Search

The main risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoke (CS). It can alter many immune cells functions such as phagocytosis, efferocytosis and cytokine production. Cytokines play a role in the orchestration of inflammation in COPD. The JAK/STAT pathways are among the most important signalling components of cytokines. The objective of this work was to investigate the role of the JAK/STAT pathway with regard to cytokine release and microsphere uptake capacity (to minimize the non-specific scavenging) in human monocyte-derived-macrophages (MDMs). The MDMs were stimulated by cigarette smoke extract (CSE) alone or in combination with lipopolysaccharide (LPS). CSE alone was not associated with significant changes in the cytokine, with the exception of IL-8/CXCL8 production. However, CSE disturbed cytokine production in LPS-stimulated MDMs. CSE increase CXCL-8 and CCL2 release in LPS-stimulated monocyte-derived macrophages and suppressed the production of IL-6 and CXCL1 in these cells. CSE also decreased microsphere uptake capacity by MDMs. Then, CSE + LPS-stimulated MDMs were treated with two different JAK inhibitors. AG490 (specific inhibitor of JAK2) and ruxolitinib (inhibitor of JAK1 and JAK2). JAK/STAT inhibitors, particularly ruxolitinib, attenuated in cytokine production without completely inhibiting when compared with dexamethasone. On the other hand, the cells exposed to dexamethasone are nearly unable to capture the microspheres, while both JAK inhibitors do not affect the uptake capacity. In summary, our results showed the versatility of ruxolitinib which might bring a better balance disturbance of cytokine release and uptake capacity. The information regarding the distinctive effect of JAK/STAT inhibitors may be useful in the development of novel treatments for COPD.

International audience

International audience; Inflammasomes are protein complexes that produce IL-1β 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-1β). We found that two cytokines were involved in these changes IL-1β regulated MMP-3 and IL-1β 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.

International audience

International audience; The amanitins (namely α- and β-amanitin) contained in certain mushrooms are bicyclic octapeptides that, when ingested, are responsible for potentially lethal hepatotoxicity. M101 is an extracellular hemoglobin extracted from the marine worm Arenicola marina. It has intrinsic Cu/Zn-SOD-like activity and is currently used as an oxygen carrier in organ preservation solutions. Our present results suggest that M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development.

International audience; Inflammatory lung disease results in a high global burden of death and disability. There are no effective treatments for the most severe forms of many inflammatory lung diseases, such as chronic obstructive pulmonary disease, emphysema, corticosteroid-resistant asthma, and coronavirus disease 2019; hence, new treatment options are required. Here, we review the role of oxidative imbalance in the development of difficult-to-treat inflammatory lung diseases. The inflammation-induced overproduction of reactive oxygen species (ROS) means that endogenous antioxidants may not be sufficient to prevent oxidative damage, resulting in an oxidative imbalance in the lung. In turn, intracellular signaling events trigger the production of proinflammatory mediators that perpetuate and aggravate the inflammatory response and may lead to tissue damage. The production of high levels of ROS in inflammatory lung diseases can induce the phosphorylation of mitogen-activated protein kinases, the inactivation of phosphoinositide 3-kinase (PI3K) signaling and histone deacetylase 2, a decrease in glucocorticoid binding to its receptor, and thus resistance to glucocorticoid treatment. Hence, antioxidant treatment might be a therapeutic option for inflammatory lung diseases. Preclinical studies have shown that antioxidants (alone or combined with anti-inflammatory drugs) are effective in the treatment of inflammatory lung diseases, although the clinical evidence of efficacy is weaker. Despite the high level of evidence for the efficacy of antioxidants in the treatment of inflammatory lung diseases, the discovery and clinical investigation of safer, more efficacious compounds are now a priority.

