Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal peptides when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes and alpha-defensins, or cryptdins, account for 70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A and muramyl dipeptide elicit cryptdin secretion. Live fungi and protozoa, however, do not stimulate degranulation. Thus intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens, and discharge microbicidal peptides at effective concentrations accordingly.

AbstractAllogeneic hematopoietic stem cell transplantation (SCT) is a curative therapy for various hematologic disorders. Graft-versus-host disease (GVHD) and infections are the major complications of SCT, and their close relationship has been suggested. In this study, we evaluated a link between 2 complications in mouse models. The intestinal microbial communities are actively regulated by Paneth cells through their secretion of antimicrobial peptides, α-defensins. We discovered that Paneth cells are targeted by GVHD, resulting in marked reduction in the expression of α-defensins, which selectively kill noncommensals, while preserving commensals. Molecular profiling of intestinal microbial communities showed loss of physiologic diversity among the microflora and the overwhelming expansion of otherwise rare bacteria Escherichia coli, which caused septicemia. These changes occurred only in mice with GVHD, independently on conditioning-induced intestinal injury, and there was a significant correlation between alteration in the intestinal microbiota and GVHD severity. Oral administration of polymyxin B inhibited outgrowth of E coli and ameliorated GVHD. These results reveal the novel mechanism responsible for shift in the gut flora from commensals toward the widespread prevalence of pathogens and the previously unrecognized association between GVHD and infection after allogeneic SCT.

Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

The release of endogenous antimicrobial peptides by mammalian epithelial cells contributes to innate mucosal immunity (1, 2). The crypts of Lieberkü hn in the small intestine of most mammals contain Paneth cells that secrete ␣-defensins (cryptdins), lysozyme, secretory phospholipase A 2 , xanthine oxidase, CD95 ligand, CD15, and tumor necrosis factor-␣ as components of apically oriented secretory granules (3-10). Although certain Paneth cell ␣-defensins have been detected in mouse skin and testis (11,12) and in human oropharyngeal and urogenital mucosa (13,14), in the small intestine, ␣-defensins are specific to Paneth cells (9). Exposure of Paneth cells to cholinergic agonists or bacterial stimuli elicits granule discharge into the crypt lumen (15), and carbamylcholine mediates secretion via increased cytosolic Ca 2ϩ (16). Regardless of how mouse Paneth cell secretion is stimulated, cryptdins constitute ϳ70% of the released bactericidal activity, and the concentration of cryptdins is estimated to be 25 mM at the point of secretion in the crypt lumen (15).␣-Defensins are processed from inactive proforms by specific proteolytic cleavage steps. Both neutrophil and Paneth cell ␣-defensins derive from ϳ10-kDa prepropeptides that contain canonical signal sequences, acidic proregions, and an ϳ3.5-kDa mature ␣-defensin peptide in the C-terminal portion of the precursor. For example, maturation of myeloid pro-␣-defensins appears to involve two primary cleavage steps, and most ␣-defensins in mature phagocytic leukocytes are completely processed (17)(18)(19)(20). In a heterologously expressed human neutrophil pro-␣-defensin, deletions in the prosegment adjacent to the proregion-defensin junction impairs post-translational processing in 32DCL3 cells (19).In mouse Paneth cells, matrix metalloproteinase-7 (MMP-7 1 ; matrilysin) mediates the processing and activation of ␣-defensins from 8.4-kDa proforms (21). MMP-7 gene disruption ablates procryptdin processing, resulting in accumulation of cryptdin precursors and the absence of activated mature cryptdin peptides in the small intestine (21). Lacking functional cryptdin peptides, MMP-7-null mice have a defect in clearance of intestinal infections, and they succumb more rapidly and to lower doses of virulent Salmonella typhimurium compared with control mice (21). Thus, the cryptdin deficiency resulting from defective procryptdin activation is associated with a measurable deficit in mucosal immunity and increased risk of systemic disease.In this study, cryptdin biosynthesis was investigated by characterizing details of intracellular procryptdin processing in

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Hayase et al. show that R-Spondin1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spondin1 or recombinant α-defensin prevents dysbiosis mediated by graft-versus-host disease, representing a novel approach to restore intestinal ecosystems and homeostasis.

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