Trifolirhizin regulates the balance of Th17/Treg cells and inflammation in the ulcerative colitis mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Ulcerative colitis (UC) is a chronic and recurrent autoimmune disease, characterized by recurrence and remission of mucosal inflammation. Although the understanding of the pathogenesis of UC has been improved, effective therapeutic drugs are required for treating patients with UC. In current work, the mouse model of colitis was established. Trifolirhizin was demonstrated to improve symptom in dextran sulfate sodium (DSS)-induced colitis mice. The body weight of mice was elevated, whereas the disease activity index (DAI) was reduced. Moreover, trifolirhizin was involved in inhibition of inflammation and regulation of the balance of T helper 17 (Th 17) cells and regulatory T (Treg) cells in DSS-induced colitis mice. Further, the activation NLRP3 inflammasome was suppressed by trifolirhizin in DSS-induced colitis mice. Trifolirhizin was also identified to regulate AMP-activated protein kinase (AMPK)-thioredoxin-interacting protein (TXNIP) pathway. The trifolirhizin-mediated anti-inflammatory effect was inhibited by suppressing AMPK in DSS-induced UC mice. In summary, the research suggested that administration of trifolirhizin significantly improved the symptoms and the pathological damage in DSS-induced UC mice. Trifolirhizin regulated the balance of Th17/Treg cells and inflammation in the UC mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Immunotherapy Mechanism of Esophageal Squamous Cell Carcinoma with the Effect of STK11/AMPK Signaling Pathway.

This study was aimed at exploring the mechanism of serine threonine protein kinase 11 (STK11)/Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway after immunotherapy for esophageal squamous cell carcinoma (ESCC), providing basic information for the clinical treatment of ESCC. In this study, tissue specimens from 100 patients with ESCC who underwent surgical treatment in Taizhou People's Hospital (group A) and 20 patients with recurrent or metastatic ESCC who received second-line immunotherapy (group B) were collected. The real-time fluorescent quantitative polymerase chain reaction (PCR) (RT-qPCR) technology was used to detect the expression levels of STK11, interferon-γ (IFN-γ), interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) in the tissues. The immunohistochemical staining was used to detect the positive expression levels (PELs) of STK11 and AMPKα in the tissues, and immunofluorescence staining was used to detect the PELs Teff cells (CD3 and CD8), Treg cells (CD4 and FOXP3), and neutrophils (CD68 and CD163). RT-qPCR results showed that the expression levels of STK11 and IFN-γ in group A were obviously lower, and those of IL-6 and VEGF were much higher in contrast to group B (P < 0.05). The results of immunohistochemical staining showed that the number of STK11- and AMPKα-positive staining cells in group A was dramatically less than that in group B (P <0.05). The results of immunofluorescence staining revealed that the number of positive staining cells for Teff cells, Treg cells, and neutrophils in group A was also less dramatically than that in group B (P <0.05). In summary, immunotherapy can play a therapeutic effect on ESCC by regulating STK11/AMPK pathway and immune cell infiltration.

TUSC2 immunogene enhances efficacy of chemo-immuno combination on KRAS/LKB1 mutant NSCLC in humanized mouse model.

KRAS/LKB1 (STK11) NSCLC metastatic tumors are intrinsically resistant to anti-PD-1 or PD-L1 immunotherapy. In this study, we use a humanized mouse model to show that while carboplatin plus pembrolizumab reduce tumor growth moderately and transiently, the addition of the tumor suppressor gene TUSC2, delivered systemically in nanovesicles, to this combination, eradicates tumors in the majority of animals. Immunoprofiling of the tumor microenvironment shows the addition of TUSC2 mediates: (a) significant infiltration of reconstituted human functional cytotoxic T cells, natural killer cells, and dendritic cells; (b) induction of antigen-specific T cell responses; (c) enrichment of functional central and memory effector T cells; and (d) decreased levels of PD-1+ T cells, myeloid-derived suppressor cells, Tregs, and M2 tumor associated macrophages. Depletion studies show the presence of functional central and memory effector T cells are required for the efficacy. TUSC2 sensitizes KRAS/LKB1 tumors to carboplatin plus pembrolizumab through modulation of the immune contexture towards a pro-immune tumor microenvironment.