International audience; AIM: Hydroxycinnamic acids their derivatives have various pharmacological properties. The hydroxycinnamic acid derivatives, methyl cinnamate, trans-cinnamic, and p-coumaric acids have been the object of study in the treatment of skin wounds. However, it is unclear whether these derivatives exert a direct beneficial effect on fibroblast function. In this study, we evaluated the effects of methyl cinnamate, trans-cinnamic, and p-coumaric acids on fibroblast migration in vitro. MATERIALS AND METHODS: NIH 3T3 and L929 fibroblast cell lines were exposed to each drug at several concentrations and the effect on cell viability, cell cycle, and extracellular matrix production were assessed by MTT assay, flow cytometry, and immunofluorescence staining, respectively. The effect on cell migration was examined using scratch assay. RESULTS: The results showed that hydroxycinnamic acid derivatives not affect cell viability, but increase fibroblast migration in the in vitro scratch-wound healing assay. They also induced an increase in S and G2/M phases accompanied by a decrease in the G0/G1 phase of the cell cycle. The cell proliferation inhibitor mitomycin C abolished the effect induced by p-coumaric acid and methyl cinnamate, indicating that only the trans-cinnamic acid stimulated migration. A transwell migration assay confirmed that trans-cinnamic acid-treated fibroblasts exhibited increased migration compared with untreated cells. trans-Cinnamic acid-induced fibroblast migration was decreased by PKA inhibitor and p38-MAPK inhibitor but not by JNK inhibitor. Additionally, trans-cinnamic acid-treated fibroblasts showed an increase in the production of laminin and collagen type I. CONCLUSION: Our study showed that trans-cinnamic acid improves fibroblast migration and modulates extracellular matrix synthesis, indicating its potential for accelerating the healing process.

International audience; Uvaol is a natural pentacyclic triterpene that is widely found in olives and virgin olive oil, exerting various pharmacological properties. However, information remains limited about how it affects fibroblasts and endothelial cells in events associated with wound healing. Here, we report the effect of uvaol in the in vitro and in vivo healing process. We show the positive effects of uvaol on migration of fibroblasts and endothelial cells in the scratch assay. Protein synthesis of fibronectin and laminin (but not collagen type I) was improved in uvaol-treated fibroblasts. In comparison, tube formation by endothelial cells was enhanced after uvaol treatment. Mechanistically, the effects of uvaol on cell migration involved the PKA and p38-MAPK signaling pathway in endothelial cells but not in fibroblasts. Thus, the uvaol-induced migratory response was dependent on the PKA pathway. Finally, topical treatment with uvaol caused wounds to close faster than in the control treatment using experimental cutaneous wounds model in mice. In conclusion, uvaol positively affects the behavior of fibroblasts and endothelial cells, potentially promoting cutaneous healing.

International audience; Alcohol consumption is considered to be the third leading cause of death in the United States. In addition to its direct toxicity, ethanol has two contrasting effects on the immune system the nucleotide oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome is inhibited by acute ethanol exposure but activated by chronic ethanol exposure. Purinergic receptors (especially the P2X7 receptor) are able to activate the NLRP3 inflammasome, and are involved in many ethanol-related diseases (such as gout, pulmonary fibrosis, alcoholic steatohepatitis and certain cancers). We hypothesized that ethanol regulates purinergic receptors and thus modulates the NLRP3 inflammasome's activity. In experiments with monocyte-derived macrophages, we found that interleukin (IL)-1β secretion was inhibited after 7h of exposure (but not 48 h of exposure) to ethanol. The disappearance of ethanol's inhibitory effect on IL-1β secretion after 48 h was not mediated by the upregulated production of IL-1β, IL-1α, IL-6 or the inflammasome components NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase 1. P2X7R expression was upregulated by ethanol, whereas expression of the P2X4 and P2X1 receptors was not. Taken as a whole, our results suggest that ethanol induces NLRP3 inflammasome activation by upregulating the P2X7 receptor. This observation might have revealed a new mechanism for inflammation in ethanol-related diseases.

Introduction Chronic Obstructive pulmonary disease (COPD) is a lung disease characterized by chronic inflammation, and mucus hypersecretion. Currently, curative treatment does not exist, and some patients develop resistance to corticosteroid drugs. Gene therapy could be a novel approach to treat the disease. However, the effectiveness of gene vectors in the airways is decreased by the presence of mucus that acts as a barrier. The aim of this study is to develop an in vitro model, able of cytokine production and mucus secretion; in the perspective of testing new drug including mucolytic gene therapy vectors. Methods We used three human airway epithelial lines (A549, Calu-3 and NCI-H292) stimulated with different concentrations of CSE (Cigarette Smoke Extract) alone or in association with LPS (lipopolysaccharides), for 24 or 48 hours. NCI-H292 and Calu-3 cells were also co-cultured in the same conditions. Then, we evaluated MUC5AC, IL8/CXCL8, GROα/CXCL1 and MCP1/CCL2 gene expression by RT-qPCR and their secretion by ELISA. Finally, we also observed MUC5AC production from NCI-H292 by immunohistochemistry. Results CSE alone or with LPS did not impact MUC5AC gene expression, in A549 cells. In contrast, LPS (0.1 μg/mL) increased IL8/CXCL8, GROα/CXCL1 and MCP1/CCL2 release. When CSE associated with LPS, there was an additive effect on cytokine release. Regarding Calu-3 cells, treatment with CSE, LPS, or both, did not affect MUC5AC gene expression, neither cytokine secretions. For NCI-H292 cells, CSE alone increased MUC5AC gene expression and an additive effect was observed when CSE was associated with LPS. LPS at low concentrations triggered some MUC5AC and IL8/CXCL8 release, which was more important when LPS was associated with CSE; but not for GROα/CXCL1. NCI-H292 cells did not release MCP1/CCL2. In the NCI-H292 and Calu-3 co-culture, CSE and LPS increased MUC5AC gene expression, but CSE did not affect cytokine secretion. LPS alone increased IL8/CXCL8 secretion, but not GROα/CXCL1 or MCP1/CCL2. Conclusion Our results show that NCI-H292 cells appear as the best model to evaluate the efficacy of gene vectors, as they are able to produce mucins and cytokines, after CSE and LPS exposure.

