Inflamed Intestinal Tissue Research Articles

Polyclonal-to-monoclonal transition in colorectal precancerous evolution.

Unravelling the origin and evolution of precancerous lesions is crucial for effectively preventing malignant transformation, yet our current knowledge remains limited1-3. Here we used a base editor-enabled DNA barcoding system4 to comprehensively map single-cell phylogenies in mouse models of intestinal tumorigenesis induced by inflammation or loss of the Apc gene. Through quantitative analysis of high-resolution phylogenies including 260,922 single cells from normal, inflamed and neoplastic intestinal tissues, we identified tens of independent celllineages undergoing parallel clonal expansions within each lesion. We also found polyclonal origins of human sporadic colorectal polyps through bulk whole-exome sequencing and single-gland whole-genome sequencing. Genomic and clinical data support a model of polyclonal-to-monoclonal transition, with monoclonal lesions representing a more advanced stage. Single-cell RNA sequencing revealed extensive intercellular interactions in early polyclonal lesions, but there was significant loss of interactions during monoclonal transition. Therefore, our data suggest that colorectal precancer is often founded by many different lineages and highlight their cooperative interactions in the earliest stages of cancer formation. These findings provide insights into opportunities for earlier intervention in colorectal cancer.

Revitalizing gut barrier integrity: role of miR-192-5p in enhancing autophagy via Rictor in enteritis.

Intestinal inflammation and compromised barrier function are critical factors in the pathogenesis of gastrointestinal disorders. This study aimed to investigate the role of miR-192-5p in modulating intestinal epithelial barrier (IEB) integrity and its association with autophagy. A DSS-induced colitis model was used to assess the effects of miR-192-5p on intestinal inflammation. In vitro experiments involved cell culture and transient transfection techniques. Various assays, including dual-luciferase reporter gene assays, quantitative real-time PCR, Western blotting, and measurements of transepithelial electrical resistance, were performed to evaluate changes in miR-192-5p expression, Rictor levels, and autophagy flux. Immunofluorescence staining, H&E staining, TEER measurements, and FITC-dextran analysis were also used. Our findings revealed a reduced expression of miR-192-5p in inflamed intestinal tissues, correlating with impaired IEB function. Overexpression of miR-192-5p alleviated TNF-induced IEB dysfunction by targeting Rictor, resulting in enhanced autophagy flux in enterocytes (ECs). Moreover, the therapeutic potential of miR-192-5p was substantiated in colitis mice, wherein increased miR-192-5p expression ameliorated intestinal inflammatory injury by enhancing autophagy flux in ECs through the modulation of Rictor. Our study highlights the therapeutic potential of miR-192-5p in enteritis by demonstrating its role in regulating autophagy and preserving IEB function. Targeting the miR-192-5p/Rictor axis is a promising approach for mitigating gut inflammatory injury and improving barrier integrity in patients with enteritis.NEW & NOTEWORTHY We uncover the pivotal role of miR-192-5p in fortifying intestinal barriers amidst inflammation. Reduced miR-192-5p levels correlated with compromised gut integrity during inflammation. Notably, boosting miR-192-5p reversed gut damage by enhancing autophagy via suppressing Rictor, offering a potential therapeutic strategy for fortifying the intestinal barrier and alleviating inflammation in patients with enteritis.

Self-crosslinking hyaluronic acid hydrogel as an enteroprotective agent for the treatment of inflammatory bowel disease

The pathological changes in inflammatory bowel disease (IBD) include the disruption of intestinal barrier function and the infiltration of pathogenic microbes. The application of an artificial protective barrier at the site of inflammation can prevent bacterial infiltration, promote epithelial cell migration, and accelerate wound healing. In this study, dopamine-modified hyaluronic acid (HA-DA) was developed as a bioadhesive self-cross-linkable hydrogel, which acted as an enteroprotective agent to promote the healing of inflamed intestinal tissue. The adhesion strength HA-DA to mouse colon was 3.81-fold higher than HA. Moreover, HA-DA promoted Caco-2 cell proliferation and migration as well as had a strong physical barrier effect after gelation. After oral administration, the HA-DA reduced weight loss and attenuated impaired goblet cell function in mice with dextran sodium sulfate-induced IBD. In addition, HA-DA promoted restoration of the epithelial barrier by the upregulation of tight junction proteins. The results reported herein substantiated that self-cross-linkable hydrogel-based enteroprotective agents are a promising approach for the treatment of IBD.

