Acute Dextran Sodium Sulfate Research Articles

P068 Discovery of IBD3540: A Novel Gut-Restricted Glutamate Carboxypeptidase II Inhibitor with Preclinical Anti-Colitis Activity

Abstract Background Glutamate carboxypeptidase II (GCPII) is robustly overexpressed in both Crohn’s disease and ulcerative colitis and is a promising therapeutic target for IBD. We have previously published that systemically or enema administered small molecule GCPII inhibitors have efficacy in multiple mouse colitis models. While this is a highly encouraging activity profile for a candidate IBD drug, an oral dose route would be strongly preferred. Thus, here we develop and characterize a new class of GCPII inhibitors designed to be gut-restricted for oral administration, with the intention of selecting a lead inhibitor for further clinical development. Methods Anti-inflammatory secondary bile acids lithocholic acid, deoxycholic acid and ursodeoxycholic acid were conjugated to GCPII inhibitor, 2-PMPA, yielding three novel constructs. These conjugates were screened for oral activity in murine dextran sulfate sodium (DSS) colitis, followed by detailed characterization of the most active inhibitor to: measure GCPII inhibition (IC50), confirm colon target engagement, evaluate plasma and colon pharmacokinetic profiles, compare efficacy versus standard-of-care agents, evaluate mechanisms of anti-inflammatory activity in vivo, confirm efficacy in a second colitis model (IL10-/-), and assess safety in standard preclinical assays. Results The deoxycholic acid 2-PMPA conjugate, IBD3540, was identified as the lead inhibitor (IC50 = 4nM). Oral IBD3540 was robustly efficacious in acute DSS colitis, where it dose-dependently inhibited colon GCPII, attenuated both gross disease activity index and blinded colon histopathology scores, and had improved efficacy relative to both sulfasalazine and tofacitinib when compared head-to-head. Mechanistically, we determined that IBD3540 attenuated pathogenic monocytic inflammation in the colon, as measured by flow cytometry, and also decreased colon pro-inflammatory cytokine content. We confirmed efficacy of IBD3540 in spontaneously occurring, chronic, IL10-/- colitis, where treatment initiated 4 weeks after the onset of colon inflammation improved multiple disease endpoints. Further, in preclinical safety screens, inclusive of CYP inhibition/induction assays, Eurofins SafetyScreen44TM, and AMES/hERG mutagenicity testing, no concerns were identified. Conclusion IBD3540 is a novel, gut-restricted, GCPII inhibitor with potent oral anti-colitis efficacy in both acute (DSS) and chronic (IL10-/-) mouse colitis models and a promising preclinical safety profile. Further development of this mechanistically unique candidate IBD drug is warranted.

Beneficial and anti-inflammatory effects of formulated prebiotics, probiotics, and synbiotics in normal and acute colitis mice

In inflammatory bowel disease (IBD), an altered gut microbiota is observed and is a proposed causal factor for compromised intestinal integrity and disease progression. Therefore, maintenance and modulation of the gut microbiome may be beneficial to health and for treatment of IBD. Prebiotics, probiotics, and synbiotics have been suggested to positively impact the gut microbiota and thus we aimed to evaluate the effects of a formulated prebiotic mixture (Pre), probiotic mixture (Pro), and synbiotics (Syn) in normal mice, and their therapeutic effects in acute dextran sulfate sodium (DSS)-induced mice. We observed significant reductions in plasma IL-6 levels and increased intestinal occludin expression in both models after treatment. In DSS-induced mice, treatment modulated gut microbiota, improved gut integrity, upregulated anti-inflammatory cytokines, and suppressed plasma pro-inflammatory mediators, potentially via inhibition of IL-6/STAT3 signaling. Our results demonstrate the beneficial effects of Pre, Pro, and Syn consumption and their potential therapeutic effects in IBD.

Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

Background and AimsCurrent management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts.MethodsWe first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities.ResultsIn the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor–specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation.ConclusionsIt is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

The Effects of Sishen Wan on T Cell Responses in Mice Models of Ulcerative Colitis Induced by Dextran Sodium Sulfate.

Currently, it is unclear whether Sishen Wan (SSW) could modulate the balance of Th1 cells, Th17 cells, and Tregs and we evaluated the effects of SSW on T cell responses in mice models of ulcerative colitis (UC). The mice models of acute UC (4% dextran sodium sulfate (DSS), 8 days) and chronic UC (3% DSS, 16 days) with SSW were assayed. Colon tissues were collected for immunohistochemical analysis, enzyme linked immunosorbent assay (ELISA), and flow cytometry (FCM). The expressions of cytokines associated with Tregs, transcription factors of Th17 cells, the frequencies of Th1 cells, Th17 cells, and Tregs, and the functional plasticity of Th17 cells were detected. The frequency of IFN-γ+ T cells was not changed significantly with SSW treatment in acute DSS. In chronic models, the frequency of IFN-γ+ T cells was downregulated with SSW. Meanwhile, the levels of RORγt and the frequency of IL-17A+ Th17 cells showed no significant differences after SSW treatment. Despite no significant effect on the transdifferentiation of Th17 cells in chronic UC models, SSW transdifferentiated Th17 cells into IL-10+ Th17 cells and downregulated IFN-γ+ Th17 cells/IL-10+ Th17 cells in acute DSS. Moreover, there were no significant changes of cytokines secreted by Tregs in acute DSS after SSW treatment, but SSW facilitated the expressions of IL-10 and IL-35, as well as development of IL-10+ Tregs in chronic DSS. SSW showed depressive effects on the immunoreaction of Th17 cells and might promote the conversion of Th17 cells into IL-10+ Th17 cells in acute UC, while it inhibited the excessive reaction of Th1 cells, facilitated the development of Tregs, and enhanced the anti-inflammatory effects in chronic UC.

Ingested nano- and microsized polystyrene particles surpass the intestinal barrier and accumulate in the body

Plastic pollution is a major global challenge of our times, baring a potential threat for the environment and the human health. The increasing abundance of nanoplastic (NP) and microplastic (MP) particles in the human diet might negatively affect human health since they - particularly in patients suffering from inflammatory bowel disease (IBD) – might surpass the intestinal barrier. To investigate whether ingested plastic particles cross the intestinal epithelium and promote bowel inflammation, mice were supplemented with NP or MP polystyrene (PS) particles for 24 or 12 weeks before inducing acute or chronic dextran sodium sulfate (DSS) colitis with continuous plastic administration. Although ingested PS particles accumulated in the small intestine and organs distant from the gastrointestinal tract, PS ingestion did not affect intestinal health nor did it promote colitis severity. Although the lack of colitis-promoting effects of small PS particles might be a relief for IBD patients, potential accumulative effects of ingested plastic particles on the gastrointestinal health cannot be excluded.

IL-33 Drives Expansion of Type 2 Innate Lymphoid Cells and Regulatory T Cells and Protects Mice From Severe, Acute Colitis.

The hallmarks of inflammatory bowel disease are mucosal damage and ulceration, which are known to be high-risk conditions for the development of colorectal cancer. Recently, interleukin (IL)-33 and its receptor ST2 have emerged as critical modulators in inflammatory disorders. Even though several studies highlight the IL-33/ST2 pathway as a key factor in colitis, a detailed mode of action remains elusive. Therefore, we investigated the role of IL-33 during intestinal inflammation and its potential as a novel therapeutic target in colitis. Interestingly, the expression of IL-33, but not its receptor ST2, was significantly increased in biopsies from the inflamed colon of IBD patients compared to non-inflamed colonic tissue. Accordingly, in a mouse model of Dextran Sulfate Sodium (DSS) induced colitis, the secretion of IL-33 significantly accelerated in the colon. Induction of DSS colitis in ST2-/- mice displayed an aggravated colon pathology, which suggested a favorable role of the IL 33/ST2 pathway during colitis. Indeed, injecting rmIL-33 into mice suffering from acute DSS colitis, strongly abrogated epithelial damage, pro-inflammatory cytokine secretion, and loss of barrier integrity, while it induced a strong increase of Th2 associated cytokines (IL-13/IL-5) in the colon. This effect was accompanied by the accumulation of regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) in the colon. Depletion of Foxp3+ Tregs during IL-33 treatment in DSS colitis ameliorated the positive effect on the intestinal pathology. Finally, IL-33 expanded ILC2s, which were adoptively transferred to DSS treated mice, significantly reduced colonic inflammation compared to DSS control mice. In summary, our results emphasize that the IL-33/ST2 pathway plays a crucial protective role in colitis by modulating ILC2 and Treg numbers.

