Microflora trigger colitis in mice deficient in selenium-dependent glutathione peroxidase and induce Gpx2 gene expression

Selenium-dependent glutathione peroxidase isoenzymes-1 and -2 are the major glutathione-dependent H2O2-reducing activities in the epithelium of the mid- to lower gastrointestinal tract. The two isoenzymes protect mice against ileocolitis. We have found that luminal microflora are required for colitis to develop in mice deficient in GPX-1 and GPX-2 activity (GPX-DKO). Within 7 days of association with microflora, previously asymptomatic germ-free GPX-DKO mice developed severe acute colitis while their littermates with at least one wild-type Gpx1 or Gpx2 gene remained virtually symptom-free. Microflora also affected Gpx2 gene expression. Gpx2, but not Gpx1, mRNA levels were elevated 4-5 fold in the ileum and colon in conventionally reared or microflora-associated adult mice compared with germ-free mice. Since the gastrointestinal tract microflora undergo major changes 2-3 weeks after birth, from relatively benign to a potentially stressful composition, we examined postnatal Gpx2 gene expression. The jejunal and ileal GPX-2 activity levels were low in two to three week-old mice and increased 5-7 fold during the next two weeks. GPX-2 activity levels were correlated with the mRNA levels. Colon Gpx2 mRNA levels held steady at about 50% of adult levels from 12-21 days of age but were several times higher than ileal levels. Our results suggest that ileal Gpx2 mRNA and GPX-2 activity levels are induced by luminal microflora. This response is consistent with a role for GPX as an anti-inflammatory activity.     

Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria

Normal luminal bacteria and their products play a role in experimental colitis and inflammatory bowel disease. However, what molecules from what cells are responsible for mounting and maintaining the mucosal defense against luminal flora is still uncertain. The aim of this study was to identify epithelial gene products involved in mucosal defense and inflammation associated with ubiquitous enteric bacteria. Germ-free ICR mice were given an oral bacterial suspension prepared from conventional components (bacterial reconstitution). Small intestinal and colonic epithelial cells were isolated from bacteria-reconstituted, germ-free, and specific pathogen-free mice. Differential gene expression was investigated by differential display, Northern blot, and sequence analysis. Bacterial reconstitution resulted in acute but self-limited colitis. In epithelial cells, we observed the induction of small intestine-specific genes of the cryptdin family and colon-specific expression of serum amyloid A1 gene. This novel approach allows the identification of known and novel gene products involved in mucosal defense against luminal microorganisms and the associated inflammatory response.     

Activation of Paneth cell alpha-defensins in mouse small intestine.

Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.     

Differential expression of spleen tyrosine kinase Syk isoforms in tissues: effects of the microbial flora

Spleen tyrosine kinase (Syk) is expressed widely in hematopoietic and non-hematopoietic cells. The widespread distribution of Syk and its involvement in host defense and allergic reactions, prompted us analyze the influence of microbial exposure on Syk expression. We compared the distribution of Syk in various tissues of germ-free and conventional mice using immunohistochemistry, Western blot analysis and real time RT-PCR. Total Syk expression was similar between germ-free and conventional mice. Since it has been claimed that Syk isoforms are differentially expressed, we studied the distribution and abundance of Syk (L) and Syk (S) isoforms in tissues from these mice. In contrast to previous reports, we found broad tissue expression of Syk (S). Interestingly, in germ-free mice the amount of Syk (S) but not Syk L protein was selectively increased in lung and spleen. In summary, our study reveals new and broad tissue expression of both Syk isoforms and demonstrates that lack of microbial flora results in selectively increased expression of Syk (S) isoform in lung and spleen.Florentina Duta, Marina Ulanova and Daniel Seidel are joint first authors. Ulrich Steinhoff and A. Dean Befus are joint last authors.M. Ulanova received a postdoctoral fellowship from the Canadian Society of Allergy and Clinical Immunology/Merck Frosst and from the Alberta Heritage Foundation for Medical Research.     

