Mouse Paneth Cell Research Articles

Radio-protective effect of sulfated polysaccharide purified from Ecklonia cava against small intestinal stem cells of γ-ray irradiated mice

Our previous research on sulfated polysaccharide purified from Ecklonia cava, a brown alga found in Jeju island, Korea, showed that sulfated polysaccharides modulate the apoptotic threshold of intestinal cells, thereby preventing intestinal damage caused by ionizing radiation. In this study, we investigated the ability of sulfated polysaccharide to augment restoration of small intestinal stem cells from γ-ray-induced damage. In our results, sulfated polysaccharide treatment increased the numbers of Ki-67-positive cells as well as inducible nitric oxide synthase (iNOS)-expressing cells in the small intestine compared with those of irradiated only mice. Meanwhile, exposure to irradiation increased the number of paneth cells, which are frequently associated with intestinal inflammation, whereas sulfated polysaccharide treatment reduced the number of paneth cells in the small intestinal crypt. Conclusively, our data suggest that reduction of iNOS-expressing cells and paneth cells in sulfated polysaccharide-treated mice contributes to the inhibition of radiation-induced intestinal inflammation.

Critical design aspects involved in the study of Paneth cells and the intestinal microbiota

Paneth cells are long-lived secretory cells that reside in the base of the crypts of Lieberkühn of the small intestine. They produce an arsenal of molecules that are involved in numerous biological processes, ranging from the control of gut microbial populations to supporting the intestinal stem cell niche. Because of these important functions, Paneth cell abnormalities are becoming implicated in a variety of disease processes. As such, it is necessary to establish parameters that will allow for the comprehensive study of Paneth cells in health and disease. In this addendum, we highlight critical design aspects involved in the study of Paneth cells and their downstream effects on the intestinal microbiota. The importance of this approach is demonstrated by our recent findings that Nod2 does not regulate mouse Paneth cell antimicrobial function, in contrast to previous reports. This work defines key issues to consider when studying Paneth cells in mouse systems.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8±5.2ng/ml in proximal small intestine to 54.3±10.3ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Mouse Paneth cell antimicrobial function is independent of Nod2

ObjectiveAlthough polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression...

Redundant Sources of Wnt Regulate Intestinal Stem Cells and Promote Formation of Paneth Cells

Wnt signaling regulates multiple aspects of intestinal physiology, including stem cell maintenance. Paneth cells support stem cells by secreting Wnt, but little is known about the exact sources and primary functions of individual Wnt family members. We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice, which lack secretory cells. Unexpectedly, Wnt3 was dispensable for maintenance of intestinal stem cells in mice, indicating a redundancy of Wnt signals. By contrast, cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt, or coculture with mesenchymal cells, restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures, whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt.

CRS-peptides: unique defense peptides of mouse Paneth cells.

The intestine is the most densely colonized site in both mice and man. Recent data suggest that the intestinal flora is, in part, controlled by antimicrobial substances secreted by the intestinal epithelium. The defense system of the small intestine includes a protective mucus layer, a high turnover of epithelial cells, and a regulated secretion of effector molecules, notably antimicrobial peptides. Human and mouse small intestines share many similarities in their intestinal defense micro-organization, including the secretion of the well-known α-defensins. Mice, however, produce an additional unique antimicrobial peptide family, the CRS (cryptdin-related sequences)-peptides, not found in man. This review comprises a detailed presentation of the peptide-based defense of the gut, with specific emphasis on the CRS-peptide family. The first part presents the current knowledge of the CRS-peptide family's biochemical characteristics and nomenclature, and the second part is devoted to the possible role of this family in the homeostasis of the gut.

The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensincryptdin-4 (Crp4) and peptide variants with mutations at Arg7 or Glu15 residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(150 substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance todegradation by proteinases.