Enhanced Susceptibility of Galectin-1 Deficient Mice to Experimental Colitis.

Galectin-1 is a β-galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with β-galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice (Lgals1−/− mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1−/− mice involved an altered Th17/Th1 profile of effector CD4+ T cells. Furthermore, increased frequencies of Foxp3+CD4+ regulatory T cells in colon lamina propria in Lgals1−/− mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1− / − mice was alleviated by adoptive transfer of wild type Foxp3+CD4+ regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation.

P016 SZN-1326, a Wnt signal activator, is more efficacious than cyclosporine A in an acute DSS model

Abstract Background Current Inflammatory bowel disease (IBD) treatments focus on inhibition of excessive inflammation, and clinical remission rates have reached a plateau. There is a clear unmet need for agents that directly repair and regenerate the intestinal epithelial barrier as mucosal healing has been associated with reduced hospitalizations and long-term remission. Wnt/β-catenin signaling promotes intestinal stem cell renewal and is crucial for intestinal epithelial homeostasis and regeneration. Fzd5, a Wnt receptor, is highly expressed in intestinal epithelium. We have engineered a FZD5,8 and LRP6 bi-specific IgG1 antibody, SZN-1326, which potently induced Wnt signaling in a Super Top Flash (STF) Luciferase reporter assay, stimulated intestinal organoid growth, increased Wnt target gene Axin2 expression in the colon tissue and ameliorated Dextran Sulfate Sodium (DSS)-induced colitis in mice. The objective of the current studies was to compare the efficacy of SZN-1326, a Wnt signal activator, to Cyclosporine A, which has been shown to have activity in an acute DSS-induced colitis mouse model. Methods To induce acute colitis, 7- to 8-week-old female C57BL/6 mice were given drinking water containing 4.0% (w/v) DSS for 7 days followed by 1.0% DSS for 3 days. Groups of mice were treated either with an isotype control antibody (anti-GFP), one intraperitoneal (IP) injection of SZN-1326 at 2, 6, 20 mg/kg on day 4 or two injections of SZN-1326 at 1, 3, 10 mg/kg on days 4 and 7, or treated daily with Cyclosporine A by oral gavage at 80 mg/kg on days 0–9, and the mice were taken down on day 10. Results Mice subjected to DSS developed severe colitis characterized by profound and sustained weight loss and bloody diarrhea, resulting in the increase of disease activity index (DAI). SZN-1326 treatments improved body weight, decreased fecal score, and reduced DAI much more effectively than Cyclosporine A. Histological evaluation showed SZN-1326 treatments repaired the damaged colon epithelium, decreased colon histology scores of inflammation, mucosal erosion, and goblet cell loss, at doses as low as 1 mg/kg dosed twice or a single dose of 2 mg/kg. In addition, SZN-1326 treatments significantly decreased serum levels of inflammatory cytokines, Lipocalin-2, IL-6 and IL-8, while Cyclosporine A treatment led only to a reduction of Lipocalin-2. Conclusion In an acute mouse IBD model, SZN-1326 at various dosing regimen stimulated intestinal epithelial regeneration, induced mucosal healing and restored the epithelial barrier resulting in reduced inflammation, improved body weight and reduced disease activity. In contrast, Cyclosporine A showed only a mild effect on reducing DAI and lipocalin-2.

Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing.

Epicertin (EPT) is a recombinant variant of the cholera toxin B subunit, modified with a C-terminal KDEL endoplasmic reticulum retention motif. EPT has therapeutic potential for ulcerative colitis treatment. Previously, orally administered EPT demonstrated colon epithelial repair activity in dextran sodium sulfate (DSS)-induced acute and chronic colitis in mice. However, the oral dosing requires cumbersome pretreatment with sodium bicarbonate to conserve the acid-labile drug substance while transit through the stomach, hampering its facile application in chronic disease treatment. Here, we developed a solid oral formulation of EPT that circumvents degradation in gastric acid. EPT was spray-dried and packed into enteric-coated capsules to allow for pH-dependent release in the colon. A GM1-capture KDEL-detection ELISA and size-exclusion HPLC indicated that EPT powder maintains activity and structural stability for up to 9 months. Capsule disintegration tests showed that EPT remained encapsulated at pH 1 but was released over 180 min at pH 6.8, the approximate pH of the proximal colon. An acute DSS colitis study confirmed the therapeutic efficacy of encapsulated EPT in C57BL/6 mice upon oral administration without gastric acid neutralization pretreatment compared to vehicle-treated mice (p < 0.05). These results provide a foundation for an enteric-coated oral formulation of spray-dried EPT.

Combination of Vedolizumab With Tacrolimus Is More Efficient Than Vedolizumab Alone in the Treatment of Experimental Colitis.

Vedolizumab is a widely used and safe therapy in inflammatory bowel disease, particularly in ulcerative colitis (UC), making it a promising candidate for enhanced efficacy by combining it with additional immunomodulatory medications. In this study, we studied the impact of vedolizumab monotreatment vs vedolizumab coadministration with other immunomodulatory drugs on intestinal inflammation and intestinal immune cells in vivo. Colon tissue from human patients with UC with active disease or in remission with or without vedolizumab treatment was stained by immunohistochemistry. We reconstituted NOD-SCID-SGM3 mice with human CD34+ cells and treated them with dextran sodium sulfate to induce acute colitis. Mice were treated with vedolizumab alone, or in combination with tacrolimus, ozanimid, or tofacitinib. Vedolizumab reduced the number of CD3+ T cells and CD68+ monocytes/macrophages in the colon of patients with UC with active disease. Vedolizumab moderately decreased immune cell numbers in acute dextran sodium sulfate-induced colitis. The combination of vedolizumab with tacrolimus further reduced the number of infiltrating CD3+ T cells and CD68+ monocytes/macrophages and was superior in ameliorating intestinal inflammation when compared to vedolizumab monotreatment. In contrast, cotreatment using vedolizumab with ozanimod or tofacitinib had no additive effect. Our data show that vedolizumab reduces the number of innate and adaptive immune cells in the mucosa of patients with UC. Further, the combination of vedolizumab with tacrolimus was more efficient to reduce immune cell numbers and to increase therapeutic efficacy than vedolizumab monotreatment. This finding indicates that combination treatment using these two drugs may be beneficial for patients who do not respond to vedolizumab monotherapy.

Matrix Metalloproteinase MMP-12 Promotes Macrophage Transmigration Across Intestinal Epithelial Tight Junctions and Increases Severity of Experimental Colitis.