Dynamic changes in extracellular vesicle-associated miRNAs elicited by ultrasound in inflammatory bowel disease patients

Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to molecular shifts of EV contents, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the inflammation-specific molecular shifts in EVs like altered EV-miRNA expression, which in turn can be detected in the peripheral blood. 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 min after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing. Candidate inducible EV-miRNAs were functionally assessed in vitro by transfection of miRNA mimics and qPCR of predicted target genes. Serum EV-miRNA concentration at baseline correlated with disease severity, as determined by clinical activity scores and sonographic findings. Three miRNAs (miR-942-5p, mir-5588, mir-3195) were significantly induced by sonography. Among the significantly regulated EV-miRNAs, miR-942-5p was strongly induced in higher grade intestinal inflammation and correlated with clinical activity in Crohn’s disease. Prediction of target regulation and transfection of miRNA mimics inferred a role of this EV-miRNA in regulating barrier function in inflammation. Induction of mir-5588 and mir-3195 did not correlate with inflammation grade. This proof-of-concept trial highlights the principle of induced molecular shifts in EVs from inflamed tissue through transabdominal ultrasound. These inducible EVs and their molecular cargo like miRNA could become novel biomarkers for intestinal inflammation in IBD.

Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Effective enhancement of the immunomodulatory capacity of canine adipose-derived mesenchymal stromal cells on colitis by priming with colon tissue from mice with colitis.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) in inflammatory bowel disease is not completely known and is not consistent. Priming with inflammatory cytokines has been proposed to adapt MSCs to an inflammatory environment to have them ready to counteract it, but may have undesirable effects on MSCs, such as increased immunogenicity. In this study, we hypothesized that priming MSCs with inflamed intestinal tissue would more effectively enhance their therapeutic effect on intestinal inflammation. The capacity of canine adipose-derived MSCs (cADSCs) primed with colon tissue homogenates from mice with experimentally induced colitis or a combination of tumor necrosis factor-α and interferon-γ to inhibit T-cell proliferation was analyzed, along with their own apoptosis, proliferation, cell surface marker expression, and transcriptome. In addition, colitis mice were treated with the primed cADSCs to assess colitis severity and immune cell profile. Priming with cytokines induced apoptosis, decreased cell proliferation, and major histocompatibility complex-II gene expression in cADSCs, but these adverse effects were mild or absent with colitis-tissue priming. cADSCs primed with colitis tissue reduced the severity of colitis via the induction of M2 macrophages and T-regulatory cells and suppression of T-helper (Th)1/Th17-cell responses, and their effects were comparable to those of cytokine-primed cells. Our results emphasize the importance of the activation of MSCs by the appropriate microenvironment to maximize their therapeutic effect.

Emerging Self‐Assembled Nanoparticles Constructed from Natural Polyphenols for Intestinal Diseases

Intestinal diseases like inflammatory bowel disease (IBD) and colorectal cancer originate from inflammation and disruption of mucosal barriers. Polyphenols can mitigate intestinal inflammation through antioxidant, anti‐inflammatory, and microbiome modulation effects. However, the poor solubility and stability of polyphenols restrict therapeutic delivery. Self‐assembly provides a nanoscale platform to overcome these limitations. Polyphenol‐based nanoparticles (PNPs) are formed via coordination of polyphenols with metals like iron, copper, and zinc based on the catechol/galloyl groups. Templeted assembly with amphiphilic block copolymers can also direct polyphenol self‐assembly into nanostructures. PNPs prepared by these mild, aqueous methods exhibit enhanced stability, pH‐responsive disassembly, high cargo‐loading capacity, and targeted accumulation in inflamed intestinal tissues. PNPs can load with hydrophobic polyphenols, drugs, genes, proteins, or probiotics and demonstrate therapeutic potential in preclinical IBD, colorectal cancer, and microbiome disorder models. Ongoing challenges include augmenting prebiotic effects, multidrug encapsulation, and engineering PNPs as biotherapeutics. Future directions involve tailored polyphenol–polymer covalent assemblies and investigating PNPs interactions with enterocytes, immune cells, and microbiota. Overall, PNPs prepared by facile self‐assembly combine the bioactivities of polyphenols with advanced delivery functionality, presenting new opportunities for combination and microbiota‐based therapies for complex intestinal diseases.

