The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3ß kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3ß axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

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.

Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10+ B Cells.

The results of recent studies have shown that granulocytic-myeloid derived suppressor cells (G-MDSCs) can secrete exosomes that transport various biologically active molecules with regulatory effects on immune cells. However, their roles in autoimmune diseases such as rheumatoid arthritis remain to be further elucidated. In the present study, we investigated the influence of exosomes from G-MDSCs on the humoral immune response in murine collagen-induced arthritis (CIA). G-MDSCs exosomes-treated mice showed lower arthritis index values and decreased inflammatory cell infiltration. Treatment with G-MDSCs exosomes promoted splenic B cells to secrete IL-10 both in vivo and in vitro. In addition, a decrease in the proportion of plasma cells and follicular helper T cells was observed in drainage lymph nodes from G-MDSCs exosomes-treated mice. Moreover, lower serum levels of IgG were detected in G-MDSCs exosomes-treated mice, indicating an alteration of the humoral environment. Mechanistic studies showed that exosomal prostaglandin E2 (PGE2) produced by G-MDSCs upregulated the phosphorylation levels of GSK-3ß and CREB, which play a key role in the production of IL-10+ B cells. Taken together, our findings demonstrated that G-MDSC exosomal PGE2 attenuates CIA in mice by promoting the generation of IL-10+ Breg cells.

Development of an immunogenomic landscape for the competing endogenous RNAs network of peri-implantitis.

BACKGROUND: Peri-implantitis is an inflammation that occurs around the implant, resulting in varying degrees of inflammatory damage to the soft and hard tissues. The characteristic criterion is the loss of the supporting bone in an inflammatory environment. However, the specific mechanisms and biomarkers involved in peri-implantitis remain to be further studied. Recently, competing endogenous RNAs (ceRNA) and immune microenvironment have been found to play a more important role in the inflammatory process. In our study, we analyzed the expression of immune related microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and message RNAs (mRNAs) in peri-implantitis by analyzing GSE33774 and GSE57631. METHODS: In this study, we explored the expression profile data of immune-related lncRNAs, miRNAs and mRNAs, and constructed immune-related ceRNA network involved in the pathogenesis of peri-implantitis. In addition, the CIBERSORT was used to evaluate the content of immune cells in normal tissues and peri-implantitis to detect the immune microenvironment of peri-implantitis. RESULTS: In the analysis, 14 DElncRNAs, 16 DEmiRNAs, and 18 DEmRNAs were used to establish an immune related ceRNA network and the immune infiltration patterns associated with peri-implantitis was discovered. Through the mutual verification of the two datasets, we found that GSK3B and miR-1297 may have important significance in the immune microenvironment and pathogenesis of peri-implantitis and GSK3B was closely related to four types of immune cells, especially with the highest correlation with resting mast cells (P = 0.0003). CONCLUSIONS: Through immune-related ceRNA network, immune-related genes (IRGs) and immune cell infiltration can further comprehensively understand the pathogenesis of peri-implantitis, which built up an immunogenomic landscape with clinical significance for peri-implantitis.

CKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia.

BACKGROUND: Chemokine-like factor 1 (CKLF1) is a chemokine increased significantly in ischemic brain poststroke. It shows chemotaxis effects on various immune cells, but the mechanisms of CKLF1 migrating neutrophils are poorly understood. Recent studies have provided evidence that CC chemokine receptor 5 (CCR5), a receptor of CKLF1, is involved in ischemic stroke. PURPOSES: To investigate the effects of HIF-1α guided AAV in ischemic brain, investigating the outcome of stroke, and examining the involvement of CKLF1/CCR5 axis in recruitment of neutrophils. RESULTS: HIF-1α guided AAV knocked down CKLF1 in ischemic area and alleviated brain damage of rats. CKLF1 migrated neutrophils through CCR5, worsening inflammatory responses. Akt/GSK-3ß pathway may involve in CKLF1/CCR5 axis guided neutrophils chemotaxis. CONCLUSIONS: CKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia. CKLF1/CCR5 axis may be a useful target for stroke therapy.

Role of the PI3K/AKT (mTOR and GSK3ß) signalling pathway and photobiomodulation in diabetic wound healing.

Activated phosphatidylinositol 3 kinase/Protein kinase B (PI3K/AKT) signalling with increased or reduced mTOR and GSK3ß activity influences the wound repair process. Diabetic wounds, usually ulcerated, are characterised by reduced growth factors and cellular performance. The occurrence of diabetic ulcers is linked to peripheral arterial disease, neuropathy, and wound contamination. Lasers or light emitting diodes (LEDs) provide photon energy with therapeutic benefits (Photobiomodulation-PBM), and has been broadly commended to quicken diabetic wound healing. PBM is efficient in the visible red and near-infrared electromagnetic spectrum, and fluencies ranging from 2 to 6 J/cm2. However, cellular and molecular mechanisms induced by PBM are not fully understood. In this review we discuss PBM and the PI3K/AKT pathway with specific focus on the mTOR and GSK3ß downstream activity in diabetic wound healing.

