PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation.

Uncontrolled overactivation of autophagy may lead to autophagic cell death, suppression of which is a pro-survival strategy for tumors. However, mechanisms involving key regulators in modulating autophagic cell death remain poorly defined. Here, we report a novel long noncoding RNA, p53 upregulated regulator of p53 levels (PURPL), functions as an oncogene to promote cell proliferation, colony formation, migration, invasiveness, and inhibits cell death in melanoma cells. Mechanistic studies showed that PURPL promoted mTOR-mediated ULK1 phosphorylation at Ser757 by physical interacting with mTOR and ULK1 to constrain autophagic response to avoid cell death. Loss of PURPL led to AMPK-mediated phosphorylation of ULK1 at Ser555 and Ser317 to over-activate autophagy and induce autophagic cell death. Our results identify PURPL as a key regulator to modulate the activity of autophagy initiation factor ULK1 to repress autophagic cell death in melanoma and may represent a potential intervention target for melanoma therapy.

Fetuin-A exerts a protective effect against experimentally induced intestinal ischemia/reperfusion by suppressing autophagic cell death.

Intestinal tissue is highly susceptible to ischemia/reperfusion injury in many hazardous health conditions. The anti-inflammatory and antioxidant glycoprotein fetuin-A showed efficacy in cerebral ischemic injury; however, its protective role against intestinal ischemia/reperfusion remains elusive. Therefore, this study investigated the protective role of fetuin-A supplementation against intestinal structural changes and dysfunction in a rat model of intestinal ischemia/reperfusion. We equally divided 72 male rats into control, sham, ischemia/reperfusion, and fetuin-A-pretreated ischemia/reperfusion (100 mg/kg/day fetuin-A intraperitoneally for three days prior to surgery and a third dose 1 h prior to the experiment) groups. After 2 h of reperfusion, the jejunum was dissected and examined for spontaneous contractility. A jejunal homogenate was used to assess inflammatory and oxidative stress enzymes. Staining of histological sections was carried out with hematoxylin, eosin and Masson's trichrome stain for evaluation. Immunohistochemistry was performed to detect autophagy proteins beclin-1, LC3, and p62. This study found that fetuin-A significantly improved ischemia/reperfusion-induced mucosal injury by reducing the percentage of areas of collagen deposition, increasing the amplitude of spontaneous contraction, decreasing inflammation and oxidative stress, and upregulating p62 expression, which was accompanied by beclin-1 and LC3 downregulation. Our findings suggest that fetuin-A treatment can prevent ischemia/reperfusion-induced jejunal structural and functional changes by increasing antioxidant activity and regulating autophagy disturbances observed in the ischemia/reperfusion rat model. Furthermore, fetuin-A may provide a protective influence against intestinal ischemia/reperfusion complications.

Beclin1­armed oncolytic Vaccinia virus enhances the therapeutic efficacy of R­CHOP against lymphoma in vitro and in vivo.

Non­Hodgkin lymphoma (NHL) is a form of lymphoid malignancy, with diffuse large B cell lymphoma (DLBCL) being the most common NHL isoform. Approximately half of patients with DLBCL are successfully cured via first­line Rituximab, Cyclophosphamide, Epirubicin, Vindesine, Prednisolone (R­CHOP) treatment. However, 30­40% of patients with DLBCL ultimately suffer from treatment­refractory or relapsed disease. These patients often suffer from high mortality rates owing to a lack of suitable therapeutic options, and all patients are at a high risk of serious treatment­associated dose­dependent toxicity. As such, it is essential to develop novel treatments for NHL that are less toxic and more efficacious. Oncolytic Vaccinia virus (OVV) has shown promise as a means of treating numerous types of cancer. Gene therapy strategies further enhance OVV­based therapy by improving tumor cell recognition and immune evasion. Beclin1 is an autophagy­associated gene that, when upregulated, induces excess autophagy and cell death. The present study aimed to develop an OVV­Beclin1 therapy capable of inducing autophagic tumor cell death. OVV­Beclin1 was able to efficiently kill NHL cells and to increase the sensitivity of these cells to R­CHOP, thereby decreasing the dose­dependent toxic side effects associated with this chemotherapeutic regimen. The combination of OVV­Beclin1 and R­CHOP also significantly improved tumor growth inhibition and survival in a BALB/c murine model system owing to the synergistic induction of autophagic cell death. Together, these findings suggest that OVV­Beclin1 infection can induce significant autophagic cell death in NHL, highlighting this as a novel means of inducing tumor cell death via a mechanism that is distinct from apoptosis and necrosis.

Hydroxychloroquine can potentially interfere with immune function in COVID-19 patients: Mechanisms and insights.