Chronic obstructive pulmonary disease (COPD) is an incurable and progressive disease. Emphysema is the principal manifestation of COPD, and the main cause of this condition is cigarette smoke (CS). Natural products have shown antioxidant and anti-inflammatory properties that can prevent acute lung inflammation and emphysema, but there are few reports in the literature regarding therapeutic approaches to emphysema. We hypothesized that supplementation with natural extracts would repair lung damage in emphysema caused by CS exposure. Mice were exposed to 60 days of CS and then treated or not with three different natural extracts (mate tea, grape and propolis) orally for additional 60 days. Histological analysis revealed significant improvements in lung histoarchitecture, with recovery of alveolar spaces in all groups treated with natural extracts. Propolis was also able to recovery alveolar septa and elastic fibers. Propolis also increased MMP-2 and decreased MMP-12 expression, favoring the process of tissue repair. Additionally, propolis recruited leukocytes, including macrophages, without ROS release. These findings led us to investigate the profile of these macrophages, and we showed that propolis could promote macrophage alternative activation, thus increasing the number of arginase-positive cells and IL-10 levels and favoring an anti-inflammatory microenvironment. We further investigated the participation of Nrf2 in lung repair, but no Nrf2 translocation to the nucleus was observed in lung cells. Proteins and enzymes related to Nrf2 were not altered, other than NQO1, which seemed to be activated by propolis in a Nrf2-independent manner. Finally, propolis downregulated IGF1 expression. In conclusion, propolis promoted lung repair in a mouse emphysema model via macrophage polarization from M1 to M2 in parallel to the downregulation of IGF1 expression in a Nrf2-independent manner. (C) 2017 Elsevier Ltd. All rights reserved.

International audience; Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation.

International audience; Acute lung injury (ALI) remains to cause a high rate of mortality in critically ill patients. It is known that inflammation is a key factor in the pathogenesis of lipopolysaccharide (LPS)-induced ALI, which makes it a relevant approach to the treatment of ALI. In this study, we evaluated the potential of nasally instilled p-coumaric acid to prevent LPS-induced ALI in mice, by evaluating its effects on cellular and molecular targets involved in inflammatory response via in vitro and in silico approaches. Our results demonstrated that p-coumaric acid reduced both neutrophil accumulation and pro-inflammatory cytokine abundance, and simultaneously increased IL-10 production at the site of inflammation, potentially contributing to protection against LPS-induced ALI in mice. In the in vitro experiments, we observed inhibitory effects of p-coumaric acid against IL-6 and IL-8 production in stimulated A549 cells, as well as reactive oxygen species generation by neutrophils. In addition, p-coumaric acid treatment decreased neutrophil adhesion on the TNF-α-stimulated endothelial cells. According to the in silico predictions, p-coumaric acid reached stable interactions with both the ATP-binding site of IKKβ as well as the regions within LFA-1, critical for interaction with ICAM-1, thereby suppressing the production of proinflammatory mediators and hindering the neutrophil infiltration, respectively. Collectively, these findings indicate that p-coumaric acid is a promising anti-inflammatory agent that can be used for developing a pharmaceutical drug for the treatment of ALI and other inflammatory disorders.

