Arianne L. Theiss

Invasive Escherichia coli are a feature of Crohn's disease.

Crohns disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, ±5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, ±0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, ±4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-α expression in macrophage cell lines (2604.8 pg/105 cells, ±447.4; 2,402.6 pg/105 cells, ±476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/105 cells, ±226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/105 cells, ±58.0 vs 920.5 pg/105 cells, ±94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.

Prohibitin protects against oxidative stress in intestinal epithelial cells

Prohibitin (PHB) is an evolutionarily conserved and ubiquitously expressed protein whose expression or function in intestinal diseases is not known. In this study, we examined the expression and role of PHB in oxidative stress associated with inflammatory bowel disease. Our results show that PHB primarily localizes to the mitochondria in intestinal epithelial cells. Its expression is down‐regulated during active human Crohns disease, experimental colitis in vivo, and oxidative stress in vitro. PHB overexpression increases the expression of glutathione‐S‐transferase π and protects from oxidant‐induced depletion of glutathione. Finally, PHB overexpression decreases accumulation of reactive oxygen metabolites, as well as increased permeability induced by oxidative stress in intestinal epithelial cells. Together, these results suggest that PHB constitutes a previously unrecognized cellular defense against oxidant injury. Thus, strategies to modulate PHB levels may constitute a novel therapeutic approach for intestinal inflammatory diseases, wherein oxidative stress plays a critical role in tissue injury and inflammation. Theiss, A. L., Idell, R. D., Srinivasan, S., Klapproth, J.‐M., Jones, D. P., Merlin, D., Sitaraman, S. V. Prohibitin protects against oxidative stress in intestinal epithelial cells. FASEB J. 21, 197–206 (2007)

The role and therapeutic potential of prohibitin in disease

Prohibitin 1 (PHB1), a pleiotropic protein in the cell, has been implicated in the regulation of proliferation, apoptosis, transcription, mitochondrial protein folding, and as a cell-surface receptor. This diverse array of functions of PHB1 is attributed to the cell type studied and its subcellular localization. This review discusses recent data that indicate a diverse role of PHB1 in disease pathogenesis and suggest that targeting PHB1 may be a potential therapeutic option for treatment of diseases including cancer, inflammatory bowel disease, insulin resistance/type 2 diabetes, and obesity. These diseases are associated with increased oxidative stress and mitochondrial dysfunction and therefore, the role of PHB1 in both responses will also be discussed.

Functional TNFα Gene Silencing Mediated by Polyethyleneimine/TNFα siRNA Nanocomplexes in Inflamed colon

During inflammatory bowel disease, TNFα is the major pro-inflammatory cytokine mainly secreted from macrophages and dendritic cells. Here, we have demonstrated that TNFα siRNA/polyethyleneimine loaded into polylactide at an optimal concentration of 20 g/L nanoparticles covered with polyvinyl alcohol are efficiently taken up by inflamed macrophages and inhibit TNFα secretion by the macrophages. Those nanoparticles have a diameter of ∼380 nm and zeta potential of -8 mV at pH 7.2, and are non-cytotoxic. Complexation, interactions and protection from RNAse between TNFα siRNA and polyethyleneimine were higher than those using chitosan. Importantly, complexation between TNFα siRNA and polyethyleneimine facilitated higher rates of siRNA loading into nanoparticles, compared to Chi or free siRNA mixed with Lipofectamine. Oral administration of encapsulated TNFα siRNA-loaded nanoparticles specifically reduced the TNFα expression/secretion in colonic tissue in LPS-treated mice. In conclusion, we have shown: (1) that proposed TNFα siRNA-loaded NPs are prepared via a non-denaturing synthetic process; (2) a high encapsulation rate of TNFα siRNA complexed to polyethyleneimine into NPs; (3) effective enzymatic protection of TNFα siRNA by polyethyleneimine; (4) non-cytotoxicity and biodegradability of nanoparticles loaded with polyethyleneimine/TNFα siRNA; and (5) in vitro and in vivo significant anti-inflammatory effects at low TNFα siRNA dose that is specific and restricted to the colonic cells. Our results collectively indicate that polyethyleneimine/TNFα siRNA nanocomplexes represent an efficient therapeutic option for diseases such as IBD.

