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Background & Aims: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells., Methods: Kcnk9 -/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro., Results: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9 -/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3 -/- mice were associated with an ameliorated course of DSS-induced colitis., Conclusions: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3 -/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, we established a doxycycline-inducible human tumor necrosis factor alpha (TNFα)-transgenic mouse line, ihTNFtg. Non-induced young and elderly mice showed low but constitutive expression of hTNFα due to promoter leakiness. The persistently present hTNFα stimulated endogenous pro-inflammatory mouse mS100A8/A9 alarmins. Secreted mS100A8/A9 in turn induced the expression and release of mouse mTNFα. The continuous upregulation of pro-inflammatory mTNFα and mS100A8/A9 proteins, due to their mutual expression dependency, gradually led to increased levels in colon tissue and blood. This finally exceeded the threshold levels tolerated by the healthy organism, leading to the onset of intestinal inflammation. Here, recombinant hTNFα functioned as an initial trigger for the development of chronic inflammation. Crossing ihTNFtg mice with S100A9 KO mice lacking active S100A8/A9 alarmins or with Rag1 KO mice lacking T and B lymphocytes completely abrogated the development of colonic inflammation, despite the still leaky hTNFα promoter. Furthermore, both the intensity of the immune response and the strength of immunosuppressive Treg induction was found to depend on the major histocompatibility complex (MHC) genetic composition. In summary, the onset of intestinal inflammation in elderly mice depends on at least four factors that have to be present simultaneously: TNFα upregulation, S100A8/A9 protein expression, functional T lymphocytes and genetic composition, with the MHC haplotype being of central importance. Only joint action of these factors leads to chronic intestinal inflammation, while absence of any of these determinants abrogates the development of the autoimmune disorder. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

The role of neutrophils in the pathogenesis of inflammatory bowel disease (IBD) is still only incompletely understood. Here, we evaluated target-specific fluorescence-mediated tomography (FMT) for visualization of neutrophil infiltration in murine experimental DSS-induced colitis. Colitis was assessed using clinical, endoscopic, and histopathological parameters. Intestinal neutrophil infiltration was determined at day 0, 4, and 10 by targeted FMT after injection of a neutrophil-specific fluorescence-labelled monoclonal antibody (Gr-1). Complementary, immunofluorescence tissue sections with Gr-1 and ELISA-based assessment of tissue myeloperoxidase (MPO) served as the gold standard for the quantification of neutrophil infiltration. Colitic animals showed decreasing body weight, presence of fecal occult blood, and endoscopic signs of inflammation. FMT revealed a significantly increased level of fluorescence only four days after colitis induction as compared to pre-experimental conditions (pmol tracer 73.2 ± 18.1 versus 738.6 ± 80.7; p < 0.05), while neither body weight nor endoscopic assessment showed significant changes at this early time. Confirmatory, post-mortem immunofluorescence studies and measurements of tissue MPO confirmed the presence of increased neutrophil infiltration in colitic mice compared to controls. Concluding, Gr-1 targeted FMT can detect early colonic infiltration of neutrophils in experimental colitis even before clinical symptoms or endoscopic alterations occur. Therefore, FMT might be an important tool for repetitive and non-invasive monitoring of inflammatory cell infiltrate in intestinal inflammation.

Background: The transmembrane heparan sulfate proteoglycan Syndecan-4 (Sdc4) plays an important role in the regulation of various inflammatory disorders. However, the involvement of Sdc4 in intestinal inflammation remains unknown. Therefore, we assessed the impact of Sdc4 deficiency on experimental colitis and epithelial wound healing in vitro and in vivo., Methods: Dextran sulfate sodium (DSS)-induced colitis was monitored in wild type and Sdc4-deficient (Sdc4-/-) mice by assessment of body weight, histology, inflammatory cellular infiltration, and colon length. Syndecan-4 expression was measured by immunohistochemistry, Western blot, and quantitative real-time PCR. Epithelial permeability was evaluated by Evans blue measurements, Western blot, and immunohistological analysis of tight junction protein expression. Impact of Sdc4 on epithelial wound healing was determined by scratch assay in vitro and by colonoscopy following mechanical wounding in vivo., Results: In Sdc4-/- mice, colitis-like symptoms including severe weight loss, shortened colon length, histological damage, and invasion of macrophages and granulocytes were markedly aggravated compared with wild type (WT) animals. Moreover, colonic epithelial permeability in Sdc4-/- mice was enhanced, while tight junction protein expression decreased. Furthermore, Sdc4-/- colonic epithelial cells had lower cell proliferation and migration rates which presented in vivo as a prolonged intestinal wound healing phenotype. Strikingly, in WT animals, Sdc4 expression was reduced during colitis and was elevated during recovery., Conclusions: The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