While until recently reactive oxygen species (ROS) were thought to mainly act as agents of cell damage, there is growing information on the role of ROS as mediators of signaling to regulate cellular homeostasis. In an article published in Cell Metabolism (2018 November 28; 6:764–775. doi: 10.1016/j.cmet.2018.07.012), Dogan and colleagues reported a notable decline of the clinical and biochemical phenotype in a double mutant model of cytochromec oxidase-defective mitochondrial myopathy and alternative oxidase (AOX). AOX directly oxidizes ubiquinone, preserving electron flow from carriers NADH and FADH2, and abolishes the contribution of complexes III and IV to membrane potential in the mitochondrial respiratory chain. Although AOX can limit the generation of ROS and preserve redox homeostasis, thereby maintaining tricarboxylic acid cycle activity, the authors highlight that antioxidants can inhibit the homeostatic response to bioenergetic failure by modulating mitochondrial biogenesis. The result supports that interruption of ROS signaling might negatively impact the induction of mitochondrial biogenesis and antioxidant gene expression. This prevents cellular processes that are subject to redox regulation for oxidative damage chain, thereby leading to mitochondrial dysfunction.

The liver is the major site of first-pass metabolism and, accordingly, is highly exposed to oxidative injury caused by reactive intermediates, resulting in the stimulation of different biological targets. Diseases such as nonalcoholic steatohepatitis, which is characterized histologically by hepatic steatosis, necroinflammation, and progressive substitution of the functioning hepatic parenchyma by fibrotic tissue, are widely related to oxidative stress, although the mechanisms are not completely understood. A rational attempt to comprehend the pathways underlying redox-mediated fibrogenic signaling may be investigating the adaptive responses to oxidative stress by interacting with the antioxidant response. The expression of a variety of downstream targets aimed at cytoprotection, primarily mediated through antioxidant response elements, are largely under the control of nuclear factor E2-related factor 2 (Nrf2). In this study, the regulation of the cellular response to oxidative stress was determined in the presence of Nrf2 activators or Nrf2-null mice influencing lipid metabolism and targeted cytoprotection of hepatocytes during inflammation/fibrosis. These interactions participate in a multi-tiered, integrated reaction to chemical stress, in which Nrf2 signaling pathway can be considered as a key factor in orchestrating adaptive responses in liver disease.

International audience; Fatty livers are susceptible to factors that cause inflammation and fibrosis, but fat deposition and the inflammatory response can be dissociated. While nonalcoholic fatty liver disease (NAFLD), caused by pathologic fat accumulation inside the liver, can remain stable for several years, in other cases NAFLD progresses to nonalcoholic steatohepatitis (NASH), which is characterized by fat accumulation and inflammation and is not a benign condition. In this review, we discuss the NASH host cells and microbial mechanisms that stimulate inflammation and predispose the liver to hepatocyte injury and fibrotic stages via increased lipid deposition. We highlight the interactions between intestine-derived bacterial products, such as lipopolysaccharide, and nutritional models of NAFLD and/or obese individuals. The results of modulating enteric microbiota suggest that gut-derived endotoxins may be essential determinants of fibrotic progression and regression in NASH.

International audience; Background: β2-adrenoceptor agonists have been shown to reduce the lipopolysaccharide (LPS)-induced cytokine release by human monocyte-derived macrophages (MDMs). We compare the expression of β2-adrenoceptors and the inhibitory effect of formoterol and salmeterol on the LPS-induced release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and a range of chemokines (CCL2, 3, 4, and IL-8) by human lung macrophages (LMs) and MDMs. Methods: LMs were isolated from patients undergoing resection and MDMs were obtained from blood monocytes in the presence of GM-CSF. LMs and MDMs were incubated in the absence or presence of formoterol or salmeterol prior to stimulation with LPS. The effects of formoterol were also assessed in the presence of the phosphodiesterase inhibitor roflumilast. Results: LPS-induced cytokine production was higher in LMs than in MDMs. Salmeterol and formoterol exerted an inhibitory effect on the LPS-induced production of TNF-α, IL-6, CCL2, CCL3, and CCL4 in MDMs. In contrast, the β2-adrenoceptor agonists were devoid of any effect on LMs - even in the presence of roflumilast. The expression of β2-adrenergic receptors was detected on Western blots in MDMs but not in LMs. Conclusions: Concentrations of β2-adrenoceptor agonists that cause relaxation of the human bronchus can inhibit cytokine production by LPS-stimulated MDMs but not by LMs. © 2017 The Author(s).

