Emma Folch-Puy

Oxygen Free Radicals and the Systemic Inflammatory Response

The generation of oxygen free radicals is known to be involved in the development of the systemic inflammatory response syndrome. In addition to their actions as noxious mediators generated by inflammatory cells, these molecules play also a crucial role contributing to the onset and progression of inflammation in distant organs. In the early stages of the process, free radicals exert their actions via activation of nuclear factors, as NFκB or AP‐1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion molecules. Finally, free radicals exert their toxic effects at the site of inflammation by reacting with different cell components, inducing loss of function and cell death. This review focuses on progress in the understanding the different actions of free radicals at the sequential stages of the development of the systemic inflammatory response. IUBMB Life, 56: 185‐191, 2004

Effect of simultaneous inhibition of TNF-α production and xanthine oxidase in experimental acute pancreatitis: The role of mitogen activated protein kinases

Objective:To assess the effects of inhibiting both tumor necrosis factor (TNF)-α production and xanthine oxidase activity on the inflammatory response, mitogen-activated protein kinase (MAPK) activation and mortality in necrotizing acute pancreatitis in rats. Summary Background Data:Pancreatic injury triggers 2 major pathways involved in the systemic effects of severe acute pancreatitis: pro-inflammatory cytokines and oxidative stress. Methods:Pancreatitis was induced by intraductal infusion of 3.5% sodium taurocholate. We examined whether treatment with oxypurinol, a specific inhibitor of xanthine oxidase, and/or pentoxifylline, an inhibitor of TNF-α production, affects pancreatic damage, ascites, lung inflammation, and MAPK phosphorylation. Results:Oxypurinol prevented p38 phosphorylation in the pancreas and partially avoided the rise in lung myeloperoxidase activity. Pentoxifylline prevented erk 1/2 and JNK phosphorylation in the pancreas, and it partially reduced ascites and the rise in lung myeloperoxidase activity. Combined treatment with oxypurinol and pentoxifylline almost completely abolished ascites, MAPK phosphorylation in the pancreas, and the increase in lung myeloperoxidase activity. Histology revealed a reduction in pancreatic and lung damage. These changes were associated with a significant improvement of survival. Conclusions:Simultaneous inhibition of TNF-α production and xanthine oxidase activity greatly reduced local and systemic inflammatory response in acute pancreatitis and decreased mortality rate. These effects were associated with blockade of the 3 major MAPKs.

An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation.

KLF11 is a biochemical paradigm for a subset of proteins that repress transcription via a Mad1‐like mSin3A interacting domain (SID). The biological role of these proteins and the significance of their biochemical activity, however, remain to be established. We report that KLF11 is downregulated in human cancers, inhibits cell growth in vitro and in vivo, and suppresses neoplastic transformation. Transgenic mice for KLF11 display a downregulation of genes encoding the oxidative stress scavengers SOD2 and Catalase1. Chromatin immunoprecipitation assays confirm that, indeed, these genes are bonafide targets of KLF11. KLF11 expression renders cells more sensitive to oxidative drugs, an effect that is rescued by infection with recombinant adenoviruses expressing SOD2 and Catalase1. KLF11‐regulated functions require the Mad1‐like SID, indicating that these target genes involved in these phenomena are regulated via this corepressor system. These results demonstrate that SID‐containing KLF repressor proteins can inhibit cell growth and neoplastic transformation, and outline for the first time cellular and molecular mechanisms by which these functions may be achieved.

Pancreatitis-associated protein I suppresses NF-κB activation through a JAK/STAT-mediated mechanism in epithelial cells

Pancreatitis-associated protein I (PAP I), also known as HIP, p23, or Reg2 protein, has recently been implicated in the endogenous regulation of inflammation. Although it was initially characterized as a protein that is overexpressed in acute pancreatitis, PAP I has also been associated with a number of inflammatory diseases, such as Crohn’s disease. Knowing that PAP I and IL-10 responses share several features, we have used a pancreatic acinar cell line (AR42J) to assess the extent to which their expression is reciprocally regulated, and whether the JAK/STAT and NF-κB signaling pathways are involved in the suppression of inflammation mediated by PAP I. We observed that PAP I is induced in epithelial cells by IL-10 and by PAP I itself. In contrast, we found phosphorylation and nuclear translocation of STAT3 and induction of suppressor of cytokine signaling 3 in response to PAP I exposure. Finally, a JAK-specific inhibitor, tyrphostin AG490, markedly prevented PAP I-induced NF-κB inhibition, pointing to a cross-talk between JAK/STAT3 and NF-κB signaling pathways. Together, these findings indicate that PAP I inhibits the inflammatory response by blocking NF-κB activation through a STAT3-dependent mechanism. Important functional similarities to the anti-inflammatory cytokine IL-10 suggest that PAP I could play a role similar to that of IL-10 in epithelial cells.