Background: To facilitate onsite decision-making during endoscopy, both accurate detection and in vivo characterization of preneoplasia are prerequisites. However, no endoscopy technique is available that meets both demands satisfactorily. We evaluated endothelin-receptor A (ETAR)-guided fluorescence endoscopy (FE) in vivo and fluorescence reflectance imaging (FRI) ex vivo for detection and characterization of early dysplastic colitis-associated colonic lesions., Methods: Colorectal cancerogenesis was investigated in the inflammatory driven AOM-DSS model and spontaneous adenoma development in ApcMin mice. A Cy5.5-labeled nonpeptidic ETAR-specific imaging probe was injected intravenously to assess tumor development in vivo by white light endoscopy (WLE) and FE. Ex vivo tumors were evaluated by FRI, histological examination, and western blot analysis. In addition, tissue samples from patients with colitis-associated malignant and nonmalignant mucosal alterations were analyzed. Specificity experiments were performed using an unspecific Cy3.5-glycine tracer., Results: Overall, 62 adenomas were observed. FE was able to detect and quantify ETAR expression targeting the ETAR-specific photoprobe. A significantly higher fluorescent contrast was detected in colonic adenomas compared to adjacent nonmalignant mucosa by FE (64.3 ± 7.9 vs. 56.6. ± 7.0; P < 0.001). These results were confirmed by FRI examination, immunochemistry, and western blot analysis. Additionally, ETAR expression in samples from human patients with colitis-associated cancer was highly elevated compared to nonmalignant alterations. Specificity experiments indicated a high binding-specificity of the applied ETAR photoprobe (1.4 ± 0.3 vs. 2.5 ± 0.7; P < 0.001)., Conclusions: We introduced ETAR guided FE in mice for successful in vivo detection and characterization of colorectal neoplasia on a molecular level., (© 2017 Crohn’s & Colitis Foundation of America. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Aim: To study mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) and vascular endothelial growth factor (VEGF)-targeted contrast enhanced ultrasound (CEUS) for the assessment of murine colitis and carcinogenesis., Methods: C57BL/6 mice were challenged with 3% dextran sodium-sulfate (DSS) for three, six or nine days to study the development of acute colitis. Ultrasound was performed with and without the addition of unspecific contrast agents. MAdCAM-1-targeted contrast agent was used to detect and quantify MAdCAM-1 expression. Inflammatory driven colorectal azoxymethane (AOM)/DSS-induced carcinogenesis was examined on day 42 and 84 using VEGF-targeted contrast agent. Highly specific tissue echogenicity was quantified using specialized software. Sonographic findings were correlated to tissue staining, western blot analysis and immunohistochemistry to quantify the degree of inflammation and stage of carcinogenesis., Results: Native ultrasound detected increased general bowel wall thickening that correlated with more progressed and more severe DSS-colitis (healthy mice: 0.3 mm ± 0.03 vs six days DSS: 0.5 mm ± 0.2 vs nine days DSS: 0.6 mm ± 0.2, P < 0.05). Moreover, these sonographic findings correlated well with clinical parameters such as weight loss ( r 2 = 0.74) and histological damage ( r < 0.01). In acute DSS-induced murine colitis, CEUS targeted against MAdCAM-1 detected and differentiated stages of mild, moderate and severe colitis 2 = 0.86) ( P < 0.01). In acute DSS-induced murine colitis, CEUS targeted against MAdCAM-1 detected and differentiated stages of mild, moderate and severe colitis via calculation of mean pixel contrast intensity in decibel (9.6 dB ± 1.6 vs 12.9 dB ± 1.4 vs 42 d, 18.2 dB ± 3.3 P 84 d, 18.6 dB ± 4.9, vs 42 d, 18.2 dB ± 3.3 vs 42 d after DSS start: 2.4 ± 1.6 P 84 d after DSS start: 3.5 ± 1.3, vs 42 d after DSS start: 2.4 ± 1.6 vs 84 d after DSS start: 3.5 ± 1.3, P < 0.01)., Conclusion: Molecularly targeted CEUS is a highly specific and non-invasive imaging modality, which characterizes murine intestinal inflammation and carcinogenesis in vivo ., Competing Interests: Conflict-of-interest statement: None of the authors has received fees for serving as a speaker or received research funding from with regard to this study. None of the authors owns stocks and/or shares with regards to this study. None of the authors owns patents with regard to this study.

