Françoise Mercier-Nomé

Acute oral exposure to ChCl : urea DES induces gut microbiota dysbiosis, disrupts the bile acid-FXR-liver axis, and triggers subclinical hepatic inflammation, revealing critical toxicological endpoints overlooked by classical safety markers.

Background: PDE2 (phosphodiesterase 2) is upregulated in human heart failure. Cardiac PDE2‐transgenic mice are protected against contractile dysfunction and arrhythmias in heart failure but whether an acute elevation of PDE2 could be of therapeutic value remains elusive. This hypothesis was tested using cardiac PDE2 gene transfer in preclinical models of heart failure. Methods and Results: C57BL/6 male mice were injected with serotype 9 adeno‐associated viruses encoding for PDE2A. This led to a ≈10‐fold rise of PDE2A protein levels that affected neither cardiac structure nor function in healthy mice. Two weeks after inoculation with serotype 9 adeno‐associated viruses, mice were implanted with minipumps delivering either NaCl, isoproterenol (60 mg/kg per day), or isoproterenol and phenylephrine (30 mg/kg per day each) for 2 weeks. In mice injected with serotype 9 adeno‐associated viruses encoding for LUC (luciferase), isoproterenol or isoproterenol+phenylephrine infusion induced left ventricular hypertrophy, decreased ejection fraction unveiled by echocardiography, and promoted fibrosis and apoptosis assessed by Masson's trichrome and Tunel, respectively. Furthermore, inotropic responses to isoproterenol of ventricular cardiomyocytes isolated from isoproterenol+phenylephrine‐LUC mice loaded with 1 μmol/L Fura‐2AM and stimulated at 1 Hz to record calcium transients and sarcomere shortening were dampened. Spontaneous calcium waves at the cellular level were promoted as well as ventricular arrhythmias evoked in vivo by catheter‐mediated ventricular pacing after isoproterenol (1.5 mg/kg) and atropine (1 mg/kg) injection. However, increased PDE2A blunted these adverse outcomes evoked by sympathomimetic amines. Conclusions: Cardiac gene therapy with PDE2A limits left ventricle remodeling, dysfunction, and arrhythmias evoked by catecholamines, providing evidence that increasing PDE2A activity acutely could prevent progression toward heart failure.

Chemotherapy is primarily administered intravenously (IV), but this route poses significant challenges (e.g., high costs, patient discomfort, logistical difficulties, side effects such as infections from catheter use). Although oral and subcutaneous (SC) routes are preferred for their convenience and have the potential for better patient comfort and cost reduction, oral chemotherapy faces issues like poor bioavailability and adherence, while SC delivery is unsuitable for irritant or vesicant drugs due to local toxicity. To overcome these limitations, the polymer prodrug strategy has been explored, where drugs are linked to a polymer, reducing toxicity and enhancing drug delivery. Recent work has focused on creating water-soluble polymer prodrugs for SC delivery of paclitaxel (Ptx), a hydrophobic and vesicant drug, which was successfully conjugated to polyacrylamide (PAAm), a very hydrophilic biocompatible polymer, resulting in safer SC injection and enhanced therapeutic efficacy in tumor-bearing mice. However, this strategy's potential depends on adapting it to other vesicant anticancer drugs. Making the polymer degradable for facilitated excretion would also be a key improvement. In this work, this approach has been successfully extended to gemcitabine (Gem), a widely used but irritant anticancer drug, and to a degradable PAAm-based promoiety, having cleavable ester groups in the main chain. The resulting Gem-based prodrugs featured upper critical solution temperature to ensure complete solubility at the temperature of the SC tissue, sustained Gem release, significant degradation under physiological conditions, improved systemic toxicity and absence of local toxicity compared to free Gem. Remarkably, Gem-PAAm polymer prodrugs exhibited significant anticancer efficacy in mice bearing Mia Pa-Ca 2 tumors, outperforming Gemzar®, the commercial formulation of Gem. These advances suggest the potential of these hydrophilic polymer prodrugs to transform SC chemotherapy, enabling the use of a broader range of anticancer drugs while reducing side effects and improving patient outcomes.

