Éditeur(s) : HAL CCSD Landes Bioscience
Résumé : International audience
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Autophagy

hal-00735751
https://hal.archives-ouvertes.fr/hal-00735751
PUBMED : 22966490

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Hepatocyte death is a central event during liver disease progression, in which immune cells play key roles by activating members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), including TNFR1 (TNFRSF1A), Fas (TNFRSF6) and TRAIL-R2 (TNFRSF10B). Receptor Interacting Protein Kinase 1 (RIPK1) emerged as a signaling node downstream of these receptors. In the case of TNFR1, RIPK1 has been demonstrated to paradoxically serve as a scaffold to promote the survival of hepatocytes and as a kinase to kill them. To evaluate whether RIPK1 also protects hepatocytes from death in response to FasL or TRAIL, we took advantage of liver parenchymal cell-specific Ripk1 knockout mice (Ripk1 (LPC-KO)). We found that Ripk1 (LPC-KO) mice, as well as primary hepatocytes derived from them, were more susceptible to Fas-mediated apoptosis than their respective WT counterparts. Fas-induced hepatocyte death was independent of TNF-α signaling. Interestingly, while TRAIL administration did not induce hepatitis in Ripk1 (LPC-KO) mice or in their WT counterparts, its combination with IFN-γ only induced TNF-α dependent apoptosis in the Ripk1 (LPC-KO) mice. Together, our data demonstrate the protective role of RIPK1 downstream of Fas and highlight the general protective function of RIPK1 in hepatocytes exposed to inflammatory conditions, where TNF-α, FasL and/or TRAIL are present.
ISSN: 2045-2322

hal-01579612
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01579612
DOI : 10.1038/s41598-017-09789-8
PUBMED : 28835677

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Background & Aims: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. Methods: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1(LPC-KO)). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-alpha during LPS-mediated liver injury. Results: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. Conclusions: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. Lay summary: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
ISSN: 0168-8278

hal-01532060
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01532060
DOI : 10.1016/j.jhep.2017.01.005
PUBMED : 28088582