Matrix metalloproteinases [MMPs] play an important role in extracellular matrix regulation during cell growth and wound healing. Increased expression of MMP-12 [human macrophage elastase] has been reported in inflammatory bowel disease [IBD] which is characterised by the loss of epithelial tight junction [TJ] barrier function and an excessive inflammatory response. The aim of this study was to investigate the role of MMP-12 in intestinal TJ barrier function and inflammation. Wild type [WT] and MMP-12-/- mice were subjected to experimental acute or chronic dextran sodium sulphate [DSS] colitis. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon and ex vivo by Ussing chamber studies. DSS administration increased colonic permeability through modulation of TJ proteins and also increased MMP-12 expression in the colonic mucosa of WT mice. The acute as well as chronic DSS-induced increase in colonic TJ permeability and the severity of DSS colitis was found to be markedly attenuated in MMP-12-/- mice. The resistance of MMP-12-/- mice to DSS colitis was characterised by reduced macrophage infiltration and transmigration, and reduced basement membrane laminin degradation. Further in vitro and in vivo studies show that macrophage transmigration across the epithelial layer is MMP-12 dependent and the epithelial TJ barrier is compromised during macrophage transmigration. Conclusions: Together, these data demonstrate that MMP-12 mediated degradation of basement membrane laminin, macrophage transmigration, and associated loss of intestinal TJ barrier are key pathogenic factors for intestinal inflammation.

DSS-induced colitis is associated with adipose tissue dysfunction and disrupted hepatic lipid metabolism leading to hepatosteatosis and dyslipidemia in mice

Considering high prevalence of non-alcoholic fatty liver diseases (NAFLD) in patients with inflammatory bowel disease (IBD), this study aimed to elucidate molecular mechanisms for how intestinal inflammatory conditions are causally linked to hepatic steatosis and dyslipidemia. Both younger and older mice treated with acute or chronic dextran sodium sulfate (DSS) developed colitis, which was evidenced by weight loss, colon length shortening, and elevated disease activity index and inflammation score. They also showed decreased expression of intestinal barrier function-related proteins and elevated plasma lipopolysaccharide level, indicating DSS-induced barrier dysfunction and thereby increased permeability. Interestingly, they displayed phenotypes of hepatic fat accumulation and abnormal blood lipid profiles. This DSS-induced colitis-associated lipid metabolic dysfunction was due to overall disruption of metabolic processes including fatty acid oxidation, lipogenesis, lipolysis, reverse cholesterol transport, bile acid synthesis, and white adipose tissue browning and brown adipose tissue thermogenesis, most of which are mediated by key regulators of energy homeostasis such as FGF21, adiponectin, and irisin, via SIRT1/PGC-1α- and LXRα-dependent pathways. Our study suggests a potential molecular mechanism underlying the comorbidity of NAFLD and IBD, which could provide a key to understanding how the two diseases are pathogenically linked and discovering critical therapeutic targets for their treatment.