Ghrelin Alleviates Endoplasmic Reticulum Stress in MC3T3E1 Cells by Inhibiting AMPK Phosphorylation.

Ghrelin is a gastric endocrine peptide that has been found to be involved in the process of energy homeostasis and bone physiology in recent years. To explore the effects of ghrelin on endoplasmic reticulum stress (ERS) in MC3T3E1 cells and its possible mechanism, an ERS model was induced by tunicamycin (TM) in the osteoblast line MC3T3E1. TM at 1.5 µg/mL was selected as the experimental concentration found by CCK8 assay. Through the determination of apoptosis, reactive oxygen species production, and endoplasmic reticulum stress-related gene expression, we found that ERS induced by TM can be relieved by ghrelin in a concentration-dependent manner (P < 0.001). Compared with the TM group, ghrelin reduced the expression of ERS-related marker genes induced by TM. Compared with the GSK621 + TM group without ghrelin pretreatment, the mRNA expression of genes in the ghrelin pretreatment group decreased significantly (P < 0.001). The results of protein analysis showed that the levels of BIP, p-AMPK, and cleaved-caspase3 in the TM group increased significantly, while the levels decreased after ghrelin pretreatment. In group GSK621 + TM compared with group GSK621 + ghrelin+TM, ghrelin pretreatment significantly reduced the level of p-AMPK, which is consistent with the trend of the ERS-related proteins BIP and cleaved-caspase3. In conclusion, ghrelin alleviates the ERS induced by TM in a concentration-dependent manner and may or at least partly alleviate the apoptosis induced by ERS in MC3T3E1 cells by inhibiting the phosphorylation of AMPK.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

Oncogenic activity and cellular functionality of melanoma associated antigen A3.

Cancer testis antigen Melanoma associated antigen A3 (MAGE-A3) has been subject of research for many years. Being expressed in various tumor types and influencing proliferation, metastasis, and tumor pathogenicity, MAGE-A3 is an attractive target for cancer therapy, particularly because in healthy tissues, MAGE-A3 is only expressed in testes and placenta. MAGE-A3 acts as a cellular master regulator by stimulating E3 ubiquitin ligase tripartite motif-containing protein 28 (TRIM28), resulting in regulation of various cellular targets. These include tumor suppressor protein p53 and cellular energy sensor AMP-activated protein kinase (AMPK). The restricted expression of MAGE-A3 in tumor cells makes MAGE-A3 an attractive target for vaccine-based immune therapy. However, although phase I and phase II clinical trials involving MAGE-A3-specific immunotherapeutic interventions were promising, large phase III studies failed. This article gives an overview about the role of MAGE-A3 as a cellular master switch and discusses approaches to improve MAGE-A3-based immunotherapies.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2.

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Neutralizing interferon-α blocks inflammation-mediated vascular injury via PI3K and AMPK in systemic lupus erythematosus.

Plasmacytoid dendritic cells (pDCs) play a key role in the initiation and amplification of systemic lupus erythematosus (SLE)-associated vascular injury. In this study, we found that dsDNA induced dose- and time-dependent increase in IFN-α and Toll-like receptor 7 (TLR7), TLR9 and IRF7 expression in pDCs. Co-cultured circulating endothelial cells (ECs) with activated pDCs significantly decreased proliferation, tube formation and migration in ECs. The elevated level of cellular IFN-α increased cell adhesion, promoted cell apoptosis, induced cell senescence and arrested cells at G0/G1 phase of endothelial progenitor cells (EPCs). Additionally, the co-culture system activated MAPK and inactivated PI3K. Pristane was used to establish a in vivo SLE-like mouse model. Importantly, we showed that INF-α-neutralizing antibody (IFN-α-NA) rescued all the changes induced by IFN-α in vitro and prevented vascular injury in pristane-induced SLE model in vivo. In conclusion, we confirmed that activated pDCs promoted vascular damage and the dysfunction of ECs/EPCs via IFN-α production. IFN-α-neutralizing antibody may be a clinical implication for preventing vascular injury. PI3K signalling and AMPK signalling were associated with SLE-associated vascular functions.

Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade.

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.

The AMPK modulator metformin as adjunct to methotrexate in patients with rheumatoid arthritis: A proof-of-concept, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Metformin (MET) may exert anti-rheumatic effects and reduce cartilage degradation through its immunomodulatory and anti-inflammatory actions. METHODS: This was a double-blind placebo-controlled study, 120 adult patients with active rheumatoid arthritis (RA) were randomized to receive MET (1000 mg) or placebo daily with methotrexate (MTX, 7.5 mg/week) for 12 weeks. American College of Rheumatology (ACR)20, ACR50, and ACR70 response rates, Disease Activity Score in 28 joints (DAS-28), and drug safety were the efficacy endpoints. Serum levels of TNF-α, IL-1ß, IL-6, IL-10, IL-17A, NF-κB, TGG-ß1, MDA together with gene expression of AMPK and IGF-IR were assessed before and after the therapy. RESULTS: A total of 80.8% of the patients in the MET group, compared with 54.7% in placebo group, met the criteria of ACR20 response after 12 weeks (P = 0.001). Statistically significant enhancements in the DAS28-3 (CRP) were observed after 4 and 8 weeks for the MET group compared with placebo and were sustained after 12 weeks. MET group showed statistically significant increase in percentage of patients achieving DAS remission after 12 weeks (P = 0.015). Significant improvements in ACR50, ACR70, Health Assessment Questionnaire Disability Index (HAQ-DI), and DAS28-3 (CRP) were also reported. MET was well-tolerated, and no serious adverse effects were reported in both groups. Furthermore, the MET group was superior in improving the measured parameters compared to the placebo. CONCLUSIONS: MET improved the anti-rheumatic effect of MTX; suggesting it to be a beneficial adjuvant in patients with RA. Trial registration ID: NCT04068246.

The SGLT2 inhibitor empagliflozin negatively regulates IL-17/IL-23 axis-mediated inflammatory responses in T2DM with NAFLD via the AMPK/mTOR/autophagy pathway.

Empagliflozin is a SGLT2 inhibitor that reduces the concentration of blood glucose by inhibiting glucose reabsorption and promoting glucose excretion. Interestingly, empagliflozin also has some additional benefits, including cardiovascular protection, decreasing uric acid levels and improving NAFLD-related liver injury. However, the specific mechanism by which empagliflozin ameliorates NAFLD-related liver injury, especially how empagliflozin regulates hepatic immune inflammatory responses, is still unknown. In this study, male C57BL/6J mice were fed a high-fat diet and injected with streptozotocin to establish an animal model of T2DM with NAFLD. Then, diabetic mice with NAFLD were administered empagliflozin by gavage. We found that empagliflozin ameliorated liver injury and lipid metabolism disorder in T2DM mice with NAFLD. Empagliflozin significantly enhanced autophagy in hepatic macrophages via the AMPK/mTOR signalling pathway. After blocking autophagy and AMPK activity, empagliflozin could not prevent NAFLD-related liver injury. Furthermore, the expression levels of IL-17/IL-23 axis-related molecules were inhibited by empagliflozin through enhancing macrophage autophagy. Inhibition of IL-17/IL-23 axis activity attenuated liver injury in T2DM mice with NAFLD. In summary, these results suggested that empagliflozin could significantly ameliorate NAFLD-related liver injury, through enhancing hepatic macrophage autophagy via the AMPK/mTOR signalling pathway and further inhibiting IL-17/IL-23 axis-mediated inflammatory responses. This study provides a theoretical basis for the rational application of empagliflozin to treat T2DM with NAFLD and improve the quality of life of T2DM patients with NAFLD, which will have social benefits.

