Oncolytic Adenovirus for the Targeting of Paclitaxel-Resistant Breast Cancer Stem Cells.

Adjuvant systemic therapies effectively reduce the risk of breast cancer recurrence and metastasis, but therapy resistance can develop in some patients due to breast cancer stem cells (BCSCs). Oncolytic adenovirus (OAd) represents a promising therapeutic approach as it can specifically target cancer cells. However, its potential to target BCSCs remains unclear. Here, we evaluated a Cox-2 promoter-controlled, Ad5/3 fiber-modified OAd designed to encode the human sodium iodide symporter (hNIS) in breast cancer models. To confirm the potential of OAds to target BCSCs, we employed BCSC-enriched estrogen receptor-positive (ER+) paclitaxel-resistant (TaxR) cells and tumorsphere assays. OAd-hNIS demonstrated significantly enhanced binding and superior oncolysis in breast cancer cells, including ER+ cells, while exhibiting no activity in normal mammary epithelial cells. We observed improved NIS expression as the result of adenovirus death protein deletion. OAd-hNIS demonstrated efficacy in targeting TaxR BCSCs, exhibiting superior killing and hNIS expression compared to the parental cells. Our vector was capable of inhibiting tumorsphere formation upon early infection and reversing paclitaxel resistance in TaxR cells. Importantly, OAd-hNIS also destroyed already formed tumorspheres seven days after their initiation. Overall, our findings highlight the promise of OAd-hNIS as a potential tool for studying and targeting ER+ breast cancer recurrence and metastasis.

Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification.

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

Exercise of high intensity ameliorates hepatic inflammation and the progression of NASH.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH). Notably, immune cell-driven inflammation is a key mechanism in the transition from fatty liver to the more serious NASH. Although exercise training is effective in ameliorating obesity-related diseases, the underlying mechanisms of the beneficial effects of exercise remain unclear. It is unknown whether there is an optimal modality and intensity of exercise to treat NAFLD. The objective of this study was to determine whether high-intensity interval training (HIIT) or moderate-intensity continuous training (MIT) is more effective at ameliorating the progression of NASH. METHODS: Wild-type mice were fed a high-fat, high-carbohydrate (HFHC) diet for 6 weeks and left sedentary (SED) or assigned to either an MIT or HIIT regimen using treadmill running for an additional 16 weeks. MIT and HIIT groups were pair-fed to ensure that energy intake was similar between the exercise cohorts. To determine changes in whole-body metabolism, we performed insulin and glucose tolerance tests, indirect calorimetry, and magnetic resonance imaging. NASH progression was determined by triglyceride accumulation, expression of inflammatory genes, and histological assessment of fibrosis. Immune cell populations in the liver were characterized by cytometry by time-of-flight mass spectrometry, and progenitor populations within the bone marrow were assessed by flow cytometry. Finally, we analyzed the transcriptional profile of the liver by bulk RNA sequencing. RESULTS: Compared with SED mice, both HIIT and MIT suppressed weight gain, improved whole-body metabolic parameters, and ameliorated the progression of NASH by reducing hepatic triglyceride levels, inflammation, and fibrosis. However, HIIT was superior to MIT at reducing adiposity, improving whole-body glucose tolerance, and ameliorating liver steatosis, inflammation, and fibrosis, without any changes in body weight. Improved NASH progression in HIIT mice was accompanied by a substantial decrease in the frequency of pro-inflammatory infiltrating, monocyte-derived macrophages in the liver and reduced myeloid progenitor populations in the bone marrow. Notably, an acute bout of MIT or HIIT exercise had no effect on the intrahepatic and splenic immune cell populations. In addition, bulk mRNA sequencing of the entire liver tissue showed a pattern of gene expression confirming that HIIT was more effective than MIT in improving liver inflammation and lipid biosynthesis. CONCLUSIONS: Our data suggest that exercise lessens hepatic inflammation during NASH by reducing the accumulation of hepatic monocyte-derived inflammatory macrophages and bone marrow precursor cells. Our findings also indicate that HIIT is superior to MIT in ameliorating the disease in a dietary mouse model of NASH.

Microbiota-Driven Activation of Intrahepatic B Cells Aggravates NASH Through Innate and Adaptive Signaling.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis is rapidly becoming the leading cause of liver failure and indication for liver transplantation. Hepatic inflammation is a key feature of NASH but the immune pathways involved in this process are poorly understood. B lymphocytes are cells of the adaptive immune system that are critical regulators of immune responses. However, the role of B cells in the pathogenesis of NASH and the potential mechanisms leading to their activation in the liver are unclear. APPROACH AND RESULTS: In this study, we report that NASH livers accumulate B cells with elevated pro-inflammatory cytokine secretion and antigen-presentation ability. Single-cell and bulk RNA sequencing of intrahepatic B cells from mice with NASH unveiled a transcriptional landscape that reflects their pro-inflammatory function. Accordingly, B-cell deficiency ameliorated NASH progression, and adoptively transferring B cells from NASH livers recapitulates the disease. Mechanistically, B-cell activation during NASH involves signaling through the innate adaptor myeloid differentiation primary response protein 88 (MyD88) as B cell-specific deletion of MyD88 reduced hepatic T cell-mediated inflammation and fibrosis, but not steatosis. In addition, activation of intrahepatic B cells implicates B cell-receptor signaling, delineating a synergy between innate and adaptive mechanisms of antigen recognition. Furthermore, fecal microbiota transplantation of human NAFLD gut microbiotas into recipient mice promoted the progression of NASH by increasing the accumulation and activation of intrahepatic B cells, suggesting that gut microbial factors drive the pathogenic function of B cells during NASH. CONCLUSION: Our findings reveal that a gut microbiota-driven activation of intrahepatic B cells leads to hepatic inflammation and fibrosis during the progression of NASH through innate and adaptive immune mechanisms.

