Paneth cells as a site of origin for intestinal inflammation.

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1(HM) mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn's disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1(ΔIEC)) or autophagy function (Atg16l1(ΔIEC) or Atg7(ΔIEC)) in intestinal epithelial cells results in each other's compensatory engagement, and severe spontaneous Crohn's-disease-like transmural ileitis if both mechanisms are compromised. Xbp1(ΔIEC) mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn's disease as a specific disorder of Paneth cells.

Upregulation of TGF-beta1 expression may be necessary but is not sufficient for excessive scarring.

Transforming growth factor beta 1 (TGF-beta1) upregulation has been implicated in hypertrophic scars and keloids, but it is unclear if it is the cause or an effect of excessive scar formation. In this study, we overexpressed TGF-beta1 in fibroblasts and characterized its role. Normal human dermal fibroblasts were genetically modified to overexpress TGF-beta1 as the wild-type latent molecule or as a mutant constitutively active molecule. TGF-beta1 secretion was measured, as were the effects of TGF-beta1 upregulation on cell proliferation, expression of smooth muscle cell alpha actin (SMC alpha-actin) and ability to contract collagen lattices. Fibroblasts were implanted intradermally into athymic mice and tissue formation was analyzed over time by histology and immunostaining. Gene-modified fibroblasts secreted approximately 20 times the TGF-beta1 released by control cells, but only cells expressing mutant TGF-beta1 secreted it in the active form. Fibroblasts expressing the active TGF-beta1 gene had increased levels of SMC alpha-actin and enhanced ability to contract a collagen lattice. After intradermal injection into athymic mice, only fibroblasts expressing active TGF-beta1 formed "keloid-like" nodules containing collagen, which persisted longer than implants of the other cell types. We conclude that upregulation of TGF-beta1 by fibroblasts may be necessary, but is not sufficient for excessive scarring. Needed are other signals to activate TGF-beta1 and prolong cell persistence.

Trehalose uptake through P2X7 purinergic channels provides dehydration protection.

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.