Anti-inflammatory effects of Platycodin D on dextran sulfate sodium (DSS) induced colitis and E. coli Lipopolysaccharide (LPS) induced inflammation.

Platycodin D (PLD) is a saponin found in Platycodon grandiflorum, which has been reported to have anti-inflammatory effects. However, the effects of PLD on ulcerative colitis (UC) remain unknown. In this study, PLD showed the potential to reduce inflammation, ameliorate intestinal damage, and maintain intestinal integrity in DSS-induced colitis. However, the beneficial effect of PLD was reduced when macrophages were depleted, indicating the key role of macrophages in the beneficial effect of PLD in DSS-induced colitis. Meanwhile, we found that PLD inhibited the expression of M1 markers and promoted the expression of M2 markers in colon. Similarly, we found PLD significantly attenuated the levels of pro-inflammatory cytokines, increased the level of anti-inflammatory cytokine and altered macrophage proportions in LPS-stimulated RAW 264.7 cells in vitro. Moreover, treating LPS-stimulated RAW 264.7 cells with PLD increased the activation of the PI3K/Akt signaling pathway and decreased activation of NF-κB pathway. Furthermore, we found that the anti-inflammatory and macrophage polarization regulatory effects of PLD was Adenosine 5'-monophosphate-activated protein kinase (AMPK)-dependent. These results indicate that PLD attenuates DSS-induced colitis and LPS-induced inflammation, and the mechanism behind the phenomenon may be regulating macrophage polarization via activation of AMPK. Our study provides a theoretical basis for PLD to be used as a potential treatment of colitis.

AMPK induces regulatory innate lymphoid cells after traumatic brain injury.

The CNS is regarded as an immunoprivileged organ, evading routine immune surveillance; however, the coordinated development of immune responses profoundly influences outcomes after brain injury. Innate lymphoid cells (ILCs) are cytokine-producing cells that are critical for the initiation, modulation, and resolution of inflammation, but the functional relevance and mechanistic regulation of ILCs are unexplored after acute brain injury. We demonstrate increased proliferation of all ILC subtypes within the meninges for up to 1 year after experimental traumatic brain injury (TBI) while ILCs were present within resected dura and elevated within cerebrospinal fluid (CSF) of moderate-to-severe TBI patients. In line with energetic derangements after TBI, inhibition of the metabolic regulator, AMPK, increased meningeal ILC expansion, whereas AMPK activation suppressed proinflammatory ILC1/ILC3 and increased the frequency of IL-10-expressing ILC2 after TBI. Moreover, intracisternal administration of IL-33 activated AMPK, expanded ILC2, and suppressed ILC1 and ILC3 within the meninges of WT and Rag1-/- mice, but not Rag1-/- IL2rg-/- mice. Taken together, we identify AMPK as a brake on the expansion of proinflammatory, CNS-resident ILCs after brain injury. These findings establish a mechanistic framework whereby immunometabolic modulation of ILCs may direct the specificity, timing, and magnitude of cerebral immunity.

Impressic Acid Attenuates the Lipopolysaccharide-Induced Inflammatory Response by Activating the AMPK/GSK3ß/Nrf2 Axis in RAW264.7 Macrophages.

