Interleukin-27 promotes autophagy in human serum-induced primary macrophages via an mTOR- and LC3-independent pathway.
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ABSTRACT:
SUBMITTER: Laverdure S
PROVIDER: S-EPMC8295388 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.
| S-EPMC7865382 | biostudies-literature
Project description:Nucleus pulposus (NP) cells reside in the avascular and hypoxic microenvironment of intervertebral discs. Importantly, many activities related to survival and function of NP cells are controlled by the HIF-family of transcription factors. We hypothesize that NP cells adapt to their hypoxic niche through modulation of macroautophagy/autophagy. In various cell types, hypoxia induces autophagy in a HIF1A-dependent fashion; however, little is known about hypoxic regulation of autophagy in NP cells. Hypoxia increases the number of autophagosomes as seen by TEM analysis and LC3-positive puncta in NP cells. Hypoxic induction of autophagy was also demonstrated by a significantly higher number of autophagosomes and smaller change in autolysosomes in NP cells expressing tandem-mCherry-EGFP-LC3B. Increased LC3-II levels were not accompanied by a concomitant increase in BECN1 or the ATG12-ATG5 complex. In addition, ULK1 phosphorylation at Ser757 and Ser777 responsive to MTOR and AMPK, respectively, was not affected in hypoxia. Interestingly, when MTOR activity was inhibited by rapamycin or Torin1, LC3-II levels did not change, suggesting a novel MTOR-independent regulation. Noteworthy, while silencing of HIF1A affected hypoxic induction of BNIP3, it did not affect LC3-II levels, indicating hypoxia-induced autophagy is HIF1-independent. Importantly, there was no change in the number of LC3-positive autophagosomes in NP-specific Hif1a null mice. Finally, inhibition of autophagic flux did not affect the glycolytic metabolism of NP cells, suggesting a possible nonmetabolic role of autophagy. Taken together, our study for the first time shows that NP cells regulate autophagy in a noncanonical fashion independent of MTOR and HIF1A signaling.
| S-EPMC5082780 | biostudies-literature
Project description:Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Replication of EAV, however, was not affected in autophagy-deficient cells lacking autophagy-related protein 7 (ATG7). Nevertheless, colocalization of DMVs and LC3 was still observed in these knockout cells, which only contain the nonlipidated form of LC3. Although autophagy is not required, depletion of LC3 markedly reduced the replication of EAV. EAV replication could be fully restored in these cells by expression of a nonlipidated form of LC3. These findings demonstrate an autophagy-independent role for LC3 in EAV replication. Together with the observation that EAV-induced DMVs are also positive for ER degradation-enhancing ?-mannosidase-like 1 (EDEM1), our data suggested that this virus, similarly to the distantly-related mouse hepatitis coronavirus, hijacks the ER-derived membranes of EDEMosomes to ensure its efficient replication.
| S-EPMC3552881 | biostudies-other
Project description:BackgroundGrowth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.Methodology/principal findingsIn the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.Conclusions/significanceThus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.
| S-EPMC3351469 | biostudies-literature
Project description:Interleukin-27 (IL-27) is a pleiotropic cytokine but its immunosuppressive effects predominate during many in vivo immunological challenges. Despite this, evidence from tumor cell line transfer models suggested that IL-27 could promote immune responses in the tumor context. However, the role of IL-27 in immunity against tumors that develop in situ and in tumor immunosurveillance remain undefined. In this study, we demonstrate that tumor development and growth are accelerated in IL-27 receptor ? (Il27ra)-deficient mice. Enhanced tumor growth in both carcinogen-induced fibrosarcoma and oncogene-driven mammary carcinoma was associated with decreased interferon-? production by CD4 and CD8 T cells and increased numbers of regulatory T-cells (Treg). This is the first study to show that IL-27 promotes protective immune responses against endogenous tumors, which is critical as the basis for future development of an IL-27 based therapeutic agent.
| S-EPMC3595259 | biostudies-literature
Project description:Astragalus membranaceus (AM), used in traditional Chinese medicine, has been shown to enhance immune functions, and recently, its anti-inflammatory effects were identified. However, the mechanisms of action remain unclear. Most studies have shown that autophagy might be involved in the immune response of the body, including inflammation. Here, we developed an inflammatory model by stimulating macrophages with lipopolysaccharides (LPS) to explore the anti-inflammatory effect and mechanisms of AM injection from the perspective of the regulation of autophagy. Immunoblot, immunofluorescence, and ELISA were used to determine the effects of AM injection on the production of interleukin-6 (IL-6) and alterations of autophagy markers. It was found that AM injection reduced the expression of IL-6 in LPS-stimulated macrophages and reversed the LPS-induced inhibition of cellular autophagy. After treatment with inhibitors of signaling pathways, it was shown that LPS downregulated autophagy and upregulated the production of IL-6 in macrophages via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. AM injection reversed the effects of LPS by activating the AMP-activated protein kinase (AMPK) instead of inhibiting Akt. These results were further confirmed by testing activators and siRNA silencing of AMPK. Hence, these 2 distinct signaling molecules appear to exert opposite effects on mTOR, which integrates information from multiple upstream signaling pathways, negatively regulating autophagy. In addition, we demonstrated that autophagy might play a key role in regulating the production of IL-6 by testing activators and inhibitors of autophagy and siRNA silencing of ATG5. These findings showed that AM injection might enhance autophagy by activating AMPK and might further play a repressive effect on the LPS-stimulated expression of IL-6. This study explored the relationship between autophagy, signaling pathways, and the production of inflammatory factors in a model of endotoxin infection and treatment with AM injection.
| S-EPMC7364262 | biostudies-literature
Project description:Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.
