Kainate receptor channel opening and gating mechanism.

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1-4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5-11. Although structures of kainate receptor domains and subunit assemblies are available12-18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1.

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

Interleukin 28A aggravates Con A-induced acute liver injury by promoting the recruitment of M1 macrophages.

Immune-mediated acute hepatic injury is characterized by the destruction of a large number of hepatocytes and severe liver function damage. Interleukin-28A (IL-28A), a member of the IL-10 family, is notable for its antiviral properties. However, despite advances in our understanding of IL-28A, its role in immune-mediated acute injury remains unclear. The present study investigated the role of IL-28A in concanavalin A (Con A)-induced acute immune liver injury. After Con A injection in mice, IL-28A level significantly increased. IL-28A deficiency was found to protect mice from acute liver injury, prolong survival time, and reduce serum aspartate aminotransferase and alanine aminotransferase levels. In contrast, recombinant IL-28A aggravated liver injury in mice. The proportion of activated M1 macrophages was significantly lower in the IL-28A-deficiency group than in the wild-type mouse group. In adoptive transfer experiments, M1 macrophages from WT could exacerbate mice acute liver injury symptoms in the IL-28A deficiency group. Furthermore, the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-12, IL-6, and IL-1ß, by M1 macrophages decreased significantly in the IL-28A-deficiency group. Western blotting demonstrated that IL-28A deficiency could limit M1 macrophage polarization by modulating the nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK), and interferon regulatory factor (IRF) signaling pathways. In summary, IL-28A deletion plays an important protective role in the Con A-induced acute liver injury model and IL-28A deficiency inhibits the activation of M1 macrophages by inhibiting the NF-κB, MAPK, and IRF signaling pathways. These results provide a potential new target for the treatment of immune-related hepatic injury.

DNAM-1 Immunoreceptor Protects Mice from Concanavalin A-Induced Acute Liver Injury by Reducing Neutrophil Infiltration.

DNAX accessory molecule-1 (DNAM-1; CD226) is an activating immunoreceptor on T cells and NK cells. The interaction of DNAM-1 with its ligand CD155 expressed on hematopoietic and nonhematopoietic cells plays an important role in innate and adaptive immune responses. In this study, we investigated the role of the DNAM-1-CD155 axis in the pathogenesis of T cell-mediated Con A-induced acute liver injury. Unexpectedly, DNAM-1-deficient (Cd226-/-) mice exhibited more severe acute liver injury and higher concentrations of IL-6 and TNF-α than did wild-type (WT) mice after Con A injection. We found that a larger number of neutrophils infiltrated into the liver of Cd226-/- mice compared with WT mice after Con A injection. Depletion of neutrophils ameliorated liver injury and decreased IL-6 and TNF-α in Cd226-/- mice after Con A injection, suggesting that neutrophils exacerbate the liver injury in Cd226-/- mice. Hepatocytes produced more significant amounts of CXCL1, a chemoattractant for neutrophils, in Cd226-/- mice than in WT mice after Con A injection. In the coculture of hepatocytes with liver lymphocytes, either DNAM-1 deficiency in liver lymphocytes or CD155 deficiency in hepatocytes promoted CXCL1 production by hepatocytes. These results suggest that the interaction of DNAM-1 with CD155 inhibits CXCL1 production by hepatocytes, leading to ameliorating acute liver injury.

ACBP/DBI protein neutralization confers autophagy-dependent organ protection through inhibition of cell loss, inflammation, and fibrosis.

Acyl-coenzyme A (CoA)-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2F77I mutation that abolishes ACBP/DBI binding to the GABAA receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b. Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

Hepatocyte estrogen sulfotransferase inhibition protects female mice from concanavalin A-induced T cell-mediated hepatitis independent of estrogens.