Background: Inflammatory bowel disease is a chronic-remittent disorder with the risk of disabling complications due to uncontrolled inflammation. Accurate biomarkers are needed to noninvasively monitor the disease course to tailor therapy. We evaluated the potential of the specific microRNA (miR)-320a to monitor disease activity in experimental colitis or patients with Crohn's disease and investigated its functional role in intestinal epithelial barrier formation., Methods: The impact of miR-320a on intestinal barrier function was tested in vitro in T84 epithelial cells by transepithelial resistance measurement and quantitative real-time polymerase chain reaction analysis on inflammatory and microbial stimulation. Experimental colitis was studied in dextran sodium sulfate colitis, T-cell transfer colitis, and IL-10 mice. Disease course was monitored by body weight measurement, colonoscopy, and histological examination. MiR-320a expression during inflammation was assessed in T84 cells, murine blood, and colonic tissue and in peripheral blood from patients with Crohn's disease with active or quiescent disease., Results: MiR-320a transfection of T84 cells reinforced barrier integrity reflected by increased transepithelial resistance (P < 0.01) and inhibited barrier-destructive enteropathogenic Escherichia coli effects resulting in increased tight junction protein JAM-A expression (P = 0.02) and decrease of barrier integrity-destabilizing miR-320a target PPP2R5B (P < 0.001). Tumor necrosis factor-α and interleukin-1β stimulation increased a miR-320a epxression in T84 cells. MiR-320a level was increased in blood samples from colitic mice and patients with Crohn's disease showing a strong correlation with disease activity (r = 0.67)., Conclusions: MiR-320a strengthens intestinal barrier function in vitro and has the potential to monitor disease activity of colitic mice. Future studies are needed to further evaluate the potential of miR-320a in patients with inflammatory bowel disease.

Background and Purpose: New therapies for inflammatory bowel disease (IBD) are highly desirable. As apolipoprotein (apo)A-I mimetic peptides are beneficial in several animal models of inflammation, we hypothesized that they might be effective at inhibiting murine colitis., Experimental Approach: Daily injections of 5A peptide, a synthetic bihelical apoA-I mimetic dissolved in PBS, or PBS alone were administered to C57BL/6 mice fed 3% (w v(-1) ) dextran sodium sulfate (DSS) in drinking water or healthy controls., Key Results: Daily treatment with 5A peptide potently restricted DSS-induced inflammation, as indicated by improved disease activity indices and colon histology, as well as decreased intestinal tissue myeloperoxidase levels and plasma TNFα and IL-6 concentrations. Additionally, plasma levels of monocyte chemoattractant protein-1 and the monocyte expression of adhesion-mediating molecule CD11b were down-regulated, pro-inflammatory CD11b(+) /Ly6c(high) monocytes were decreased, and the number of intestinal monocytes was reduced in 5A peptide-treated animals as determined by intravital macrophage-related peptide-8/14-directed fluorescence-mediated tomography and post-mortem immunhistochemical F4/80 staining. Intravital fluorescence microscopy of colonic microvasculature demonstrated inhibitory effects of 5A peptide on leukocyte adhesion accompanied by reduced plasma levels of the soluble adhesion molecule sICAM-1. In vitro 5A peptide reduced monocyte adhesion and transmigration in TNFα-stimulated monolayers of human intestinal microvascular endothelial cells. Increased susceptibility to DSS-induced inflammation was noted in apoA-I(-/-) mice., Conclusions and Implications: The 5A peptide is effective at ameliorating murine colitis by preventing intestinal monocyte infiltration and activation. These findings point to apoA-I mimetics as a potential treatment approach for IBD., (© 2016 The British Pharmacological Society.)

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary non-invasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography, discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD.

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell-induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn's disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Introduction: Leukocyte-specific transcript 1 (LST1) encoded peptides are involved in immunomodulation and nanotube-mediated cell-cell communication. The aim of this study was to assess the expression of LST1 in colonic epithelium and endothelium during intestinal inflammation., Methods: LST1 expression was evaluated by RT-PCR, FACS, western blot analysis, and immunohistochemistry in intestinal epithelial Caco-2 cells, human intestinal microvascular endothelial cells and in human histological specimens from inflammatory bowel disease (IBD) patients and non-IBD colitis patients., Results: LST1 expression was significantly increased upon proinflammatory stimulation in intestinal epithelial and endothelial cells. Furthermore, LST1 tissue expression was significantly enhanced in macroscopically inflamed colonic mucosal biopsies as compared to non-affected mucosal areas., Conclusions: This is the first report demonstrating regulated LST1 expression in human intestinal epithelial and microvascular endothelial cells and in inflamed colonic tissue from IBD patients. Proinflammatory expression of LST1 occurs in the setting of human IBD and is not restricted to immune cell populations. Future studies are needed to further elucidate the role of soluble and membrane-expressed LST1 in the regulation of mucosal intestinal immunity and inflammation as well as to reveal possible therapeutic implications.

