Murine Intestinal Tract Research Articles

Segmental patterning of microbiota and immune cells in the murine intestinal tract

ABSTRACT The intestine exhibits distinct characteristics along its length, with a substantial immune cell reservoir and diverse microbiota crucial for maintaining health. This study investigates how anatomical location and regional microbiota influence intestinal immune cell abundance. Using conventionally colonized and germ-free mice, segment-specific immune cell composition and microbial communities were assessed. Metagenomic sequencing analyzed microbiome variations, while flow cytometry and immunofluorescence examined immune cell composition. Microbiome composition varied significantly along the intestine, with diversity and abundance increasing from upper to lower segments. Immune cells showed distinct segment-specific patterning influenced by microbial colonization and localization. T cell subsets displayed varied dependence on microbiome presence and anatomical location. This study highlights locoregional differences in intestinal immune cell and microbiome composition, identifying immune subsets susceptible to microbiota presence. The findings provide context for understanding immune cell alterations in disease models.

In situ analysis of variations of arsenicals, microbiome and transcriptome profiles along murine intestinal tract

In situ-based studies on microbiome-host interactions after arsenic exposure are few. In this study, the variations in arsenics, microbiota, and host genes along murine intestinal tracts were determined after arsenic exposure for two months. There was a gradual increase in the concentration of total As (CtAs) in feces from ileum to colon, whereas CtAs in the corresponding tissues were relatively stable. Differences in arsenic levels between feces and tissues were significantly different. The proportion of arsenite (iAsⅢ) in feces gradually decreased, however, it gradually increased in tissues. After arsenic exposure, the diversity and abundance of microbial community and networks in each segment were significantly dysregulated. Notably, 328, 579 and 90 differently expressed genes were detected in ileum, cecum, and colon, respectively. In addition, microbiome and transcriptome analyses showed a significant correlation between the abundance of Faecalibaculum and expressions of Plb1, Hspa1b, Areg and Duoxa2 genes. This implies that they may be involved in arsenic biotransformation. In vitro experiments using Biofidobactrium and Lactobacillus showed that probiotics have arsenic transformation abilities. Therefore, gut microbiome may modulate arsenic accumulation, excretion and detoxification along the digestive tract. Moreover, the abundance and diversity of gut microbiome may be related to the changes in host health.

Toxic effects of the food additives titanium dioxide and silica on the murine intestinal tract: Mechanisms related to intestinal barrier dysfunction involved by gut microbiota

This study aimed to compare the effects of three food-grade particles (micro-TiO2, nano-TiO2, and nano-SiO2) on the murine intestinal tract and to investigate their potential mechanisms of action. A 28-day oral exposure murine model was established. Samples of blood, intestinal tissues and colon contents were collected for detection. The results showed that all three particles could cause inflammatory damage to the intestine, with nano-TiO2 showing the strongest effects. Exposure also led to changes in gut microbiota, especially mucus-associated bacteria. Our results suggest that the toxic effects on the intestine were due to reduced intestinal mucus barrier function and an increase in metabolite lipopolysaccharides which activated the expression of inflammatory factors downstream. In mice exposed to nano-TiO2, the intestinal PKC/TLR4/NF-κB signalling pathway was activated. These findings will raise awareness of toxicities associated with the use of food-grade TiO2 and SiO2.

Epidermal growth factor regulation by autophagy-mediated lncRNA H19 in murine intestinal tract after severe burn.

