Endoplasmic reticulum stress induces ligand-independent TNFR1-mediated necroptosis in L929 cells

Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin α production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress

A purine metabolic checkpoint that prevents autoimmunity and autoinflammation.

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation

ATG16L1 orchestrates interleukin-22 signaling in the intestinal epithelium via cGAS-STING.

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1 ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1 ΔIEC and Atg16l1 ΔIEC/Xbp1 ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1 ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium

Epithelial endoplasmic reticulum stress orchestrates a protective IgA response.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.Wellcome Trust Senior Investigator Award 106260/Z/14/Z HORIZON2020/European Research Council Consolidator Grant 64888

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Defective ATG16L1-mediated removal of IRE1α drives Crohn's disease-like ileitis.

ATG16L1$^{T300A}$, a major risk polymorphism in Crohn's disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1α. IRE1α accumulates in Paneth cells of Atg16l1$^{ΔIEC}$ mice, and humans homozygous for ATG16L1$^{T300A}$ exhibit a corresponding increase of IRE1α in intestinal epithelial crypts. In contrast to a protective role of the IRE1β isoform, hyperactivated IRE1α also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1$^{ΔIEC}$ mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1α, and optineurin deficiency amplifies IRE1α levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1$^{ΔIEC}$ mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate-induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1α aggregates during ER stress at this site.This study was supported by the European Research Council under the European Community’s Seventh Framework Program (grant FP7/2007-2013)/ERC, agreement no. 260961 to A. Kaser and grant HORIZON2020/ERC, agreement no. 648889 to A. Kaser), the Wellcome Trust (Investigator Award 106260/Z/14/Z to A. Kaser and Principal Research Fellowship 2008/Z/16/Z to D. Ron), the Cambridge Biomedical Research Centre (A. Kaser), a Medical Research Council PhD for clinicians training fellowship (grant MR/N001893/1 to J. Bhattacharyya), fellowships from the European Crohn’s and Colitis Organization (M. Tschurtschenthaler and T.E. Adolph), the Research Training Group Genes, Environment, and Inflammation supported by the Deutsche Forschungsgemeinschaft (grant RTG 1743/1 to P. Rosenstiel), the SFB877 subproject B9 and CLVIII ExC 306 Inflammation at Interfaces (P. Rosenstiel), and the National Institutes of Health (grants DK044319, DK051362, DK053056, and DK088199 to the Harvard Digestive Diseases Center and grant DK0034854 to R.S. Blumberg)

Создание мультфильма-оригами с детьми с тяжелыми нарушениями речи на уроках ручного труда в специальной коррекционной школе 5 вида

The theme of this article is the creation of a cartoon-origami with children with severe speech disorders. The main purpose of the work is to describe the process of creating a cartoon-origami with children with severe speech disorders.Тема данной статьи – создание мультфильма-оригами с детьми с тяжелыми нарушениями речи. Основная цель работы заключается в описании процесса создания мультфильма-оригами с детьми с тяжелыми нарушениями речи

Creating a multifilm on the basis of polymeric clay with children with heavy disabilities of speech in manual labor lessons in special correctional school of 5 types

The topic of this article is the creation of a cartoon based on polymer clay with children with severe speech disorders. The main objective of the work is to describe the process of creating a cartoon based on polymer clay with children with severe speech disorders

Endoplasmic reticulum stress induces ligand-independent tnfr1-mediated necroptosis in l929 cells

Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin a production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress