bluuebunny/crossref_metadata_2025 huggingface dataset:
P008 Machine learning derived histological features of epithelial injury and repair in Ulcerative Colitis biopsies correlate with disease severity
Authors: Griffin M , Zhang Y , Shah C , Shamshoian J , Mountain V , Sucipto K , Jayson C , Najdawi F
Published: January 2024

Background

Ulcerative colitis (UC) is an incurable chronic and debilitating disease. Frequent relapse is common despite available therapeutics. Endoscopically and histologically confirmed mucosal healing is the gold standard for long term remission, but a better understanding of disease pathology is needed for all patients to achieve inflammation reduction plus epithelial repair and wound healings. Here, we describe machine learning (ML) models that exhaustively quantify the inflammatory and epithelial histologic morphology of the colonic mucosa. Model-derived features are associated with Geboes Score (GS).

Methods

ML models (IBDExploreTM, PathAI, Boston) previously trained to quantify tissue features (e.g. epithelium) and cells (e.g. lymphocytes) were deployed on 447 whole slide images (WSI) of hematoxylin and eosin (H&E)-stained UC colonic mucosa from clinical and commercial sources.

Model predictions yielded >900 quantitative human interpretable features (HIFs). To identify histological features of epithelial injury and repair, further analyses focused on 212 relevant HIFs that describe the epithelial layer that were divided into 10 Feature Categories (FCs, Table 1) based on whether they describe loss of integrity, surface or crypt injury and repair.

Slide-level GS (Grade Level and Subgrade 3) were assigned to each WSI based on a median consensus scores of three expert gastrointestinal pathologists. Absolute Spearman rank correlations between individual HIFs and GS were calculated and associations between HIFs and GS were assessed at the FC level.

Results

HIFs were assessed at the FC level to identify broad trends associated with GS. Goblet cell HIFs in all epithelium and in crypt epithelium had the strongest associations, and were negatively correlated with Grade Level GS; positive correlations were found with enterocytes and loss of epithelial integrity HIFs (Figure 1A). Subgrade 3 GS was most strongly, and positively, correlated with epithelial injury and repair in the crypts, and negatively correlated with goblet cells in crypt epithelium and crypt epithelium goblet cell mucin (Figure 1B). Within FCs, the HIF driving the correlation trend was identified (Table 1). Model overlay outputs allow direct visualisation of histological features (Figure 1 C,D) illustrating disparities in the composition of GS0 and GS5 epithelium, and this is supported by the computed HIF values from each WSI.

Conclusion

Quantitative HIFs describing mucosa epithelial injury and repair strongly correlate with GS. Our models produce a granular, measurable blueprint of the epithelium that can be leveraged to inform hypotheses for therapeutic target selection, mechanism of action, and disease progression and remission.
P026 Differential effects of tofacitinib on macrophage activation may contribute to lack of response in ulcerative colitis patients
Authors: Melón-Ardanaz E , Veny M , Corraliza A M , Garrido-Trigo A , Gudiño V , Moro M , Sanzo-Machuca Á , Esteller M , Masamunt M C , Caballol B , Ordás I , Fernández-Clotet A , Ricart E , Panés J , Salas A
Published: January 2024

Background

We previously showed that ulcerative colitis (UC) patients refractory to tofacitinib present an increase in the activation and the pro-inflammatory profile of intestinal macrophages1. However, the mechanism by which tofacitinib promotes further activation of macrophages in these group of patients remains unknown.

Methods

Blood monocytes from healthy individuals were cultured in the presence of M-CSF as previously described2. The differentiated macrophages were treated with LPS (10ng/ml), TNF (20ng/ml) or IFNγ (5ng/ml) in the presence or absence of tofacitinib (300nM). The effect of tofacitinib on gene expression and protein production in response to each stimulus was measured by qPCR and ELISA, respectively. Single-cell RNAseq (scRNAseq) data generated in a cohort of UC patients treated with tofacitinib1 was compared to the bulk transcriptome of monocyte-derived macrophages stimulated with LPS +/- anti-IL-10 mAb3.

Results

Monocyte-derived macrophages treated with tofacitinib significantly reduced the expression of activation markers downstream from IFNγ or TNFα. In contrast, the response to LPS was either maintained or intensified by tofacitinib as seen by the increase in expression of inflammatory marker genes including chemokines and cytokines CXCL1, CXCL5, IL1B, TNFA and IL6, and M1-like markers INHBA and CLEC5A (Figure 1A). Protein analysis of cell supernatants confirmed these observations. Compared to IFNγ and TNF, LPS drives the production of high concentrations of the anti-inflammatory IL-10 (Figure 1B) which can act on macrophages through a JAK-dependent signaling pathway. We hypothesized that by inhibiting IL-10 autocrine signaling on LPS-activated macrophages tofacitinib would promote their hyperactivation. Indeed, the transcriptional profile of LPS-stimulated macrophages treated with a blocking anti-IL-10 antibody3 highly overlapped with those genes that were upregulated in macrophages from patients refractory to tofacitinib, including IL1B, IL6, INHBA and IDO1 (Figure 1C). This overlap was not observed in responder patients.

Conclusion

Based on the combination of biopsy scRNAseq from tofacitinib-treated patients and in vitro macrophage cultures, we suggest that lack of response to tofacitinib is driven by its effect on IL-10 signaling. While IL-10 is essential to control macrophage activation when stimulated with the pathogen molecular pattern LPS, it plays no significant role when macrophages are activated with IFNγ or TNF. Hence, we hypothesize that LPS-dependent inflammation may predispose to lack of response to tofacitinib.
P037 Modelling gut complexity and disease: development of a next-generation adult stem cell based tubular gut-on-a-chip model
Authors: Kosim K , Bokkers M , Oudshoorn J , van Heusden T , Morelli M , Kurek D , van den Broek L , Joore J , Vulto P
Published: January 2024

Background

Developing effective therapies for Inflammatory Bowel Disease (IBD) necessitates advanced in vitro models that faithfully replicate the intricacies of the gut. The current challenge is to create models that encompass the 3D morphology, heterogeneity, and boundary characteristics of intestinal tissue while also being amenable to high-throughput screening. Microfluidic techniques are emerging as vital tools to introduce physiologically relevant cues into conventional cell cultures.

Methods

We present an innovative in vitro model employing human adult stem cell-derived colon organoids cultivated as perfused epithelial tubules within the MIMETAS OrganoPlate®. This microfluidic platform facilitates the simultaneous culture of up to 64 independent gut tubules on an extracellular matrix, offering continuous media perfusion without artificial membrane interference. The barrier function of the epithelial tubules is assessed in real-time through transepithelial electrical resistance (TEER) measurements. Immunostaining and 3D reconstruction of confocal images evaluate the expression of relevant epithelial markers.

Results

The tubular epithelial model of the intestinal tract demonstrated rapid cell polarization, expression of cell-specific markers, tight junction formation, and the presence of functional transporters. TEER measurements indicated functional and reproducible barrier integrity across tubules generated from different organoid donors for at least 10 days. Incorporation of additional cell types, such as patient-derived immune cells and endothelium, marks a groundbreaking advancement in understanding disease pathogenesis. The plug-and-play modularity of these models allows for flexibility in incorporating specific cell types based on the biological pathway of interest.

Conclusion

Our next-generation gut-on-a-chip model faithfully mimics the IBD phenotype and facilitates screening for potential drug targets. Combining adult human stem cell-derived intestinal organoids with microfluidic technology provides a robust platform to study physiology and disease mechanisms in patient-specific gut models. The inclusion of IBD patient-derived cells promises further refinement, enabling a more profound exploration of disease phenotypes and treatment responsiveness. Moreover, organoids, as one of the most physiologically relevant cell sources, authentically represent patient biology, enhancing the translational potential of our findings.
P042 Significant correlation between intestinal ultrasound and magnetic resonance enterography for assessing disease activity and complications in Crohn’s disease
Authors: Bárbara A , Manso M , Bravo C , Reves J , Nascimento C , Gomes C , Torres J , Palmela C
Published: January 2024

Background

The advantages of intestinal ultrasound (IUS) over magnetic resonance enterography (MRE) include wide availability, low cost, and the possibility of being point-of-care. We here aimed to compare the performance of IUS with MRE for assessing disease activity and complications in Crohn’s disease (CD).

