Epithelial-mesenchymal transition (EMT), characterized by loss of epithelial adhesion and gain of mesenchymal features, is an important mechanism to empower epithelial cells into the motility that occurs during embryonic development and recurs in cancer and fibrosis. Whether and how EMT occurs in wound healing and fibrosis in human skin remains unknown. In this study we found that migrating epithelial cells in wound margins and deep epithelial ridges had gained mesenchymal features such as vimentin and FSP1 expression. In hypertrophic scars, EMTrelated genes were elevated along with inflammatory cytokines, indicating a causal relationship. To reconstitute EMT in vitro, normal human skin and primary keratinocytes were exposed to cytokines such as tumor necrosis factor-␣ (TNF-␣), resulting in expression of vimentin, FSP1, and matrix metalloproteinases. Moreover, TNF-␣-induced EMT was impaired by antagonists against bone morphogen proteins (BMP) 2/4, suggesting that BMP mediates the TNF-␣-induced EMT in human skin. Indeed, TNF-␣ could induce BMP-2 and its receptor (BMPR1A) in human skin and primary keratinocytes , and BMP2 could induce EMT features in skin explants and primary keratinocytes. In summary , we uncovered EMT features in both acute and fibrotic cutaneous wound healing of human skin. Moreover , we propose that the mesenchymal induction in wound healing is motivated by TNF-␣ , in part , through induction of BMP. (Am J

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3−/− mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3−/− mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

ObjectiveATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohn's disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells.DesignWe genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant.ResultsWe found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway.ConclusionsThe CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.

ObjectiveA common genetic coding variant in the core autophagy gene ATG16L1 is associated with increased susceptibility to Crohn's disease (CD). The variant encodes an amino acid change in ATG16L1 such that the threonine at position 300 is substituted with an alanine (ATG16L1 T300A). How this variant contributes to increased risk of CD is not known, but studies with transfected cell lines and gene-targeted mice have demonstrated that ATG16L1 is required for autophagy, control of interleukin-1-β and autophagic clearance of intracellular microbes. In addition, studies with human cells expressing ATG16L1 T300A indicate that this variant reduces the autophagic clearance of intracellular microbes.Design/ResultsWe demonstrate, using somatically gene-targeted human cells that the ATG16L1 T300A variant confers protection from cellular invasion by Salmonella. In addition, we show that ATG16L1-deficient cells are resistant to bacterial invasion.ConclusionsThese results suggest that cellular expression of ATG16L1 facilitates bacterial invasion and that the CD-associated ATG16L1 T300A variant may confer protection from bacterial infection.

DL. Intestinal epithelial expression of TNFAIP3 results in microbial invasion of the inner mucus layer and induces colitis in IL-10-deficient mice.