Maiko Sasaki

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.

Development of a unique small molecule modulator of CXCR4.

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

Citrobacter rodentium lifA/efa1 Is Essential for Colonic Colonization and Crypt Cell Hyperplasia In Vivo

ABSTRACT Previously, we have identified a large gene (lifA, for lymphocyte inhibitory factor A) in enteropathogenic Escherichia coli (EPEC) encoding a protein termed lymphostatin that suppresses cytokine expression in vitro. This protein also functions as an adhesion factor for enterohemorrhagic E. coli (EHEC) and Shiga toxin-producing E. coli and is alternatively known as efa1 (EHEC factor for adherence 1). The lifA/efa1 gene is also present in Citrobacter rodentium, an enteric pathogen that causes a disease termed transmissible murine colonic hyperplasia (TMCH), which induces colitis and massive crypt cell proliferation, in mice. To determine if lifA/efa1 is required for C. rodentium-induced colonic pathology in vivo, three in-frame mutations were generated, disrupting the glycosyltransferase (GlM12) and protease (PrMC31) motifs and a domain in between that does not encode any known activity (EID3). In contrast to infection with wild-type C. rodentium, that with any of the lifA/efa1 mutant strains did not induce weight loss or TMCH. Enteric infection with motif mutants GlM12 and PrM31 resulted in significantly reduced colonization counts during the entire 20-day course of infection. In contrast, EID3 was indistinguishable from the wild type during the initial colonic colonization, but cleared rapidly after day 8 of the infection. The colonic epithelium of all infected mice displayed increased epithelial regeneration. However, significantly increased regeneration was observed by day 20 only in mice infected with the wild-type in comparison to those infected with lifA/efa1 mutant EID3. In summary, lifA/efa1 is a critical gene outside the locus for enterocyte effacement that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration.

The Role of Bacteria in the Pathogenesis of Ulcerative Colitis

Factors implicated in the pathophysiology of ulcerative colitis (UC) are an abnormal immune response, defect in intestinal epithelial barrier function, and gut microbiota. Currently, it is unclear whether specific bacterial strains are responsible for the induction of intestinal inflammation, but increased bacterial tissue invasion has been described in affected UC patients. Further, a quantitative and qualitative microbial imbalance in UC, defined as dysbiosis, has been characterized by an increase in Rhodococcus spp., Shigella spp., and Escherichia spp., but a decrease in certain Bacteroides spp.. More specifically, Campylobacter spp., Enterobacteriae, and enterohepatic Helicobacter were more prevalent in tissue sample from UC patients subjected to molecular detection methods, but not controls. In addition, serologic testing identified Fusobacterim varium as a potential contributor to the intestinal inflammation in UC. Interestingly, in-situ hybridization studies have shown anti-inflammatory Lactobacillus spp. and Pediococcus spp. were absent in samples from subjects affected by UC. Therefore, dysbiosis is a factor in the pathogenesis of UC.

Krüppel-like factor 5 mediates transmissible murine colonic hyperplasia caused by Citrobacter rodentium infection.

BACKGROUND & AIMS Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. METHODS Heterozygous Klf5 knockout (Klf5(+/-)) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5(+/-) mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt. RESULTS Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5(+/-) mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5(+/-) animals as compared with WT littermates. CONCLUSION Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.

The Bacterial Virulence Factor Lymphostatin Compromises Intestinal Epithelial Barrier Function by Modulating Rho GTPases

Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins beta-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities.

Bacterial induction of proinflammatory cytokines in inflammatory bowel disease.

&NA; It has become increasingly clear that inflammatory bowel disease (IBD) develops on the background of genetic defects in the host, conveying an increased susceptibility to an environmental antigen or antigens. The environmental factor implicated in the pathophysiology of gut inflammation, which is undergoing increased scrutiny, is the intestinal flora. The intestinal flora as a whole and specific bacteria and their products have been found to trigger cytokine expression in various cell types. Consistently, multiple bacterial strains were found to induce tumor necrosis factor alpha (TNF‐&agr;) and interleukin‐8 (IL‐8) in macrophage and epithelial cell systems, respectively, in particular in Crohns disease. Interestingly, various cell types from patients with IBD display an increased susceptibility to specific bacterial products, including flagellin, pili, and lipopolysaccharides. It remains to be determined whether additional effector proteins regulate cytokine expression and the aberrant mucosal immune response in IBD. (Inflamm Bowel Dis 2010)

Crohn's Disease–associated Escherichia coli Survive in Macrophages by Suppressing NFκB Signaling

Background:Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohns disease (CD). Crohns disease–associated Escherichia coli (CDEC) is characterized by their ability to invade epithelial cells and survive and induce high concentration of TNF-&agr; from infected macrophages. However, the molecular mechanisms of CDEC survival in infected macrophages are not completely understood. Methods:Intracellular survival of CDEC strain LF82 isolated from inflamed ileum tissue, 13I isolated from inflamed colonic tissue, and control E. coli strains were tested in the murine macrophage cell line, J774A.1 by Gentamicin protection assay. Modulation of intracellular cell signaling pathways by the E. coli strains were assessed by western blot analysis and confocal microscopy. Results:13I demonstrated increased survival in macrophages with 2.6-fold higher intracellular bacteria compared with LF82, yet both strains induced comparable levels of TNF-&agr;. LF82 and 13I differentially modulated key mitogen-activated protein kinase pathways during the acute phase of infection; LF82 activated all 3 mitogen-activated protein kinase pathways, whereas 13I activated ERK1/2 pathway but not p38 and JNK pathways. Both 13I and LF82 suppressed nuclear translocation of NF&kgr;B compared with noninvasive E. coli strains during the acute phase of infection. However, unlike noninvasive E. coli strains, 13I and LF82 infection resulted in chronic activation of NF&kgr;B during the later phase of infection. Conclusions:Our results showed that CDEC survive in macrophages by initially suppressing NF&kgr;B activation. However, persistence of bacterial within macrophages induces chronic activation of NF&kgr;B, which correlates with increased TNF-&agr; secretion from infected macrophages.

Lysophosphatidic acid receptor 1 is important for intestinal epithelial barrier function and susceptibility to colitis

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

Understanding the Impact of ErbB Activating Events and Signal Transduction on Antigen Processing and Presentation: MHC Expression as a Model.

Advances in molecular pathology have changed the landscape of oncology. The ability to interrogate tissue samples for oncogene amplification, driver mutations, and other molecular alterations provides clinicians with an enormous level of detail about their patient’s cancer. In some cases, this information informs treatment decisions, especially those related to targeted anti-cancer therapies. However, in terms of immune-based therapies, it is less clear how to use such information. Likewise, despite studies demonstrating the pivotal role of neoantigens in predicting responsiveness to immune checkpoint blockade, it is not known if the expression of neoantigens impacts the response to targeted therapies despite a growing recognition of their diverse effects on immunity. To realize the promise of ‘personalized medicine’, it will be important to develop a more integrated understanding of the relationships between oncogenic events and processes governing anti-tumor immunity. One area of investigation to explore such relationships centers on defining how ErbB/HER activation and signal transduction influences antigen processing and presentation.