Methods

Prospective longitudinal study of CD patients starting anti-TNF therapy. IUS and MRE were performed at baseline (W0) and after 1 year of infliximab therapy (W54). Bowel wall thickness (BWT) of the most affected segment (MAS) was measured in both exams (average of 4 measurements) and compared. Transmural response (TR) was defined as a reduction in 25% of BWT. All MRE were centrally reviewed by a blinded radiologist.

Results

We included 48 CD patients (48% male; median age 31 years, IQR 26-44). Ileum was the most commonly affected segment in 63% of the patients. At baseline the agreement between the two methods to select the most affected segment was moderate (k=0.581, p<0.001). At W54, 50% of patients had TR in IUS and 40% in MRE. After 1 year of therapy there was a significant decrease in median BWT in both methods (IUS: 4.88mm vs 3.22mm, MRE: 8.21mm vs 4.91mm, both p<0.001). We found a significant association between the two methods in detecting intestinal complications (stricturing or penetrating disease) on both timepoints (W0: 31.25% vs 21.73%; p<0.001; W54: 17.78% vs 20.83% ; p=0.004), and also a significant moderate to high correlation between BWT measured by IUS and MRE on both timepoints (W0:r=0.34, p=0.02; W54:r=0.58; p<0.001). The correlation was higher when the MAS was the ileum (W0 and W54: r=0.65, p<0.001).

Conclusion

We found a moderate to good agreement in the selection of MAS between IUS and MRE. There was also a moderate correlation between BWT measurements and detection of complications between the two methods, which increased when the MAS was the ileum. Our results suggest that IUS, a readily available and inexpensive tool, is accurate to evaluate disease activity and treatment response, particularly in patients with ileal disease.
P043 RIPK3 – a new marker for assessment of activity in Crohn’s disease
Authors: Panayotova E , Atanassova A
Published: January 2024

Background

RIPK3 (Receptor Interacting Protein Kinase-3) is a key molecule involved in the process of necroptosis. Necroptosis is a regulated form of cell death that involves the activation of specific signaling pathways leading to cell lysis. The released endogenous molecules - DAMPs (Danger Associated Molecular Patterns) activate the immune system and lead to inflammation. This relationship suggests a role for necroptosis in the pathogenesis of IBD (Inflammatory Bowel Disease).

Methods

The aim of the study was to evaluate RIPK3 expression in patients with Crohn's disease (CD) according to disease activity and some clinical parameters. Tissue expression of RIPK3 in the inflamed areas of 85 patients with Crohn's disease was determined immunohistochemically. The CDAI (Crohn’s Disease Activity Index) was used to assess activity, and the Montreal Classification to determine disease location and bahavior.

Results

A correlation was found between RIPK3 expression and CDAI (p=0.038), with patients in remission having the lowest expression (154.67), while patients with severe activity had overexpression of the marker (180.0) (Fig.1).

According to the localization of CD, a significant difference was found in the expression of RIPK3 (p<0.05), with the highest expression of the marker in the ileum (L1) and colon (L2) with involvement of the upper GIT (L4) – L1+L4 (225.50) and L2 + L4 (218.00). Isolated involvement of terminal ileum (L1) was characterized by the lowest expression of the marker, while patients with colonic localization (L2) had high expression of RIPK3 (184.87).

Regarding disease behavior, it was found that the highest expression of the marker is in the patients with phenotype B2+B3 – both stricturing and penetrating disease, followed by inflammatory disease (B1) (Fig.2). Perianal disease further increased RIPK3 expression, with patients with concurrent perianal disease showing high expression of the marker (181.85 vs. 167.86 for those without perianal disease; p<0.05).