Inflammatory diseases are caused by excessive inflammation from pro-inflammatory mediators and cytokines produced by macrophages. The Nrf2 signaling pathway protects against inflammatory diseases by inhibiting excessive inflammation via the regulation of antioxidant enzymes, including HO-1 and NQO1. We investigated the anti-inflammatory effect of impressic acid (IPA) isolated from Acanthopanax koreanum on the lipopolysaccharide (LPS)-induced inflammation and the underlying molecular mechanisms in RAW264.7 cells. IPA attenuated the LPS-induced production of pro-inflammatory cytokines and reactive oxygen species, and the activation of the NF-κB signaling pathway. IPA also increased the protein levels of Nrf2, HO-1, and NQO1 by phosphorylating CaMKKß, AMPK, and GSK3ß. Furthermore, ML385, an Nrf2 inhibitor, reversed the inhibitory effect of IPA on LPS-induced production of pro-inflammatory cytokines in RAW264.7 cells. Therefore, IPA exerts an anti-inflammatory effect via the AMPK/GSK3ß/Nrf2 signaling pathway in macrophages. Taken together, the findings suggest that IPA has preventive potential for inflammation-related diseases.

Adiponectin restrains ILC2 activation by AMPK-mediated feedback inhibition of IL-33 signaling.

ILC2s are present in adipose tissue and play a critical role in regulating adipose thermogenesis. However, the mechanisms underlying the activation of adipose-resident ILC2s remain poorly defined. Here, we show that IL-33, a potent ILC2 activator, stimulates phosphorylation of AMPK at Thr172 via TAK1 in primary ILC2s, which provides a feedback mechanism to inhibit IL-33-induced NF-κB activation and IL-13 production. Treating ILC2s with adiponectin or an adiponectin receptor agonist (AdipoRon) activated AMPK and decreased IL-33-NF-κB signaling. AdipoRon also suppressed cold-induced thermogenic gene expression and energy expenditure in vivo. In contrast, adiponectin deficiency increased the ILC2 fraction and activation, leading to up-regulated thermogenic gene expression in adipose tissue of cold-exposed mice. ILC2 deficiency or blocking ILC2 function by neutralization of the IL-33 receptor with anti-ST2 diminished the suppressive effect of adiponectin on cold-induced adipose thermogenesis and energy expenditure. Taken together, our study reveals that adiponectin is a negative regulator of ILC2 function in adipose tissue via AMPK-mediated negative regulation of IL-33 signaling.

Corticosterone Enhances the AMPK-Mediated Immunosuppressive Phenotype of Testicular Macrophages During Uropathogenic Escherichia coli Induced Orchitis.

Testicular macrophages (TM) play a central role in maintaining testicular immune privilege and protecting spermatogenesis. Recent studies showed that their immunosuppressive properties are maintained by corticosterone in the testicular interstitial fluid, but the underlying molecular mechanisms are unknown. In this study, we treated mouse bone marrow-derived macrophages (BMDM) with corticosterone (50 ng/ml) and uncovered AMP-activated protein kinase (AMPK) activation as a critical event in M2 polarization at the phenotypic, metabolic, and cytokine production level. Primary TM exhibited remarkably similar metabolic and phenotypic features to corticosterone-treated BMDM, which were partially reversed by AMPK-inhibition. In a murine model of uropathogenic E. coli-elicited orchitis, intraperitoneal injection with corticosterone (0.1mg/day) increased the percentage of M2 TM in vivo, in a partially AMPK-dependent manner. This study integrates the influence of corticosterone on M2 macrophage metabolic pathways, phenotype, and function, and highlights a promising new avenue for the development of innovative therapeutics for orchitis patients.

AMPKα1 in B Cells Dampens Primary Antibody Responses yet Promotes Mitochondrial Homeostasis and Persistence of B Cell Memory.