Paneth cell cryptdins act in vitro as apical paracrine regulators of the innate inflammatory response

Intestinal-specific antimicrobial alpha-defensins, termed cryptdins, are secreted into the intestinal lumen by mouse Paneth cells in response to microbial pathogens. Cryptdins kill microbes by forming pores in their limiting membranes. The cryptdin isoforms 2 and 3 also can form anion-conductive pores in eukaryotic cell membranes, thus affecting cell physiology. Here, we find that when applied to apical membranes of the human intestinal cell line T84, cryptdin 3 (Cr3) induces secretion of the proinflammatory cytokine interleukin 8 (IL-8) in a dose-dependent manner. The induction of IL-8 secretion is specific to the cryptdins that form channels in mammalian cell membranes because cryptdin 4, which does not form pores in T84 cells, does not induce IL-8 secretion. Cr3 induces inflammatory cytokine secretion by activating NF-kappaB and p38 mitogen-activated protein kinase in a Ca2+-dependent manner, but influx by extra-cellular Ca2+ is not involved. Unlike other known inflammatory agonists, signal transduction by Cr3 occurs slowly, suggesting a novel mechanism of action. These results show that selective cryptdins may amplify their roles in innate immunity by acting as novel paracrine agonists to coordinate an inflammatory response with the antimicrobial secretions of Paneth cells.     

Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium

Cryptdin mRNA codes for the apparent precursor to a corticostatin/defensin-related peptide that accumulates to high levels in mouse intestinal crypt epithelium during postnatal development. The primary structure, intestinal cell distribution, and developmental appearance of cryptdin mRNA have been determined. Cryptdin mRNA is 450-480 nucleotides long. Translation of the partial cryptdin cDNA sequence reveals a 70-amino acid open reading frame that includes 32 carboxy-terminal residues that align with those in the consensus sequence, C.CR...C....ER..G.C....CCR, which is a common feature of leukocyte defensins and lung corticostatins (Selsted, M. E., D. M. Brown, R. J. DeLange, S. S. L. Harwig, and R. I. Lehrer. 1985. J. Biol. Chem. 260:4579-4584; Zhu, Q., J. Hu, S. Mulay, F. Esch, S. Shimasaki, and S. Solomon. 1988. Proc. Natl. Acad. Sci. USA. 85:592-596). In situ hybridization of cryptdin cDNA to paraformaldehyde-fixed, frozen sections of adult jejunum and ileum showed intense and specific labeling of epithelial cells in the base of all crypts. Analysis of sections from suckling mice showed that cryptdin mRNA is detectable in 10-20% of crypts in 10-d-old mice, in approximately 80% of crypts in 16-d-old mice, and in all crypts of mice 20 d and older. During the fourth week, the sequence accumulates in crypts to the maximal adult level. Cryptdin mRNA content in adult small intestine is independent both of T cell involvement and luminal bacteria. The role of cryptdin in small bowel physiology remains to be determined: cryptdin may inhibit bacterial translocation, modulate intestinal hormone synthesis, influence hormonal sensitivity of the intestinal epithelium, or exhibit a multiplicity of related activities.     

Enteric defensins: antibiotic peptide components of intestinal host defense

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.     

Regulation of Dab2 expression in intestinal and renal epithelia by development

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.     

Influence of the gut microflora on protein synthesis in tissues and in the whole body of chicks.

1. The influence of the gut microflora on protein synthesis in individual tissues and in the whole body of young chicks was investigated by the large-dose injection of [3H]phenylalanine. 2. Growth of germ-free chicks was significantly better than that of conventional controls. Wet weights of liver, spleen, duodenum, jejunum + ileum and caeca were heavier in conventional birds than in germ-free counterparts. 3. Fractional rates of protein synthesis were higher in jejunum + ileum and whole body of conventional birds than in those of germ-free birds. Amounts of protein synthesized were larger in liver, jejunum + ileum and caeca in the presence of the gut microflora. 4. When tissues were classified into gut + liver and the remainder of the carcass, in the presence of the gut microflora an enhanced protein synthesis in fractional and absolute rate was found in the gut + liver, which is in direct contact or in close association with micro-organisms, whereas virtually no effect of the gut micro-organisms was detected in the remainder of the carcass. 5. The contribution of protein synthesis of gut + liver to that of the whole body was larger in conventional chicks than in germ-free birds, whereas the reverse was true for the remainder of the carcass.     