Conclusion

High RIPK3 expression in CD patients correlated with severe disease activity as measured by CDAI, involvement of upper GIT - L1+L4 and L2+L4, stricturing with penetrating disease - B2+B3 and concurrent perianal disease.
P049 The lipidome of creeping fat in Crohn’s disease points towards a harmful microenvironment
Authors: Verstockt S , Dehairs J , Vanderhoydonc F , Ke B J , De Greef I , Sabino J , Ferrante M , Bislenghi G , D’Hoore A , Swinnen J , Verstockt B , Vermeire S
Published: January 2024

Background

A pathognomonic observation in Crohn’s disease (CD) is the existence of mesenteric adipose tissue (MAT) wrapping around the inflamed and fibrotic intestine, so-called creeping fat. The etiology of this phenomenon is unclear, and both a protective and harmful role in disease pathogenesis and progression have been proposed. Given this conflicting evidence, we aimed to further unravel the role of creeping fat by extensive profiling of its lipidome.

Methods

We prospectively included 22 CD patients who underwent an ileocecal resection with ileocolonic anastomosis (Table 1). Perioperatively paired adipose tissue samples were collected from 3 locations: (1) creeping fat, (2) MAT close to the proximal unaffected ileum, and (3) MAT far from the diseased ileum, defined as near the base of the ileocecal artery (Fig. 1A). The lipidome of all 66 samples was profiled by quantitative mass spectrometry (targeted C18 LC-MS/MS and HILIC LC-MS/MS; Lipometrix core KU Leuven). This approach enables the detection of mediator lipids, and membrane and storage lipids, of which the latter group represents more complex chemical structures and thus a higher diversity in classification and number of species. Data analysis and paired Wilcoxon rank- sum tests were performed with Python and R 4.3.2.

Results

Across all CD adipose tissue samples, we detected 38 mediator lipid species, and 456 membrane and storage lipid species . When paired-wise comparing creeping fat with MAT far from the diseased ileum, we observed a significant decrease in 16 lipid mediators (p<0.05), namely arachidonic acid (AA), docosahexaenoic acid (DHA), 4 epoxy and 10 hydroxy mediator lipids; with hydroxy mediator X being the top downregulated in creeping fat (fold change (FC)=-2.11, p=0.0003) (Fig. 1B). While AA and DHA are (semi)-essential fatty acids and function as precursors, the other 8 decreased lipids in creeping fat are known to be anti-inflammatory, and 3 (incl. mediator X) are also anti-fibrotic. The lipid mediator profile of MAT close to the proximal unaffected ileum was similar to that of creeping fat, except for a pro-inflammatory lipid being increased in creeping fat (FC=2.30, p=0.004) (Fig. 1B). Lastly, when evaluating the membrane and storage lipid abundances both at species level (sum notation; fatty acyl composition) and at class level, no paired-wise differences among the three adipose tissue locations were found (p>0.05).

Conclusion

In this pilot lipidomics study, we did not observe any changes in storage or membrane lipids in creeping fat as compared to paired unaffected MAT. In contrast, a decrease in anti-inflammatory and anti-fibrotic lipid species along with an increase in a pro-inflammatory species points towards a harmful microenvironment within creeping fat.
P057 Caudovirales may trigger molecular mimicry mechanisms in Crohn’s Disease
Authors: Errico C , Massimino L , Facoetti A , Nicolò S , Cagliani S , Spanò S , Parigi T L , Riva M , Danese S , Ungaro F
Published: January 2024

Background

The gut virome is known to play a role in the development of Crohn's disease (CD), a type of Inflammatory Bowel Disease (IBD). Several years ago, Norman and colleagues observed an expansion of Caudovirales linked to CD pathogenesis, alongside a reduction in bacterial diversity. To delve deeper, we exploited the IBD TaMMA framework and observed a significantly higher abundance of Caudovirales in the CD individuals compared to healthy controls, confirming earlier findings.

Methods

To further examine the involvement of this viral species in various mucosal cell compartments, we conducted flow cytometry cell sorting (FACS) on intestinal biopsies from CD and healthy individuals. Isolated immune and non-immune cells based on population-specific markers, including CD8 and CD4 T cells, B cells, macrophages, dendritic cells, epithelium, endothelium, and fibroblasts underwent transcriptomics and metatranscriptomics.

Results

We identified higher abundance of the Proteus virus Isfahan, belonging to Caudovirales viral order, in CD dendritic cells and CD macrophages compared to their healthy counterparts. Notably, gene ontology (GO) analysis indicated that only CD dendritic cells, fibroblasts, and epithelial cells exhibited compromised biological responses to viral insults compared to their healthy counterparts. In contrast, CD macrophages displayed upregulation of molecular pathways regulating viral processes. This suggests that virome dysbiosis in these cells may contribute to CD pathogenesis by altering their biological functions. We thus wondered whether Proteus virus Isfahan encoded proteins had similarities with the human ones, somehow establishing molecular mimicry justifying the likelihood of the autoimmune nature of CD. We observed sequence homology in the Proteus virus Isfahan and the human thymidylate synthase, RECQ-like DNA helicase, and dCMP deaminase. These viral proteins are likely to be recognized by the immune system by sharing similarities with the human homologs, thus activating autoreactive immune response and establishing the persistent chronic inflammatory environment within CD mucosa. Experiments employing T helper cells in coculture with Proteus virus Isfahan proteins-overexpressing innate immune cells are ongoing to prove the autoreactive activation of the host’s immunity.

Conclusion

Given the growing evidence of viral entities as triggers for IBD, these data could pave the way for discovering viruses directly involved in CD pathogenesis and serving as the booster for molecular mimicry mechanisms, finally providing potential targets for novel intervention strategies that may improve CD management.
P059 GSDMB triggers intestinal inflammation via regulating BHLHA15-mediated hyperactive unfolded protein response
Authors: Gong W , Zhao W
Published: January 2024

Background

Impaired intestinal epithelial barrier has been considered to be associated with an increasing variety of gastrointestinal diseases, especially inflammatory bowel disease (IBD) encompassing Crohn’s disease (CD) and ulcerative colitis (UC). Herein, we investigated the role of Gasdermin B (GSDMB) in modulating intestinal epithelial barrier integrity and proposed a promising therapeutic strategy.

Methods

We generated GSDMB (GSDMBfl/fl;Villin-Cre) intestinal epithelial-specific knock in mice to observe the functions of GSDMB in intestinal epithelial barrier. RNA-seq analysis and human and murine intestine-derived organoids were used to determine the mechanism of function of GSDMB.

Results

To interrogate the intestinal functions of GSDMB in vivo, we generated GSDMB conditional knock in mice (heterozygote: GSDMBfl/-;Villin-Cre; homozygote: GSDMBfl/fl;Villin-Cre) by crossing a mouse line containing the floxed alleles of human GSDMB and a transcriptional stop codon at the transcriptional initiation site (GSDMBfl/fl) with the mouse line expressing Cre-recombinase under control of the Villin promoter (Villin-Cre). GSDMBfl/fl;Villin-Cre mice developed spontaneous enterocolitis and exhibited aberrant intestinal barrier integrity (as shown in the figure). GSDMB prominently provoked cell death of absorptive enterocytes and affected the function of goblet cells and Paneth cells in this setting. Mechanistically, epithelial GSDMB modulated hyperactive unfolded protein response of IECs by up-regulating BHLHA15 to mediate intestinal barrier injury instead of by the invasion of pathogenic microorganisms.

Conclusion

We have uncovered an important and a previously unrecognized role of GSDMB in intestinal inflammation, which represents a potential therapeutic target for IBD.
P070 The influence of Crohn’s disease-derived blood plasma upon anabolic signaling responses to leucine stimulation in skeletal muscle cells in vitro
Authors: Fatani H , Kirkpatrick E , Atherton P , Moran G , Brook M
Published: January 2024

Background

Many Crohn’s Disease (CD) patients have low muscle mass and function (sarcopenia), yet the precise drivers of this pathophysiology are unclear. Positive muscle protein balance is driven by essential amino acids (EAA) that stimulate muscle protein synthesis (MPS), with the EAA leucine (L) playing a key role. However, if circulating factors in CD impact muscle anabolic responses to feeding remain unclear. Our aims were to use plasma from healthy human volunteers (HV) and active CD patients to investigate the impact on anabolic signaling and responses to feeding.