Experimental acute pancreatitis in PAP/HIP knock-out mice

Background and aims: PAP/HIP was first reported as an additional pancreatic secretory protein expressed during the acute phase of pancreatitis. It was shown in vitro to be anti-apoptotic and anti-inflammatory. This study aims to look at whether PAP/HIP plays the same role in vivo. Methods: A model of caerulein-induced pancreatitis was used to compare the outcome of pancreatitis in PAP/HIP−/− and wild-type mice. Results: PAP/HIP−/− mice showed the normal phenotype at birth and normal postnatal development. Caerulein-induced pancreatic necrosis was, however, less severe in PAP/HIP−/− mice than in wild-type mice, as judged by lower amylasemia and lipasemia levels and smaller areas of necrosis. On the contrary, pancreas from PAP/HIP−/− mice was more sensitive to apoptosis, in agreement with the anti-apoptotic effect of PAP/HIP in vitro. Surprisingly, pancreatic inflammation was more extensive in PAP/HIP−/− mice, as judged from histological parameters, increased myeloperoxidase activity and increased pro-inflammatory cytokine expression. This result, in apparent contradiction with the limited necrosis observed in these mice, is, however, in agreement with the anti-inflammatory function previously reported in vitro for PAP/HIP. This is supported by the observation that activation of the STAT3/SOCS3 pathway was strongly decreased in the pancreas of PAP/HIP−/− mice and by the reversion of the apoptotic and inflammatory phenotypes upon administration of recombinant PAP/HIP to PAP/HIP−/− mice. Conclusion: The anti-apoptotic and anti-inflammatory functions described in vitro for PAP/HIP have physiological relevance in the pancreas in vivo during caerulein-induced pancreatitis.

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL‐1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL‐1 solutions with or without MEL + TMZ and subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin‐1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL‐1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL‐1 alone. IGL‐1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin‐1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL‐1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.

Importance of the liver in systemic complications associated with acute pancreatitis: the role of Kupffer cells.

Although its exact nature is still unknown, acute pancreatitis progresses with a local production of inflammatory mediators, eventually leading to systemic inflammatory response syndrome. Knowing that almost all pancreatic mediators released from the pancreas to the bloodstream may pass through the liver before their dilution in the systemic circulation, it would be reasonable to assume a determinant role for this organ in the development of the inflammatory response associated with acute pancreatitis. Thus, recent studies have shown the involvement of the liver in the complex network of events triggering the multiorgan dysfunction associated with the disease. Once pancreatic mediators reach the liver, they strongly activate Kupffer cells, the resident macrophages, greatly amplifying the release of cytokines into the bloodstream and thus contributing to the systemic manifestations of acute pancreatitis. Altogether, these results show that the pancreas is not the only source of mediators that trigger the deleterious effects of acute pancreatitis, but that the liver may orchestrate the final outcome of the disease. The purpose of this review is to discuss progress in understanding the function of the liver in the early stages of the development of systemic organ dysfunction secondary to acute pancreatitis. Copyright

Oxidised lipids present in ascitic fluid interfere with the regulation of the macrophages during acute pancreatitis, promoting an exacerbation of the inflammatory response

Background: Pancreatitis-associated ascitic fluid (PAAF) plays a critical role in the pathogenesis of acute pancreatitis. Taking into consideration that damaged pancreas exudes high concentrations of lipolytic enzymes in the peritoneal cavity, large amounts of lipid metabolism derived products could occur in PAAF. In this study, we have examined the involvement of the lipid fraction of PAAF generated in the early stages of experimental acute pancreatitis. Methods: Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. After 3 h, PAAF was collected and its lipid fraction was obtained. Lipid composition and levels of lipid peroxidation were measured. Toxicity was evaluated by measuring the effects of the PAAF lipid fraction on cell viability of hepatic and macrophage cell lines. In vivo effects on the liver were also evaluated. Effects on the inflammatory response were determined by measuring the levels of nuclear factor kappa B (NFκB) activation, the effect on the inhibitory activity of 15-deoxy-PGJ2 and the possible interference on PPARγ activation. Results: High concentrations of oxidised free fatty acids were detected in PAAF. Besides the direct cell toxicity, the PAAF-derived lipid extract interfered with the anti-inflammatory pathway mediated by PPARγ. Addition of this lipid extract to macrophage cell cultures had no direct effect on the activation of NFκB, but abolished the inhibitory activity of endogenous PPARγ agonists such as 15-deoxy-PGJ2. Conclusions: Oxidised free fatty acids present in PAAF interfere with the endogenous regulatory mechanism of the inflammatory response, thus promoting an exacerbation of macrophage activation in acute pancreatitis.

Reg3β Deficiency Impairs Pancreatic Tumor Growth by Skewing Macrophage Polarization

The lectin Reg3β provides crucial protection to various tissues against inflammation, a potential risk factor for pancreatic ductal adenocarcinoma. Reg3β is also overexpressed in serum and pancreatic juice from patients with this cancer, but its function in this context remains to be elucidated. In this study, we investigated the role of Reg3β in tumor development in an orthotopic mouse model of pancreatic cancer. Reg3β deletion in mice drastically impaired pancreatic tumor growth, correlating with decreased angiogenesis and increased apoptosis of tumor cells. Moreover, Reg3β deficiency resulted in an alteration of the tumoral immune microenvironment, reflected by a decrease in the M2/M1 ratio of tumor-associated macrophages and an upregulation of CD3(+) cell infiltration. Addition of Reg3β to prestimulated RAW 264.7 or primary macrophages enhanced M2 polarization through the activation of STAT3 signaling pathway. Conditioned media from Reg3β-M2-polarized primary macrophages inhibited apoptosis and prolonged the viability of Panc02 tumor cells. Our studies reveal a novel role for Reg3β as a tumor promoter in pancreatic adenocarcinoma through the regulation of tumor stroma. Thus, inhibition of this protein may be a useful strategy in treatment of pancreatic cancer.

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)‐1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways.