Mouse Models Of Intestinal Inflammation Research Articles

Nutritional, molecular, and functional properties of a novel enzymatically hydrolyzed porcine plasma product.

In the present study, an enzymatically hydrolyzed porcine plasma (EHPP) was nutritionally and molecularly characterized. EHPP molecular characterization showed, in contrast to spray-dried plasma (SDP), many peptides with relative molecular masses (Mr) below 8,000, constituting 73% of the protein relative abundance. IIAPPER, a well-known bioactive peptide with anti-inflammatory and antioxidant properties, was identified. In vivo functionality of EHPP was tested in C. elegans and two different mouse models of intestinal inflammation. In C. elegans subjected to lipopolysaccharide exposure, EHPP displayed a substantial anti-inflammatory effect, enhancing survival and motility by 40% and 21.5%, respectively. Similarly, in mice challenged with Staphylococcus aureus enterotoxin B or Escherichia coli O42, EHPP and SDP supplementation (8%) increased body weight and average daily gain while reducing the percentage of regulatory Th lymphocytes. Furthermore, both products mitigated the increase of pro-inflammatory cytokines expression associated with these challenged mouse models. In contrast, some significant differences were observed in markers such as Il-6 and Tnf-α, suggesting that the products may present different action mechanisms. In conclusion, EHPP demonstrated similar beneficial health effects to SDP, potentially attributable to the immunomodulatory and antioxidant activity of its characteristic low Mr bioactive peptides.

Peptide-Based Hydrogel for Nanosystems Encapsulation: the Next Generation of Localized Delivery Systems for the Treatment of Intestinal Inflammations.

Conventional therapies for inflammatory bowel diseases are mainly based on systemic treatments which cause side effects and toxicity over long-term administration. Nanoparticles appear as a valid alternative to allow a preferential accumulation in inflamed tissues following oral administration while reducing systemic drug exposure. To increase their residence time in the inflamed intestine, the nanoparticles are here associated with a hydrogel matrix. A bioadhesive peptide-based hydrogel is mixed with nanoemulsions, creating a hybrid lipid-polymer nanocomposite. Mucopenetrating nanoemulsions of 100nm are embedded in a scaffold constituted of the self-assembling peptide hydrogel product PuraStat. The nanocomposite is fully characterized to study the impact of lipid particles in the hydrogel structure. Rheological measurements and circular dichroism analyses are performed to investigate the system's microstructure and physical properties. Biodistribution studies demonstrate that the nanocomposite acts as a depot in the stomach and facilitates the slow release of the nanoemulsions in the intestine. Efficacy studies upon oral administration of the drug-loaded system show the improvement of the disease score in a mouse model of intestinal inflammation.

GRANZYME A-PRODUCING CD4 T CELLS ARE DRIVERS OF IMMUNE CHECKPOINT BLOCKADE-INDUCED COLITIS

Abstract Immune checkpoint blockade (ICB) (i.e. anti-CTLA4 and/or anti-PD-1) is a type of cancer immunotherapy that is broadly and effectively used across multiple cancer types. Despite this efficacy, ~60% of individuals of ICB recipients develop autoimmune-like inflammation in the skin, lungs and intestines. Of these barrier sites, an inflamed intestine is the most common form of severe disease that requires cessation of immunotherapy and potential tumor rebound. ICB-induced colitis therefore represents a significant health concern in an increasing population of cancer patients receiving this otherwise effective immunotherapy. Previous work has shown evidence of a dysregulated intestinal CD4 T cell response in ICB colitis patients, however, how CD4 T cells might contribute to disease has remained unclear. Here, we identify a unique subset of T helper (Th) 1-like CD4 T cells that express the serine protease granzyme A (GZMA) that are specifically elevated in ICB-induced colitis patients, suggesting a pathogenic role for GZMA-expressing Th cells in disease. As mouse models for ICB-induced colitis are virtually non-existent, we have profiled existing mouse models of intestinal inflammation for the presence of colonic GZMA-expressing CD4 T cells for more in-depth functional studies. Using this approach, we have identified the T cell transfer model of colitis as a model that mimics the GZMA CD4 T cell phenotype observed in human ICB colitis patients. Within this model, GZMA-expressing CD4 T cells arise early in the intestines after transfer and only minimally express other CD4 T helper (Th) cell lineage cytokines (i.e. IFN-γ or IL-17) indicating that these cells may represent a unique Th cell subtype that are associated with intestinal disease. Indeed, transfer of GZMA-deficient CD4 T cells into colitis-prone mice resulted in reduced intestinal inflammation and damage, despite efficient trafficking of these cells to the intestines. Mechanistically, CD4 T cell-derived GZMA promoted an exaggerated intestinal epithelial cell response to the microbiota, but was not required for immune-editing of the microbiota in this setting. Taken together, our data indicate that GZMA-producing Th cells are likely major contributors to ICB-induced colitis and may represent a valuable therapeutic target for limiting intestinal disease during ICB therapy.

Dietary sodium butyrate and forskolin promote cell proliferation to resist Citrobacter rodentium infection by lysozyme upregulation

The invasion of pathogens derived from diet or the environment triggers intestinal inflammation, which is a result of the host intestinal barrier damage and immune dysfunction, such as the decrease of host defense peptides (HDPs) and inhibition of cell proliferation. Herein, the siRNA interference demonstrated that a critical lysozyme, which was mainly induced by dietary supplementations of sodium butyrate and forskolin, improved cell proliferation by inhibiting p38 MAPK and JNK signaling pathways and reinforced the intestinal barrier. Moreover, we established a mouse model of intestinal inflammation with Citrobacter rodentium (C. rodentium). It was found that NaBu and FSK stimulated intestinal epithelial proliferation to repair barrier damage, and performed stronger disease resistance to C. rodentium infection on account of the lysozyme upregulation in colon. Therefore, this study revealed that NaBu and FSK prevented or alleviated the development of foodborne infections by promoting lysozyme expression, which may be mediated by the effect of lysozyme on promoting cell proliferation and improving barrier function.

Moluodan promotes DSS-induced intestinal inflammation involving the reprogram of macrophage function and polarization

Ethnopharmacological relevanceMoluodan (MLD) is a traditional Chinese medicine that is composed of 18 herbal medicines based on traditional Chinese medicine theory and practice. It has long been used in treating chronic gastritis and its components were traditionally used in dealing with intestinal inflammation. However, its specific pharmacological mechanism is still unclear. Aim of the studyThe upper and lower digestive tract diseases are correlated. In clinical practice, some chronic gastritis patients are also accompanied by intestinal inflammation. Due to the unclear pharmacological mechanism of MLD and its effect on intestinal inflammation, there is doubt whether MLD is still suitable for this type of patient. Therefore, this study aims to elucidate the pharmacological mechanism of MLD and identify its effect in the mouse model of intestinal inflammation. Materials and methodsMice intestinal inflammation model was induced by 2.5% dextran sulfate sodium (DSS). The mice were given different concentrations of MLD via oral gavage (0.25, 0.5 g/kg b.w.). Pharmacodynamic indicators were assessed including body weight, colon length, disease activity index (DAI), bloody stool score, inflammatory factors, histological change, etc. RAW264.7 macrophage cells were used for in vitro experiments that illuminated the role of MLD in reprogramming macrophage function and polarization. RT-qPCR and western blots were performed to measure the mRNA and protein levels of macrophage polarization marker and effector molecules. The functions of polarized macrophages were tested using ROS detection probes, Edu assay and wound healing assay. ResultsThe administration of MLD exhibited obvious hemostatic effects, while unexpectedly accentuating various aspects of the DSS-induced intestinal inflammation in mice, including increased body weight loss and colon shortening, elevated disease activity index, and intensified colonic tissue damage. Additionally, MLD treatment induced more severe inflammatory cell infiltration and higher proinflammatory cytokines expression in colon tissue. Further results showed that MLD promoted M1 macrophage polarization and stimulated its proinflammatory cytokines expression, while only slightly affecting the function of M2 macrophage. Western blot analysis revealed that MLD induced the phosphorylation of AKT and NF-κB. The polarization of M1 macrophages induced by MLD was inhibited by either an Akt inhibitor or a NF-κB inhibitor. ConclusionsAlthough MLD has an obvious hemostatic effect, it generally promoted the severity of DSS-induced colitis in mice by facilitating macrophage polarization toward the M1 phenotype through the AKT/NF-κB pathway. Our study suggested that MLD may not be suitable for colitis, especially during the acute inflammation stage.