Modeling IBD in the mouse through random germline mutagenesis

Inflammatory bowel diseases (IBD), including both ulcerative colitis and Crohn's disease, have strong genetic components. In mice, several mutations are known to favor IBD or to inhibit intestinal inflammation. But a comprehensive picture of the pathogenesis of IBD cannot be assembled based on the limited information so far available from genetic analyses. To unveil new genes important for the pathogenesis of IBD, we took an unbiased, hypothesis-free view of IBD, using germline mutagenesis to analyze the phenomenon in mice. We screen for mutations that cause susceptibility to IBD on a defined genetic background using the Dextran sodium sulfate (DSS) colitis model, positionally clone these mutations, and form hypotheses concerning the participation of each validated gene in the disease pathogenesis. A total of 5846 mice have been screened by placing them on 1% DSS in drinking water for a period of 7 days. During this time period all animals are weighed daily, and extreme outliers are retrieved for further study. A total of 16 transmissible mutations causing hypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into 5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelial cells. Wound closure also additionally requires water transport via aquaporin 3, signaling via the EGF receptor pathway, and an intact unfolded protein response, which depends on Mbtps1, also known as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 also enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes with unknown functions have been identified causing increased susceptibility to DSS colitis. One of the mutant mice identified in this screen was homozygous for a mutation in an uncharacterized gene. The gene is predicted to encode a protein with an unstructured cytoplasmic N-terminal domain and a C-terminal domain containing five transmembrane α helices connected by short loops. The mutation lies in the splice acceptor site of intron 3 of the seven exon gene, and affects a thymine base five nucleotides from the next exon. cDNA sequencing revealed that the mutation results in skipping of exon 4. Splicing of exon 3 to exon 5 creates a frameshift and a premature stop codon. The Klein-Zschocher mouse showed severe weight loss after administration of 1% DSS in the drinking water. Periodic acid-Schiff stain staining of small intestines and colons of Klein-Zschocher mice revealed reduced numbers of Paneth and goblet cells in naïve mutant mice. Furthermore, electron microscopy of the small intestine and colon of naive Klein-Zschocher mice showed abnormal vesicles in Paneth and goblet cells of the mutant mice. Defective Paneth cells of Klein-Zschocher mutant mice show enhanced autophagy as demonstrated by LC3 Western blotting. In addition, the intestinal permeability was increased 85% in Klein-Zschocher mice demonstrating epithelial barrier dysfunction in these mice. Disturbed epithelial integrity together with abnormal Paneth and goblet cells explain the increased susceptibility in the DSS colitis model. Taken together, using random mutagenesis and the DSS mouse model we found new genes and new functions of known genes that might play a role in IBD. A coherent picture of the essential events required for homeostasis has begun to emerge.

Host-Derived IL-22 Limits Graft Versus Host Disease and Protects the Intestinal Stem Cell Niche