Emerging evidence indicates that metabolic programs regulate B cell activation and Ab responses. However, the metabolic mediators that support the durability of the memory B cell and long-lived plasma cell populations are not fully elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine kinase that integrates cellular energy status and nutrient availability to intracellular signaling and metabolic pathways. In this study, we use genetic mouse models to show that loss of ΑMPKα1 in B cells led to a weakened recall Ab response associated with a decline in the population of memory-phenotype B cells. AMPKα1-deficient memory B lymphocytes exhibited aberrant mitochondrial activity, decreased mitophagy, and increased lipid peroxidation. Moreover, loss of AMPKα1 in B lymphoblasts was associated with decreased mitochondrial spare respiratory capacity. Of note, AMPKα1 in B cells was dispensable for stability of the bone marrow-resident, long-lived plasma cell population, yet absence of this kinase led to increased rates of Ig production and elevated serum Ab concentrations elicited by primary immunization. Collectively, our findings fit a model in which AMPKα1 in B cells supports recall function of the memory B cell compartment by promoting mitochondrial homeostasis and longevity but restrains rates of Ig production.

Phloretin Modulates Human Th17/Treg Cell Differentiation In Vitro via AMPK Signaling.

OBJECTIVE: We conducted studies to explore the effect of phloretin on glucose uptake, proliferation, and differentiation of human peripheral blood CD4+ T cells and investigated the mechanism of phloretin on inducing Th17/Treg development. METHODS: Naïve CD4+ T cells were purified from peripheral blood of healthy volunteers, stimulated with anti-CD3/CD28 antibodies, and polarized in vitro to generate Th17 or Treg cells. Glucose uptake, proliferation, cell cycle, protein expression (phospho-Stat3, phospho-Stat5), and Th17 and Treg cell numbers were analyzed by flow cytometry. AMP-activated protein kinase (AMPK) signaling was analyzed by western blot. Results and Discussion. Phloretin could inhibit the glucose uptake and proliferation of activated CD4+ T cells. The proliferation inhibition was due to the G0/G1 phase arrest. Phloretin decreased Th17 cell generation and phospho-Stat3 expression as well as increased Treg cell generation and phospho-Stat5 expression in the process of inducing Th17/Treg differentiation. The phosphorylation level of AMPK was significantly enhanced, while the phosphorylation level of mTOR was significantly decreased in activated CD4+ T cells under phloretin treatment. The AMPK signaling inhibitor compound C (Com C) could neutralize the effect of phloretin, while the agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) could impact the Th17/Treg balance similar to phloretin during Th17/Treg induction. CONCLUSION: Our results suggest that phloretin can mediate the Th17/Treg balance by regulating metabolism via the AMPK signal pathway.

Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells.

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.

Mitochondrial-targeted ubiquinone alleviates concanavalin A-induced hepatitis via immune modulation.

BACKGROUND: Despite knowledge regarding the effects of antioxidants in ameliorating oxidative damage, evidence concerning their effects on activated immune cells is lacking. Here, a concanavalin A (Con A)-induced hepatitis mouse model was used to investigate the protective effects and immune regulatory mechanisms of mitochondrial-targeted ubiquinone (MitoQ). METHODS: Wild-type (WT) and CD1d-knockout (CD1d-/-, NKT cell deficient) mice were pretreated with MitoQ and then intravenously injected with a sublethal dose of Con A. Serum transaminase and inflammatory cytokine levels were tested. Immune cell functions and AMPK/mTORC1 pathway activation in liver tissue were also evaluated. RESULTS: NKT cells were critical for extensive pro-inflammatory cytokine production and prolonged liver injury upon Con A challenge, while IFN-γ-producing non-NKT cells played an important role during the hyperacute phase. MitoQ treatment not only ameliorated NKT cell-independent hyperacute hepatitis within 12 h post Con A administration but also alleviated NKT cell-dependent extended liver injury at 24 h. The underlying mechanisms involved an inhibition of the heightened activation of iNKT cells and conventional T cells, suppression of the excessive production of IFN-γ, TNF-α and IL-6, and modulation of aberrant AMPK and mTORC1 pathways. CONCLUSION: MitoQ efficiently alleviates Con A-induced hepatitis through immune regulation, suggesting a new therapeutic approach for immune-mediated liver injury by targeting mitochondrial ROS.