Epithelially Restricted Interferon Epsilon Protects Against Colitis

Background & AimsType I interferon (T1IFN) signalling is crucial for maintaining intestinal homeostasis. We previously found that the novel T1IFN, IFNε, is highly expressed by epithelial cells of the female reproductive tract, where it protects against pathogens. Its function has not been studied in the intestine. We hypothesise IFNε is important in maintaining intestinal homeostasis. MethodsWe characterised IFNε expression in mouse and human intestine by immunostaining and studied its function in the DSS colitis model using both genetic knock-outs and neutralising antibody. ResultsWe demonstrate that IFNε is expressed in human and mouse intestinal epithelium and expression is lost in inflammation. Furthermore, we show IFNε limits intestinal inflammation in mouse models. Regulatory T cell (Treg) frequencies were paradoxically decreased in DSS-treated IFNε-/- mice, suggesting a role for IFNε in maintaining the intestinal Treg compartment. Colitis was ameliorated by transfer of wild-type Tregs into IFNε-/- mice. This demonstrates that IFNε supports intestinal Treg function. ConclusionsOverall, we have shown IFNε expression in intestinal epithelium, and its critical role in gut homeostasis. Given its known role in the female reproductive tract, we now show IFNε has a protective role across multiple mucosal surfaces.

NF-κB p65 and SETDB1 expedite lipopolysaccharide-induced intestinal inflammation in mice by inducing IRF7/NLR-dependent macrophage M1 polarization

Macrophages exhibit distinct phenotypes that are pro-inflammatory (M1) or anti-inflammatory (M2) in response to inflammation. In this study, we tried to identify the roles and mechanisms of interferon regulatory factor 7 (IRF7) in modulating the phenotypes of macrophages in lipopolysaccharide (LPS)-induced intestinal inflammation. The mouse model of intestinal inflammation was induced by lipopolysaccharide (LPS), and mouse bone marrow-derived macrophages (BMDMs) and mouse intestinal epithelial cells were selected for experimental verification in vitro. Results demonstrated that IRF7 was highly expressed in the mouse model of intestinal inflammation, while IRF7 deficiency repressed macrophage M1 polarization and attenuated intestinal inflammation in mice. p65 and SET domain bifurcated 1 (SETDB1) synergistically promoted histone 3 lysine 4 trimethylation (H3K4me3) methylation to elevate IRF7 expression, which activated the Nod-like receptor (NLR) pathway to induce macrophage M1 polarization. Through this mechanism, IRF7 in BMDMs functioned to accelerate intestinal epithelial cell apoptosis and their release of pro-inflammatory proteins. Furthermore, the promoting effect of p65 and SETDB1 on LPS-induced intestinal inflammation was validated in vivo. To sum up, NF-κB p65 and SETDB1 facilitated IRF7-mediated macrophage M1 polarization, thereby aggravating the LPS-induced intestinal inflammation. Hence, this study highlights the appealing value of these factors as anti-inflammatory targets.

Cell-autonomous Hedgehog signaling controls Th17 polarization and pathogenicity

Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.

Impact of Epithelial Cell Shedding on Intestinal Homeostasis.