CFTR基因在小鼠肠道组织中的差异表达

目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组（每组8只）：对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/（kg·bw）]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子（Cl-）分泌中发挥主要作用,结肠的作用最弱。     

Gas supersaturation in the cecal wall of mice due to bacterial CO2 production.

PCO2 in the lumen and serosa of cecum and jejunum was measured in mice. The anesthetic used was a fentanyl-fluanisone-midazolam mixture. PCO2 was recorded in vivo and postmortem. PCO2 was 409 +/- 32 Torr (55 +/- 4 kPa) in the cecal lumen and 199 +/- 22 Torr (27 +/- 3 kPa) on the serosa in normal mice. Irrigation of the cecum resulted in serosal and luminal PCO2 levels of 65-75 Torr. Cecal PCO2 was significantly lower in germ-free mice (65 +/- 5 Torr). Cecal PCO2 increased significantly after introduction of normal bacterial flora into germ-free mice. Introduction of bacterial monocultures into germ-free mice had no effect. After the deaths of the mice, cecal PCO2 increased rapidly in normal mice. The intestinal bacteria produced the majority of the cecal PCO2, and the use of tonometry in intestinal segments with a high bacterial activity should be interpreted with caution. We propose that serosal PCO2 levels >150-190 Torr (20-25 kPa) in the cecum of mice with a normal circulation may represent a state of gas supersaturation in the cecal wall.     

Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.     

Regional variation in ontogeny of class II antigens in enterocytes of mouse small intestine.

The ontogeny of major histocompatibility class II antigens in small intestine enterocytes of postnatal C3H/He mice was investigated. Cryosections of duodenal, jejunal, and ileal segments from 7-, 14-, 16-, 20-, 21-, 23-, 25-, 27-, 28-day-old and 7-week-old mice were stained for the class II antigens with MRC OX6 monoclonal antibodies by peroxidase-antiperoxidase labelling. In adults, the duodenum exhibited least expression of class II antigens that increased progressively towards the ileum. The expression in the villous epithelium was first seen in the duodenum and jejunum 21 days after birth but the ileal enterocytes did not exhibit any class II antigens. The earliest appearance (21 days postnatal) of class II antigens in the enterocytes coincides with the age of weaning which suggests that immunologic stimulation by ingested antigens after weaning may influence expression of these antigens. At day 28 after birth, the duodenum and jejunum expressed levels comparable to those in the adults. The first expression of the antigens seen in the ileum was at day 28 postpartum. Crypt epithelium of the three regions of the small intestine showed expression similar to that of corresponding regional villous enterocytes. We conclude that there is an age-dependent regional variation in the expression of class II antigens in enterocytes, and the expression increases with age. The variation in expression of the class II antigens in enterocytes of postnatal mice is attributed to the developmental status of the tissue. The nature of postnatal expression of the antigens is important since an early appearance of these antigens may have implications in autoimmunity.     

Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion.     

Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.     

.肽链延长因子eEF1A-1和eEF1A-2在小鼠神经元中的表达特征

研究2种肽链延长因子(eEF1A-1,eEF1A-2)在不同发育阶段的小鼠神经元中的表达特征,探 讨其调控机制.应用Western印迹和组织免疫荧光技术分析两蛋白质在不同基因型小鼠(n =10)神经细胞中的表达水平和分布.结果表明,在胚胎期和幼龄期的野生型小鼠神经元胞 质中,eEF1A-1呈高水平表达并随发育而下降,于出生后26 d时停止表达;而eEF1A-2蛋白于出生后7 d开始表达并呈上调趋势,出生后20 d时达到最高水平,其后一直保持稳定表达,2种肽链延长因子在野生型小鼠神经元中的表达随发育而呈相反变化.eEF1A_2基因突变小鼠无eEF1A-2蛋白,eEF1A_1蛋白的表达模式与野生型小鼠基本类似,但出生后26 d时仍有微量表达.2种肽链延长因子在野生型小鼠发育阶段的表达水平变化受内在机制调控,不直接受各自表达水平的影响;eEF1A_2蛋白与神经元生理功能的维持有密切关系.