The recent global pandemic due to COVID-19 is caused by a type of coronavirus, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Despite rigorous efforts worldwide to control the spread and human to human transmission of this virus, incidence and death due to COVID-19 continue to rise. Several drugs have been tested for treatment of COVID-19, including hydroxychloroquine. While a number of studies have shown that hydroxychloroquine can prolong QT interval, potentially increasing risk of ventricular arrhythmias and Torsade de Pointes, its effects on immune cell function have not been extensively examined. In the current review, an overview of coronaviruses, viral entry and pathogenicity, immunity upon coronavirus infection, and current therapy options for COVID-19 are briefly discussed. Further based on preclinical studies, we provide evidences that i) hydroxychloroquine impairs autophagy, which leads to accumulation of damaged/oxidized cytoplasmic constituents and interferes with cellular homeostasis, ii) this impaired autophagy in part reduces antigen processing and presentation to immune cells and iii) inhibition of endosome-lysosome system acidification by hydroxychloroquine not only impairs the phagocytosis process, but also potentially alters pulmonary surfactant in the lungs. Therefore, it is likely that hydroxychloroquine treatment may in fact impair host immunity in response to SARS-CoV-2, especially in elderly patients or those with co-morbidities. Further, this review provides a rationale for developing and selecting antiviral drugs and includes a brief review of traditional strategies combined with new drugs to combat COVID-19.

Hyperglycemia aggravates acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via the inhibition of AMPK/mTOR-mediated autophagy induction.

Although the detrimental effects of diabetes mellitus/hyperglycemia have been observed in many liver disease models, the function and mechanism of hyperglycemia regulating liver-resident macrophages, Kupffer cells (KCs), in thioacetamide (TAA)-induced liver injury remain largely unknown. In this study, we evaluated the role of hyperglycemia in regulating NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome activation by inhibiting autophagy induction in KCs in the TAA-induced liver injury model. Type I diabetes/hyperglycemia was induced by streptozotocin treatment. Compared with the control group, hyperglycemic mice exhibited a significant increase in intrahepatic inflammation and liver injury. Enhanced NLRP3 inflammasome activation was detected in KCs from hyperglycemic mice, as shown by increased gene induction and protein levels of NLRP3, cleaved caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain and interleukin-1ß, compared with control mice. NLRP3 inhibition by its antagonist CY-09 effectively suppressed inflammasome activation in KCs and attenuated liver injury in hyperglycemic mice. Furthermore, inhibited autophagy activation was revealed by transmission electron microscope detection, decreased LC3B protein expression and p-62 protein degradation in KCs isolated from TAA-stressed hyperglycemic mice. Interestingly, inhibited 5' AMP-activated protein kinase (AMPK) but enhanced mammalian target of rapamycin (mTOR) activation was found in KCs from TAA-stressed hyperglycemic mice. AMPK activation by its agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or mTOR signaling knockdown by small interfering RNA restored autophagy activation, and subsequently, inhibited NLRP3 inflammasome activation in KCs, leading to ultimately reduced TAA-induced liver injury in the hyperglycemic mice. Our findings demonstrated that hyperglycemia aggravated TAA-induced acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via inhibiting AMPK/mTOR-mediated autophagy. This study provided a novel target for prevention of toxin-induced acute liver injury under hyperglycemia.

Targeting autophagy is a promising therapeutic strategy to overcome chemoresistance and reduce metastasis in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy, mainly affecting children and adolescents. Currently, surgical resection combined with adjuvant chemotherapy has been standardized for OS treatment. Despite great advances in chemotherapy for OS, its clinical prognosis remains far from satisfactory; this is due to chemoresistance, which has become a major obstacle to improving OS treatment. Autophagy, a catabolic process through which cells eliminate and recycle their own damaged proteins and organelles to provide energy, can be activated by chemotherapeutic drugs. Accumulating evidence has indicated that autophagy plays the dual role in the regulation of OS chemoresistance by either promoting drug resistance or increasing drug sensitivity. The aim of the present review was to demonstrate thatautophagy has both a cytoprotective and an autophagic cell death function in OS chemoresistance. In addition, methods to detect autophagy, autophagy inducers and inhibitors, as well as autophagy­mediated metastasis, immunotherapy and clinical prognosis are also discussed.

Sourcing the immune system to induce immunogenic cell death in Kras-colorectal cancer cells.

BACKGROUND: Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations. METHODS: Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts. RESULTS: We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa ßGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response. CONCLUSIONS: Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer.

Distinct Tissue-Specific Roles for the Disease-Associated Autophagy Genes ATG16L2 and ATG16L1.

