Helen M. Becker

Protein tyrosine phosphatase nonreceptor type 2 regulates autophagosome formation in human intestinal cells

Background: Autophagy is a process of central importance for maintaining cell homeostasis, survival, and the regulation of inflammation. Recent studies associated variants within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2), and autophagy genes, such as autophagy‐related 16‐like 1 (ATG16L1), with chronic inflammatory disorders, such as Crohns disease (CD). We show that PTPN2 regulates autophagy in human intestinal epithelial cells (IEC) and primary colonic lamina propria fibroblasts (CLPF). Methods: Protein analysis in IEC and CLPF was performed by western blotting. Autophagososme formation was assessed by LC3B immunofluorescence or immunohistochemistry. Human intestinal tissue samples were obtained from noninflammatory bowel disease (IBD) control or from CD patients and genotyped for disease‐associated PTPN2 or ATG16L1 variations. Results: Knockdown of PTPN2 causes impaired autophagosome formation and dysfunctional autophagy resulted in increased levels of intracellular Listeria monocytogenes (LM) and elevated IEC apoptosis in response to tumor necrosis factor (TNF) and interferon gamma (IFN‐&ggr;). Similar findings were observed in primary CLPF derived from CD patients carrying the CD‐associated PTPN2 variant. Presence of the ATG16L1 variant prevented the cytokine‐induced rise in PTPN2 protein, finally resulting in impaired LC3B‐II levels in IEC. Actively inflamed intestinal biopsies from CD patients carrying either ATG16L1 or PTPN2 genetic variants revealed aberrant LC3B expression patterns when compared with samples from non‐IBD control patients. Conclusions: Our results demonstrate that PTPN2 regulates autophagosome formation in human intestinal cells. We provide a model of how a dysfunction of the CD susceptibility genes, PTPN2 and/or ATG16L1, may contribute to the onset and perpetuation of chronic intestinal inflammation. (Inflamm Bowel Dis 2011;)

Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome

Objective Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. Design Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. Results Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1β and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. Conclusion These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.

BCL-2 Modifying Factor (BMF) Is a Central Regulator of Anoikis in Human Intestinal Epithelial Cells

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf−/−) with dextran sulfate sodium. Colonic crypt length in Bmf−/− mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf−/− mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival.

Probiotic Escherichia coli Nissle 1917 and Commensal E. coli K12 Differentially Affect the Inflammasome in Intestinal Epithelial Cells

Background: The probiotic bacterial strain Escherichia coli Nissle 1917 (EcN) is used for the treatment of ulcerative colitis (UC), diarrhea and constipation. Its beneficial effects in the treatment of UC have been demonstrated in several controlled clinical studies; however, the mechanism of action on the cellular level is still not completely clear. The intracellular pattern recognition receptor NLRP3 is expressed in intestinal epithelial cells (IEC), activates caspase-1 within the inflammasome complex and has been implicated to play a role in the etiology of inflammatory bowel diseases. Methods: Probiotic EcN and commensal E. coli K12 were applied to IEC in vitro. Inflammasome activation, interleukin (IL)-18 release and caspase-1 activation were determined by coimmunoprecipitation, Western blot and ELISA. Apoptosis was investigated by Western blot. Results: Incubation of Caco-2 cells with EcN resulted in lower inflammasome activation and subsequent secretion of mature IL-18 as compared to the commensal strain K12. Induction of apoptosis as determined by cleavage of caspase-3 and poly (ADP-ribose) polymerase were lower in EcN-stimulated cells. Autophagy was induced by both bacterial strains, but to a higher extent by K12. Conclusion: These findings indicate that genetically very similar E. coli strains differ markedly in their ability to activate the inflammasome.