BACKGROUND: The heart expresses 2 main subtypes of cAMP-dependent protein kinase (PKA; type I and II) that differ in their regulatory subunits, RIα and RIIα. Embryonic lethality of RIα knockout mice limits the current understanding of type I PKA function in the myocardium. The objective of this study was to test the role of RIα in adult heart contractility and pathological remodeling. METHODS: We measured PKA subunit expression in human heart and developed a conditional mouse model with cardiomyocyte-specific knockout of RIα (RIα-icKO). Myocardial structure and function were evaluated by echocardiography, histology, and ECG and in Langendorff-perfused hearts. PKA activity and cAMP levels were determined by immunoassay, and phosphorylation of PKA targets was assessed by Western blot. L-type Ca ²⁺ current ( I _Ca,L ), sarcomere shortening, Ca ²⁺ transients, Ca ²⁺ sparks and waves, and subcellular cAMP were recorded in isolated ventricular myocytes (VMs). RESULTS: RIα protein was decreased by 50% in failing human heart with ischemic cardiomyopathy and by 75% in the ventricles and in VMs from RIα-icKO mice but not in atria or sinoatrial node. Basal PKA activity was increased ≈3-fold in RIα-icKO VMs. In young RIα-icKO mice, left ventricular ejection fraction was increased and the negative inotropic effect of propranolol was prevented, whereas heart rate and the negative chronotropic effect of propranolol were not modified. Phosphorylation of phospholamban, ryanodine receptor, troponin I, and cardiac myosin-binding protein C at PKA sites was increased in propranolol-treated RIα-icKO mice. Hearts from RIα-icKO mice were hypercontractile, associated with increased I _Ca,L, and [Ca ²⁺ ] _i transients and sarcomere shortening in VMs. These effects were suppressed by the PKA inhibitor, H89. Global cAMP content was decreased in RIα-icKO hearts, whereas local cAMP at the phospholamban/sarcoplasmic reticulum Ca ²⁺ ATPase complex was unchanged in RIα-icKO VMs. RIα-icKO VMs had an increased frequency of Ca ²⁺ sparks and proarrhythmic Ca ²⁺ waves, and RIα-icKO mice had an increased susceptibility to ventricular tachycardia. On aging, RIα-icKO mice showed progressive contractile dysfunction, cardiac hypertrophy, and fibrosis, culminating in congestive heart failure with reduced ejection fraction that caused 50% mortality at 1 year. CONCLUSIONS: These results identify RIα as a key negative regulator of cardiac contractile function, arrhythmia, and pathological remodeling.

Enterococcus faecalis is a bacterial species present at a subdominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is one of the rare pathobionts identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unraveling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.

Reshaping the intestinal microbiota by the ingestion of fiber, such as pectin, improves alcohol-induced liver lesions in mice by modulating bacterial metabolites, including indoles, as well as bile acids (BAs). In this context, we aimed to elucidate how oral supplementation of pectin affects BA metabolism in alcohol-challenged mice receiving feces from patients with alcoholic hepatitis. Pectin reduced alcohol liver disease. This beneficial effect correlated with lower BA levels in the plasma and liver but higher levels in the caecum, suggesting that pectin stimulated BA excretion. Pectin modified the overall BA composition, favoring an augmentation in the proportion of hydrophilic forms in the liver, plasma, and gut. This effect was linked to an imbalance between hydrophobic and hydrophilic (less toxic) BAs in the gut. Pectin induced the enrichment of intestinal bacteria harboring genes that encode BA-metabolizing enzymes. The modulation of BA content by pectin inhibited farnesoid X receptor signaling in the ileum and the subsequent upregulation of Cyp7a1 in the liver. Despite an increase in BA synthesis, pectin reduced BA serum levels by promoting their intestinal excretion. In conclusion, pectin alleviates alcohol liver disease by modifying the BA cycle through effects on the intestinal microbiota and enhanced BA excretion.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Pectin, a soluble fiber, improves non-alcoholic fatty-liver disease (NAFLD), but its mechanisms are unclear. We aimed to investigate the role of pectin-induced changes in intestinal microbiota (IM) in NAFLD. We recovered the IM from mice fed a high-fat diet, treated or not with pectin, to perform a fecal microbiota transfer (FMT). Mice fed a high-fat diet, which induces NAFLD, were treated with pectin or received a fecal microbiota transfer (FMT) from mice treated with pectin before (preventive FMT) or after (curative FMT) being fed a high-fat diet. Pectin prevented the development of NAFLD, induced browning of adipose tissue, and modified the IM without increasing the abundance of proteobacteria. Preventive FMT also induced browning of white adipose tissue but did not improve liver steatosis, in contrast to curative FMT, which induced an improvement in steatosis. This was associated with an increase in the concentration of short-chain fatty acids (SCFAs), in contrast to preventive FMT, which induced an increase in the concentration of branched SCFAs. Overall, we show that the effect of pectin may be partially mediated by gut bacteria.

Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that several ligands of the chemokine receptor Cxcr2 were up-regulated in the PyMT (polyoma middle T oncogene) model of breast cancer. Interestingly, the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2−/− animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2−/− TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs. The transcriptomic analysis of PyMT-Cxcr2−/− TANs showed that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. In particular, PyMT-Cxcr2−/− TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects.