CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8⁺ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8⁺ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8⁺ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8⁺T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8⁺ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8⁺ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8⁺ T-cell survival. mTOR may be a possible target to reverse CD8⁺ T-cell immune dysfunction in lethal fungal sepsis.     

Saikosaponin-d increases radiation-induced apoptosis of hepatoma cells by promoting autophagy via inhibiting mTOR phosphorylation

Objective: The aim of this study was to analyze the effects of saikosaponin-d (SSd) on autophagy activity and radiosensitivity of hepatoma cells, and to elucidate its related molecular mechanisms.Methods: The growth of SMMC-7721 and MHCC97L hepatoma cells were detected by clonal formation and survival fraction. Flow cytometry was used to detect the changes of apoptosis of hepatoma cells. The morphological changes of autophagy of hepatoma cells were observed by transmission electron microscopy and were further quantitatively detected by laser confocal microscopy. The expressions of related proteins were detected by Western blotting.Results: SSd can significantly increase the apoptosis of hepatoma cells induced by radiation and inhibit the proliferation of hepatoma cells. The addition of the autophagy inhibitor chloroquine (CQ) or an mTOR agonist (MHY1485), which could reduce the promoting effect of SSd on radiation-induced apoptosis and inhibitory effect on the proliferation of hepatoma cells. Transmission electron microscopy and confocal microscopy results also showed that the number of autophagosomes was significantly higher in the radiation and SSd co-treatment group than in the radiotherapy or SSd alone group; however, the effect of SSd on autophagy in hepatoma cells was decreased after adding MHY1485, siRNA-P53 or AMPK inhibitor (Compound C). Western blot analysis showed that after the addition of SSd, the phosphorylation of mTOR was significantly decreased by radiation, the expression of the autophagy-related proteins LC3-II and Beclin-1 was increased, p62 was decreased, and the expression of cleaved caspase-3 and cleaved PARP was enhanced; this effect of SSd was partially reversed after the addition of MHY1485, siRNA-P53 or Compound C.Conclusions: SSd increases radiation-induced apoptosis of hepatoma cells by promoting autophagy via inhibiting mTOR phosphorylation and providing a possible potential approach for radiosensitization therapy of liver cancer.     

Induced regulatory T cells suppress Tc1 cells through TGF-β signaling to ameliorate STZ-induced type 1 diabetes mellitus

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.     

Rebamipide potentially mitigates methotrexate-induced nephrotoxicity via inhibition of oxidative stress and inflammation: A molecular and histochemical study

Methotrexate (MTX) is a widely used chemotherapeutic agent; nevertheless, the nephrotoxicity associated with its use has limited its clinical use. Rebamipide (REB) is a gastro-protective agent with diverse promising biological activities. Here, we investigated the renoprotective effects of REB against MTX-induced nephrotoxicity in rats. Male Wistar rats were allocated into four groups: the normal control group, the REB group (100 mg kg⁻¹ day⁻¹, PO, for 12 days), the MTX group (which received a single injection of 20 mg/kg, ip), and the REB + MTX group (which received 100 mg kg⁻¹ day⁻¹ REB for 7 days before and 5 days after being injected with 20 mg/kg MTX). Interestingly, MTX triggered kidney injury, characterized by renal dysfunction along with histopathological alterations. Moreover, increased reactive oxygen species level and inflammatory response were detected in the kidney of MTX-treated rats. However, REB prevented MTX-induced oxidative kidney injury and boosted an antioxidant balance. Mechanistically, REB markedly activated the NRF-2 protein and upregulated the expression of both SIRT-1 and FOXO-3 genes. Additionally, REB administration strongly inhibited the inflammatory response by downregulating both NF-κB-p65 and TLR-4. Finally, the coadministration of REB and MTX activated the mTOR/PI3K/AKT signaling pathway. Simultaneously, REB treatment attenuated the reduction in glomerular size, the widening of the capsular spaces, and the tubular cell damage due to MTX administration. Taken together, these results indicate the potential of REB as adjuvant therapy to prevent nephrotoxicity in patients receiving MTX treatment.     

