BACKGROUND:Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) encompasses a spectrum of liver conditions, primarily driven by metabolic factors and characterized by steatosis. Extracellular vesicles and particles (EVPs) are emerging as biomarkers for liver diseases as they reflect the state of their cells of origin and carry molecular cargo that can influence disease progression. We aimed to explore the role of EVPs with MASLD progression, smoking impact and EVPs effect on macrophages. METHODS:We analyzed EVPs using nanoparticle tracking analysis, transmission electron microscopy and flow cytometry. Cytokine expressions were quantified in EVPs-exposed macrophages. RESULTS:We showed that EVP number is higher in patients with steatohepatitis and they were correlated with the severity of liver inflammation and steatosis, as well as with biological markers. In particular CD63+-ASGR1+-EVPs were notably more prevalent in blood of patients with steatohepatitis. Active smoking further increased EVP numbers and CD63+-ASGR1+-EVPs in MASLD patients. Functionally, exposure of macrophages to CD63+-EVPs from MASLD patients induced TGF-β and TIMP-1 secretion. CONCLUSION:This study highlights the potential of EVs as biomarkers for MASLD progression and their role in mediating disease mechanisms via intercellular communication, notably by their potential to contribute to fibrogenic signaling. Although exploratory, the study also highlights the potential impact of the exposome, particularly smoking, on MASLD pathogenesis.

OBJECTIVE:Obesity and overweight are associated with low-grade inflammation induced by adipose tissue expansion and perpetuated by altered intestinal homeostasis, including increased epithelial permeability. Intestinal epithelium functions are supported by intestinal epithelial cells (IEC) mitochondria function. However, diet-induced obesity (DIO) may impair mitochondrial activity of IEC and consequently, intestinal homeostasis. The aim of the project was to determine whether DIO alters the mitochondrial function of IEC, and what are the consequences on intestinal homeostasis. METHODS:C57Bl/6J mice were fed a control diet for 22 weeks or a high fat diet (58 kcal% fat). Bioenergetic adaptations of IEC were evaluated on isolated crypts and villi from mouse jejunum. To determine the link between mitochondrial function and alterations of intestinal homeostasis in response to lipid overload, we used the jejunal epithelial cell line IPEC-J2 in vitro and mouse jejunum organoids. RESULTS:Here, we report that DIO in mice induced lipid metabolism adaptations favoring lipid storage in IEC together with reduced number, altered dynamics and diminished oxidative phosphorylation activity of IEC mitochondria. Using the IPEC-J2 cell line, we showed that IEC lipid metabolism and oxidative stress machinery adaptations preceded mitochondrial bioenergetic ones. Moreover, we unraveled the intricate link between IEC energetic status and proliferation / differentiation balance since enhancing mitochondrial function with the AMPK activator AICAR in jejunal organoids reduced proliferation and initiated IEC differentiation and conversely. We confirmed that the reduced IEC mitochondrial function observed in DIO mice was associated with increased proliferation and reduced differentiation, promoting expression of the permissive Cldn2 in the jejunal epithelium of DIO mice. CONCLUSIONS:Our study provides new insights into metabolic adaptations of IEC in obesity by revealing that excess lipid intake diminishes mitochondrial number in IEC, reducing IEC differentiation that contribute to increased epithelial permeability.

A positive association between human exposure to environmental pollutants and progression from benign hepatic steatosis to advanced chronic liver diseases has been documented. Among chemicals found in air pollution, polycyclic aromatic hydrocarbons (PAHs) are of particular concern, due to their omnipresence in the environment. Ingestion of contaminated food is the primary route of exposure. Previous studies on the ability of PAHs to induce the pathological progression of liver steatosis have been limited to the analysis of individual PAHs. The aim of this study was therefore to examine the effects of a mixture of PAHs whose composition closely recapitulates that of contaminated food. The PAH mixture elicited both a steatohepatitis-like state in steatotic WIF-B9 hepatocytes (100 nM for 72 hours) and the progression of steatohepatitis in rats fed a lipid-enriched diet (0.8 mg/kg for 90 days). The PAH mixture induced transient necroptosis at 5 hours followed by a gradual increase in cellular apoptosis. PAH metabolism-dependent necroptosis appeared to be responsible for the development of the secondary apoptosis. Hepatocyte exposure induced a necroptosis-dependent release of extracellular vesicles (EVs), that appeared to be protective against necroptosis; however, those necroptotic EVs triggered apoptosis in recipient hepatocytes. Blocking of ASGR EV receptors with asialofetuin inhibited the interaction of EVs with hepatocytes and hence apoptosis. In conclusion, EV release seems to be crucial to avoid necroptosis, but inhibition of EV uptake can protect against apoptosis.