Methods

HV were 32 ± 4 y with a BMI 24 ± 1 and CD 26 ± 3 y with a BMI 23 ± 1 disease duration 5 ± 1 y faecal calprotectin 720 ± 12 ug/g. C2C12 myotubes were treated with 4 conditions of media conditioned with 5% plasma from HV or CD, in addition to being treated ± 5mM L for 4 hrs. Protein content was quantified via nanodrop and muscle anabolic signaling measured via immunoblotting for p-mTORC1 (Ser2448), p-4E-BP1 (Thr37/46) and p-P70S6K1 (Thr389).

Results

Immunoblotting revealed no difference in mTORC1 phosphorylation between HV and CD (HV:18.9 ± 2.7 AU, CD: 19.9 ± 3.2 AU), with no change in response to L treatment (HV:18.0 ± 2.4 AU, CD: 16.6 ± 2.3 AU). There was no difference in P70S6K1 phosphorylation between HV and CD (HV: 33.7 ± 3.2 AU, CD: 28.0 ± 4.7 AU) with both groups showing significant increases in phosphorylation with L treatment (HV: 56.4 ± 4.2 AU (P<0.05), CD: 54.5 ± 9.2 AU (P<0.001)). There was no difference in 4EBP1 phosphorylation between HV and CD (HV: 49.8 ± 6.4 AU, CD: 64.6 ± 7.5 AU) with only HV showing a significant increase in phosphorylation with L (HV: 105.5 ± 8.7 AU (P<0.0001), CD: 47.8 ± 5.3 AU). There was difference in total protein concentration between HV and CD, or with L treatment.

Conclusion

We found CD derived blood plasma did not impact the leucine stimulated increase in P70S6K1 phosphorylation but did result in blunted phosphorylation of 4EBP1. These disparities in anabolic signaling indicate that systemic factors in CD may impact muscle anabolic responses to feeding. Yet, the relationship between anabolic markers and rates of MPS remains complex.
P079 Low-intensity Pulsed Ultrasound reflects the status of mucosal injury and repair through stage-specific miRNAs in experimental model of Ulcerative Colitis
Authors: Pineda Chavez S E , Carriles Linares A , Rizzo G , Wozny M , Cafarelli A , Mori G , Sorriento A , Loy L , Dal Buono A , Gabbiadini R , Bezzio C , Lucchetti D , Sgambato A , Ricotti L , Repici A , Armuzzi A , Vetrano S
Published: January 2024

Background

Diagnosis and therapeutic efficacy monitoring of Ulcerative Colitis (UC) are based on a multidisciplinary approach comprising symptomatology and invasive endoscopic assessment coupled to the histopathological characterization of gut tissue biopsies. New technologies that non-invasively reflect tissue-specific changes remains an unmet need. Here we exploited Low Intensity Pulsed Ultrasound (LIPUS) to induce tissue stage-specific miRNAs in circulating blood as a non-invasive approach to reflect the status of the colonic mucosa in experimental model of colitis.

Methods

Based on our previous study, LIPUS was applied at a frequency of 38 kHz and at an intensity of 0 (as control) or 150 mW/cm2 for 3 min, using a device dedicated for in vivo studies. Acute and chronic colitis were induced in C57BL/6N mice (5 animals/group) by administration of 2 and 2.5% ad libitum dextran sodium sulfate (DSS) respectively in their drinking water for 7 days for the acute and three cycles of 7 days for the chronic model. miRNA array profiling was performed on extracellular vesicles (EVs) isolated from plasma of both acute and chronic models, at both active and remission state, 2 h after LIPUS stimulation. miRNA were correlated to the mucosal status by multiplexed immunofluorescence staining using Opal Multiplex IHC (CD4, CD8, CD68, pan-cytokeratin and CD31 markers) and EVs characterized by MACSplex surface epitope detection.

Results

An enrichment of EVs characterized mainly by stromal and immune cell surface markers was found in both acute and chronic inflammatory states, LIPUS stimulation significantly modulated miRNA profiles and clearly discriminated between active acute and chronic phases. 61 miRNAs in acute and 28 in chronic colitis were specifically and significantly upregulated by LIPUS in the active phase, with a fold change ranging from 2 to 1500 compared to control. Both acute and chronic disease showed a miRNA profile coherent with the active inflammatory state of intestinal mucosa. Only 10 and 23 miRNAs were upregulated in acute and chronic remission respectively. The enhancement of miR-18a-3p and miR-301b-3p in the recovery phases correlated to instetin with reduced immune cell infiltration and epithelial layer regeneration.