International audience; Alveolar macrophages play a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Monocytes are recruited from blood during inflammation and then mature into alveolar macrophages. The aim of this study was to investigate the effect of cigarette smoke (CS) at different times in lung macrophages and monocytes from blood and bone marrow in mice. Male mice (C57BL/6, n = 45) were divided into groups: control, CS 5 days, CS 14 days and CS 30 days. Five days' CS exposure induced a pronounced influx of neutrophils and macrophages in the lung associated with increased levels of keratinocyte chemoattractant (KC), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and matrix metalloproteinase (MMP)-12. After 14 days of CS exposure, neutrophil recruitment and cytokine production were greatly reduced. Moreover, chronic CS exposure led to increased recruitment of macrophages (with high expression of CD206), transforming growth factor-β (TGF-β) production as well as no detection of TNF-α, interleukin (IL)-6 and KC. CS can also change the monocyte phenotype in the blood and bone marrow, with an increase in Ly6C(low) cells. These results show for the first time that CS can change not only macrophage polarization but also monocyte. These results suggest that continued recruitment of Ly6C(low) monocytes may help the distinct renewing macrophage M2 population required for COPD progression.

Emphysema is an incurable and progressive disease, and cigarette smoke (CS) is the main risk factor. Natural extracts have shown antioxidant and anti-inflammatory properties that can prevent acute lung inflammation and emphysema, but there are almost no reports in literature about therapeutics approaches to emphysema. We hypothesized if supplementation with propolis would repair lung damage in emphysema caused by CS exposure. C57BL/6 mice were exposed to 60 days of CS and then treated or not with propolis extract orally for more 60 days. Histological analysis revealed significant improvement in lung histoarchiteture, with recover of alveolar spaces, septa and elastic fibers in CS+propolis group. This natural extract also was able to increase MMP-2 and decrease MMP-12 expression, favoring the process of tissue repair. Propolis was also capable of recruiting leukocytes, among them macrophages, without ROS release. It leads us to investigate the profile of these macrophages and we showed that propolis could promote macrophage alternative activation and increase IL-10 levels, developing an anti-inflammatory microenvironment. We further investigated Nrf2 participation in lung repair promoted by propolis, but we showed that propolis did not stimulated Nrf2 translocation to nucleus. Proteins and enzymes related to Nrf2 was not altered, other than NQO1, that can be activate by propolis in a Nrf2-indepent manner. Propolis also acts downregulating IGF1. Beneficial effects of propolis supplementation in emphysema treatment likely result from it capacity of act in IGF1 downregulation and MMPs and change macrophage profile, in a Nrf2-independent manner.

International audience; 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)-1β, 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.

International audience; Adenosine triphosphate is involved in many metabolic reactions, but it has also a role as a cellular danger signal transmitted through purinergic receptors (PRs). Indeed, adenosine 5′-triphosphate (ATP) can bind to PRs which are found in the membrane of many cell types, although the relative proportions of the receptor subtypes differ. PRs are classified according to genetic and pharmacological criteria and especially their affinities for agonists and their transduction mechanism (i.e. as metabotropic P2YRs or ionotropic P2XRs). Extracellular ATP release by activated or necrotic cells may activate various PRs and especially P2X7R, the best-characterized PR, on immune cells. P2X7R is known to regulate the activation of the Nod-like receptor (NLR)-family protein, NLRP3 inflammasome, which permit the release of IL-1β, a potent pro-inflammatory cytokine. The P2X7R/NLRP3 pathway is involved in many inflammatory diseases, such as gout, and in fibrosis diseases associated with inflammatory process, liver or lung fibrosis. Some authors imaging also a real promising therapeutic potential of P2X7R blockage. Thus, several pharmaceutical companies have developed P2X7R antagonists as novel anti-inflammatory drug candidates. Clinical trials of the efficacy of these antagonists are now underway. A better understanding of the P2X7R/NLRP3 signalling pathways permits the identification of targets and the development of a new class of drugs able to inhibit the fibrogenesis process and collagen deposition.

There is evidence that diets rich in salt or simple sugars as fructose are associated with abnormalities in blood pressure regulation. However, the mechanisms underlying pathogenesis of salt- and fructose-induced kidney damage and/or consequent hypertension yet remain largely unexplored. Here, we tested the role of oxidative state as an essential factor along with high salt and fructose treatment in causing hypertension. Fischer male rats were supplemented with a high-fructose diet (20% in water) for 20 weeks and maintained on high-salt diet (8%) associate in the last 10 weeks. Fructose-fed rats exhibited a salt-dependent hypertension accompanied by decrease in renal superoxide dismutase activity, which is the first footprint of antioxidant inactivation by reactive oxygen species (ROS). Metabolic changes and the hypertensive effect of the combined fructose-salt diet (20 weeks) were markedly reversed by a superoxide scavenger, Tempol (10 mg/kg, gavage); moreover, Tempol (50 mM) potentially reduced ROS production and abolished nuclear factor-kappa B (NF-κB) activation in human embryonic kidney HEK293 cells incubated with L-fructose (30 mM) and NaCl (500 mosmol/kg added). Taken together, our data suggested a possible role of oxygen radicals and ROS-induced activation of NF-κB in the fructose- and salt-induced hypertension associated with the progression of the renal disease.