Succinate dehydrogenase inhibitors (SDHi) are fungicides used worldwide to control the proliferation of fungi in crops. They act by blocking the activity of succinate dehydrogenase (SDH), a universal enzyme involved in mitochondrial functions and metabolism. While SDH-encoding genes are tumour suppressors, which loss-of-function mutations predispose to different types of rare tumors in humans, the consequences of chemical inactivation of SDH by SDHi remain largely unknown, particularly regarding their carcinogenic potential. Here, we investigated the metabolic and cellular impact of SDHi on human non-cancer and transformed colon cells. We show that SDHi inhibit SDH activity and increase the level of succinate, known to act as an oncometabolite in SDH-deficient cancers. SDHi exposure also induces a Warburg-like metabolic reprogramming typical of cancer cells, associated with transcriptomic and morphological changes promoting cell migration and invasion. These effects are enhanced in transformed colon cells carrying mutations in colorectal cancer (CRC) driver genes. These findings provide the first evidence that SDHi-mediated chemical inactivation of SDH mimics some metabolic and phenotypic features previously described in human tumors with SDH genetic deficiencies. Given that loss of SDH expression in CRC patients correlates with a poor prognosis, these patients could represent a population sensitive to SDHi exposure. Therefore, it would be wise to include them in biomonitoring programs. Finally, our work highlights the need to improve regulatory assessment procedures to take better account of SDHi mode of action, by developing relevant tests to cover the multiple key events linked to SDH inactivation and assess the resulting mitochondrial toxicity.

Human hepatocellular carcinomas (HCCs) with cancer stem cell (CSC) features are a subclass of therapeutically challenging cancers. We recently showed that retrodifferentiation of hepatic cancer cells into CSC-like cells leads to metabolic reprogramming and chemoresistance. The molecular mechanisms whereby differentiated cancer cells switch towards a CSC phenotype are poorly understood. By studying metabolic reprogramming associated with HCC cell plasticity, we identified an unsuspected role of peroxisome proliferator-activated receptor (PPAR)γ in hepatic CSC phenotype acquisition. Gene expression and metabolic analyses performed throughout the cell differentiation/retrodifferentiation process of human HepaRG and HBG-BC2 HCC cells show that metabolic reprogramming in hepatic CSCs is associated with a fragmented mitochondrial network, decreased respiration, de novo lipogenesis, and fatty acid oxidation, but increased glycolysis and lipid storage. Mitochondrial genes downregulated in HepaRG-CSCs are also downregulated in the STEM HCC subclass. While PPARα is the main isoform in differentiated hepatic cells, we find high PPARγ expression in hepatic CSCs. Accordingly, nuclear localization of PPARγ is detected in human HCC tumors, and PPARγhigh/PPARαlow expression is associated with the STEM HCC subclass and a poor outcome in human HCC cohorts. PPARγ silencing or/and inhibition of its target gene pyruvate dehydrogenase kinase 4 reactivates cell respiration, increases reactive oxygen species production and sensitizes hepatic CSCs to chemotherapy. Conversely, PPARα activation synergizes with chemotherapy to induce cell death. Targeting PPARγ, a key regulator of metabolic reprogramming and stemness in hepatic CSCs, or modulating the PPARγ/PPARα balance that finely tunes the differentiation/retrodifferentiation process in HCC deserves further investigation for anti-tumor therapy.

Although polycyclic aromatic hydrocarbons (PAHs) are a public health concern in many countries, the cellular response at concentrations representative of human exposure remains poorly defined. At the cellular level, because of their function and composition, mitochondria are privileged targets of these contaminants. PAHs such as benzo[a]pyrene (B[a]P) can compromise mitochondrial functions. Using an integrated approach, we examined how low-dose B[a]P disrupts the three determinants of mitochondrial homeostasis (biogenesis, mitophagy, and dynamics) and triggers apoptosis. We assessed the cellular response to a low-dose of B[a]P (100 nM) in the human microvascular endothelial cell line HMEC-1. Endothelial cells are sensitive to PAHs and display marked reactivity to effectors of B[a]P toxicity, such as oxidative stress. We show that B[a]P doesn’t modify mRNA expression of mitochondrial biogenesis markers but causes a blockage of autophagic flux. In parallel, we observe early and transient mitochondrial elongation accompanied by decreased expression of the fission factors DRP1 and MFF, and increased intracellular concentration of ATP. These events constitute cellular signatures of stress-induced mitochondrial hyperfusion (SIMH), a mechanism of resistance to apoptosis. B[a]P eventually causes apoptosis of HMEC-1 cells, which is mitochondria-dependent, caspase-independent, requires B[a]P bioactivation by CYP1 family enzymes, and is only detectable after 24 h of exposure when SIMH markers decline. Our results show that endothelial cells can mobilize SIMH to delay and limit B[a]P-induced apoptosis. We confirm that mitochondria, at human-relevant doses, are early sensitive targets of B[a]P and show that changes in mitochondrial dynamics constitute an early readout of cellular responses to B[a]P.

Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.

sprG1/SprF1 is a type I toxin-antitoxin system located on Staphylococcus aureus prophage. It has previously been shown that the two toxins, SprG131 and SprG144, encoded by the sprG1 gene, are two membrane-associated peptides structured in a single α-helix. Overexpression of these two peptides leads to growth inhibition and even S. aureus death. In this study, we investigated the involvement of each peptide in this toxicity, the sequence requirements necessary for SprG131 toxicity, and the mechanism of action of these two peptides. Our findings show that both peptides, when expressed individually, are able to stop growth, with higher toxicity observed for SprG131. The combination of a hydrophobic domain and a charged domain located only at the C-terminus is necessary for this toxicity, likely to retain the orientation of the transmembrane domain. A net cationic charge for SprG131 is not essential to induce a growth defect in S. aureus. Furthermore, we established a chronology of toxic events following overexpression to gain insights into the mode of action of SprG144 and SprG131. We demonstrated that mesosome-like structures are already formed when membrane is depolarized, about 20 min after peptides induction. This membrane depolarization occurs concomitantly with a depletion of intracellular ATP, leading to S. aureus growth arrest. Moreover, we hypothesized that SprG144 and SprG131 do not form large pores in the S. aureus membrane, as ATP is not excreted into the extracellular medium, and membrane permeabilization is delayed relative to membrane depolarization. The next challenge is to identify the conditions under which SprG144 and SprG131 are naturally expressed, and to uncover their potential roles during staphylococcal growth, colonization, and infection.

The gut microbiota contributes to human health and disease; however, the mechanisms by which commensal bacteria interact with the host are still unclear. To date, a number of in vitro systems have been designed to investigate the host-microbe interactions. In most of the intestinal models, the enteroendocrine cells, considered as a potential link between gut bacteria and several human diseases, were missing. In the present study, we have generated a new model by adding enteroendocrine cells (ECC) of L-type (NCI-H716) to the one that we have previously described including enterocytes, mucus, and M cells. After 21 days of culture with the other cells, enteroendocrine-differentiated NCI-H716 cells showed neuropods at their basolateral side and expressed their specific genes encoding proglucagon (GCG) and chromogranin A (CHGA). We showed that this model could be stimulated by commensal bacteria playing a key role in health, Roseburia intestinalis and Bacteroides fragilis, but also by a pathogenic strain such as Salmonella Heidelberg. Moreover, using cell-free supernatants of B. fragilis and R. intestinalis, we have shown that R. intestinalis supernatant induced a significant increase in IL-8 and PYY but not in GCG gene expression, while B. fragilis had no impact. Our data indicated that R. intestinalis produced short chain fatty acids (SCFAs) such as butyrate whereas B. fragilis produced more propionate. However, these SCFAs were probably not the only metabolites implicated in PYY expression since butyrate alone had no effect. In conclusion, our new quadricellular model of gut epithelium could be an effective tool to highlight potential beneficial effects of bacteria or their metabolites, in order to develop new classes of probiotics.

Background TiO 2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO 2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO 2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO 2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO 2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO 2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 µg/cm 2 . To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF–SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO 2 NMs for up to 2 weeks were also studied. Results Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 µg/cm 2 . In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. Conclusion These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO 2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term.

Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex and restores normal ATP production levels. Altogether, these data provide evidence for a role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.

Exposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl3. Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and Al2O3 NMs (0.03 to 80 μg/cm2). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively. The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al0 NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl3. None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation.

Polyphasic taxonomic analysis was performed on a novel bacterium, designated UR159T, isolated in 2016 from human blood of a septic patient hospitalized in France. Preliminary 16S rRNA gene sequence-based phylogenetic analysis indicated that strain UR159T belonged to the family Flavobacteriaceae, forming a distinct phyletic line distantly related (<94% sequence similarity) to known species of the family. Further phenotypic, chemotaxonomic and genomic analyses were performed. Cells were non-motile, oxidase-negative, catalase-positive Gram-negative rods. It was strictly aerobic yielding yellow-pigmented colonies, and was metabolically rather inert. Major fatty acids were iso-branched fatty acids, predominantly iso-C15:0 (55.5%) and iso-C17:1ω9c (8.8%). Whole genome sequencing revealed a 2.3-Mbp genome encoding a total of 2262 putative genes with a genomic DNA G+C content at 37.6mol%. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain UR159T and the most closely related members of the Flavobacteriaceae family were <75% and <39%, respectively, much below the established cut-offs for ANI (<95–96%) and isDDH (<70%) for species and genus delineation. Average Amino Acid Identity (AAI) percentages were also estimated and were lower than 65% (cut-off proposed for genus delineation for uncultivated prokaryotes) in all cases, except for F. marinum that was just at the limit (65.1%). Based on these findings, we propose it as a new genus and species, Avrilella dinanensis gen. nov., sp. nov. (type strain UR159T=CIP 111616T=DSM 105483T).

Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches.