Paloma Martín-Sanz

Bio: Paloma Martín-Sanz is an academic researcher from Spanish National Research Council. The author has contributed to research in topics: Hepatocyte & Proinflammatory cytokine. The author has an hindex of 35, co-authored 116 publications receiving 9589 citations. Previous affiliations of Paloma Martín-Sanz include Carlos III Health Institute & University of Barcelona.

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

Abstract: 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 flux 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, 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. 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

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

Abstract: 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.

Substrate Fate in Activated Macrophages: A Comparison between Innate, Classic, and Alternative Activation

Abstract: Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-(13)C(2)]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-gamma or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1alpha activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1alpha, the switch of PFK2 isoenzymes still occurs in LPS/IFN-gamma-activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1alpha-independent mechanisms.

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

Abstract: The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Inhibition of IκB Kinase and IκB Phosphorylation by 15-Deoxy-Δ12,14-Prostaglandin J2 in Activated Murine Macrophages

Abstract: Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

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

Abstract: 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 flux 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, 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. 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm

Abstract: Plasticity is a hallmark of cells of the myelomonocytic lineage. In response to innate recognition or signals from lymphocyte subsets, mononuclear phagocytes undergo adaptive responses. Shaping of monocyte-macrophage function is an essential component of resistance to pathogens, tissue damage and repair. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. A better understanding of the molecular basis of myelomonocytic cell plasticity will open new vistas in immunopathology and therapeutic intervention.

Succinate is an inflammatory signal that induces IL-1β through HIF-1α

Abstract: Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.