Oral liposomal delivery of an activatable budesonide prodrug reduces colitis in experimental mice

Inflammatory bowel disease (IBD) is one of the most common intestinal disorders, with increasing global incidence and prevalence. Numerous therapeutic drugs are available but require intravenous administration and are associated with high toxicity and insufficient patient compliance. Here, an oral liposome that entraps the activatable corticosteroid anti-inflammatory budesonide was developed for efficacious and safe IBD therapy. The prodrug was produced via the ligation of budesonide with linoleic acid linked by a hydrolytic ester bond, which was further constrained into lipid constituents to form colloidal stable nanoliposomes (termed budsomes). Chemical modification with linoleic acid augmented the compatibility and miscibility of the resulting prodrug in lipid bilayers to provide protection from the harsh environment of the gastrointestinal tract, while liposomal nanoformulation enables preferential accumulation to inflamed vasculature. Hence, when delivered orally, budsomes exhibited high stability with low drug release in the stomach in the presence of ultra-acidic pH but released active budesonide after accumulation in inflamed intestinal tissues. Notably, oral administration of budsomes demonstrated favorable anti-colitis effect with only ∼7% mouse body weight loss, whereas at least ∼16% weight loss was observed in other treatment groups. Overall, budsomes exhibited higher therapeutic efficiency than free budesonide treatment and potently induced remission of acute colitis without any adverse side effects. These data suggest a new and reliable approach for improving the efficacy of budesonide. Our in vivo preclinical data demonstrate the safety and increased efficacy of the budsome platform for IBD treatment, further supporting clinical evaluation of this orally efficacious budesonide therapeutic.

P655 MadCAM1 Negativity of Lamina Propria Endothelial Cells is Associated with Non-Response to Vedolizumab in Inflammatory Bowel Disease Patients

Abstract Background Vedolizumab (VDZ), a monoclonal antibody against α4β7 integrin has been shown to be effective in inducing and maintaining remission in inflammatory bowel disease (IBD). By blocking α4β7, it is preventing the homing of lymphocytes through binding to mucosal vascular adressin cell adhesion molecule 1 (MadCAM1) localised on the intestinal endothelial cells. We have previously reported that MadCAM1 is expressed to various extent on lamina propria endothelial cells of IBD patients and that this expression is stable over time. The aim of the present study was to analyse whether expression of MadCAM1 was related to the response to VDZ in IBD patients. Methods All IBD patients treated with VDZ in one referral IBD centre were included. The biopsies or resection specimen from the inflamed intestinal tissue were stained by immunohistochemistry for MadCAM1 expression. Clinical response to vedolizumab was assessed in patients with the minimal treatment duration of 10 weeks and the response rate between patients with positive and negative MadCAM1 were analyzed by Fisher`s exact test. Results In total, 92 IBD patients were treated with VDZ; for 83 of them intestinal biopsies or surgical specimen were available for assessment of MadCAM1 expression. In four patients (5%) no expression of MadCAM1 was detected, remaining 79 patients expressed MadCAM1 to various extent. Overal, 30 patients (36%) were non-responders to VDZ treatment. With regards to MadCAM1 expression, 67% of patients expressing MadCAM1 responded to VDZ (53 patients) while none of the patients negative for MadCAM1 responded to treatment (p=0.0171). Conclusion Lack of MadCAM1 expression in intestinal endothelium is associated with clinical non-response to vedolizumab.

Inhibition of Soluble Stem Cell Factor Promotes Intestinal Mucosal Repair.

Incidences of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are escalating worldwide and can be considered a global public health problem. Given that the gold standard approach to IBD therapeutics focuses on reducing the severity of symptoms, there is an urgent unmet need to develop alternative therapies that halt not only inflammatory processes but also promote mucosal repair. Previous studies have identified increased stem cell factor (SCF) expression in inflamed intestinal mucosal tissues. However, the role that SCF plays in mediating intestinal inflammation and repair has not been explored. Changes in the expression of SCF were evaluated in the colonic tissue of healthy mice and during dextran sodium sulfate (DSS)-induced colitis. Furthermore, mucosal wound healing and colitis severity were analyzed in mice subjected to either mechanical biopsy or DSS treatment, respectively, following intestinal epithelial cell-specific deletion of SCF or anti-SCF antibody administration. We report robust expression of SCF by intestinal epithelial cells during intestinal homeostasis with a switch to immune cell-produced SCF during colitis. Data from mice with intestinal epithelial cell-specific deletion of SCF highlight the importance of immune cell-produced SCF in driving the pathogenesis of colitis. Importantly, antibody-mediated neutralization of total SCF or the specific SCF248 isoform decreased immune cell infiltration and enhanced mucosal wound repair following biopsy-induced colonic injury or DSS-induced colitis. These data demonstrate that SCF functions as a pro-inflammatory mediator in mucosal tissues and that specific neutralization of SCF248 could be a viable therapeutic option to reduce intestinal inflammation and promote mucosal wound repair in individuals with IBD.

Inflammation-targeting polyamine nanomedicines for the treatment of ulcerative colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by immune system dysfunction. Despite the availability of various anti-inflammatory drugs, they exhibit low therapeutic efficacy with systemic side effects. In this study, we developed oral anti-inflammatory polyamine-based nanomedicines for the treatment of ulcerative colitis. Polyamine-bearing nanoparticles were prepared by the self-assembly of hyaluronic acid in organic solvents and crosslinking with branched oligoethyleneimine. Polyamine nanoparticles were found to suppress excessive inflammatory responses by scavenging the reactive oxygen species (ROS). Moreover, these nanoparticles inhibited enzymatic degradation and targeting of inflamed intestinal tissues. Additionally, they suppressed the inflammatory responses and recovered the pathological disorders in the colon of an ulcerative colitis mouse model. Therefore, polyamine-based nanomedicines exhibit great potential as biocompatible ROS-scavenging drugs for the treatment of IBD.

Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces.

Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces1-4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses5-7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines8,9 and the release of tissue-protective amphiregulin (AREG)10-12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions13-15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.

NF-κB-coupled IL17 mediates inflammatory signaling and intestinal inflammation in Artemia sinica

Nuclear factor-κB (NF-κB) plays a role as a rheostatic transcription factor in regulating intestinal inflammation, and its disruption or constitutive activation leads to inflammation and injury. However, the molecular mechanisms of NF-κB regulation remain largely unknown. In this study, the NF-κB-regulated host defenses against pathogen infections and facilitation of IL17 expression during stimulation with different bacteria were investigated. Intestinal inflammation was induced by dextran sulfate sodium, and NF-κB activity was inhibited in an intestinal injury model. Mannose receptor C type, ABF1/2, serpin B13, lysozyme, and β-arrestin were significantly controlled by NF-κB in the inflamed intestinal tissue. High levels of NF-κB activation resulted in less pervasive intestinal damage and the maintenance of intestinal barrier integrity. Intestinal injury robustly increased the expression of IL17. NF-κB activation was enhanced by IL17 deficiency in the intestinal injury model. IL17 inhibition aggravated intestinal inflammation, leading to loss of epithelial architecture and the infiltration of inflammatory cells. These data suggest that NF-κB and IL17 play key mediator roles in the maintenance of gut epithelial integrity and immune homeostasis.

Interleukin 21‐induced Mitochondrial Dysfunction Drives Regulatory T Cell Inflammatory Response during Intestinal Inflammation

BackgroundSignificant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL‐21), which is known to support T helper (Th) cell function, is highly expressed within inflamed intestinal tissues of IBD patients compared to healthy controls. In addition, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD. Given that healthy Tregs are critical for self‐tolerance and prevention of IBD, we investigated the metabolic role of IL‐21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolism pathways during IBD pathogenesis.MethodsHuman Tregs as well as relevant control effector Th cells were generated from naïve CD4+ T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted metabolic transcriptional profiling. Immune phenotyping was assessed by fluorescence‐activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry‐based metabolomics. Ultrastructural analysis of mitochondria was performed by confocal and transmission electron microscopy. Intestinal inflammation was induced in Rag1‐/‐ (T and B cell deficient) mice by the adoptive transfer of pathogenic naïve CD4+ T cells.ResultsAcute IL‐21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed an IL‐21‐mediated increase in the expression of genes associated with glycolysis and pathways known to support anabolic and OXPHOS metabolism, thus resembling a hypermetabolic state. Furthermore, IL‐21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of effector Th cell‐associated cytokines such as interferon γ, tumor necrosis factor, IL‐17A, and IL‐17F. Exploring the mechanisms underlying IL‐21‐induced effects, we found significant disruption of mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent pyruvate entry into the mitochondria. IL‐21‐induced GSK3β activation was accompanied by a marked increase in intracellular and extracellular metabolites such as pyruvate and lactate, as assessed by metabolomics. Importantly, GSK3 inhibition or supplementation with mitochondrial membrane‐permeable methyl pyruvate broadly abrogated metabolic wiring of and inflammatory responses by IL‐21‐stimulated Tregs and effector Th cells. Collectively, these results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4+ T cells. Lastly, GSK3 inhibition prevented pathogenic CD4+ T cell‐induced colitis in mice as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines.ConclusionsIL‐21 potently engages human Tregs in a hypermetabolic state that augments inflammatory cytokine production via induction of mitochondrial dysfunction. Therefore desensitizing CD4+ T cells to detrimental cues, such as IL‐21, may also augment Treg function during human IBD.