Inhibition of GSK-3ß alleviates cerebral ischemia/reperfusion injury in rats by suppressing NLRP3 inflammasome activation through autophagy.

The nod-like receptor protein 3 (NLRP3) inflammasome has a critical role in cerebral ischemic injury, and autophagy is related to activation of the inflammasome under oxidative stress conditions. However, it is unclear how NLRP3 inflammasome activation is regulated. Glycogen synthase kinase 3ß (GSK-3ß) emerged as an important risk factor for brain ischemia reperfusion injury, and GSK-3ß inhibits autophagic activity in many diseases. In this study, we examined whether NLRP3 inflammasome-derived inflammation could be ameliorated by GSK-3ß inhibition in a cerebral ischemia reperfusion injury model and assessed whether autophagy is involved in this process. To establish ischemic reperfusion injury, we used a middle cerebral artery occlusion-reperfusion (MCAO/R) model in rats. A chemical inhibitor (SB216763) and GSK-3ß siRNA were used to suppress GSK-3ß activation and GSK-3ß expression in vivo. The results demonstrated that SB216763 and GSK-3ß siRNA improved neurological scores, reduced cerebral infarct volume, and decreased the levels of NLRP3 inflammasome, cleaved-caspase-1, IL-1ß, and IL-18. Inhibiting GSK-3ß activation enhanced autophagic activity (ratio of LC3B-II/LC3B-I and p62/SQSTM1), whereas treating with an autophagy inhibitor (3-MA) abrogated the inhibitory effect on NLRP3 inflammasome activation after GSK-3ß inhibition. These results suggest that inhibiting GSK-3ß downregulates NLRP3 inflammasome expression by increasing autophagic activity in cerebral ischemia reperfusion injury. GSK-3ß might be an attractive specific target and that it functions by regulating the NLRP3 inflammasome.

Discovery and anti-inflammatory evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3ß (GSK-3ß).

Glycogen synthase kinase-3ß (GSK-3ß) has been identified to promote inflammation and its inhibitors have also been proven to treat some inflammatory mediated diseases in animal models. Non-ATP competitive inhibitors inherently have better therapeutical value due to their higher specificity than ATP competitive ones. In this paper, we designed and synthesized a series of new BTZ derivatives as non-ATP competitive GSK-3ß inhibitors. Kinetic analysis revealed two typical compounds 6j and 3j showed the different non-ATP competitive mechanism of substrate competition or allosteric modulation to GSK-3ß, respectively. As expected, the two compounds showed good specificity in a panel test of 16 protein kinases, even to the closest enzymes, like CDK-1/cyclin B and CK-II. The in vivo results proved that both compounds can greatly attenuate the LPS-induced acute lung injury (ALI) and diminish inflammation response in mice by inhibiting the mRNA expression of IL-1ß and IL-6. Western blot analysis demonstrated that they negatively regulated GSK-3ß, and the mechanism of the observed beneficial effects of the inhibitors may involve both the increased phosphorylation of the Ser9 residue on GSK-3ß and protein expression of Sirtuin 1 (SIRT1). The results support that such novel BTZ compounds have a protective role in LPS-induced ALI, and might be attractive candidates for further development of inflammation pharmacotherapy, which greatly thanks to their inherently high selectivities by the non-ATP competitive mode of action. Finally, we proposed suggesting binding modes by Docking study to well explain the impacts of compounds on the target site.

Characterization and function of GSK3ß from Litopenaeus vannamei in WSSV infection.

GSK3ß, a serine/threonine protein kinase, is a crucial regulator in several signaling pathway and plays a vital role in multiple cellular processes including cell proliferation, growth, apoptosis and immune response. In this study, a GSK3ß homolog from L. vannamei, designed as LvGSK3ß, was characterized. Sequence analysis showed that LvGSK3ß possessed a highly similarity with GSK3ß from other species, which contained a catalytic domain and serine/threonine phosphorylation sites. To analyze the role of LvGSK3ß in the process of white spot syndrome virus (WSSV) infection, real-time quantitative PCR and western blot assays were performed. The results showed that the transcription and expression levels of LvGSK3ß were inhibited upon WSSV challenge, accompanied with down-regulated phosphorylation levels. When LvGSK3ß was silenced, the transcription of WSSV gene ie1 was inhibited, and the apoptosis of hemocytes induced by WSSV was up-regulated remarkably as well. In addition, inactivation of LvGSK3ß could also depress virus infection that further validated the results. Conclusively, LvGSK3ß was an important protein for shrimp immunomodulation, and shrimp might promote the apoptosis to restrain WSSV infection by inhibition of LvGSK3ß. The study will be helpful for understanding the molecular mechanism of host-virus interaction.

GSK-3ß activation index is a potential indicator for recurrent inflammation of chronic rhinosinusitis without nasal polyps.

Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK-3) was quantitatively evaluated in patients with CRSsNP (n = 20) and healthy controls (n = 20). The mRNA levels of GSK-3α and GSK-3ß were examined by qPCR, the immunoreactivities of GSK-3ß and nuclear factor-κB (NF-κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK-3ß, phospho-GSK-3ß (p-GSK-3ß, s9) and NF-κB were examined using Western blot analysis. We found that GSK-3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK-3ß mRNA and protein levels. However, when compared with healthy control group, the GSK-3ß activation index, defined as the ratio of GSK-3ß over p-GSK-3ß, was significantly decreased, whereas the NF-κB protein abundance was significantly increased in CRSsNP group (P < 0.05). Strikingly, the GSK-3ß activation index, was highly correlated with NF-κB protein level, as well as CT scores in CRSsNP group (P < 0.05). It was also highly correlated with the mRNA expressions of inflammation-related genes, including T-bet, IFN-γ and IL-4 in CRSsNP group (P < 0.05). Our findings suggest that GSK-3ß activation index, reflecting the inhibitory levels of GSK-3ß through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK-3ß may play a pivotal role in the pathogenesis of CRSsNP.

Enhanced neuroprotection of anthocyanin-loaded PEG-gold nanoparticles against Aß1-42-induced neuroinflammation and neurodegeneration via the NF-KB /JNK/GSK3ß signaling pathway.

Amyloid-beta (Aß1-42) plaques and neurofibrillary tangles (NFTs) are the main hallmarks considered to be associated with neuroinflammation in Alzheimer's disease (AD). Recently, nanoparticle-based targeted drug delivery approaches have been found to be a useful tool in the neurotherapeutics field. Therefore, we examined and compared the neuroprotective effect of anthocyanins alone and anthocyanin-loaded poly (ethylene glycol)-gold nanoparticles (PEG-AuNPs) in Aß1-42-injected mouse and in vitro models of AD. We determined that anthocyanins alone or conjugated with PEG-AuNPs (AnPEG-AuNPs) reduced Aß1-42-induced neuroinflammatory and neuroapoptotic markers via inhibiting the p-JNK/NF-κB/p-GSK3ß pathway in both in vivo and in vitro AD models. However, anthocyanins loaded with PEG-AuNPs were more effective compared to anthocyanins alone. Taken together, these results demonstrate that PEG-coated gold anthocyanins nanoparticles could be a new therapeutic agent in the field of nanomedicine to prevent neurodegenerative diseases such as AD.

Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3ß/Foxp3 Axis.

Previous studies mainly focused on the role of the epidermal growth factor receptor (EGFR) in tumor cells, whereas the effects of the EGFR on immune responses has not been determined. Our study shows that the EGFR signaling pathway play a role in the regulation of regulatory T cells (Treg cells) in cancer patients. The EGF-like growth factor Amphiregulin (AREG) protein was frequently up-regulated in a tissue microarray, which was associated with worse overall survival. Additionally, in sera, tissue specimens, and effusions of lung or gastric cancer patients, up-regulated AREG protein enhanced the suppressive function of Treg cells. AREG maintained the Treg cell suppressive function via the EGFR/GSK-3ß/Foxp3 axis in vitro and in vivo Furthermore, inhibition of EGFR by the tyrosine kinase inhibitor gefitinib restored the activity of GSK-3ß and attenuated Treg cell function. ß-TrCP was involved in GSK-3ß-mediated Foxp3 degradation, and mass spectrometry identified Lys356 as the ubiquitination site of Foxp3 by ß-TrCP. These findings demonstrate the posttranslational regulation of Foxp3 expression by AREG in cancer patients through AREG/EGFR/GSK-3ß signaling, which could lead to Foxp3 protein degradation in Treg cells and a potential therapeutic target for cancer treatment.

Non-tumor tissue derived interleukin-17B activates IL-17RB/AKT/ß-catenin pathway to enhance the stemness of gastric cancer.

Inflammation is a critical component involved in tumor progression. Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has been associated with the progression of cancers. However, the role of IL-17B/IL-17RB (IL-17 receptor B) signaling to stemness of gastric cancer remains unknown. Here, we confirmed that the expression of IL-17RB in gastric cancer tissues was significantly increased, that overexpression was associated with poor prognosis of gastric cancer patients, and that overexpression was positively correlated with some stemness markers. Interestingly, the expression of IL-17B was upregulated in patient serum rather than gastric tumor tissues. Furthermore, exogenous rIL-17B significantly promoted the stemness of gastric cancer cells depending on IL-17RB and induced the expression of IL-17RB. Simultaneously, the expression of phosphorylated AKT, GSK-3ß, and ß-catenin as well as the nuclear translocation of ß-catenin were significantly increased in the MGC-803 cell in a dose-dependent manner, when treated with rIL-17B. The AKT inhibitor, LY294002, and the knockdown of AKT expression reversed the rIL-17B-induced upregulation of ß-catenin and some stemness markers. Together, our results indicate that the IL-17B/IL-17RB signal can promote the growth and migration of tumor cells, and upregulate cell stemness through activating the AKT/ß-catenin pathway in gastric cancer, suggesting that IL-17RB may be a novel target in human gastric cancer therapy.