A45 A NOVEL PH-SENSITIVE OPIOID ANALGESIC THAT SELECTIVELY INHIBITS NOCICEPTION IN DSS-INDUCED COLITIS

Abstract Background Opioid drugs are used to treat pain in inflammatory bowel disease (IBD) but their side effects can cause serious morbidity. Therefore, we tested a novel opioid analgesic, ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenylpropionamide (NFEPP) which selectively activates peripheral µ-opioid receptors at acidic pH, as occurs in inflamed tissue. Aims Evaluate whether NFEPP causes analgesia in the inflamed colon of DSS-colitis mice using both in vitro and in vivo techniques. Methods To measure the visceral motor reflex (VMR) in response to colorectal distention, EMG electrodes connected to a telemetric transmitter were implanted in mice (c57BL/6), after 10 days recovery acute dextran sodium sulfate (DSS) colitis was induced (5 days 2.5% DSS, 2 days water). VMR was measured 30 min after s.c. injection of vehicle or 0.2 mg/kg of NFEPP or fentanyl. Motility was assessed by fecal pellet count 1 hour after NFEPP. Colonic tissue pH was evaluated using the SNARF-4F-5 carboxylic acid probe. Excitability of mouse dorsal root ganglia (DRG) neurons was measured by recording the rheobase (minimum input current to fire an action potential) after superfusion of NFEPP (300 nM, 10 min) or vehicle at pH 6.5 or 7.4. Colonic afferent nerve responses to probing with a von Frey filament (1 gm) were examined before and after exposure to NFEPP (300 nM, 5 min superfusion) at pH 6.5 and 7.4 respectively. The data was analyzed with Welch’s t-test, 1- or 2-way ANOVA with post hoc Dunnett or Bonferroni or Tukey’s test. Results NFEPP significantly inhibited the VMR in response to distension in mice with colitis compared to vehicle (decreased response by 65%, P&amp;lt;0.001). NFEPP had no effect in control mice. Conversely, fentanyl caused a similar decreased response in both groups (DSS 79% and control 67%, P&amp;lt;0.001). Pelleting was not affected by NFEPP injection in either group compared to vehicle. The pH measurement revealed a more acidic environment in DSS colonic tissue (ΔpH0.37±0.14, P&amp;lt;0.05) compared to controls. In patch-clamp studies, NFEPP decreased DRG excitability at pH 6.5 compared to the baseline and vehicle (increased rheobase 53.84%, P&amp;lt;0.01 and 36.36%, P&amp;lt;0.05 respectively) but had no effect at pH 7.4. In colonic afferent nerve recordings, NFEPP significantly attenuated afferent responses (28.9% P&amp;lt;0.01) to probing at pH 6.5 but also had no effect at pH 7.4. Conclusions This pH-selective opioid agonist significantly inhibits pain at the site of inflammation where the tissue pH is acidic but has no effect in tissues where the pH is in the physiological range. Thus, NFEPP could be an effective opioid analgesic in IBD while being devoid of any unwanted side effects. Funding Agencies CCC

Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BackgroundImpaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis.MethodsWe developed an enzyme‐linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO‐C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included.ResultsIn the acute and chronic dextran sulphate sodium‐induced rat colitis model, the PRO‐C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO‐C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO‐C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO‐C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05).ConclusionPRO‐C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO‐C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

Energy Drink Administration Ameliorates Intestinal Epithelial Barrier Defects and Reduces Acute DSS Colitis.

The rise in the prevalence of inflammatory bowel diseases in the past decades coincides with changes in nutritional habits, such as adaptation of a Western diet. However, it is largely unknown how certain nutritional habits, such as energy drink consumption, affect intestinal inflammation. Here, we assessed the effect of energy drink supplementation on the development of intestinal inflammation in vitro and in vivo. HT-29 and T84 intestinal epithelial cells and THP-1 monocytic cells were treated with IFNγ in presence or absence of different concentrations of an energy drink. Colitis was induced in C57BL/6 mice by addition of dextran sodium sulfate (DSS) to drinking water with or without supplementation of the energy drink. Energy drink supplementation caused a dose-dependent decrease in IFNγ-induced epithelial barrier permeability, which was accompanied by upregulation of the pore-forming protein claudin-2. Administration of the energy drink reduced secretion of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α from HT-29, T84, and THP-1 cells. In vivo, energy drink administration reduced clinical symptoms of DSS-induced colitis and epithelial barrier permeability. Endoscopic and histologic colitis scores and expression of pro-inflammatory cytokines were significantly reduced by energy drink co-administration. Energy drink consumption seems to exert an unexpected anti-inflammatory effect in vitro and in vivo in our experimental setting. However, our experimental approach focuses on intestinal inflammation and neglects additional effects of energy drink consumption on the body (eg, on metabolism or sleep). Therefore, the translation of our findings into the human situation must be taken with caution.

Transgenic overexpression of ITGB6 in intestinal epithelial cells exacerbates dextran sulfate sodium-induced colitis in mice.