Structure-based design and biological evaluation of novel mTOR inhibitors as potential anti-cervical agents

The mammalian target of rapamycin (mTOR) is a critical component of the PI3K-AKT signaling pathway. It is highly activated in cervical cancer, which continues to pose an important clinical challenge with an urgent need for new and improved therapeutic approaches. Herein, we describe the structure-based drug discovery and biological evaluation of a series of mTOR kinase inhibitors as potential anti-cervical cancer agents. The results of enzymatic activity assays supported C3 as a potential mTOR inhibitor, which exhibited high inhibitory activity with an IC₅₀ of 1.57 μM. Molecular docking and dynamics simulation were conducted to predict the binding patterns, suggesting relationships between structure and activity. The anti-proliferative assay against diverse cancer cell lines was displayed subsequently, revealing that C3 exhibited significant proliferation inhibition against cervical cancer cell HeLa (IC₅₀ = 0.38 μM) compared with other cell lines. Moreover, C3 could effectively reduce the expression of phospho-ribosomal S6 protein (p-S6) in HeLa cells in a dose-dependent manner. Noteworthily, mTOR signaling and other cellular pathways might contribute to the significant effect of C3 against cervical cancer simultaneously. These data indicated that C3 represented a good lead molecule for further development as a therapeutic agent for cervical cancer treatment.     

De novo mTOR inhibitor‐based immunosuppression in ABO‐incompatible kidney transplantation

ABO‐incompatible (ABOi) kidney transplantation (KTx) has become an accepted therapeutic option in renal replacement therapy for patients without a blood group‐compatible living donor. Using different desensitization strategies, most centers apply B‐cell depletion with rituximab and maintenance immunosuppression (IS) with tacrolimus and mycophenolic acid. This high load of total IS leads to an increased rate of surgical complications and virus infections in ABOi patients. Our aim was to establish ABOi KTx using an immunosuppressive regimen, which is effective in preventing acute rejection without increasing the risk for viral infections. Therefore, we selected a de novo immunosuppressive protocol with low‐dose calcineurin inhibitor and the mTOR inhibitor everolimus for our ABOi program. Here, we report the first 25 patients with a complete three‐yr follow‐up treated with this regimen. Three‐yr patient survival and graft survival were 96% and 83%. The rate of acute T‐cell‐mediated rejections was low (12%). Cytomegalovirus (CMV) infection was evident in one patient only (4%). Surgical complications were common (40%), but mild in 80% of cases. We demonstrate that ABOi KTx with a de novo mTOR inhibitor‐based regimen is feasible without severe surgical or immunological complications and a low rate of viral infections.     

Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

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Rapamycin ameliorates the CTLA4‐Ig–mediated defect in CD8+ T cell immunity during gammaherpesvirus infection

Latent viral infections are a major concern among immunosuppressed transplant patients. During clinical trials with belatacept, a CTLA4‐Ig fusion protein, patients showed an increased risk of Epstein–Barr virus‐associated posttransplant lymphoproliferative disorder, thought to be due to a deficient primary CD8⁺ T cell response to the virus. Using a murine model of latent viral infection, we observed that rapamycin treatment alone led to a significant increase in virus‐specific CD8⁺ T cells, as well as increased functionality of these cells, including the ability to make multiple cytokines, while CTLA4‐Ig treatment alone significantly dampened the response and inhibited the generation of polyfunctional antigen‐specific CD8⁺ T cells. However, the addition of rapamycin to the CTLA4‐Ig regimen was able to quantitatively and qualitatively restore the antigen‐specific CD8⁺ T cell response to the virus. This improvement was physiologically relevant, in that CTLA4‐Ig treated animals exhibited a greater viral burden following infection that was reduced to levels observed in untreated immunocompetent animals by the addition of rapamycin. These results reveal that modulation of T cell differentiation though inhibition of mTOR signaling can restore virus‐specific immune competence even in the absence of CD28 costimulation, and have implications for improving protective immunity in transplant recipients.