Autoimmune hepatitis (AIH) is a typical T cell-mediated chronic liver disease with a higher incidence in females. However, the molecular mechanism for the female predisposition is poorly understood. Estrogen sulfotransferase (Est) is a conjugating enzyme best known for its function in sulfonating and deactivating estrogens. The goal of this study is to investigate whether and how Est plays a role in the higher incidence of AIH in females. Concanavalin A (ConA) was used to induce T cell-mediated hepatitis in female mice. We first showed that Est was highly induced in the liver of ConA-treated mice. Systemic or hepatocyte-specific ablation of Est, or pharmacological inhibition of Est, protected female mice from ConA-induced hepatitis regardless of ovariectomy, suggesting the effect of Est inhibition was estrogen independent. In contrast, we found that hepatocyte-specific transgenic reconstitution of Est in the whole-body Est knockout (EstKO) mice abolished the protective phenotype. Upon the ConA challenge, EstKO mice exhibited a more robust inflammatory response with elevated production of proinflammatory cytokines and changed liver infiltration of immune cells. Mechanistically, we determined that ablation of Est led to the hepatic induction of lipocalin 2 (Lcn2), whereas ablation of Lcn2 abolished the protective phenotype of EstKO females. Our findings demonstrate that hepatocyte Est is required for the sensitivity of female mice to ConA-induced and T cell-mediated hepatitis in an estrogen-independent manner. Est ablation may have protected female mice from ConA-induced hepatitis by upregulating Lcn2. Pharmacological inhibition of Est might be a potential strategy for the treatment of AIH.

CD1d protects against hepatocyte apoptosis in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, a well-defined form of cell death in non-alcoholic steatohepatitis (NASH), is considered the primary cause of liver inflammation and fibrosis. However, the mechanisms underlying the regulation of hepatocyte apoptosis in NASH remain largely unclear. We explored the anti-apoptotic effect of hepatocyte CD1d in NASH. METHODS: Hepatocyte CD1d expression was analyzed in patients with NASH and mouse models. Hepatocyte-specific gene overexpression or knockdown and anti-CD1d crosslinking were used to investigate the anti-apoptotic effect of hepatocyte CD1d on lipotoxicity-, Fas-, and concanavalin (ConA)-mediated liver injuries. A high-fat diet, a methionine-choline-deficient diet, a Fas agonist, and ConA were used to induce lipotoxic and/or apoptotic liver injuries. Palmitic acid was used to mimic lipotoxicity-induced apoptosis in vitro. RESULTS: We identified a dramatic decrease in CD1d expression in hepatocytes of patients with NASH and mouse models. Hepatocyte-specific CD1d overexpression and knockdown experiments collectively demonstrated that hepatocyte CD1d protected against hepatocyte apoptosis and alleviated hepatic inflammation and injuries in NASH mice. Furthermore, decreased JAK2-STAT3 signaling was observed in NASH patient livers. Mechanistically, anti-CD1d crosslinking on hepatocytes induced tyrosine phosphorylation of the CD1d cytoplasmic tail, leading to the recruitment and phosphorylation of JAK2. Phosphorylated JAK2 activated STAT3 and subsequently reduced apoptosis in hepatocytes, which was associated with an increase in anti-apoptotic effectors (Bcl-xL and Mcl-1) and a decrease in pro-apoptotic effectors (cleaved-caspase 3/7). Moreover, anti-CD1d crosslinking effectively protected against Fas- or ConA-mediated hepatocyte apoptosis and liver injury in mice. CONCLUSIONS: Our study uncovered a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 axis in hepatocytes that conferred hepatoprotection and highlighted the potential of hepatocyte CD1d-directed therapy for liver injury and fibrosis in NASH, as well as in other liver diseases associated with hepatocyte apoptosis. IMPACT AND IMPLICATIONS: Excessive and/or sustained hepatocyte apoptosis is critical in driving liver inflammation and injury. The mechanisms underlying the regulation of hepatocyte apoptosis in non-alcoholic steatohepatitis (NASH) remain largely unclear. Here, we found that CD1d expression in hepatocytes substantially decreases and negatively correlates with the severity of liver injury in patients with NASH. We further revealed a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 signaling axis in hepatocytes, which confers significant protection against liver injury in NASH and acute liver diseases. Thus, hepatocyte CD1d-targeted therapy could be a promising strategy to manipulate liver injury in both NASH and other hepatocyte apoptosis-related liver diseases.