Prohibitin Is a Novel Regulator of Antioxidant Response That Attenuates Colonic Inflammation in Mice

BACKGROUND & AIMS Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice. METHODS Colitis was induced using 2 established mouse models (Salmonella typhimurium and dextran sodium sulfate) in PHB transgenic mice and wild-type littermates. Oxidative stress was determined by measuring glutathione and protein carbonyl levels in the cecum or colon. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of oxidant responses, expression, and activation, was assessed in colon mucosa and cultured intestinal epithelial cells overexpressing PHB. RESULTS Cells overexpressing PHB showed sustained Nrf2 nuclear accumulation and DNA binding during oxidant stress. PHB transgenic mice exhibited decreased oxidative stress and colitis and increased Nrf2 messenger RNA expression, nuclear protein translocation, and DNA binding compared with wild-type littermates during colitis. CONCLUSIONS These results show that PHB is a regulator of Nrf2 expression in intestinal epithelial cells during oxidative conditions and prevents inflammation-associated oxidative stress and injury through sustained activation of Nrf2. Our data show that PHB is a novel regulator of antioxidants and suggest that restoration of PHB levels represents a potential therapeutic approach in inflammatory bowel disease.

Interleukin-6 Induces Keratin Expression in Intestinal Epithelial Cells POTENTIAL ROLE OF KERATIN-8 IN INTERLEUKIN-6-INDUCED BARRIER FUNCTION ALTERATIONS

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.

Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

Expression of prohibitin 1 (PHB), a multifunctional protein in the cell, is decreased during inflammatory bowel disease (IBD). Little is known regarding the regulation and role of PHB during intestinal inflammation. We examined the effect of tumor necrosis factor alpha (TNF-alpha), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-alpha activation of nuclear factor-kappa B (NF-kappaB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-alpha decreased PHB protein and mRNA abundance in intestinal epithelial cells in vitro and in colon mucosa in vivo. Sustained expression of prohibitin in intestinal epithelial cells in vitro and in vivo (prohibitin transgenic mice, PHB TG) resulted in a marked decrease in TNF-alpha-induced nuclear translocation of the NF-kappaB protein p65, NF-kappaB/DNA binding, and NF-kappaB-mediated transcriptional activation despite robust IkappaB-alpha phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-alpha-induced increased epithelial permeability. Expression of importin alpha3, a protein involved in p50/p65 nuclear import, was decreased in cells overexpressing PHB and in colon mucosa of PHB TG mice. Restoration of importin alpha3 levels sustained NF-kappaB activation by TNF-alpha during PHB transfection. These results suggest that PHB inhibits NF-kappaB nuclear translocation via a novel mechanism involving alteration of importin alpha3 levels. TNF-alpha decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-alpha and NF-kappaB on barrier function.

Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

Introduction Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohns disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells. Methods We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. Results TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability. Conclusions Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.

Nanoparticle-based therapeutic delivery of prohibitin to the colonic epithelial cells ameliorates acute murine colitis

Background: Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF‐&kgr;B) contribute to mucosal barrier integrity and epithelial homeostasis, two key events in the pathogenesis of inflammatory bowel disease (IBD). Genetic restoration of intestinal epithelial prohibitin 1 (PHB) levels during experimental colitis reduces the severity of disease through sustained epithelial antioxidant expression and reduced NF‐&kgr;B activation. To determine the therapeutic potential of restoring epithelial PHB during experimental colitis in mice, we assessed two methods of PHB colonic mucosal delivery: adenovirus‐directed administration by enema and poly(lactic acid) nanoparticle (NPs) delivery by gavage. Methods: As a proof‐of‐principle to demonstrate the therapeutic efficacy of PHB, we utilized adenovirus‐directed administration by enema. Second, we used NPs‐based colonic delivery of biologically active PHB to demonstrate therapeutic use for human IBD. Colitis was induced by oral administration of dextran sodium sulfate (DSS) in water for 6‐7 days. Wildtype mice receiving normal tap water served as controls. Results: Both methods of delivery resulted in increased levels of PHB in the surface epithelial cells of the colon and reduced severity of DSS‐induced colitis in mice as measured by body weight loss, clinical score, myeloperoxidase activity, proinflammatory cytokine expression, histological score, and protein carbonyl content. Conclusions: This is the first study to show oral delivery of a biologically active protein by NPs encapsulated in hydrogel to the colon. Here we show that therapeutic delivery of PHB to the colon reduces the severity of DSS‐induced colitis in mice. PHB may represent a novel therapeutic target in IBD. (Inflamm Bowel Dis 2010)

Prohibitin attenuates colitis-associated tumorigenesis in mice by modulating p53 and STAT3 apoptotic responses

Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.