To investigate the regulation of epidermal growth factor (EGF) by autophagy‐mediated long non‐coding RNA (lncRNA) H19 in the intestinal tracts of severely burned mice. C57BL/6J mice received third‐degree burns to 30% of the total body surface area. Rapamycin and 3‐methyladenine (3‐MA) were used to activate and inhibit autophagy, and the changes in LC3 and Beclin1 levels were assessed by Western blotting. The effect of autophagy on lncRNA H19 was detected by qRT‐PCR. Adenovirus‐mediated overexpression of lncRNA H19 in IEC‐6 cells was used to assess the effects of lncRNA H19 on EGF and let‐7g via bioinformatics analysis, Western blotting and qRT‐PCR. let‐7g mimic/inhibitor was used to overexpress/inhibit let‐7g, and qRT‐PCR and Western blotting were used to detect the effects of let‐7g on EGF. The expression levels of LC3‐II, Beclin1 and lncRNA H19 were increased in intestinal tissues and IEC‐6 cells after rapamycin treatment but were reversed after 3‐MA treatment. LC3‐II, Beclin1 and lncRNA H19 levels increased in intestinal tissues after the burn, and these increases were more significant after rapamycin treatment but decreased after 3‐MA treatment. The lncRNA H19 overexpression in IEC‐6 cells resulted in increased and decreased expression levels of EGF and let‐7g, respectively. Furthermore, overexpression and inhibition of let‐7g resulted in decreased and increased expression of EGF, respectively. Taken together, intestinal autophagy is activated after a serious burn, which can increase the transcription level of lncRNA H19. lncRNA H19 may regulate the repair of EGF via let‐7g following intestinal mucosa injury after a burn.

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

Background & AimsShiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell–derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo.MethodsIn vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice.ResultsStx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal–epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro.ConclusionsIntestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

Deletion of the SKO1 Gene in a hog1 Mutant Reverts Virulence in Candida albicans.

Candida albicans displays the ability to adapt to a wide variety of environmental conditions, triggering signaling pathways and transcriptional regulation. Sko1 is a transcription factor that was previously involved in early hypoxic response, cell wall remodeling, and stress response. In the present work, the role of sko1 mutant in in vivo and ex vivo studies was explored. The sko1 mutant behaved as its parental wild type strain regarding the ability to colonize murine intestinal tract, ex vivo adhesion to murine gut epithelium, or systemic virulence. These observations suggest that Sko1 is expendable during commensalism or pathogenesis. Nevertheless, the study of the hog1 sko1 double mutant showed unexpected phenotypes. Previous researches reported that the deletion of the HOG1 gene led to avirulent C. albicans mutant cell, which was, therefore, unable to establish as a commensal in a gastrointestinal murine model. Here, we show that the deletion of sko1 in a hog1 background reverted the virulence of the hog1 mutant in a systemic infection model in Galleria mellonella larvae and slightly improved the ability to colonize the murine gut in a commensalism animal model compared to the hog1 mutant. These results indicate that Sko1 acts as a repressor of virulence related genes, concluding that Sko1 plays a relevant role during commensalism and systemic infection.

Oral vaccination with the porcine circovirus type 2 (PCV-2) capsid protein expressed by Lactococcus lactis induces a specific immune response against PCV-2 in mice.

Porcine circovirus type 2 (PCV2) can cause postweaning, multisystemic wasting syndrome in pigs, which leads to enormous losses in the swine industry worldwide. Here, a genetically engineered Lactococcus strain expressing the main protective antigen of PCV2, the Cap protein, was developed to act against PCV2 infection as an oral vaccine. Expression of the Cap protein was confirmed via western blot, ELISA and fluorescence microscopy. Over 90% of the recombinant pAMJ399-Cap/MG1363 survived a simulated gastrointestinal transit. It also survived the murine intestinal tract for at least 11days. Then, the safety and immunogenicity of pAMJ399-Cap/MG1363 in orally immunized mice was evaluated. The levels of the sIgA, IgG and cytokines (IL-4 and IFN-γ) obtained from the mice immunized with pAMJ399-Cap/MG1363 were significantly higher than those in the control groups. pAMJ399-Cap/MG1363 can survive in the gastrointestinal transit and effectively induce mucosal, cellular and humoral immune response against PCV2 infection via oral administration. This study demonstrates the potential of the genetically engineered Lactococcus lactis as a candidate for an oral vaccine against PCV2.

Mucus Architecture and Near-Surface Swimming Affect Distinct Salmonella Typhimurium Infection Patterns along the Murine Intestinal Tract

SummaryMucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis.

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis.