TPN10466 ameliorates Concanavalin A-induced autoimmune hepatitis in mice via inhibiting ERK/JNK/p38 signaling pathway.

Autoimmune hepatitis (AIH) eventually progresses to liver fibrosis, cirrhosis, and even hepatocellular carcinoma, causing irreversible damage to the liver. Concanavalin A-induced hepatitis in mice is a well-established model with pathophysiology similar to that of immune-mediated liver injury in human viral and autoimmune hepatitis, and it has been widely used to explore the pathogenesis and clinical treatment of human immune hepatitis. Artemisinin has been shown to exhibit anti-inflammatory effects through unclear mechanisms. In this study, we aimed to assess the effect of the artemisinin derivative TPN10466 on AIH. In vitro studies showed that TPN10466 dose dependently inhibited the percentage of IFN-γ-producing T cells. Further studies showed that TPN10466 attenuated the disease severity of AIH by downregulating the ability of lymphocytes to secrete IFN-γ and by reducing lymphocyte number in the liver. In addition, we found that TPN10466 treatment reduced T-cell responses by inhibiting JNK, ERK, and p38 pathways. In conclusion, our work suggests that TPN10466 provides protection against the autoimmune disease AIH by suppressing the inflammatory response of T cells, suggesting that TPN10466 may be a promising potential agent for the treatment of AIH.

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase.

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Glyco-DNA: Enzymatic Synthesis of Base-Modified and Hypermodified DNA Displaying up to Four Different Monosaccharide Units in the Major Groove.

A portfolio of six modified 2'-deoxyribonucleoside triphosphate (dNTP) derivatives derived from 5-substituted pyrimidine or 7-substituted 7-deazapurine bearing different carbohydrate units (d-glucose, d-galactose, d-mannose, l-fucose, sialic acid and N-Ac-d-galactosamine) tethered through propargyl-glycoside linker was designed and synthesized via the Sonogashira reactions of halogenated dNTPs with the corresponding propargyl-glycosides. The nucleotides were found to be good substrates for DNA polymerases in enzymatic primer extension and PCR synthesis of modified and hypermodified DNA displaying up to four different sugars. Proof of concept binding study of sugar-modified oligonucleotides with concanavalin A showed positive effect of avidity and sugar units count.

Effect of conA-unbound proteins from Melipona beecheii honey on the formation of Pseudomonas aeruginosa ATCC 27853 biofilm.

Pseudomonas aeruginosa is an opportunistic bacterium that can form a biofilm with the ability to colonize different surfaces and for increasing resistance to antibiotics. An alternative to solve this problem may be the use of non-glucose/mannose glycosylated proteins from Melipona beecheii honey, which are capable of inhibiting the growth of this pathogen. In this work, the antibiofilm activity of the conA-unbound protein fraction (F1) from M. beecheii was evaluated. The crude protein extract (CPE) and the F1 fraction inhibited the P. aeruginosa biofilm growth above 80% at 4 and 1.3 µg/mL, respectively. These proteins affected the structure of the biofilm, as well as fleQ and fleR gene expressions involved in the formation and regulation of the P. aeruginosa biofilm. The results demonstrated that the F1 fraction proteins of M. beecheii honey inhibit and affect the formation of the P. aeruginosa biofilm.

Electrochemical Impedimetric Platform Based on Con A@MIL-101 for Glycoprotein Detection.