The gut barrier acts as a first line of defense in the body, and plays a vital role in nutrition and immunoregulation. A layer of epithelial cells bound together via intercellular junction proteins maintains intestinal barrier integrity. Based on a tight equilibrium between cell extrusion and cell restitution, the renewal of the epithelium (epithelial turnover) permits the preservation of cell numbers. As the last step within the epithelial turnover, cell shedding occurs due to the pressure of cell division and migration from the base of the crypt. During this process, redistribution of tight junction proteins enables the sealing of the epithelial gap left by the extruded cell, and thereby maintains barrier function. Disturbance in cell shedding can create transient gaps (leaky gut) or cell accumulation in the epithelial layer. In fact, numerous studies have described the association between dysregulated cell shedding and infection, inflammation, and cancer; thus epithelial cell extrusion is considered a key defense mechanism. In the gastrointestinal tract, altered cell shedding has been observed in mouse models of intestinal inflammation and appears as a potential cause of barrier loss in human inflammatory bowel disease (IBD). Despite the relevance of this process, there are many unanswered questions regarding cell shedding. The investigation of those mechanisms controlling cell extrusion in the gut will definitely contribute to our understanding of intestinal homeostasis. In this review, we summarized the current knowledge about intestinal cell shedding under both physiological and pathological circumstances.

A38 EVALUATING GLYCOCAGED DEXAMETHASONE IN SHIP-DEFICIENT MICE

Abstract Background Corticosteroids systemically reduce immune system activity thereby limiting inflammation in the GI tract. Corticosteroid use is limited by deleterious systemic effects including immunosuppression and adverse effects on growth and development (Giles et al., 2018). The metabolism of dietary polysaccharides, such as plant cell wall xyloglucan, by gut symbionts has been demonstrated to be mediated by niche members of the human gut microbiota (Larsbrink et al., 2014). By caging corticosteroids like dexamethasone (DEX) with xyloglucans to be released by microbiota at the site of disease, there is potential to increase efficacy and limit adverse effects. Mice deficient in Src homology 2 domain-containing inositol polyphosphate 5’-phosphatase (SHIP-/-) develop spontaneous CD-like ileal inflammation (Ngoh et al., 2016). We have used SHIP-/- mice to compare the efficacy of DEX versus caged DEX to reduce intestinal inflammation. Aims Caged DEX will reduce CD-like ileal inflammation in SHIP-/- mice at a lower dose than uncaged DEX because it is released and acts at the site of inflammation. To assess this hypothesis we propose 3 aims: Measure decaging activity along the GI tract of SHIP-/- mice ex vivo Determine the minimum effective dose of DEX required to reduce ileal inflammation in SHIP-/- mice Determine if equimolar and lower concentrations of caged DEX can reduce ileal inflammation in SHIP-/- mice Methods Intestinal contents and feces were harvested from SHIP-/- mice. Decaging activity was measured ex vivo, using caged resorufin (a fluorescent reporter). SHIP-/- mice were orally gavaged with dexamethasone (or vehicle), or the molar equivalent of caged dexamethasone from 6–8 weeks of age. At 8 weeks of age, gross and histopathology were assessed and IL-1β concentrations were measured in full-thickness tissue homogenates. Results SHIP-/- mice demonstrated decaging activity in the cecal, colon, and fecal contents assayed. DEX treatment (3 or 1 mg/kg) of SHIP-/- mice eliminated CD-like gross pathology and histopathology, and reduced IL-1β concentrations in full-thickness ileal tissue homogenates from SHIP-/- mice compared to untreated or vehicle-treated mice. DEX did not have any harmful effects on the intestines of SHIP+/+ mice. 1 or 0.3 mg/kg molar equivalent of caged DEX (3-fold lower concentrations) eliminated CD-like gross pathology in SHIP-/- mice compared to treatment with vehicle control. Additionally, the mean IL-1β concentration in tissue homogenates of mice treated with the molar equivalent of 1 mg/kg caged DEX was reduced Conclusions SHIP-/- mice express decaging activity are effectively treated by DEX, and caged DEX shows similar efficacy at reducing SHIP-/- ileal pathology at lower concentrations than uncaged DEX. In future experiments, we will examine caged DEX at a lower concentration, and in 2nd mouse model of intestinal inflammation. Funding Agencies GlycoNet