N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche, and migration up the crypt–villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Surface display of PbrR on Escherichia coli and evaluation of the bioavailability of lead associated with engineered cells in mice

Human exposure to lead mainly occurs by ingestion of contaminated food, water and soil. Blocking lead uptake in the gastrointestinal tract is a novel prevention strategy. Whole-cell biosorbent for lead was constructed with PbrR genetically engineered on the cell surface of Escherichia coli (E. coli), a predominant strain among intestinal microflora, using lipoprotein (Lpp)-OmpA as the anchoring protein. In vitro, the PbrR displayed cells had an enhanced ability for immobilizing toxic lead(II) ions from the external media at both acidic and neutral pH, and exhibited a higher specific adsorption for lead compared to other physiological two valence metal ions. In vivo, the persistence of recombinant E. coli in the murine intestinal tract and the integrity of surface displayed PbrR were confirmed. In addition, oral administration of surface-engineered E. coli was safe in mice, in which the concentrations of physiological metal ions in blood were not affected. More importantly, lead associated with PbrR-displayed E. coli was demonstrated to be less bioavailable in the experimental mouse model with exposure to oral lead. This is reflected by significantly lower blood and femur lead concentrations in PbrR-displayed E. coli groups compared to the control. These results open up the possibility for the removal of toxic metal ions in vivo using engineered microorganisms as adsorbents.

Dietary Fructose Metabolism By Splanchnic Organs: Size Matters

The initial metabolism of fructose is thought to primarily take place in the liver. Using stable isotope labeling combined with tissue and arterio-venous sampling, Jang etal. (2018) demonstrate that in mice, the small intestine is the primary site of fructose metabolism. This raises important questions about fructose handling in humans.

Lactobacillus johnsonii ameliorates intestinal, extra-intestinal and systemic pro-inflammatory immune responses following murine Campylobacter jejuni infection

Campylobacter jejuni infections are progressively increasing worldwide. Probiotic treatment might open novel therapeutic or even prophylactic approaches to combat campylobacteriosis. In the present study secondary abiotic mice were generated by broad-spectrum antibiotic treatment and perorally reassociated with a commensal murine Lactobacillus johnsonii strain either 14 days before (i.e. prophylactic regimen) or 7 days after (i.e. therapeutic regimen) peroral C. jejuni strain 81–176 infection. Following peroral reassociation both C. jejuni and L. johnsonii were able to stably colonize the murine intestinal tract. Neither therapeutic nor prophylactic L. johnsonii application, however, could decrease intestinal C. jejuni burdens. Notably, C. jejuni induced colonic apoptosis could be ameliorated by prophylactic L. johnsonii treatment, whereas co-administration of L. johnsonii impacted adaptive (i.e. T and B lymphocytes, regulatory T cells), but not innate (i.e. macrophages and monocytes) immune cell responses in the intestinal tract. Strikingly, C. jejuni induced intestinal, extra-intestinal and systemic secretion of pro-inflammatory mediators (such as IL-6, MCP-1, TNF and nitric oxide) could be alleviated by peroral L. johnsonii challenge. In conclusion, immunomodulatory probiotic species might offer valuable strategies for prophylaxis and/or treatment of C. jejuni induced intestinal, extra-intestinal as well as systemic pro-inflammatory immune responses in vivo.

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota.

BackgroundWithin seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction.Methodology/Principal findingsSecondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum.Conclusion/SignificanceWith respect to the intestinal microbiota composition “humanized” mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation.

Intestinal Preparation Techniques for Histological Analysis in the Mouse.

The murine intestinal tract represents a difficult organ system to study due to its long convoluted tubular structure, narrow diameter, and delicate mucosa which undergoes rapid changes after sampling prior to fixation. These features do not make for easy histological analysis as rapid fixation in situ, or after simple removal without careful dissection, results in poor postfixation tissue handling and limited options for high quality histological sections. Collecting meaningful quantitative data by analysis of this tissue is further complicated by the anatomical changes in structure along its length. This article describes two methods of intestinal sampling at necropsy that allow systematic histological analysis of the entire intestinal tract, either through examination of cross sections (circumferences) by the gut bundling technique or longitudinal sections by the adapted Swiss roll technique, together with basic methods for data collection. © 2016 by John Wiley & Sons, Inc.

Antioxidative effects in vivo and colonization of Lactobacillus plantarum MA2 in the murine intestinal tract.

Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibet kefir grains, which possess several excellent properties and functions. We previously demonstrated the antioxidant activities of this bacterium in vitro. However, the maintenance and survival of L. plantarum MA2 inside the murine intestinal tract, where it exerts its probiotic properties, and whether its effects are elicited directly on the host remain unknown. Therefore, this study investigated the mechanisms of L. plantarum MA2 in aging mice following D-galactose administration. The levels of malondialdehyde decreased significantly in the L. plantarum MA2 groups after oral ingestion compared to the D-galactose model group, and total antioxidant capacity and glutathione peroxidase and superoxide dismutase activities increased significantly in the serum and liver. We combined fluorescein isothiocyanate labeling and green fluorescent protein expression to dynamically monitor the colonization and distribution of L. plantarum MA2 in the murine intestinal tract. The results indicated that L. plantarum MA2 was detected in the ileum, colon, and feces after single and continuous oral administration at day 21 and was maintained at 10(4)-10(5)CFU/g. These results suggest that L. plantarum MA2 colonizes and survives in the murine intestinal tract to exert its antioxidative effects.

The Role of Gelatinases in Campylobacter Jejuni Infection of Gnotobiotic Mice.

Matrix metalloproteinases (MMP)-2 and -9 (also referred to gelatinases-A and -B, respectively) are upregulated in the inflamed gut of mice and men. We recently demonstrated that synthetic gelatinase blockage reduced large intestinal pro-inflammatory immune responses and apoptosis following murine Campylobacter (C.) jejuni infection. In order to address which gelatinase mediates C. jejuni-induced immune responses, gnotobiotic MMP-2–/–, MMP-9–/–, and wildtype (WT) mice were generated by broadspectrum antibiotic treatment and perorally infected with C. jejuni strain 81-176. The pathogen stably colonized the murine intestinal tract irrespective of the genotype but did not translocate to extra-intestinal compartments. At days 8 and 14 postinfection (p.i.), less pronounced colonic histopathological changes were observed in infected MMP-2–/– mice, less distinct epithelial apoptosis, but more epithelial proliferation in both MMP-2–/– and MMP-9–/– mice, as compared to WT controls. Reduced immune responses in gelatinase-deficient mice were characterized by lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa and lamina propria. The expression of IL-22, IL-18, IL-17A, and IL-1β mRNA was higher in the colon of MMP-2–/– as compared to WT mice. In conclusion, both MMP-2 and MMP-9 are differentially involved in mediating C. jejuni-induced intestinal immunopathology.

Abstract 631: Gastrointestinal toxicities of 5-FU increase the proportion of regulatory T cells in murine intestinal tract: Advantages of alternate-day S-1 administration

Abstract [Introduction] 5-fluorouracil (5-FU), a core anticancer agent used for various malignancies, induces gastrointestinal (GI) toxicities. The tegafur-based oral 5-FU prodrug S-1 is recognized to be a key drug for GI and other malignancies in Japan and some Asian countries. S-1 was developed to potentiate antitumor activity of 5-FU and to reduce gastrointestinal toxicities. However, S-1 also causes GI toxicities by the standard daily regimen. On the other hand, the alternate-day administration of S-1 has been proposed to minimize toxicities without reducing the anticancer efficacy of 5-FU. Recently, tumor immunology has greatly advanced, and the role of T cells in antitumor immunity has been well investigated. However, it remains unknown how GI toxicities of 5-FU affect T cell subsets related to antitumor immunity. [Purpose] The purpose of this study is to clarify the influence of 5-FU mediated GI toxicities on T cell subsets related to antitumor immunity. [Methods] Two regimens of S-1 were compared to evaluate the impact of GI toxicities of 5-FU on T cells using Balb/c mice. The first regimen consisted of the daily administration of S-1 (daily group) as a 5-FU GI toxicity model, and the second regimen consisted of the alternate-day administration of S-1 (alternate-day group) as a 5-FU non-GI toxicity model. S-1 was orally administered at a dose of 12 mg/kg as tegafur. We then investigated the degree of GI toxicities and T cell subsets in intra-abdominal lymphoid tissues (spleen, mesenteric lymph node and gut-associated lymphoid tissue). [Results] Only the daily group exhibited body weight loss and GI toxicities. Flow cytometric analyses demonstrated that the proportion of regulatory T cells in the intestinal tissue was 6-fold higher in the daily group than that in the control group, and the proportions of Th1 cells in the daily group showed a decreasing trend. However, the alternate-day group exhibited almost no changes in the proportions of T cell subsets. [Conclusions] GI toxicities of 5-FU increase the proportion of regulatory T cells in intestinal tissue and decrease the proportions of Th1 cells systemically, which suggests that GI toxicities of 5-FU have a negative influence on T cell-dependent antitumor immunity, and the alternate-day administration of S-1 has neither GI toxicities nor the influence on T cell subsets, which suggests that 5-FU regimens without GI toxicities are more useful than those with GI toxicities from the viewpoint of antitumor immunity. Citation Format: Taiki Kajiwara, Koh Miura, Shinobu Ohnuma, Tetsuhiko Shirasaka, Toshihiro Komura, Masahide Toshima, Atsushi Kohyama, Katsuyoshi Kudoh, Sho Haneda, Hiroaki Musha, Fuyuhiko Motoi, Yu Katayose, Takeshi Naitoh, Michiaki Unno. Gastrointestinal toxicities of 5-FU increase the proportion of regulatory T cells in murine intestinal tract: Advantages of alternate-day S-1 administration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 631. doi:10.1158/1538-7445.AM2014-631

