Reduced butyrate-producing bacteria could result in increased epithelial permeability particularly in the context of concomitant exposure to another stimulus that reduces mitochondria function. We speculate that prebiotics, the substrate for butyrate synthesis, is a valuable prophylaxis in the regulation of epithelial permeability and could be of benefit in preventing relapses in IBD.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.

We present a new role for glycogen synthase kinase (GSK) in the regulation of aggregation and chemotaxis in Dictyostelium. GSK regulates two chemotactic pathways, PIP3 and TORC2; hence, a loss of function of GSK leads to poor chemotaxis, an observation not previously seen when only one chemotactic pathway was targeted.

DM. Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-␣. Am J Physiol Gastrointest Liver Physiol 294: G669-G678, 2008. First published January 10, 2008 doi:10.1152/ajpgi.00382.2007.-A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-␣, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1␤, IL-6, and TNF-␣. Given the role of TNF-␣ in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-␣ (10 ng/ml) was added to the DNP (0.1 mM) ϩ E. coli (10 6 colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP ϩ E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-␤ signaling (pyrrolidine dithiocarbamate, NF-␤ essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-␤. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-␣ released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress. permeability; bacterial translocation; NFB; T84 cells; macrophages A NUMBER OF PUTATIVE CAUSATIVE agents/mechanisms have been proposed for the inflammatory bowel diseases (IBDs): genetic predisposition, infection, dysregulated immune reactions, triggers from the commensal flora, and a defect in epithelial permeability (44). Abnormal mitochondria have been observed in epithelial cells in tissues resected from patients with Crohn's disease (28), indicating metabolic stress (i.e., ATP depletion) in these individuals. Similarly, reductions in mitochondria acetoacetyl CoA thiolase have been shown in the epithelium of patients with ulcerative colitis, which would result in reduced ATP synthesis from butyrate, and hence these cells would experience a degree of metabolic stress (36). Metabolic stress can arise as a consequence of infection, ischemia, and inflammation itself (8, 14). Thus we hypothes...