Trans-Ned 19-Mediated Antagonism of Nicotinic Acid Adenine Nucleotide-Mediated Calcium Signaling Regulates Th17 Cell Plasticity in Mice.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing agent and its inhibition proved to inhibit T-cell activation. However, the impact of the NAADP signaling on CD4+ T-cell differentiation and plasticity and on the inflammation in tissues other than the central nervous system remains unclear. In this study, we used an antagonist of NAADP signaling, trans-Ned 19, to study the role of NAADP in CD4+ T-cell differentiation and effector function. Partial blockade of NAADP signaling in naïve CD4+ T cells in vitro promoted the differentiation of Th17 cells. Interestingly, trans-Ned 19 also promoted the production of IL-10, co-expression of LAG-3 and CD49b and increased the suppressive capacity of Th17 cells. Moreover, using an IL-17A fate mapping mouse model, we showed that NAADP inhibition promotes conversion of Th17 cells into regulatory T cells in vitro and in vivo. In line with the results, we found that inhibiting NAADP ameliorates disease in a mouse model of intestinal inflammation. Thus, these results reveal a novel function of NAADP in controlling the differentiation and plasticity of CD4+ T cells.

Rules of Engagement: Epithelial-Microbe Interactions and Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex, multifactorial disorders that lead to chronic and relapsing intestinal inflammation. The exact etiology remains unknown, however multiple factors including the environment, genetic, dietary, mucosal immunity, and altered microbiome structure and function play important roles in disease onset and progression. Supporting this notion that the gut microbiota plays a pivotal role in IBD pathogenesis, studies in gnotobiotic mice have shown that mouse models of intestinal inflammation require a microbial community to develop colitis. Additionally, antimicrobial therapy in some IBD patients will temporarily induce remission further demonstrating an association between gut microbes and intestinal inflammation. Finally, a dysfunctional intestinal epithelial barrier is also recognized as a key pathogenic factor in IBD. The intestinal epithelium serves as a barrier between the luminal environment and the mucosal immune system and guards against harmful molecules and microorganisms while being permeable to essential nutrients and solutes. Beneficial (i.e., mutualists) bacteria promote mucosal health by strengthening barrier integrity, increasing local defenses (mucin and IgA production) and inhibiting pro-inflammatory immune responses and apoptosis to promote mucosal homeostasis. In contrast, pathogenic bacteria and pathobionts suppress expression and localization of tight junction proteins, cause dysregulation of apoptosis/proliferation and increase pro-inflammatory signaling that directly damages the intestinal mucosa. This review article will focus on the role of intestinal epithelial cells (IECs) and the luminal environment acting as mediators of barrier function in IBD. We will also share some of our translational observations of interactions between IECs, immune cells, and environmental factors contributing to maintenance of mucosal homeostasis, as it relates to GI inflammation and IBD in different animal models.

Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

P008 Store-operated calcium entry controls immune cell function and activation in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), both representing major clinical challenges in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in immune cells regulating many of their functional properties. Conditional knockout mice, in which SOCE signaling components are deleted in T cells, have revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD and it remains elusive, which immune cell subsets are affected by the pharmaceutical blockade of SOCE. Therefore, we here aimed to investigate the effects of the SOCE inhibitor BTP-2 on the function and activation of lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes (LPMC) were isolated from UC and CD patients undergoing colon resection. Cells were ex -vivo stimulated with PMA/Ionomycin in the presence or absence of increasing concentrations of BTP-2, fixed and stored at -80°C until acquisition by mass cytometry. LPMCs were stained using 37 lineage and functional markers targeting B, T, NK or myeloid cells and the resulting flow cytometry standard (FCS) files were analyzed by using cytobank and the R/Bioconductor 9 packages. Additionally, Ca2+ influx measurements were performed in order to assess the impact of SOCE inhibition on the calcium homeostasis of various immune cell subsets. Results Data on B, T, NK, and myeloid cells revealed that treatment with BTP-2 causes a dose-dependent inhibition of calcium influx, demonstrating that BTP-2 is a potent inhibitor of SOCE in human LPMCs and PBMCs. By applying mass cytometry, we investigated how the gradual pharmacologic inhibition of SOCE affects the activation and pro-inflammatory functions of various immune cell subsets obtained from colon specimens of patients with UC and CD, which were characterized by the accumulation of CD4+ effector T cells, IFNγ-producing CD8+ T cells and IL-17-producing innate lymphoid cells (ILCs) compared to non-inflamed specimens. Remarkably, inhibition of SOCE attenuated the production of cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by T cells and ILCs, reduced the activation of B cells and decreased the production of IFNγ by myeloid cells without affecting the viability of primary human epithelial cells. Conclusion Our data reveal for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation

ObjectiveDysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution,...