National audience; 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-1β. 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-1β might be the new anti-inflammatory drugs.; L’immunité innée constitue la première ligne de défense de l’organisme contre les pathogènes. La réponse inflammatoire permet de lutter contre le « non soi » comme les organismes infectieux ou les particules exogènes. L’inflammasome est un complexe multiprotéique intracytoplasmique activé par des stress cellulaires ou des infections et sont responsables de la libération de cytokines pro-inflammatoires, notamment l’IL-1β. L’inflammasome NLRP3, le plus étudié des inflammasomes, est impliqué dans des pathologies inflammatoires telles que la maladie de Crohn, la polyarthrite rhumatoïde ou l’arthrite goutteuse. Les CAPS (syndromes périodiques associés à la cryopyrine) sont des pathologies spécifiques de l’inflammasome NLRP3. Le canakinumab (Ilaris®) est la seule spécialité ayant eu l’autorisation de mise sur le marché (AMM) dans le traitement des CAPS. Les biothérapies ciblant l’inflammasome NLRP3 et l’IL-1β peuvent représenter de nouvelles armes thérapeutiques anti-inflammatoires.

International audience; Matrix metalloproteinases (MMPs) are a group of proteases known to regulate the turnover of extracellular matrix and thus are suggested to be important in the process of several diseases associated with tissue remodeling. Furthermore, the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue, may be of interest as a basis for future treatment. Degradation of extracellular matrix is currently associated with structural and recruited cell activation and release of inflammatory mediators and MMPs. Indeed, a marked increase in their expression is observed associated with a variety of inflammatory diseases, including respiratory pathologies. In these conditions, we have to consider MMPs as therapeutic targets which can be inhibited by non-selective and/or selective inhibitors as anti-inflammatory compounds. The present review aims to discuss the potential interest of the inhibition of MMP in inflammatory diseases with a focus on respiratory diseases.

International audience; The anti-inflammatory effects of CI-1044 and of the other selective PDE4 inhibitors rolipram and cilomilast were investigated in Brown-Norway (BN) rats, against lipopolysaccharide-induced tumor necrosis factor alpha (TNFalpha) production in whole blood and antigen-induced lung eosinophilia. In vitro, CI-1044 inhibited TNFalpha production with an IC(50) of 0.31 microm being equipotent to Cilomilast (IC(50) = 0.26 microm) and rolipram (IC(50) = 0.11 microm). Given orally, CI-1044 inhibited ex vivo TNFalpha production with an ED(50) value of 0.4 mg/kg after single administration, whereas rolipram (ED(50) = 1.4 mg/kg) and cilomilast (ED(50) = 1.6 mg/kg) were less potent. In the same ex vivo setting, but given repeatedly, CI-1044 led to an ED(50) of 0.5 mg/kg corresponding to a plasma concentration of 82.6 ng/mL (0.22 microm). In vivo, CI-1044 prevented TNFalpha release with an ED(50) of 1 mg/kg p.o. and inhibited ovalbumin-induced lung eosinophilia following single or repeated oral administration with an ED(50) of 3.25 and 4.8 mg/kg p.o., respectively, suggesting the absence of pharmacological tolerance. CI-1044 in this model was equipotent to rolipram (81% inhibition at 10 mg/kg) but better than cilomilast (25% inhibition at 10 mg/kg). Finally, CI-1044 (10 mg/kg) inhibited inflammatory cell recruitment with a long duration of action (up to 8 h) and was still active when given post-challenge. Our data show that CI-1044 is an orally active PDE4 inhibitor that may be used as an anti-inflammatory therapy in lung inflammatory diseases.

Background: MMPs are known to regulate the turnover of extracellular matrix and have been suggested to be important in lung disease associated with tissue remodeling. Macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling in inflammatory respiratory diseases such as chronic obstructive pulmonary diseases (COPD), including emphysema. Recent studies using MMP-12 inhibitors have demonstrated a reduction in both the inflammatory process and airspace enlargement in lung tissue. Objective/methods: This review discusses the potential involvement of MMP-12 in the pathophysiological process and proposes MMP-12 as a target for inflammatory disorders of the respiratory system. Results/conclusions: MMP-12 plays a predominant role in the inflammatory process induced by cigarette smoke, and therefore is potentially an important therapeutic target for the treatment of COPD.