Oral administration of inflammatory microenvironment-responsive carrier-free infliximab nanocomplex for the targeted treatment of inflammatory bowel disease

Although the strategy for administering antibodies orally for inflammatory bowel disease (IBD) treatment has been proposed, the rational design for efficient delivery system is still a challenge, restricted by poor drug loading and nonspecific distribution. In this study, we developed an inflammatory microenvironment-responsive nanocomplex (IFXSS@HA or IFXTK@HA) for oral administration of Infliximab (IFX) for IBD treatment using self-cross-linking technology, which was developed by reactive oxygen species–responsive cross-linkers and fabricated with hyaluronic acid. The nanocomplex is a “carrier-free” antibody backpack with a high drug loading (>90%), inflammation targeting, and bioresponsive controlled release. The results of preliminary in vivo studies indicated that the nanocomplex showed an extended retention time and caused an increased antibody accumulation in the inflamed intestinal tissues based on hyaluronic acid functionalization and responsive controlled release within the inflammation site, exhibiting great efficacy by reducing intestinal inflammation and systemic exposure. This study provided a rational design for an oral delivery system of antibodies, explored the design of the combination of vehicle and functionalization, and showed a strategy for the oral administration of antibodies in IBD treatment.

PLA2-Triggered Activation of Cyclosporine-Phospholipid Prodrug as a Drug Targeting Approach in Inflammatory Bowel Disease Therapy.

Oral medication with activity specifically at the inflamed sites throughout the gastrointestinal tract and limited systemic exposure would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). For this purpose, we have designed a prodrug by linking active drug moiety to phospholipid (PL), the substrate of phospholipase A2 (PLA2). PLA2 expression and activity is significantly elevated in the inflamed intestinal tissues of IBD patients. Since PLA2 enzyme specifically hydrolyses the sn-2 bond within PLs, in our PL-based prodrug approach, the sn-2 positioned FA is replaced with cyclosporine, so that PLA2 may be exploited as the prodrug-activating enzyme, releasing the free drug from the PL-complex. Owing to the enzyme overexpression, this may effectively target free cyclosporine to the sites of inflammation. Four PL-cyclosporine prodrugs were synthesized, differing by their linker length between the PL and the drug moiety. To study the prodrug activation, a novel enzymatically enriched model was developed, the colonic brush border membrane vesicles (cBBMVs); in this model, tissue vesicles were produced from colitis-induced (vs. healthy) rat colons. PLA2 overexpression (3.4-fold) was demonstrated in diseased vs. healthy cBBMVs. Indeed, while healthy cBBMVs induced only marginal activation, substantial prodrug activation was evident by colitis-derived cBBMVs. Together with the PLA2 overexpression, these data validate our drug targeting strategy. In the diseased cBBMVs, quick and complete activation of the entire dose was obtained for the 12-carbon linker prodrug, while slow and marginal activation was obtained for the 6/8-carbon linkers. The potential to target the actual sites of inflammation and treat any localizations throughout the GIT, together with the extended therapeutic index, makes this orally delivered prodrug approach an exciting new therapeutic strategy for IBD treatment.

Prodrug-Based Targeting Approach for Inflammatory Bowel Diseases Therapy: Mechanistic Study of Phospholipid-Linker-Cyclosporine PLA2-Mediated Activation.

Therapeutics with activity specifically at the inflamed sites throughout the gastrointestinal tract (GIT) would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). We aimed to develop the prodrug approach that can allow such site-specific drug delivery. Currently, using cyclosporine as a drug of choice in IBD is limited to the most severe cases due to substantial systemic toxicities and narrow therapeutic index of this drug. Previously, we synthesized a series of a phospholipid-linker-cyclosporine (PLC) prodrugs designed to exploit the overexpression of phospholipase A2 (PLA2) in the inflamed intestinal tissues, as the prodrug-activating enzyme. Nevertheless, the extent and rate of prodrug activation differed significantly. In this study we applied in-vitro and modern in-silico tools based on molecular dynamics (MD) simulation, to gain insight into the dynamics and mechanisms of the PLC prodrug activation. We aimed to elucidate the reason for the significant activation change between different linker lengths in our prodrug design. Our work reveals that the PLC conjugate with the 12-carbon linker length yields the optimal prodrug activation by PLA2 in comparison to shorter linker length (6-carbons). This optimized length efficiently allows cyclosporine to be released from the prodrug to the active pocket of PLA2. This newly developed mechanistic approach, presented in this study, can be applied for future prodrug optimization to accomplish optimal prodrug activation and drug targeting in various conditions that include overexpression of PLA2.