Microparticles and Their Impact on Intestinal Immunity

Microparticles are small (<1 µm), nonbiological particles that are used in many areas of daily life. As food additive they are used as anticaking agents or food colorants. The most common food-derived ingested compounds are aluminium silicate and titanium dioxide (TiO2), the latter being a white pigment used in toothpaste or sugar toppings. The increasing abundance of microparticles in the Western diet raises the question of the potential risks associated with gastrointestinal diseases such as Crohn’s disease (CD). Accumulation of particles has been shown in cells of Peyer’s patches, but it is not clear whether this also has pathological effects. NLRP3 is a member of the intracellular pattern recognition receptor family and it is part of the inflammasome, a multiprotein complex containing caspase-1 which activates the proinflammatory cytokines interleukin (IL)-1β and IL-18. With regard to recent findings identifying small particles such as asbestos and monosodium urate as NLRP3 activators, TiO2 may be another potential target for inflammasome studies. We found that macrophage-like cells readily take up TiO2 after 6 h. Incubation of cells with TiO2 resulted in the assembly of NLRP3 with caspase-1. This inflammasome assembly correlated with secretion of IL-1β. In intestinal epithelial cells, TiO2 also was found to be ingested. The counting of particles localized intracellularly revealed a dose-dependent increase of TiO2-positive cells. This points to the fact that in humans with a leaky intestinal barrier (such as IBD patients), TiO2 microparticles may be taken up by macrophages and intestinal epithelial cells, may activate the inflammasome and induce IL-1β and IL-18 secretion. This may aggravate inflammation in susceptible individuals.

Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3−/− Mice

The pathogenesis of Crohns disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3−/− mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3−/− mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3−/− mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3−/− mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.

Dealing with Our ‘In-Vironment’: New Aspects of Inflammatory Bowel Disease Pathogenesis and Therapy

making it more proinflammatory or aggressive. Other factors may directly act on the intestinal barrier function. Our therapeutic approaches still mainly focus on regulating adaptive immunity. Only recently have the barrier function of the gut mucosa and the defense function of the innate immune system come into focus. A recent FALK meeting entitled ‘Dealing with our ‘In-vironment’: New Aspects in IBD Pathogenesis and Therapy’ was, therefore, centered around recent discoveries that may change future treatment strategies. In a 2-day meeting, genetic risk factors in IBD, antibacterial defense mechanisms, the epithelial barrier as a border to the in-vironment, environmental factors in pathogenesis, disease markers, new therapeutic approaches and future therapeutic direction were discussed. The articles summarized here cover the most important aspects of the meeting and highlight the role of the in-vironment in IBD pathogenesis. Gerhard Rogler , Zürich Our understanding of the pathogenesis of inflammatory bowel disease (IBD) has dramatically changed during recent years. The focus of research has shifted from adaptive immunity to genetic risk factors and now to disturbances in innate immunity and the interactions of the mucosal immune system with the content of the gut – our ‘in-vironment’. The in-vironment is a term introduced by Michael Mayerfeld Bell in his book ‘An Invitation to Environmental Sociology’. He describes it as the ‘human body, which is continuously interacting with the environment’. He points to the fact that eating and drinking are particular ways for humans to interact with their environment. The groundbreaking insights gained in the last years indicate that the genetic factors only contribute 50% to the risk of developing IBD and that environmental factors may trigger or even cause the disease. These factors mediate their effects via uptake into the human body. They may change the composition of our microbiome

T1930 Microparticles and Their Impact on Intestinal Immunity

antly decreased after 7 days TPN administration compared with controls (13.5±2.4% v.s. 24.4±4.2%), and there was a significantly increased epithelial cell proliferation in SBS group compared to the sham-operated group (36.3±7.6% v.s. 24.4±4.2%). TPN administration significantly decreased the genes mRNA expressions, which promote the cell proliferation and survival, i.e. c-Myc, cyclinD1, cyclinE andCDK4when comparedwith controls. However, those gene expressions are significant increase in SBS group compared to sham-operated group respectively (p 0.05). Conclusion: This study suggested that cell cycle regulators may play an important role in intestinal epithelial cell proliferation.