Integrins, as a large family of cell adhesion molecules, play a crucial role in maintaining intestinal homeostasis. In inflammatory bowel disease (IBD), homeostasis is disrupted. Integrin αvβ6, which is mainly regulated by the integrin β6 subunit gene (ITGB6), is a cell adhesion molecule that mediates cell‐cell and cell‐matrix interactions. However, the role of ITGB6 in the pathogenesis of IBD remains elusive. In this study, we found that ITGB6 was markedly upregulated in inflamed intestinal tissues from patients with IBD. Then, we generated an intestinal epithelial cell‐specific ITGB6 transgenic mouse model. Conditional ITGB6 transgene expression exacerbated experimental colitis in mouse models of acute and chronic dextran sulphate sodium (DSS)‐induced colitis. Survival analyses revealed that ITGB6 transgene expression correlated with poor prognosis in DSS‐induced colitis. Furthermore, our data indicated that ITGB6 transgene expression increased macrophages infiltration, pro‐inflammatory cytokines secretion, integrin ligands expression and Stat1 signalling pathway activation. Collectively, our findings revealed a previously unknown role of ITGB6 in IBD and highlighted the possibility of ITGB6 as a diagnostic marker and therapeutic target for IBD.

TOXICOLOGICAL FINDINGS OF A RECOMBINANT CHOLERA TOXIN B SUBUNIT VARIANT WITH THERAPEUTIC POTENTIAL IN ULCERATIVE COLITIS

Abstract Background The cholera toxin B subunit (CTB) is the nontoxic and homopentameric component of the holotoxin. Upon binding to GM1 ganglioside on the surface of epithelial cells, CTB mediates entry and retrograde transport through the endomembrane system and disengages the catalytic A subunit in the endoplasmic reticulum (ER). EPICERTIN (EPT) is a recombinant variant of CTB with a non-native C-terminal extension harboring an ER-retention motif, KDEL. We have found that increased ER-retention time resulting from this modification allowed EPT to induce an unfolded protein response and TGF-β signaling in colon epithelial cells, triggering wound healing activity in preclinical colitis models. The unique epithelial repair activity of EPT hints at its therapeutic potential in ulcerative colitis. Objective We aim to develop data supporting a first-in-human clinical trial with an EPT enema indicated for ulcerative colitis. Here, we evaluated the efficacy and toxicity of intrarectal (IR) administration of EPT in preclinical rodent models. Results IR administration of EPT at 0.1 and 1 µM to female C57/BL6 mice (0.6 and 6.1 μg/animal) with acute dextran sodium sulfate (DSS)-induced colitis resulted in decreased disease activity index scores and increased body weight recovery, supporting a target therapeutic dose of ≤1 µM for clinical administration. A dose-escalation study was performed following a single IR exposure at 1, 2 and 5 µM (61.4, 122.8 and 307 μg/animal) in male and female Sprague Dawley rats. No drug-related adverse effects were evident in clinical observations, including clinical pathology and gross necropsy, even at the highest dose tested. A pharmacokinetics study was performed in male and female mice dosed with a 1 or 10 µM (6.1 and 61.4 μg/animal) IV bolus of EPT. Plasma samples were collected periodically for up to 24 h postdose. EPT concentrations were highest at first collection and decreased steadily until unquantifiable by 4 h. The elimination phase half-life was 0.26 to 0.3 h. When healthy and DSS-induced colitic mice (n = 72) were dosed with 1 or 10 µM EPT IR, marginal amounts of EPT were found in only 4 plasma samples scattered across groups and time points, suggesting that systemic exposure after IR administration is negligible. Conclusion These data support further development of EPT as a potential therapeutic for ulcerative colitis.

Matricellular Protein SPARCL1 Regulates Blood Vessel Integrity and Antagonizes Inflammatory Bowel Disease.

BackgroundThe understanding of vascular plasticity is key to defining the role of blood vessels in physiologic and pathogenic processes. In the present study, the impact of the vascular quiescence marker SPARCL1 on angiogenesis, capillary morphogenesis, and vessel integrity was evaluated.MethodsAngiogenesis was studied using the metatarsal test, an ex vivo model of sprouting angiogenesis. In addition, acute and chronic dextran sodium sulfate colitis models with SPARCL1 knockout mice were applied.ResultsThis approach indicated that SPARCL1 inhibits angiogenesis and supports vessel morphogenesis and integrity. Evidence was provided that SPARCL1-mediated stabilization of vessel integrity counteracts vessel permeability and inflammation in acute and chronic dextran sodium sulfate colitis models. Structure-function analyses of purified SPARCL1 identified the acidic domain of the protein necessary for its anti-angiogenic activity.ConclusionsOur findings inaugurate SPARCL1 as a blood vessel–derived anti-angiogenic molecule required for vessel morphogenesis and integrity. SPARCL1 opens new perspectives as a vascular marker of susceptibility to colitis and as a therapeutic molecule to support blood vessel stability in this disease.

A Novel OGR1 (GPR68) Inhibitor Attenuates Inflammation in Murine Models of Colitis

Background and Aims: Local extracellular acidification is associated with several conditions, such as ischemia, cancer, metabolic disease, respiratory diseases, and inflammatory bowel disease (IBD). Several recent studies reported a link between IBD and a family of pH-sensing G protein-coupled receptors. Our previous studies point to an essential role for OGR1 (GPR68) in the modulation of intestinal inflammation and fibrosis. In the current study, we evaluated the effects of a novel OGR1 inhibitor in murine models of colitis. Methods: The effects of a novel small-molecule OGR1 inhibitor were assessed in the acute and chronic dextran sulfate sodium (DSS) murine models of colitis. Macroscopic disease indicators of intestinal inflammation were evaluated, and epithelial damage and immune cell infiltration and proliferation were assessed by immunohistochemistry. Results: The OGR1 inhibitor ameliorated clinical parameters in acute and chronic DSS-induced colitis. In mice treated with the OGR1 inhibitor, endoscopy showed no thickening and normal vascularity, while fibrin was not detected. Histopathological findings revealed a decrease in severity of colonic inflammation in the OGR1 inhibitor group when compared to vehicle-DSS controls. In OGR1 inhibitor-treated mice, staining for the macrophage marker F4/80 and cellular proliferation marker Ki-67 revealed a reduction of infiltrating macrophages and slightly enhanced cell proliferation, respectively. This was accompanied by a reduction in pro-inflammatory cytokines, TNF and IL-6, and the fibrosis marker TGF-β1. Conclusion: This is the first report providing evidence that a pharmacological inhibition of OGR1 has a therapeutic effect in murine colitis models. Our data suggest that targeting proton-sensing OGR1 using specific small-molecule inhibitors may be a novel therapeutic approach for the treatment of IBD.

The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease.

(1) Background: Hypoxia is a common feature of inflammation when hypoxia inducible factors (HIFs) adapt cells to conditions of low oxygen tension and inflammation. We studied the role of HIF-1 and HIF-2 in cells of the myeloid lineage in a mouse model of acute colitis. (2) Methods: Mice with and without a conditional knockout for either Hif-1a or Hif-2a or Hif-1a and Hif-2a in cells of the myeloid lineage were treated with 2.5% dextran sodium sulfate (DSS) for 6 days to induce an acute colitis. We analyzed the course of inflammation with respect to macroscopic (disease activity index) and microscopic (histology score and immunohistochemical staining of immune cells) parameters and quantified the mRNA expression of cytokines and chemokines in the colon and the mesenteric lymph nodes. (3) Results: A conditional knockout of myeloid Hif-1a ameliorated whereas the knockout of Hif-2a aggravated murine DSS colitis by increased recruitment of neutrophils to deeper layers of the colon. This led to higher expression of Il6, Ifng, Cd11c, Cd4, and Cd8 in the colon but also induced anti-inflammatory mediators such as Foxp3 and Il10. A conditional knockout of Hif-1a and Hif-2a did not show any differences compared to wildtype mice. (4) Conclusions: Myeloid HIF-1α and HIF-2α play opposing roles in acute DSS colitis. Thus, not only a cell type specific, but also the isoform specific modulation of HIFs needs to be addressed in attempts to modify HIF for therapeutic purposes.