| S-EPMC6319544 | biostudies-literature
Project description:p62 protein has been implicated in bone metastasis and is a multifunctional adaptor protein usually correlated with autophagy. Herein, we investigated p62 expression and its prognostic significance in bone metastasis of lung adenocarcinoma, and analyzed whether the mechanism involved depends on autophagy. mRNA and protein expression of p62, LC3B and Beclin 1 were detected by reverse transcription-quantitative PCR and western blotting, respectively, in fresh bone metastasis tissues (n=6 cases) and normal cancellous bone tissues (n=3 cases). The association between p62 and LC3B expression and patient prognosis was subsequently analyzed in 62 paraffin-embedded bone metastasis specimens by immunohistochemistry assay. Small interfering RNA (siRNA) was employed to downregulate p62 expression in SPC-A-1 and A549 cells. Cell proliferation and migration ability were tested by CCK8, CCF and Transwell assays respectively. Autophagy was induced by Rapamycin or inhibited by Atg 7 knockout/Chloroquine in A549 cells and p62 and LC3II/I expression were analyzed. After subcutaneous inoculation or intracardial injection of A549 cells into nude mice, the effect of p62 downregulation in vivo was analyzed by histopathological examination. The results showed that p62, LC3B and Beclin 1 mRNA and protein were all overexpressed in bone metastasis tissues (all P<0.01). Patient samples with high p62 expression levels were significantly associated with more bone lesions (>3), shorter overall survival rates and shorter progression free survival rates compared with patients having lower p62 expression (P=0.014, P=0.003, P=0.048, respectively). Cox regression analysis identified p62 expression as an independent prognostic indicator of overall survival of patients with bone metastasis (P=0.007). In vitro p62 downregulation inhibited SPC-A-1 and A549 cells migration but had no effect on cell proliferation. After autophagy induction or inhibition, p62 expression involved in autophagy flux and changed inconsistently according to the switch of LC3I to LC3II in different autophagy conditions. In vivo p62 downregulation had no effect on growth of subcutaneous tumor. Lung or bone metastasis lesion was not found in all mice model. These findings suggested that p62 overexpression promotes tumor cell invasion out of LC3-dependent autophagy, which could be used a potential prognostic biomarker and therapeutic target for bone metastasis of lung adenocarcinoma.
| S-EPMC8175982 | biostudies-literature
Project description:Antibody-mediated lymphoablation is used in solid organ and stem cell transplantation and autoimmunity. Using murine anti-thymocyte globulin (mATG) in a mouse model of heart transplantation, we previously reported that the homeostatic recovery of CD8+ T cells requires help from depletion-resistant memory CD4+ T cells delivered through CD40-expressing B cells. This study investigated the mechanisms by which B cells mediate CD8+ T cell proliferation in lymphopenic hosts. While CD8+ T cell recovery required MHC class I expression in the host, the reconstitution occurred independently of MHC class I, MHC class II, or CD80/CD86 expression on B cells. mATG lymphoablation upregulated the B cell expression of several cytokine genes, including IL-15 and IL-27, in a CD4-dependent manner. Neither treatment with anti-CD122 mAb nor the use of IL-15Rα-/- recipients altered CD8+ T cell recovery after mATG treatment, indicating that IL-15 may be dispensable for T cell proliferation in our model. Instead, IL-27 neutralization or the use of IL-27Rα-/- CD8+ T cells inhibited CD8+ T cell proliferation and altered the phenotype and cytokine profile of reconstituted CD8+ T cells. Our findings uncover what we believe is a novel role of IL-27 in lymphopenia-induced CD8+ T cell proliferation and suggest that targeting B cell-derived cytokines may increase the efficacy of lymphoablation and improve transplant outcomes.
| S-EPMC6483639 | biostudies-literature
Project description:Cancer cells with a less differentiated stem-like phenotype are more resistant to therapeutic manipulations than their differentiated counterparts, and are considered as one of the main causes of cancer persistence and relapse. As such, induction of differentiation in cancer stem-like cells (CSLCs) has emerged as an alternative strategy to enhance the efficacy of anticancer therapies. CSLCs are metabolically distinct from differentiated cells, and any aberration from the intrinsic metabolic state can induce differentiation of CSLCs. Therefore, metabolism-related molecular targets, with a capacity to promote differentiation within CSLCs, are of therapeutic importance. Here, we demonstrate that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme catalyzing the synthesis of amino acid serine, is important for maintaining the poorly differentiated, stem-like state of CSLCs. Our data shows that PHGDH deficiency impairs the tumorsphere formation capacity in embryonal carcinoma stem-like cells (ECSLCs), breast cancer stem-like cells (BCSLCs) and patient-derived brain tumor-initiating cells (BTICs), which is accompanied by the reduced expression of characteristic stemness-promoting factors, such as Oct4, Nanog, Sox-2, and Bmi-1. Mechanistically, PHGDH deficiency in ECSLCs promotes differentiation to various lineages via degradation of Oct4 and by increasing the stability of differentiation marker ?3-tubulin. Furthermore, PHGDH inhibition promotes p-mTOR independent but Beclin-1-dependent autophagy, independent of apoptosis. When studied in combination, the inhibition of both PHGDH and p-mTOR in ECSLCs causes further augmentation of autophagy, and additionally promotes apoptosis, demonstrating the clinical applicability of PHGDH-based manipulations in cancer therapies. Recapitulating these in vitro findings in CSLC models, the intratumoral PHGDH expression in patient-derived tumors is positively correlated with the mRNA levels of stemness factors, especially Oct4, and cancer patients co-expressing high levels of PHGDH and Oct4 display significantly lower survival than those with low PHGDH/Oct4 co-expression. Altogether, this study identifies a clinically-relevant role for PHGDH in the regulation of stemness-differentiation axis within CSLCs.
| S-EPMC6155240 | biostudies-literature