An electrochemical impedimetric biosensing platform with lectin as a molecular recognition element has been established for the sensitive detection of glycoproteins, a class of important biomarkers in clinical diagnosis. One of the representative metal-organic framework materials, MIL-101(Cr)-NH2, was utilized as the supporting matrix, and its amino groups served as the anchors to immobilize the lectins of concanavalin A (Con A), constituting Con A@MIL-101(Cr)-NH2 for the determination of invertase (INV) as a model glycoprotein. The Con A concentration, immobilization time, and incubation time with INV were optimized. Under the optimal conditions, the degree of impedance increase was linearly proportional to the logarithm of INV concentration between 1.0 × 10-16 and 1.0 × 10-11 M, affording a limit of detection as low as 3.98 × 10-18 M. Good specificity, stability, reproducibility, and repeatability were demonstrated for the fabricated biosensing platform. Moreover, real mouse serum samples were spiked with different concentrations of INV. Excellent recoveries were obtained, which demonstrated the biosensing platform's capability of analyzing glycoproteins within a complex matrix.

Ganoderma lucidum dry extract supplementation modulates T lymphocyte function in older women.

Ganoderma lucidum (a mushroom used in traditional Chinese medicine) compounds may attenuate ageing-related physiological changes and restore normal immunity. However, studies on the physiological effects of Ganoderma lucidum dry extract food supplements are few. Therefore, here, we aimed to investigate the effects of Ganoderma lucidum dry extract food supplement on the lymphocyte function of older women. This was a double-blind clinical trial (n 60) with a final 39 older volunteers, divided into two groups Ganoderma lucidum (n 23) and placebo (n 16). The Ganoderma lucidum group received 2000 mg/d of Ganoderma lucidum dry extract for 8 weeks. We used flow cytometry to determine the lymphocyte profile. CD4+ lymphocyte gene expression was evaluated by real-time polymerase chain reaction. We observed that in the Ganoderma lucidum group, concanavalin A stimulation increased lymphocyte proliferation. Further, we observed an increase in expression of Forkhead box P3, transforming growth factor-beta, IL-10, IL-6, retinoic acid receptor-related orphan receptor gamma, GATA-binding protein 3 and interferon gamma genes in the Ganoderma lucidum group. Furthermore, in the Ganoderma lucidum group, ionomycin and phorbol 12-myristate 13-acetate stimulation led to decrease in Th17+ cells and increase in Th2+ cells. Thus, in older women, Ganoderma lucidum regulates T lymphocyte function leading to a predominant anti-inflammatory action but does not induce T lymphocyte proliferation through CD28 signalling pathway.

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis.

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

The Protective Role of IL-36/IL-36R Signal in Con A-Induced Acute Hepatitis.

The IL-36 family, including IL-36α, IL-36ß, IL-36γ, and IL-36R antagonist, belong to the IL-1 superfamily. It was reported that IL-36 plays a role in immune diseases. However, it remains unclear how IL-36 regulates inflammation. To determine the role of IL-36/IL-36R signaling pathways, we established an acute hepatitis mouse model (C57BL/6) by i.v. injection of the plant lectin Con A. We found that the levels of IL-36 were increased in the liver after Con A injection. Our results demonstrated the infiltrated neutrophils, but not the hepatocytes, were the main source of IL-36 in the liver. Using the IL-36R-/- mouse model (H-2b), we surprisingly found that the absence of IL-36 signals led to aggravated liver injury, as evidenced by increased mortality, elevated serum alanine aminotransferase and aspartate aminotransferase levels, and severe liver pathological changes. Further investigations demonstrated that a lack of IL-36 signaling induced intrahepatic activation of CD4+ and CD8+ T lymphocytes and increased the production of inflammatory cytokines. In addition, IL-36R-/- mice had reduced T regulatory cell numbers and chemokines in the liver. Together, our results from the mouse model suggested a vital role of IL-36 in regulating T cell function and homeostasis during liver inflammation.

Discovery of adamantane-type polycyclic polyprenylated acylphloroglucinols that can prevent concanavalin A-induced autoimmune hepatitis in mice.