Background: Macrophage elastase (MMP-12) is involved in the inflammatory process of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate in mice the effect of MMP-12 inhibition on the inflammatory process induced by cigarette smoke (CS) or by lipopolysaccharide (LPS) exposure of the airways. Experimental approach: C57BL/6 mice were given, orally, either the selective MMP-12 inhibitor AS111793 (3, 10, 30 and 100 mg kg−1), the PDE-4 inhibitor roflumilast (3 mg kg−1) or vehicle, then exposed to CS (for 3 days) or to LPS (100 μg mL−1, 30 min). Subsequent to the last smoke or LPS exposure, bronchoalveolar lavages (BAL) were performed and lungs were removed and homogenized to analyze various markers of inflammation at appropriate times. Key results: Inhibition of MMP-12 by AS111793 (10 and 30 mg kg−1) was associated with a reduction of the increase in neutrophil number in BAL fluids after 4 days and of macrophages after 11 days. On day 4, AS111793 also significantly reduced all the inflammation markers that had increased after CS exposure, including soluble TNF receptors I and II, MIP-1γ, IL-6 and pro-MMP-9 activity in BAL fluids, and KC/CXCL1, fractalkine/CX3CL1, TIMP-1 and I-TAC/CXCL11 in lung parenchyma. In contrast, inhibition of MMP-12 did not reduce neutrophil influx, pro-MMP-9 activity or KC/CXCL1 release in BAL fluids of mice exposed to LPS. Conclusion: Inhibition of MMP-12 with AS111793, reduced the inflammatory process associated with exposure of mice to CS, strongly suggesting a specific involvement of MMP-12 in lung inflammation following CS exposure. British Journal of Pharmacology (2008) 154, 1206–1215; doi:10.1038/bjp.2008.180; published online 19 May 2008

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.

International audience; Benzo(a)pyrene (BP) is an environmental contaminant known to favor airway inflammation likely through up-regulation of pro-inflammatory cytokines. The present study was designed to characterize its effects toward interleukin-8 (IL-8), a well-established pulmonary inflammatory cytokine. In primary human macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion levels in a dose-dependent manner. Such an up-regulation was likely linked to aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was reduced after AhR expression knock-down through RNA interference. Moreover, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation experiments showed BP-triggered binding of AhR to a consensus xenobiotic responsive element (XRE) found in the human IL-8 promoter. Finally, BP administration to mice led to over-expression of keratinocyte chemoattractant (KC), the murine functional homologue of IL-8, in lung. It also triggered the recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was however fully abolished in the presence of a chemical antagonist of the KC/IL-8 receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of KC in BP-induced lung inflammation. Overall, these data highlight an AhR-dependent regulation of IL-8 in response to BP that likely contributes to the airway inflammatory effects of this environmental chemical.

Macrophage elastase (recombinant human matrix metalloproteinase-12, rhMMP-12), was instilled in mouse airways, inducing an early inflammatory response characterized by neutrophil recruitment and cytokine release in the bronchoalveolar lavage (BAL) fluids, followed by a delayed macrophage recruitment. We investigated the role played by alveolar macrophages and neutrophils in the delayed macrophage influx induced by rhMMP-12 (8 x 10(-3) U/mouse) in A/J mice. Mice depleted of circulating neutrophils, using a cytotoxic antibody, did not present an increase in neutrophil numbers in bronchoalveolar lavage fluids, 4 h and 24 h after rhMMP-12 instillation but the macrophage recruitment was not modified as compared to control mice at 7 days. Similar results were obtained using mice when the gene for neutrophil elastase was knocked out. Intranasal instillation of clodronate liposomes, 72 h prior to rhMMP-12 instillation, induced macrophage depletion which did not modify the macrophage recruitment at 7 days. Moreover, the stimulation of mouse macrophages by rhMMP-12 did not elicit the release of cytokines in culture supernatants. These results indicate that resident alveolar macrophages and recruited neutrophils do not play a role in the delayed macrophage recruitment induced by rhMMP-12.

International audience; 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. © 2016 The Author(s).