Abstract 309Despite decades of intensive research, graft vs. host disease (GVHD) remains a major complication of allogeneic transplantation. Much progress has been made toward understanding GVHD pathophysiology and the mechanisms regulating the donor immune response, however no factors have been identified to regulate the response of transplant recipients to GVHD and its concomitant damage. Furthermore, virtually all strategies available to reduce clinical GVHD do so by limiting the donor immune system at the expense of therapeutic graft vs. leukemia/lymphoma (GVL) responses. IL-22 is a recently characterized cytokine that is produced by both helper T cells and innate lymphoid cells (ILC). Expression of its receptor (IL-22R) is generally limited to epithelial and other non-hematopoietic cells, and it has demonstrated a protective role for intestinal epithelium during experimental colitis, providing a signal for intestinal epithelial cell survival, proliferation, and wound healing.We investigated the role of IL-22 in GVHD during MHC-mismatched C57BL/6 (B6) into BALB/c murine bone marrow transplantation (BMT). Elimination of IL-22 with anti-IL-22 neutralizing antibody lead to increased GVHD mortality post-BMT in comparison to isotype control (p<.01). Interestingly, use of IL-22 knockout (KO) donor marrow or T cells had no impact on survival or on the ability to clear A20 lymphoma post-BMT, but IL-22 KO BMT recipients demonstrated significantly increased mortality during GVHD (p<.01). The critical role of recipient-derived IL-22 was confirmed in a minor antigen-mismatched LP into B6 BMT model (Figure 1). IL-22 KO recipients again demonstrated significantly increased mortality during GVHD (p<.001) and histopathologic evidence of GVHD in the small (p<.01) and large (p<.001) intestine and liver (p<.001). Furthermore, BMT into hematopoietic IL-22 KO chimeras also demonstrated increased GVHD mortality (p<.05) and clinical scoring (p<.001), indicating that the source of protective IL-22 is residual host-derived radio-resistant hematopoietic cells.IL-22 ELISA on tissue homogenates following BMT with wild type donors and recipients indicated that IL-22 levels are increased three weeks post-BMT in both small and large intestine (p<.05), as well as following sublethal radiation without transplantation (p<.05 small intestine; p<.01 large) in comparison to normal controls. However, IL-22 was significantly reduced in both tissues during GVHD (2 weeks post-BMT in small intestine, Figure 2A; 3 weeks post-BMT in large, p<.05), suggesting that IL-22 was produced by residual host hematopoietic cells that were being eliminated during GVHD. Consistent with this, we identified IL-22-producing host ILC (CD45+CD3−RORg+) in small intestine lamina propria two weeks post-BMT, whereas no IL-22 production could be identified in CD3+ or RORg− subsets, and these IL-22-producing ILC were significantly reduced during GVHD (p<.05, Figure 2B). Interestingly, although host IL-22 deficiency was associated with increased GVHD morbidity, mortality, and pathology, no major differences were observed in donor lymphocyte infiltration of recipient intestines, frequencies of donor lymphoid subsets, or donor T cell inflammatory cytokines post-BMT. In contrast, IL-22R expression on recipient intestinal epithelium was increased post-BMT, and GVHD in IL-22 KO recipients led to increased damage of the intestinal epithelium as measured by FITC-dextran absorption (p<.05). Additionally, IL-22R expression was identified by immunohistochemistry in intestinal crypts where the stem/progenitor cell niche is located, and use of LGR5-LacZ reporter mice indicated that intestinal stem cells (ISC) were indeed targets of GVHD. Finally, histologic assessment of the ISC niche demonstrated exacerbated loss of both ISC (p<.001, Figure 2C) and Paneth cells (p<.05) in IL-22 KO mice during GVHD. [Display omitted] [Display omitted] In summary, IL-22 is produced post-BMT by host ILC that are eliminated during GVHD, and IL-22 deficiency increases GVHD morbidity and mortality. While IL-22 deficiency did not significantly alter the donor immune response, it did lead to increased GVHD pathology, loss of epithelial integrity, and damage to the ISC niche. IL-22 is thus critical for protection of host epithelium during GVHD but is not involved in GVL. This may be exploited in the future to reduce clinical GVHD without limiting the curative potential of the transplant. Disclosures:Fouser:Pfizer: Employment.

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24(+) cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Bactericidal Activity of Mouse α-Defensin Cryptdin-4 Predominantly Affects Noncommensal Bacteria

Mouse Paneth cell α-defensins, termed cryptdins, are secreted into the intestinal lumen, have microbicidal activity, and contribute to intestinal innate immunity. Among them, cryptdin-4 (Crp4) has the most potent microbicidal activity. In the intestinal lumen, commensal bacteria colonize and elicit beneficial effects in the host. However, the effects of Crp4 against commensal bacteria are poorly understood. Thus, we investigated the bactericidal activities of Crp4 against commensal bacteria compared to noncommensal bacteria. Oxidized Crp4 showed only minimal or no bactericidal activity against 8 out of 12 commensal bacterial species, including Bifidobacterium bifidum and Lactobacillus casei. We further addressed a role of the conserved disulfide bonds of Crp4 by analyzing reduced Crp4 (r-Crp4). r-Crp4 demonstrated significantly greater bactericidal activities against 7 of 12 commensal bacteria than did oxidized Crp4. Oxidized Crp4 and r-Crp4 elicited equivalently potent bactericidal activities against 11 of the 11 noncommensal bacteria tested, such as Salmonella enterica serovar Typhimurium,and against 5 of 12 commensal bacteria. Furthermore, when r-Crp4 was exposed to a processing enzyme of cryptdins, i.e. MMP-7, r-Crp4 was degraded and the bactericidal activities disappeared. These findings suggest that Crp4 has selective bactericidal activities against intestinal microbiota and that the activities are dependent on the disulfide bonds.