P090 Unraveling the rewiring of cytokine signalling in Inflammatory Bowel Disease using a novel network biology approach

Abstract Background Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of chronic immune-mediated disorders such as inflammatory bowel disease (IBD). By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available resources and studies modelling cytokine-cytokine interactions that would be important for better understanding the dysregulation of cytokine signalling in IBD, and revealing potential new therapeutic targets for patients. Methods Based on the workflow of a cytokine signalling resource, CytokineLink, we developed previously, in this effort we built novel interaction networks using single-cell expression data from patients with ulcerative colitis (UC) and Crohn’s disease (CD). We defined two meta-networks for each disease: a cell–cell communication network connected by cytokines; and a cytokine–cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Results The rewiring of cell–cell and cytokine–cytokine interaction networks between healthy, non-inflamed and inflamed intestinal tissues in IBD was measured using the generated meta-networks. Cytokine-cytokine interaction networks revealed that in active UC, the Th1 cytokine IFNg and the chemokines CCL28 and CCL14 were central to cytokine-mediated communication networks. In CD, IL12B and IL13 formed the central cytokines in inflamed ileal tissues. Cell–cell interaction networks identified likely main cell types mediating the signalling of these key cytokines in non-inflamed and inflamed intestinal tissues: mast cells being the main source of IL13 in both IBD patients and healthy individuals. Conclusion We generated interaction networks aimed at capturing the most prevalent cell–cell and cytokine–cytokine interactions using disease specific single-cell expression data. Using the generated interaction networks we identified the condition-specific rewiring of cytokine mediated signalling in IBD, and key cytokines that could be targeted for ameliorating inflammatory responses. The data can be used by the community as a platform for hypothesis-generation. Potential use cases are deconvoluting the complexities of cytokine signalling in IBD to reveal potential cytokine drug targets, or predicting the downstream effects of drugs on cytokine responses.

P034 DISRUPTive liquid biopsies in Inflammatory Bowel Disease (DISRUPT-IBD): Release of exosomal and molecular tissue signals by transabdominal ultrasound?

Abstract Background A major drawback in diagnosis and monitoring of IBD are insufficient biomarkers that do not correlate well with tissue disease activity. Inflammation and cellular stress lead to an increased release of exosomes (Lipinski et al, 2019, EMBO J) carrying inflammatory signal proteins as cargo (Wozniak et al, 2020, J Cell Biol). Interestingly, colorectal cancer cells strongly release tumor markers after stimulation by sonography waves, probably through the disruptive energy of standard ultrasound (D’Souza et al, 2009, PNAS). We thus hypothesize that application of ultrasound waves on inflamed intestinal tissue in active IBD induces the release of tissue-specific exosomes that can be detected in the peripheral blood and used as novel biomarkers. Methods 26 patients with different degrees of disease activity and various ongoing therapies with IBD were included in a pilot trial. Serum was taken before and 30 minutes after diagnostic transabdominal ultrasound. Serum exosomes were precipitated (using ExoQuick) and exosomal (exo-)miRNA were isolated for miRNA sequencing using SeraMir protocol. Exosomal proteins were isolated for quantification of inflammatory markers (as exosomal cargo). Results We found a correlation between exo-miRNA concentration and disease severity, determined by clinical assessment scores and sonographic findings. Exo-miRNA sequencing revealed a select few miRNAs to be induced by sonography. However, the overall composition of annotated miRNA transcripts did not separate samples according to sampling timepoints. Among these “inducible” exo-miRNA, we found hsa-miR-942, which is involved in cell cycle and Wnt signaling in intestinal epithelial cells, to be significantly induced in higher grade intestinal inflammation. The exosomal protein concentrations did not correlate with disease severity at baseline, but were increased after sonography in severe intestinal inflammation. We did not find markers of the inflammasome apparatus as inducible exosomal cargos. Conclusion Sonography-induced exosomal markers, e.g., exo-miRNAs, could become novel biomarkers for intestinal inflammation in IBD. Further exploration of the functional role of inducible biomarkers and continued investigation of exosomal miRNA and protein profiling will be performed.

P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis

Abstract Background Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown. Methods Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry. Results The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105). Conclusion Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.