Urinary Excretion of Iohexol as a Permeability Marker in a Mouse Model of Intestinal Inflammation: Time Course, Performance and Welfare Considerations.

Simple SummaryIn mammals, different diseases are associated with intestinal changes that may cause an increase in gut permeability. Intestinal permeability tests allow the evaluation of intestinal damage in humans, veterinary patients and laboratory animal models. When used in mouse models, these tests require that animals are singly housed in metabolic cages with a wire-grid floor to collect urine samples. This raises welfare concerns. Iohexol meets several criteria for an ideal intestinal permeability marker and has recently been used in several species. Here, we examined the performance of an intestinal permeability test using iohexol administered by mouth and following excretion over 24 h in urine. As a model, we chose immunodeficient mice with intestinal inflammation induced by adoptive transfer of effector/memory T cells. We collected urine samples at seven time points to profile the urinary excretion of iohexol, in addition to intestinal tissue samples for histological assessment. We conclude that a 6 h cumulative urine sample may be sufficient to evaluate small intestinal permeability in this mouse model and increased urinary excretion of iohexol is correlated with increased severity of duodenitis. The welfare of mice housed in metabolic cages could be improved by reducing the cage periods from 24 to 6 h.Intestinal permeability (IP) tests are used to assess intestinal damage in patients and research models. The probe iohexol has shown advantages compared to 51Cr-EDTA or absorbable/nonabsorbable sugars. During IP tests, animals are housed in metabolic cages (MCs) to collect urine. We examined the performance of an iohexol IP test in mice. Rag1-/- (C57BL/6) mice of both sexes were divided into controls or treatment groups, the latter receiving injections of effector/memory T cells to induce intestinal inflammation. After two, four and five weeks (W), a single dose of iohexol was orally administered. Urine was collected seven times over 24 h in MCs. Iohexol concentration was measured by ELISA. Intestinal histological damage was scored in duodenal sections. In control and treated mice of both sexes, urinary excretion of iohexol peaked at 4 h. From W2 to W4/W5, urinary iohexol excretion increased in treated mice of both sexes, consistent with development of duodenitis in this model. Positive correlations were observed between the urinary excretion of iohexol in W4/W5 and the histological severity of duodenitis in treated male mice. We conclude that a 6 h cumulative urine sample appears sufficient to evaluate small IP to iohexol in this mouse model, improving animal welfare by reducing cage periods.

Mouse Models of Intestinal Fibrosis.

Mouse models are essential for investigation of underlying disease mechanisms that drive intestinal fibrosis, as well as assessment of potential therapeutic approaches to either prevent or resolve fibrosis. Here we describe several common mouse models of intestinal inflammation and fibrosis, including chemically driven colitis models, a bacterially triggered colitis model, and spontaneous intestinal inflammation in genetically susceptible mouse strains. Detailed protocols are provided for dextran sodium sulfate (DSS) colitis, 2,4,6-trinitro-benzene sulfonic acid (TNBS) colitis, adherent-invasive Escherichia coli (AIEC)-triggered colitis, the interleukin-10 knockout (IL-10KO) mouse model of spontaneous colitis, and the SAMP/YitFc model of spontaneous ileocolitis.

The Polyphosphate Kinase of Escherichia coli Is Required for Full Production of the Genotoxin Colibactin.

