Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes

Autophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPP(V717I) and HsMAPT(P301L), exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPP(V717I) and HsMAPT(P301L) with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies

Specific targeting of the NRF2/β-TrCP axis promotes beneficial effects in NASH

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease that compromises liver function, for which there is not a specifically approved medicine. Recent research has identified transcription factor NRF2 as a potential therapeutic target. However, current NRF2 activators, designed to inhibit its repressor KEAP1, exhibit unwanted side effects. Alternatively, we previously introduced PHAR, a protein-protein interaction inhibitor of NRF2/β-TrCP, which induces a mild NRF2 activation and selectively activates NRF2 in the liver, close to normal physiological levels. Herein, we assessed the effect of PHAR in protection against NASH and its progression to fibrosis. We conducted experiments to demonstrate that PHAR effectively activated NRF2 in hepatocytes, Kupffer cells, and stellate cells. Then, we used the STAM mouse model of NASH, based on partial damage of endocrine pancreas and insulin secretion impairment, followed by a high fat diet. Non-invasive analysis using MRI revealed that PHAR protects against liver fat accumulation. Moreover, PHAR attenuated key markers of NASH progression, including liver steatosis, hepatocellular ballooning, inflammation, and fibrosis. Notably, transcriptomic data indicate that PHAR led to upregulation of 3 anti-fibrotic genes (Plg, Serpina1a, and Bmp7) and downregulation of 6 pro-fibrotic (including Acta2 and Col3a1), 11 extracellular matrix remodeling, and 8 inflammatory genes. Overall, our study suggests that the mild activation of NRF2 via the protein-protein interaction inhibitor PHAR holds promise as a strategy for addressing NASH and its progression to liver fibrosisThis research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (grants PID2019-110061RB-I00, PID-2021-122766OB-100 and PDC2021-121421-I00, PDC2022-133765-I00, MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe” by the European Union.), CIBERdem and CIBERned (ISCIII), and The Autonomous Community of Madrid (grant P2022/BMD-7230). RFG enjoyed a FPI contract of MINECO (FPI-2017). DCS is a holder of a FPI contract of MICINN (Ministry of Science and Innovation, FPI-2020, PRE2020-091886). JJV is holder of a FPU contract of MIU (Ministry of Universities, FPU2020, FPU20/03326

Experimental Meningococcal Sepsis in Congenic Transgenic Mice Expressing Human Transferrin

Severe meningococcal sepsis is still of high morbidity and mortality. Its management may be improved by an experimental model allowing better understanding of its pathophysiology. We developed an animal model of meningococcal sepsis in transgenic BALB/c mice expressing human transferrin. We studied experimental meningococcal sepsis in congenic transgenic BALB/c mice expressing human transferrin by transcriptional profiling using microarray analysis of blood and brain samples. Genes encoding acute phase proteins, chemokines and cytokines constituted the largest strongly regulated groups. Dynamic bioluminescence imaging further showed high blood bacterial loads that were further enhanced after a primary viral infection by influenza A virus. Moreover, IL-1 receptor–associated kinase–3 (IRAK-3) was induced in infected mice. IRAK-3 is a negative regulator of Toll-dependant signaling and its induction may impair innate immunity and hence result in an immunocompromised state allowing bacterial survival and systemic spread during sepsis. This new approach should enable detailed analysis of the pathophysiology of meningococcal sepsis and its relationships with flu infection

Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Mitochondrial Dynamics and Activity in Legionella-Infected Cells

International audienceThe study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages

Sorting of Phagocytic Cells Infected with Legionella pneumophila

International audienceThe ability of Legionella pneumophila to colonize host cells and to form a replicative vacuole depends on its ability to counteract the host cell response by secreting more than 300 effectors. The host cell responds to this bacterial invasion with extensive intracellular signaling to counteract the replication of the pathogen. When studying L. pneumophila infection in vitro, only a small proportion of the cell lines or primary cells used to analyze the host response are infected; the study of such a mixed cell population leads to unprecise results. In order to study the multitude of pathogen-induced phenotypic changes occurring in the host cell, the separation of infected from uninfected cells is a top priority. Here we describe a highly efficient FACS-derived protocol to separate cells infected with a L. pneumophila strain encoding a fluorescent protein. Indeed, the highly infected, homogenous cell population obtained after sorting is the best possible starting point for the studies of infection-induced effects