c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

NK Cell Anti-Tumor Surveillance in a Myeloid Cell-Shaped Environment

NK cells are innate lymphoid cells endowed with cytotoxic capacity that play key roles in the immune surveillance of tumors. Increasing evidence indicates that NK cell anti-tumor response is shaped by bidirectional interactions with myeloid cell subsets such as dendritic cells (DCs) and macrophages. DC-NK cell crosstalk in the tumor microenvironment (TME) strongly impacts on the overall NK cell anti-tumor response as DCs can affect NK cell survival and optimal activation while, in turn, NK cells can stimulate DCs survival, maturation and tumor infiltration through the release of soluble factors. Similarly, macrophages can either shape NK cell differentiation and function by expressing activating receptor ligands and/or cytokines, or they can contribute to the establishment of an immune-suppressive microenvironment through the expression and secretion of molecules that ultimately lead to NK cell inhibition. Consequently, the exploitation of NK cell interaction with DCs or macrophages in the tumor context may result in an improvement of efficacy of immunotherapeutic approaches

Granzyme A and CD160 expression delineates ILC1 with graded functions in the mouse liver

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA+ ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm+ cells skewed toward a GzmA–CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

International audienceIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field