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.

Bacterial Type I CRISPR-Cas systems influence inflammasome activation in mammalian host by promoting autophagy.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR-Cas) systems in prokaryotes function at defending against foreign DNAs, providing adaptive immunity to maintain homeostasis. CRISPR-Cas may also influence immune regulation ability in mammalian cells through alterations of pathogenic extent and nature. Recent research has implied that Type I CRISPR-Cas systems of Pseudomonas aeruginosa strain UCBPP-PA14 impede recognition by Toll-like receptor 4, and decrease pro-inflammatory responses both in vitro and in vivo. However, the molecular mechanism by which CRISPR-Cas systems affect host immunity is largely undemonstrated. Here, we explored whether CRISPR-Cas systems can influence autophagy to alter the activation of inflammasome. Using the wild-type PA14 and total CRISPR-Cas region deletion (∆TCR) mutant strain, we elucidated the role and underlying mechanism of Type I CRISPR-Cas systems in bacterial infection, and showed that CRISPR-Cas systems impacted the release of mitochondrial DNA and induction of autophagy. CRISPR-Cas deficiency led to an increase of mitochondrial DNA release, a decrease in autophagy, an increase of inflammasome activation and, ultimately, an elevation of pro-inflammatory response. Our findings illustrate a new important mechanism by which Type I CRISPR-Cas systems control their virulence potency to evade host defense.

Ursolic acid exhibits anti-inflammatory effects through blocking TLR4-MyD88 pathway mediated by autophagy.

There is an urgent need for effective treatments to reduce the large and growing burden of acute kidney injury (AKI) and its consequences. Inflammation is believed to play a vital role in the pathophysiology of AKI. Macrophage autophagy is considered protective against inflammation. Previous study discovered that ursolic acid (UA), a natural pentacyclic triterpene carboxylic acid found in many plants as apples, bilberries, cranberries and so on, promoted cancer cell autophagy. In the present study, we aimed to explore the effect of UA on ameliorating AKI and the role of macrophage autophagy in the context of inflammation. The data from in vivo experiments showed that pretreatment of mice with UA significantly suppressed xylene-induced ear oedema as well as protected against LPS-induced AKI. Related mechanisms were further studied through in vitro experiment. As expected, UA decreased inflammatory factors TNF-α, IL-6 and IL-1ß secretion in macrophages in response to lipopolysaccharide (LPS) stimulation. Furthermore, UA blocked LPS-induced TLR4/MyD88 pathway. More importantly, enhanced autophagy of macrophages by UA through increasing the expression of both LC3B and Beclin-1 led to alter macrophage function. What is more, similar to UA, autophagy inhibitor 3-MA obviously decreased inflammation factors releases hinting the vital role of autophagy in regulating inflammation. In all, above study suggested that UA is a potential anti-inflammatory natural compound for treating AKI by inducing autophagy.

The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses.

Hantavirus infection, which causes severe zoonotic diseases with high mortality in humans, has become a global public health concern. Here, we demonstrate that Hantaan virus (HTNV), the prevalent prototype of the hantavirus in Asia, can restrain innate immune responses by manipulating host autophagy flux. HTNV induces complete mitophagy at the early stage of infection but incomplete autophagy at the late stage, and these responses involve the viral glycoprotein (Gn) and nucleocapsid protein (NP), respectively. Gn translocates to mitochondria and interacts with TUFM, recruiting LC3B and promoting mitophagy. Gn-induced mitophagy inhibits type I interferon (IFN) responses by degrading MAVS. Additionally, we found that NP competes with Gn for binding to LC3B, which inhibits Gn-mediated autophagosome formation, and interacts with SNAP29, which prevents autophagosome-lysosome fusion. Thus, NP disturbs the autophagic degradation of Gn. These findings highlight how hantaviruses repurpose host autophagy and evade innate immune responses for their life cycle and pathogenesis.

DNA-stimulated cell death: implications for host defence, inflammatory diseases and cancer.

The immune system detects disturbances in homeostasis that occur during infection, sterile tissue damage and cancer. This initiates immune responses that seek to eliminate the trigger of immune activation and to re-establish homeostasis. At the same time, these mechanisms can also play a crucial role in the progression of disease. The occurrence of DNA in the cytosol constitutes a potent trigger for the innate immune system, governing the production of key inflammatory cytokines such as type I interferons and IL-1ß. More recently, it has become clear that cytosolic DNA also triggers other biological responses, including various forms of programmed cell death. In this article, we review the emerging literature on the pathways governing DNA-stimulated cell death and the current knowledge on how these processes shape immune responses to exogenous and endogenous challenges.