Influence of inflammasome pathway activation in macrophages on the matrix metalloproteinase expression of human hepatic stellate cells.

Inflammasomes are protein complexes that produce IL-1ß in response to damage or pathogens. As such, inflammasomes are involved in several types of hepatic fibrosis. However, the mechanisms by which these complexes drive the liver's fibrogenic status remain unclear. We co-cultured differentiated macrophages (the THP-1 cell line or human monocyte-derived macrophages (MDMs)) with human hepatic fibroblasts (either the LX-2 cell line or primary human hepatic stellate cells (HSCs)). The inflammasome pathway was activated with lipopolysaccharide (LPS) and monosodium urate (MSU) crystals, and the HSCs' responses were analyzed. Our results show that co-culture of HSCs with THP-1 cells upregulated transcription of the genes coding for metalloproteinase (MMP)-3 and MMP-9. After inflammasome pathway activation, the HSCs' phenotype was the same in the presence of THP-1 cells or MDMs (i.e. upregulation of MMP-3, MMP-9, and the pro-inflammatory cytokine IL-1ß). We found that two cytokines were involved in these changes: IL-1ß regulated MMP-3 and IL-1ß mRNA expression, whereas TNF-α regulated MMP-9 mRNA expression. Experiments with primary cells revealed that a general inflammatory environment is responsible for the downregulation of pro-fibrotic markers. Our present results suggest that inflammasome pathway activation in macrophages leads to a pro-inflammatory environment for HSCs leading to MMP/TIMP imbalance and enhanced fibrolytic properties.

Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell.

Previous studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation.

The release of pro-inflammatory cytokines is mediated via mitogen-activated protein kinases rather than by the inflammasome signalling pathway in keratinocytes.

Toll-like receptors (TLRs) are expressed in the skin and airway epithelial tissues, which are the most important sites of host-pathogen interactions. TLRs recognize the 3-D structures of pathogen-associated molecules and are therefore useful markers of the innate immune response. Here, we investigated the role of lipopolysaccharides and monosodium urate (MSU) crystals in the activation of the TLR and NOD-like receptor (NLR) pathways in human keratinocytes. Analysis of the inflammasome compounds revealed that NOD-like receptor P3 and TLR4, both of which are components of inflammasome complexes involved in the activation of interleukin (IL)-1ß, were not expressed in keratinocytes. Transcriptomic analysis showed that the combination of MSU and lipopolysaccharide priming did not elicit significant results compared to MSU treatment, which induced the expression of TLR2, IL-6 and IL-8/chemokine (C-X-C motif) ligand 8 CXCL8 in the keratinocyte cell line HaCaT. Furthermore, MSU promoted the phosphorylation of extracellular signal-regulated kinase 1/2 and MAPK14/p38α mitogen-activated protein kinases. We concluded that MSU stimulates a pro-inflammatory response in keratinocytes via mitogen-activated protein kinase pathway to induce production of IL-8/CXCL8 chemokine (C-X-C motif) ligand 8 and TLR2.

Involvement of matrix metalloproteinases (MMPs) and inflammasome pathway in molecular mechanisms of fibrosis.

Fibrosis is a basic connective tissue lesion defined by the increase in the fibrillar extracellular matrix (ECM) components in tissue or organ. Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of ECM and so they are suggested to be important in tissue remodelling observed during fibrogenic process associated with chronic inflammation. Tissue remodelling is the result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components markedly controlled by the MMPs/TIMP imbalance. We previously showed an association of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP-9/TIMP-1 ratio. Using the carbon tetrachloride (CCl4) preclinical model of liver fibrosis in mice, we observed a significant increase in collagen deposition with increased expression and release of tissue inhibitors of metalloproteinase (TIMP)-1 both at 24 h and 3 weeks later. This suggests an early altered regulation of matrix turnover involved in the development of fibrosis. We also demonstrated an activation of NLRP3-inflammasome pathway associated with the IL-1R/MyD88 signalling in the development of experimental fibrosis both in lung and liver. This was also associated with an increased expression of purinergic receptors mainly P2X7 Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of tissue remodelling and fibrosis.

Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1ß or TNF-α Release from Human Hepatic Stellate Cells.

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and ß, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-ß1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and ß, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-ß1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-ß1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1ß, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

[The NLRP3 inflammasome: Physiopathology and therapeutic application]. / L'inflammasome NLRP3 : physiopathologie et application thérapeutique.

The innate immune system constitutes the first line of host defense against pathogens. "Nonself", such as exogenous particles or pathogens, triggers an inflammatory response. Inflammasomes are molecular platforms activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as IL-1ß. Activation of the NLRP3 inflammasome pathway, the most extensively studied, appears to be the corner stone of many inflammatory diseases, including Crohn's disease, rheumatoid arthritis and gout. Cryopyrine-associated periodic syndromes (CAPS) are NLRP3 inflammasome-associated diseases. Canakinumab (Ilaris(®)) is the only drug approved for CAPS treatment in France. Targeted therapy against NLRP3 inflammasome and IL-1ß might be the new anti-inflammatory drugs.

IL-1ß production is dependent on the activation of purinergic receptors and NLRP3 pathway in human macrophages.

The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 µg/ml), a 6 h treatment with MSU crystals (250, 500, and 1000 µg/ml) induced the MDMs to release IL-1ß, IL-1α, and IL-6 in a dose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1ß in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 µg/ml) induced significantly greater expression of NLRP3 and IL-1ß than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1ß production and pro-IL-1ß cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1ß and other M1 macrophage cytokines (such as IL-1α, IL-6, and TNF-α) from MDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1ß from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.

Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1ß, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1ß from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1ß mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1ß, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1ß from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury.