitis is a progressive inflammatory disorder that can lead to liver failure. Endothelial permeability is the vital pathophysiological change involved in infiltrating inflammatory factors. DDX24 has been implicated in immune signaling. However, the precise role of DDX24 in immune-mediated hepatitis remains unclear. Here, we investigate the phenotype of endothelium-targeted Ddx24 conditional knockout mice with Concanavalin A (ConA)-induced hepatitis.

Mice with homozygous endothelium-targeted Ddx24 conditional knockout (Ddx24flox/flox; Cdh5-Cre+) were established using the CRISPR/Cas9 mediated Cre-loxP system. We investigated the biological functions of endothelial cells derived from transgenic mice and explored the effects of Ddx24 in mice with ConA-induced hepatitis in vivo. The mass spectrometry was performed to identify the differentially expressed proteins in liver tissues of transgenic mice.

We successfully established mice with endothelium-targeted Ddx24 conditional knockout. The results showed migration and tube formation potentials of murine aortic endothelial cells with DDX24 silencing were significantly promoted. No differences were observed between Ddx24flox/flox; Cdh5-Cre+ and control regarding body weight and length, pathological tissue change and embryogenesis. We demonstrated Ddx24flox/flox; Cdh5-Cre+ exhibited exacerbation of ConA-induced hepatitis by up-regulating TNF-α and IFN-γ. Furthermore, endothelium-targeted Ddx24 conditional knockout caused vascular hyper-permeability in ConA-injected mice by down-regulating vascular integrity-associated proteins. Mechanistically, we identified Ddx24 might regulate immune-mediated hepatitis by inflammation-related permeable barrier pathways.

These findings prove that endothelium-targeted Ddx24 conditional knockout exacerbates ConA-induced hepatitis in mice because of vascular hyper-permeability. The findings indicate a crucial role of DDX24 in regulating immune-mediated hepatitis, suggesting DDX24 as a potential therapeutic target in the disorder.

Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear.

To determine the role and mechanism of UGT1A1 in liver damage progression.

We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study.

Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism.

UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.

Immune responses in the liver are related to the development and progression of liver failure, and precise prediction of their behavior is important. Deconvolution is a methodology for estimating the immune cell proportions from the transcriptome, and it is mainly applied to blood-derived samples and tumor tissues. However, the influence of tissue-specific modeling on the estimation results has rarely been investigated. Here, we constructed a system to evaluate the performance of the deconvolution method on liver transcriptome data. We prepared seven mouse liver injury models using small-molecule compounds and established a benchmark dataset with corresponding liver bulk RNA-Seq and immune cell proportions. RNA-Seq expression for nine leukocyte subsets and four liver-associated cell types were obtained from the Gene Expression Omnibus to provide a reference. We found that the combination of reference cell sets affects the estimation results of reference-based deconvolution methods and established a liver-specific deconvolution by optimizing the reference cell set for each cell to be estimated. We applied this model to independent datasets and showed that liver-specific modeling is highly extrapolatable. We expect that this approach will enable sophisticated estimation from rich tissue data accumulated in public databases and to obtain information on aggregated immune cell trafficking.

Purinergic P2X4 receptor (P2X4R) has been shown to have immunomodulatory properties in infection, inflammation, and organ damage including liver regeneration and fibrosis. However, the mechanisms and pathophysiology associated with P2X4R during acute liver injury remain unknown. We used P2X4R-/- mice to explore the role of P2X4R in three different models of acute liver injury caused by concanavalin A (ConA), carbon tetrachloride, and acetaminophen. ConA treatment results in an increased expression of P2X4R in the liver of mice, which was positively correlated with higher levels of aspartate aminotransferase and alanine aminotransferase in the serum. However, P2X4R gene ablation significantly reduced the severity of acute hepatitis in mice caused by ConA, but not by carbon tetrachloride or acetaminophen. The protective benefits against immune-mediated acute hepatitis were achieved via modulating inflammation (Interleukin (IL)-1β, IL-6, IL-17A, interferon-γ, tumor necrosis factor-α), oxidative stress (malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase), apoptosis markers (Bax, Bcl-2, and Caspase-3), autophagy biomarkers (LC3, Beclin-1, and p62), and nucleotide oligomerization domain-likereceptorprotein 3(NLRP3) inflammasome-activated pyroptosis markers (NLRP3, Gasdermin D, Caspase-1, ASC, IL-1β). Additionally, administration of P2X4R antagonist (5-BDBD) or agonist (cytidine 5'-triphosphate) either improved or worsened ConA-induced autoimmune hepatitis, respectively. This study is the first to reveal that the absence of the P2X4 receptor may mitigate immune-mediated liver damage, potentially by restraining inflammation, oxidation, and programmed cell death mechanisms. And highlight P2X4 receptor is essential for ConA-induced acute hepatitis.