Colibactin induces DNA damage in mammalian cells and has been linked to the virulence of Escherichia coli and the promotion of colorectal cancer (CRC). By looking for mutants attenuated in the promoter activity of clbB encoding one of the key enzymes for the production of colibactin, we found that a mutant of the gene coding for the polyphosphate kinase (PPK) produced less colibactin than the parental strain. We observed this phenotype in different strains ranging from pathogens responsible for meningitis, urinary tract infection, or mouse colon carcinogenesis to the probiotic Nissle 1917. We confirmed the role of PPK by using an inhibitor of PPK enzymatic activity, mesalamine (also known as 5-aminosalicylic acid). Interestingly, mesalamine has a local anti-inflammatory effect on the epithelial cells of the colon and is used to treat inflammatory bowel disease (IBD). Upon treatment with mesalamine, a decreased genotoxicity of colibactin-producing E. coli was observed both on epithelial cells and directly on purified DNA. This demonstrates the direct effect of mesalamine on bacteria independently from its anti-inflammatory effect on eukaryotic cells. Our results suggest that the mechanisms of action of mesalamine in treating IBD and preventing CRC could also lie in the inhibition of colibactin production. All in all, we demonstrate that PPK is required for the promoter activity of clbB and the production of colibactin, which suggests that PPK is a promising target for the development of anticolibactin and antivirulence strategies.IMPORTANCE Colibactin-producing E. coli induces DNA damage in eukaryotic cells and promotes tumor formation in mouse models of intestinal inflammation. Recent studies have provided strong evidence supporting the causative role of colibactin in human colorectal cancer (CRC) progression. Therefore, it is important to understand the regulation of the production of this genotoxin. Here, we demonstrate that polyphosphate kinase (PPK) is required for the promoter activity of clbB and the production of colibactin. Interestingly, PPK is a multifunctional player in bacterial virulence and stress responses and has been proposed as a new target for developing antimicrobial medicine. We observed inhibition of colibactin production by using a previously identified PPK inhibitor (i.e., mesalamine, an anti-inflammatory drug commonly prescribed for inflammatory bowel diseases). These data brought us a new perspective on the regulatory network of colibactin production and provided us a clue for the development of anticolibactin strategies for CRC treatment/prophylaxis.

Study on the additive protective effect of PGLYRP3 and Bifidobacterium adolescentis Reuter 1963 on severity of DSS-induced colitis in Pglyrp3 knockout (Pglyrp3 −/−) and wild-type (WT) mice

Background and AimsPglyrp3 is a bactericidal innate immunity protein known to sustain the habitual gut microbiome and protect against experimental colitis. Intestinal inflammation and metaflammation are commonly associated with a marked reduction of commensal bifidobacteria. Whether Pglyrp3 and bifidobacteria interact synergistically or additively to alleviate metaflammation is unknown. We investigated the extent to which Pglyrp3 and bifidobacteria regulate metaflammation and gut bacterial dysbiosis in DSS-induced mouse models of intestinal inflammation. Material & Methods8–10 weeks old male mice were used. In both WT and Pglyrp3 −/− experiments, the mice were randomly divided into three groups of 16 mice per group: (1) a control group receiving sterile tap water, (2) an experimental group receiving sterile tap water supplemented with only 5% DSS, and (3) an experimental group receiving sterile tap water supplemented with 5% DSS and 1 × 109 CFU/ml of Bifidobacterium adolescentis (B.a.) for 7 days. Wild-type (WT) littermates of the respective gene (i.e. Pglyrp3) were used as controls throughout the study. Clinical signs of general health and inflammation were monitored daily. Faecal pellet samples were analysed by qRT-PCR for microbial composition. Histology of relevant organs was carried out on day 8. Metabolic parameters and liver inflammation were determined in serum samples. ResultsIntestinal inflammation in mice of group 2 were significantly increased compared to those of control group 1. There was a significant difference in mean scores for inflammation severity between DSS-treated WT and DSS-treated Pglyrp3 −/− mice. Buildup of key serum metabolic markers (cholesterol, triglyceride and glucose) was set off by colonic inflammation. qRT-PCR quantification showed that DSS significantly decreased the Clostridium coccoides and Bifidobacterium cell counts while increasing those of Bacteroides group in both WT and Pglyrp3 −/− mice. These manifestations of DSS-induced dysbiosis were significantly attenuated by feeding B.a. Both the local and systemic ill-being of the mice alleviated when they received B.a. DiscussionThis study shows that Pglyrp3 facilitates recognition of bifidobacterial cell wall-derived peptidoglycan, thus leading additively to a reduction of metaflammation through an increase in the number of bifidobacteria, which were able to mitigate intestinal immunopathology in the context of Pglyrp3 blockade.