International audience; Uvaol, a triterpene present in olives and virgin olive oil, has been shown to possess anti-inflammatory properties and antioxidant effects. However, until now, no studies have demonstrated its potential effects on allergic inflammation. The aim of this study was to evaluate the anti-inflammatory effects of uvaol in a mouse model of allergy characterized by eosinophil-dominant inflammation in actively sensitized mice. The anti-inflammatory effect of uvaol was analyzed in two murine models of allergic inflammation (pleurisy and asthma). In these models, Swiss mice were sensitized and challenged with ovalbumin (OVA). In the pleurisy model, the pleural eosinophilic inflammation and IL-5 concentrations were examined 24 h after the OVA challenge, while in the asthma model were examined the airway inflammation via bronchoalveolar lavage (BAL) fluid cytology and lung histopathology analyses. Our results showed that uvaol decreased the accumulation of eosinophils and the concentration of IL-5 in pleural effluent. Uvaol also demonstrated important anti-inflammatory activity by inhibiting production of IL-5 and influx of leukocytes, mainly of eosinophils, in BAL fluid, but without interfering with levels of reactive oxygen species in leukocytes. Moreover, the eosinophil infiltration, mucus production, number of alveoli that collapsed, and IL-5 levels in the lung were clearly decreased by uvaol treatment. These findings indicate that uvaol can be a good candidate for the treatment of allergic inflammation by inhibiting eosinophil influx and IL-5 production in ovalbumin-induced allergy

International audience; 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 β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. 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-β1 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-1β, 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

International audience; Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production

The nerve growth factor (NGF) is a neurotrophic factor essential for the development and survival of neurons. It has also been identified as a mediator of inflammation and can cause airway hyperresponsiveness [Frossard et al., Eur. J. Pharmacol. 500, 453 (2004)]. Evidence in rodents suggests a link between tachykinins, the sensory nerves, and NGF. Recent evidence shows that NGF is released by the proinflammatory cytokine interleukin-1beta and induces hyperresponsiveness to the tachykinin NK1 receptor agonist [Sar(9),Met(O(2))(11)]SP in isolated human bronchi. The aim of this study was to determine the role of sensory nerves through the effect of the tachykinin NK3 receptor antagonist SR142801 in the interleukin-1beta effects and/or the NGF-induced airway hyperresponsiveness. SR142801 (0.1 microM) abolished the interleukin-1beta (10 ng/ml, 21 degrees C, 15 h)-induced increased NGF release from isolated human bronchi in vitro (P0.05). In organ bath studies, SR142801 also abolished the interleukin-1beta-induced airway hyperresponsiveness to [Sar(9),Met(O(2))(11)]SP (0.1 microM) (P0.05). SR142801 also inhibited the NGF-induced airway hyperresponsiveness (P0.01). This study suggests tachykininergic sensory nerves to be involved in the interleukin-1beta-induced NGF release and airway hyperresponsiveness.

Direct instillation of a recombinant human form of MMP-12 (rhMMP-12) in mice airways elicited an early inflammatory response characterized by neutrophil influx, cytokine release and gelatinase activation followed by a delayed response, mainly characterized by macrophage recruitment. As this experimental model of lung inflammation partially mimics some features of chronic obstructive pulmonary disease (COPD), we have investigated the effects of treatment by anti-inflammatory compounds, dexamethasone and rolipram and a non-specific matrix metalloproteinase (MMP) inhibitor, marimastat. The compounds were administrated orally, 1 h before rhMMP-12 instillation (8 x 10(-3) U/mouse). Total and differential cell counts were evaluated in the bronchoalveolar lavage fluids. Cytokines and MMP-9 were quantified in bronchoalveolar lavage fluids and in lung homogenate supernatants. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.1 and 0.3 mg/kg) were able to decrease significantly neutrophil recruitment at 4 and 24 h after rhMMP-12 instillation, but only marimastat (30 and 100 mg/kg) was effective at decreasing the macrophage recruitment occurring at day 7. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.3 mg/kg) reduced significantly IL-6, KC/CXCL1, MIP-1alpha/CCL3 and MMP-9 levels in bronchoalveolar lavage fluid. Similar results were obtained in lung homogenates except with rolipram. Dexamethasone and rolipram were able to inhibit the early inflammatory response but were ineffective to limit the macrophage influx. In contrast, marimastat was able to reduce early and late response. These data indicate that MMP-12 instillation in mice could highlight some of the inflammatory response seen in COPD and could be used for the pharmacological evaluation of new anti-inflammatory mechanisms of action.

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-1β. 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-1β might be the new anti-inflammatory drugs.

We report here the complete genome sequence of a noninvasive strain of Streptococcus pyogenes M/ emm28 , isolated from perianal dermatitis in a child. The genome is composed of 1,950,454 bp, with a G+C content of 38.2%, and it has 1,925 identified coding sequences and harbors two intact prophages and a new integrating conjugative element (ICE).

Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turnover of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. These lead to the concept that modulation of airway remodelling, including excessive proteolysis damage of the tissue, may be of interest for future treatment. There is a great need for relevant models and more information is required regarding the involvement of MMPs in chronic obstructive pulmonary disease.