Immunomodulatory T cells play a pivotal role in protection against (auto)immune-mediated diseases that open perspectives for therapeutic modulation. However, how immune regulatory networks operate in vivo is less understood. To this end, we focused on FOXP3+CD4+CD25+ regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells, two lymphocyte populations that independently regulate adaptive and innate immune responses. In vitro, a functional interplay between Tregs and iNKT cells has been described, but whether Tregs modulate the function and phenotype of iNKT cell subsets in vivo and whether this controls iNKT-mediated autoimmunity is unclear. Taking advantage of the conditional depletion of Tregs, we examined the in vivo interplay between iNKT and Treg cells in steady state and in preclinical models of liver and gut autoimmunity. Under non-inflamed conditions, Treg depletion enhanced glycolipid-mediated iNKT cell responses, with a general impact on Type 1, 2 and 17 iNKT subsets. Moreover, in vivo iNKT activation in the absence of Tregs suppressed the induction of iNKT anergy, consistent with a reduction in programmed cell death receptor 1 (PD-1) expression. Importantly, we unveiled a clear role for an in vivo Treg-iNKT crosstalk both in concanavalin A-induced acute hepatitis and oxazolone-induced colitis. Here, the absence of Tregs led to a markedly enhanced liver and gut pathology, which was not observed in iNKT-deficient mice. Taken together, these results provide evidence for a functional interplay between regulatory T cell subsets critical in controlling the onset of autoimmune disease.

Epstein-Barr virus (EBV) is often kept silent and asymptomatic; however, its reactivation induces a chronic and/or recurrent infection that is associated with numerous diseases, including cancer and inflammation-related disorders. As no specific treatment is currently available, the immune factors-based micro-immunotherapy (MI) medicine 2LEBV® could be considered a valuable therapeutic option to sustain the immune system in EBV reactivation.

The present work aimed to investigate, for the first time, the effect of 2LEBV® in several in vitro models of uninfected immune-related cells.

2LEBV® displayed phagocytosis-enhancing capabilities in granulocytes. In human peripheral blood mononuclear cells (PBMCs), it increased the intra- and extra-cellular expression of interleukin (IL)-2. Moreover, it modulated the secretion of other cytokines, increasing IL-4, IL-6, and tumor necrosis factor-α levels or lowering other cytokines levels such as IL-9. Finally, 2LEBV® reduced the expression of human leukocyte antigen (HLA)-II in endothelial cells and macrophages.

Although these data are still preliminary and the chosen models do not consider the underlying EBV-reactivation mechanisms, they still provide a better understanding of the mechanisms of action of 2LEBV®, both at functional and molecular levels. Furthermore, they open perspectives regarding the potential targets of 2LEBV® in its employment as a therapeutic intervention for EBV-associated diseases.

The nuclear receptor liver receptor homolog-1 (LRH-1) has been shown to promote apoptosis resistance in various tissues and disease contexts; however, its role in liver cell death remains unexplored. Hepatocyte-specific deletion of LRH-1 causes mild steatosis and inflammation but unexpectedly shields female mice from tumor necrosis factor (TNF)-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes show markedly attenuated estrogen receptor alpha and elevated nuclear factor κB (NF-κB) activity, while LRH-1 overexpression inhibits NF-κB activity. This inhibition relies on direct physical interaction of LRH-1's ligand-binding domain and the Rel homology domain of NF-κB subunit RelA. Mechanistically, increased transcription of anti-apoptotic NF-κB target genes and the proteasomal degradation of pro-apoptotic BCL-2 interacting mediator of cell death prevent mitochondrial apoptosis and ultimately protect mice from TNF-induced liver damage. Collectively, our study emphasizes LRH-1 as a critical, sex-dependent regulator of cell death and inflammation in the healthy and diseased liver.

Liverworts provide valuable ecological services to improve the sustainability of agriculture, encompassing soil health maintenance and natural pest management. Some liverworts have potential applications in medicine and as food additives. Twenty-two novel diterpenoids (anajoerins A-V), of which anajoerins B-G are rearranged labdanes featuring an unprecedented 6/5 fused ring system, were isolated from the Chinese liverwort Anastrophyllum joergensenii Schiffn. The absolute configurations of all compounds were identified based on high-resolution electrospray ionization mass spectroscopy data, NMR spectra, and ECD calculations. Plausible biogenetic pathways for unprecedented rearranged labdanes were proposed. Seven diterpenoids exhibited anti-inflammatory activity by reducing nitric oxide production in LPS-stimulated RAW264.7 murine macrophages in a dose-dependent manner with IC50s between 9.71 and 56.56 μM. All tested compounds showed no cytotoxicity at the tested concentrations. Western blot analyses of NF-κB p65 downregulation showed that anajoerin L could inhibit the NF-κB signaling pathway. Furthermore, anajoerin L also suppressed the secretion of the ConA-induced proinflammatory cytokines IFN-γ, TNF-α, and IL-6.

Autoimmune hepatitis (AIH), characterized by immune-driven liver destruction and cytokine production, is a progressive inflammatory liver condition that may progress to hepatic cirrhosis or tumors. However, the underlying mechanism is not well understood, and the treatment options for this disease are limited. Pemetrexed (PEM), a clinically used anti-folate drug for treating various tumors, was found to inhibit the nuclear factor (NF)-κB signaling pathways that exert an important role in the development of AIH. Here, we investigated the impact of PEM on immune-mediated hepatic injuries using a murine model of Concanavalin A (Con A)-induced hepatitis, a well-established model for AIH. Mice received intraperitoneal PEM injections 3 times at 12-hour intervals, and two hours later, they were challenged with Con A. Liver samples and serum were collected after 10 h. The results indicate that PEM significantly improved mouse survival rates and lowered serum transaminase levels. Moreover, PEM effectively alleviated oxidative stress, reduced histopathological liver damage, and mitigated hepatocyte apoptosis. Notably, it reduced the activation of M1-type macrophages in the liver. The expression of proinflammatory cytokines and genes associated with M1 macrophages, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-12, IL-1β, and inducible nitric oxide synthase (iNOS), was also decreased. Finally, the results indicated that PEM regulates M1 macrophage activation by modulating the NF-κB signaling pathways. Overall, these results demonstrate that PEM effectively guards against immune-mediated hepatic injuries induced by Con A by inhibiting M1 macrophage activation through the NF-κB signaling pathways and indicate the potential of PEM as a practical treatment option for AIH in clinical settings.

Autoimmune hepatitis is a serious hepatic disorder with unknown nosogenesis, and natural products have been deemed to be one of the most significant sources of new drugs against this disease. Prenyllongnols A-D (1-4), four undescribed prenylated acylphloroglucinols, were isolated from Hypericum longistylum. Compounds 1-4 exhibited remarkable immunosuppressive activities in murine splenocyte proliferation under the induction of concanavalin A (Con A), and IC50 values ranged from 2.98 ± 0.21 to 6.34 ± 0.72 μM. Furthermore, in a Con A-challenged autoimmune hepatitis mouse model, the mice in the group that were pretreated with isolate 2 significantly ameliorated liver injury and decreased proinflammatory cytokine production. Notably, natural product 2 was the first prenylated acylphloroglucinol to protect against concanavalin A-induced autoimmune hepatitis. This finding underscores the potential of prenylated acylphloroglucinol-type metabolites as promising candidates for designing novel immunosuppressors in the quest for new antiautoimmune hepatitis drugs.

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.