Scope: In previous studies, we could show that the B vitamin nicotinamide (NAM) enhanced antimicrobial activity of neutrophils. Here, we assessed the effects of NAM in two models of experimental colitis., Methods and Results: Colitis was induced in C57BL/6 mice either by oral infection with Citrobacter rodentium or by DSS (dextran sodium sulphate) administration, and animals were systemically treated with NAM. Ex vivo bacterial clearance was assessed in murine and human whole blood, as well as isolated human neutrophils. In C. rodentium-induced colitis, NAM treatment resulted in markedly decreased systemic bacterial invasion, histological damage and increased fecal clearance of C. rodentium by up to 600-fold. In contrast, NAM had no effect when administered to neutrophil-depleted mice. Ex vivo stimulation of isolated human neutrophils, as well as murine and human whole blood with NAM led to increased clearance of C. rodentium and enhanced expression of antimicrobial peptides in neutrophils. Moreover, NAM treatment significantly ameliorated the course of DSS colitis, as assessed by body weight, histological damage and myeloperoxidase activity., Conclusion: Pharmacological application of NAM mediates beneficial effects in bacterial and chemically induced colitis. Future studies are needed to explore the clinical potential of NAM in the context of intestinal bacterial infections and human inflammatory bowel disease (IBD)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report on a 24-year-old male patient with history of bloody diarrhea, abdominal pain and vomiting. Endoscopy revealed massive ulcerative discontinuous proctosigmoiditis with deep, sharply demarcated epithelial denudations and enterotoxigenic methicillin-resistant Staphylococcus aureus (MRSA) was detected in mucosal biopsies. After treatment with linezolide and steroids, a significant amelioration of colitis was detected and testing for MRSA became negative. In face of the case presented here, we suggest that in patients with refractory inflammatory bowel disease (IBD), microbiological assessment should be performed to detect a possible Staphylococcus aureus infection in order to initiate an antimicrobial treatment in addition to IBD-specific treatment.

Unlabelled: In patients with inflammatory bowel disease (IBD) and in murine IBD models, mucosal disease activity is routinely assessed by endoscopy and histologic evaluation. This information is valuable for monitoring treatment response, with mucosal healing being a major treatment goal. The aim of this study was to evaluate the translational potential of noninvasive (18)F-FDG PET/CT for the assessment of mucosal damage in murine dextran sodium sulfate (DSS) colitis and human IBD., Methods: After induction of DSS colitis, (18)F-FDG uptake was serially assessed from colonic volumes of interest defined on PET/CT scans and intraindividually correlated to histologic findings and to infiltrating cell types. In addition, (18)F-FDG PET/CT scans of 25 Crohn disease patients were analyzed, and colonic (18)F-FDG uptake was correlated to endoscopically assessed damage., Results: At days 4 and 7 after DSS induction, colonic (18)F-FDG uptake was significantly increased, with a distinct peak in the medial colon. (18)F-FDG uptake strongly correlated with histologic epithelial damage. Additionally, (18)F-FDG uptake increased in the bone marrow in the course of the disease, correlating with an increase in intestinal (18)F-FDG uptake. Histology and fluorescence-activated cell sorting analysis of the bone marrow of DSS mice revealed an increased number of immature neutrophils, whereas mucosal polymerase chain reaction suggested a correlation of (18)F-FDG uptake to T cell infiltration. In accordance with the results of (18)F-FDG PET/CT in DSS colitis, an increased (18)F-FDG uptake was found in 87% of deep mucosal ulcerations in IBD patients, whereas mild endoscopic lesions were detected only by (18)F-FDG PET/CT in about 50% of patients assessed., Conclusion: (18)F-FDG PET/CT is a noninvasive method for evaluation of both experimental colitis and Crohn disease patients and thereby offers promising translational potential.

Treatment options for inflammatory bowel disease (IBD) are incompletely helpful, and surgery is often needed. One promising class of future therapeutic agents for IBD is melanocortin-related peptides, which exhibit potent immunomodulatory effects. We investigated KdPT, a tripeptide derivative of the C-terminus of α-melanocyte-stimulating hormone, as an anti-inflammatory small molecule in vivo and in vitro. Intestinal inflammation was studied after oral administration of dextran sodium sulfate and in IL-10 gene-deficient mice. The effects of KdPT on key colonic epithelial cell functions were studied in vitro and in vivo by evaluating proliferation, wound healing, transepithelial resistance, and expression of tight junction proteins. Melanin assays were performed to determine the melanotropic effects of KdPT. KdPT-treated animals showed markedly reduced severity of inflammation in both colitis models. In colonic epithelial cells, KdPT increased proliferation, accelerated closure of wounds, and improved transepithelial electrical resistance after stimulation with interferon-γ/tumor necrosis factor-α. Moreover, treatment with KdPT also prevented the loss of tight junction protein expression and improved barrier function in vivo. KdPT acted independently of IL-1 receptor type I in vivo and did not affect melanogenesis in vitro. KdPT is capable of attenuating the course of experimental colitis in different models and maintains epithelial cell function. Furthermore, KdPT does not induce pigmentation, emphasizing the potential of this small molecule for the future treatment of IBD., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)