CD1d Expression in Paneth Cells and Rat Exocrine Pancreas Revealed by Novel Monoclonal Antibodies Which Differentially Affect NKT Cell Activation

BackgroundCD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated.Methodology/Principal FindingsTwo mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb.Conclusions/SignificanceThe two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d−/− mice, which resulted in an altered composition of the gut flora.

Introduction of protein transduction domains to the N‐terminus of β‐defensins influences microbicidal activity

Protein transduction domains (PTDs) enable protein fusion partners to transmigrate the cell membrane of mammalian cell lines to the cytosol. To test whether conjugation of PTDs to α‐defensins enhances microbicidal activity by membrane disruptive mechanisms, we prepared recombinant α‐defensins modified with N terminal PTDs. Rhesus myeloid α‐defensin‐4 (RMAD‐4) and mouse Paneth cell α‐defensin cryptdin‐4 (Crp4) were expressed in E. coli, introducing TAT (YGRKKRRQRRR) and nona‐Arg (9R) at both peptide N‐termini. In in vitro bactericidal peptide assays against E. coli, L. monocytogenes, V. cholerae, S. Typhimurium and S. aureus, both PTD‐conjugated peptides exhibited markedly increased activities when compared to the parent molecules. For example, at 0.75 μM, TAT‐RMAD‐4 effectively killed 99.9% of S. Typhimurium CS022 in contrast to RMAD‐4 which had no microbicidal effect at this concentration. Measurements of induced permeabilization of live E. coli cells showed that PTD‐modified peptides were significantly more membrane disruptive. Cytotoxicity assays on RAW264.7 cells showed that PTD fusion to RMAD‐4 and Crp4 imparted cytotoxic activity to the peptides at levels &gt;10 μM. β‐Defensins with N‐terminal PTDs have enhanced antibacterial activities, suggesting potential applications against intracellular infections. Supported by NIH grants AI059346 and DK044632.

Diversity and activation of rhesus Paneth cell α‐defensins

Paneth cells (PCs) occur in small intestinal crypts from which they secrete α‐defensins at high levels. To investigate rhesus macaque enteric α‐defensin (REDs) diversity, ileal mucosal protein and mRNAs were characterized. Analyses of ileum α‐defensin cDNA and gene sequences identified 20 distinct RED cDNAs and 7 RED genes not represented in the cDNA population. Certain deduced RED proteins coded by abundant mRNAs have mutations at canonical α‐defensin structural features. α‐Defensin proteins isolated from ileum occur predominantly as proREDs with negligible processed REDs detected by acid‐urea PAGE and MALDI‐TOF MS. In vitro, purified natural and recombinant proREDs were activated by trypsin to yield structurally intact, mature RED peptides. Because mature RED peptides are lacking from ileal PCs and mouse PC α‐defensins persist in colonic lumen (J. Biol. Chem. 284: 27848–27856, 2009), colonic luminal contents were assayed for evidence of post‐secretory proRED processing. Mature, intact RED peptides were evident in large bowel lumen, far downstream from their small intestinal site of secretion, and they were identified as α‐defensins by Edman sequencing. The processed state of α‐defensins in rhesus PCs resembles that in human PCs and differs from the mouse PCs, where activated, mature α‐defensins are abundant in PC dense core granules. Supported by NIH grants AI059346 and DK044632.

Elevated Expression of Paneth Cell CRS4C in Ileitis-prone SAMP1/YitFc Mice: REGIONAL DISTRIBUTION, SUBCELLULAR LOCALIZATION, AND MECHANISM OF ACTION

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete host defense peptides and proteins, including alpha-defensins, as mediators of innate immunity. Mouse Paneth cells also express alpha-defensin-related Defcr-rs genes that code for cysteine-rich sequence 4C (CRS4C) peptides that have a unique CPX triplet repeat motif. In ileitis-prone SAMP1/YitFc mice, Paneth cell levels of CRS4C mRNAs and peptides are induced more than a 1000-fold relative to non-prone strains as early as 4 weeks of age, with the mRNA and peptide levels highest in distal ileum and below detection in duodenum. CRS4C-1 peptides are found exclusively in Paneth cells where they occur only in dense core granules and thus are secreted to function in the intestinal lumen. CRS4C bactericidal peptide activity is membrane-disruptive in that it permeabilizes Escherichia coli and induces rapid microbial cell K(+) efflux, but in a manner different from mouse alpha-defensin cryptdin-4. In in vitro studies, inactive pro-CRS4C-1 is converted to bactericidal CRS4C-1 peptide by matrix metalloproteinase-7 (MMP-7) proteolysis of the precursor proregion at the same residue positions that MMP-7 activates mouse pro-alpha-defensins. The absence of processed CRS4C in protein extracts of MMP-7-null mouse ileum demonstrates the in vivo requirement for intracellular MMP-7 in pro-CRS4C processing.

Inhibition of bactericidal activity is maintained in a mouse α-defensin precursor with proregion truncations

α-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-α-defensin proCrp4(20–92), anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-α-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the α-defensin component of the precursor would alter proCrp4 inhibition. Accordingly, two proCrp4 deletion variants, (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4, truncated in a manner corresponding to deletions between MMP-7 cleavage sites, were prepared and assayed for bactericidal peptide activity. Consistent with the properties of full-length proCrp4(20–92), (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4 were processed effectively by MMP-7, lacked bactericidal activity at high peptide levels over a 3h exposure period, and failed to induce permeabilization of live Escherichia coliin vitro. Thus, bringing the inhibitory proregion domain into greater proximity with the Crp4 component of the precursor did not alter the activity of this pro-α-defensin. Therefore, the conserved distance that separates inhibitory acidic proregion residues from the Crp4 peptide is not critical to maintaining proCrp4(20–92) in an inactive state.

Electropositive Charge in α-Defensin Bactericidal Activity: Functional Effects of Lys-for-Arg Substitutions Vary with the Peptide Primary Structure

Cationic amino acids contribute to alpha-defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charges at neutral pH, alpha-defensins contain an average of nine Arg residues per Lys residue. To investigate the role of high alpha-defensin Arg content, all Arg residues in mouse Paneth cell alpha-defensin cryptdin 4 (Crp4) and rhesus myeloid alpha-defensin 4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4, respectively. Lys-for-Arg replacements in Crp4 attenuated bactericidal activity and slowed the kinetics of Escherichia coli ML35 cell permeabilization, and (R/K)-Crp4 required longer exposure times to reduce E. coli cell survival. In marked contrast, Lys substitutions in RMAD-4 improved microbicidal activity against certain bacteria and permeabilized E. coli more effectively. Therefore, Arg-->Lys substitutions attenuated activity in Crp4 but not in RMAD-4, and the functional consequences of Arg-->Lys replacements in alpha-defensins are dependent on the peptide primary structure. In addition, the bactericidal effects of (R/K)-Crp4 and (R/K)-RMAD-4 were more sensitive to inhibition by NaCl than those of the native peptides, suggesting that the high Arg content of alpha-defensins may be under selection to confer superior microbicidal function under physiologic conditions.

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.

A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells

Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Positive selection of arginine in α‐defensin: Attenuation of bactericidal activity by Lys→Arg substitutions is dependent on peptide primary structure

Mammalian α‐defensins are endogenous cationic antimicrobial peptides that contain an average of nine times more Arg than Lys. To investigate the functional role of such high Arg content, Arg residues in the mouse Paneth cell α‐defensin cryptdin‐4 (Crp4) and rhesus myeloid α‐defensin RMAD4 were completely replaced with Lys to prepare (R/K)‐Crp4 and (R/K)‐RMAD4, respectively. Arg mutagenesis of Crp4 attenuated peptide bactericidal activity against several species and the kinetics of (R/K)‐Crp4‐mediated E. coli ML35 cell permeabilization were markedly slower than Crp4, needing a 20 min peptide exposure to detect permeabilization. In contrast to the attenuating effects of Arg to Lys replacements on Crp4, corresponding mutations in RMAD4 improved both microbicidal activity against certain bacterial species and the ability to permeabilize live E. coli. Therefore, although Arg→Lys substitutions in Crp4 delay E. coli cell permeabilization and extend exposure times required to induce bacterial cell death, comparable mutagenesis of RMAD4 does not attenuate either activity. Collectively, these findings are inconsistent with the positive selection of Arg in α‐defensins solely to confer selective advantage through superior antibacterial function. Supported by NIH grants DK044632 and AI059346.