Hyperadamans A-G (1-7), seven new adamantane type polycyclic polyprenylated acylphloroglucinols (PPAPs), were isolated from Hypericum wilsonii N. Robson. Structurally, 1-4 were the first adamantanes bearing an unusual 2,7-dioxabicyclo-[2.2.1]-heptane fragment, and compound 5 was the first adamantane with a rare 1,6-dioxaspiro[4.4]nonane section. Importantly, 1-7 exhibited significant immunosuppressive activity on Con A-induced T-lymphocyte proliferation in vitro, with IC50 values ranging from 3.97 ± 0.10 to 18.12 ± 1.07 µM. Pretreatment with 1 in Con A-challenged autoimmune hepatitis mice could dramatically ameliorate the levels of hepatic injury indexes (ALT and AST) and reduce the product of proinflammatory cytokines (COX-2, IL-6, IL-1ß, IL-18, IL-23A and TNF-α). Furthermore, the protective effect of 1 on the Con A-induced liver injury was corroborated by the histological analysis and the immunohistochemistry.

High-Throughput Human Complement C3 N-Glycoprofiling Identifies Markers of Early Onset Type 1 Diabetes Mellitus in Children.

Recently, it was shown that children at the onset of type 1 diabetes (T1D) have a higher proportion of oligomannose glycans in their total plasma protein N-glycome compared to their healthy siblings. The most abundant complement component, glycoprotein C3, contains two N-glycosylation sites occupied exclusively by this type of glycans. Furthermore, complement system, as well as C3, was previously associated with T1D. It is also known that changes in glycosylation can modulate inflammatory responses, so our aim was to characterize the glycosylation profile of C3 in T1D. For this purpose, we developed a novel high-throughput workflow for human C3 concanavalin A lectin affinity enrichment and subsequent LC-MS glycopeptide analysis which enables protein-specific N-glycosylation profiling. From the Danish Childhood Diabetes Register, plasma samples of 61 children/adolescents newly diagnosed with T1D and 84 of their unaffected siblings were C3 N-glycoprofiled. Significant changes of C3 N-glycan profiles were found. T1D was associated with an increase in the proportion of unprocessed glycan structures with more mannose units. A regression model including C3 N-glycans showed notable discriminative power between children with early onset T1D and their healthy siblings with area under curve of 0.879. This study confirmed our previous findings of plasma high-mannose glycan changes in a cohort of recent onset T1D cases, suggesting the involvement of C3 N-glycome in T1D development. Our C3 glycan-based discriminative model could be valuable in assessment of T1D risk in children.

MiR-326 sponges TET2 triggering imbalance of Th17/Treg differentiation to exacerbate pyroptosis of hepatocytes in concanavalin A-induced autoimmune hepatitis.

INTRODUCTION AND OBJECTIVES: MicroRNA-326 is abnormally expressed in autoimmune diseases, but its roles in autoimmune hepatitis (AIH) are unknown. In this study, we aimed to investigate the effect of miR-326 on AIH and the underlying mechanism. MATERIALS AND METHODS: Concanavalin A was administrated to induce AIH in mice and the expression levels of miR-326 and TET2 was evaluated by qRT-PCR and western blot, respectively. The percentages of Th17 and Treg cells were evaluated by flow cytometry and their marker proteins were determined by western blot and ELISA. The mitochondrial membrane potential (MMP) and ROS level were tested with the JC-1 kit and DCFH-DA assay. The binding relationships between miR-326 and TET2 were verified by dual-luciferase reporter assay. The liver tissues were stained by the HE staining. In vitro, AML12 cells were cocultured with mouse CD4+T cells. The expression levels of pyroptosis-related proteins were assessed by western blot. RESULTS: Concanavalin A triggered AIH and enhanced the expression level of miR-326 in mice. It increased both Th17/Treg ratio and the levels of their marker proteins. The expression of TET2 was decreased in AIH mice. Knockdown of miR-326 could decrease the levels of pyroptosis-related proteins, the ROS level and increase MMP. In mouse CD4+T cells, miR-326 sponged TET2 to release IL-17A. Coculture of AML12 cells with isolated CD4+T cells from miR-326 knockdown AIH mice could relieve pyroptosis. CONCLUSIONS: Knockdown of miR-326 exerted anti-pyroptosis effects via suppressing TET2 and downstream NF-κB signaling to dampen AIH. We highlighted a therapeutic target in AIH.