Clostridium ramosum promotes high-fat diet-induced obesity in gnotobiotic mouse models.

ABSTRACTThe intestines of obese humans and mice are enriched with Erysipelotrichi, a class within the Firmicutes. Clostridium ramosum, a member of the Erysipelotrichi, is associated with symptoms of the metabolic syndrome in humans. To clarify the possible obesogenic potential of this bacterial species and to unravel the underlying mechanism, we investigated the role of C. ramosum in obesity development in gnotobiotic mice. Mice were associated with a simplified human intestinal (SIHUMI) microbiota of eight bacterial species, including C. ramosum, with the SIHUMI microbiota except C. ramosum (SIHUMIw/oCra), or with C. ramosum only (Cra) and fed a high-fat diet (HFD) or a low-fat diet (LFD). Parameters related to the development of obesity and metabolic diseases were compared. After 4 weeks of HFD feeding, the mouse groups did not differ in energy intake, diet digestibility, gut permeability, and parameters of low-grade inflammation. However, SIHUMI and Cra mice fed the HFD gained significantly more body weight and body fat and displayed higher food efficiency than SIHUMIw/oCra mice fed the HFD. Gene expression of glucose transporter 2 (Glut2) in jejunal mucosa and of fatty acid translocase (CD36) in ileal mucosa was significantly increased in the obese SIHUMI and Cra mice compared with the less obese SIHUMIw/oCra mice. The data demonstrate that the presence of C. ramosum in SIHUMI and Cra mice enhanced diet-induced obesity. Upregulation of small intestinal glucose and fat transporters in these animals may contribute to their increased body fat deposition.

Impact of metal ion homeostasis of genetically modifiedEscherichia coliNissle 1917 and K12 (W3110) strains on colonization properties in the murine intestinal tract

Metal ions are integral parts of pro- as well as eukaryotic cell homeostasis. Escherichia coli proved a valuable in vitro model organism to elucidate essential mechanisms involved in uptake, storage, and export of metal ions. Given that E. coli Nissle 1917 is able to overcome murine colonization resistance, we generated several E. coli Nissle 1917 mutants with defects in zinc, iron, copper, nickel, manganese homeostasis and performed a comprehensive survey of the impact of metal ion transport and homeostasis for E. coli colonization capacities within the murine intestinal tract. Seven days following peroral infection of conventional mice with E. coli Nissle 1917 strains exhibiting defined defects in zinc or iron uptake, the respective mutant and parental strains could be cultured at comparable, but low levels from the colonic lumen. We next reassociated gnotobiotic mice in which the microbiota responsible for colonization resistance was abrogated by broad-spectrum antibiotics with six different E. coli K12 (W3110) mutants. Seven days following peroral challenge, each mutant and parental strain stably colonized duodenum, ileum, and colon at comparable levels. Taken together, defects in zinc, iron, copper, nickel, and manganese homeostasis do not compromise colonization capacities of E.coli in the murine intestinal tract.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100

AbstractAcute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin–expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.