Conclusion

An optimal LIPUS dosage that non-invasively targets the colon induces the tissue to release specific miRNAs that reflect the mucosal status. LIPUS may open a new era of non-image ultrasound diagnostics that works as disruptive liquid biopsy allowing the detection of tissue biomarkers for monitoring IBD disease progression.
P082 Downregulated apelin expression in T cells in the setting of Chronic Colitis
Authors: Inoue T , Fushimi M , Yamada D , Watabe T , Watanabe M , Okamoto R , Nagaishi T
Published: January 2024

Background

APJ is a G protein-coupled receptor and was originally found as an orphan receptor. Isolation of apelin (APL) from the alimentary tract was found to be the endogenous ligand for APJ. It has been reported that APL is upregulated in the colonic tissues of a murine model of acute colitis induced by dextran sodium sulfate (DSS), and thus it may be suggested to be associated with the pathogenesis of inflammatory bowel diseases (IBD) in humans. However, the mechanism and function of APL in the context of IBD are still not understood. Here, we analyzed APL expression in an animal model of chronic colitis to define its function in the pathogenesis of IBD.

Methods

Each cell type in the colonic tissue, including epithelial cells and lamina propria lymphocytes, were first isolated from wild type C57BL6 mice (WT) to assess APL expression. Next, naïve T cells isolated from WT were adoptively transferred into Rag deficient mice (Rag-/-) to induce chronic colitis, and then APL expression was measured either in the colonic tissue or in the isolated splenic and colonic CD4+ T cells from these T cell-reconstituted Rag-/- to compare with those of WT or Rag-/- without colitis. In addition, WT naïve T cells were differentiated into either Th1, Th2 or Th17 in vitro to analyze APL expression. Finally, the Rag-/- receiving naïve T cells were administered a synthetic APL peptide, APL-13, to assess the severity of colitis.

Results

Semi-quantitative PCR (qPCR) revealed that CD4+ T cells express relatively higher level of APL compared to other cell types including the epithelial cells in colonic tissue from WT. However, APL expression in the colonic tissues from the Rag-/- induced chronic colitis was unexpectedly downregulated compared to control groups without colitis; this is not consistent with the previous report using acute DSS colitis model. Subsequently, qPCR revealed significantly decreased APL expression in the splenic and colonic T cells from Rag-/- induced colitis compared to that of WT. APL expressions in the effecter T cells in vivo and all of the differentiated T cells in vitro were also significantly downregulated compared to naïve T cells and non-differentiated control T cells, respectively. Given these results, APL-13 was injected into the Rag-/- that underwent T cell reconstitution to antagonize the APL downregulation. This resulted in reduced severity of colitis compared to that of vehicle control-injected group.

Conclusion

These results suggest that T cells can be one of the major sources of APL in colonic tissues, and APL downregulation in effecter T cells may lead to the development of chronic colitis. In addition, this study also suggests that APL may be a novel therapeutic target for IBD.
P098 The Endoscopic Severity Score Map (ESSM): An Artificial Intelligence scoring system providing accurate, objective and localised measurements of endoscopic disease severity in ulcerative colitis
Authors: Gutierrez Becker B , Fraessle S , Yao H , Lüscher J , Girycki R , Machura B , Gośliński J , Czornik J , Pitura M , Arús-Pous J , Fisher E , Bojic D , Richmond D , Bigorgne A , Prunotto M
Published: January 2024

Background

Commonly used scoring schemes as the Mayo Endoscopic Subscore (MES) account for disease severity only at specific (i.e., the worst) segments and do not capture disease extent. However, for an accurate assessment of disease severity in patients with ulcerative colitis (UC), the measure of the precise extent of disease activity is necessary. Alternative systems that include disease extent have been proposed (Balint 2018), but their implementation is prohibited by the time and cost constraints of comprehensively scoring each location along the entire colon. Here, we present the Endoscopic Severity Score Map (ESSM), a scoring system based on Artificial Intelligence, capable of providing an assessment of disease severity and extent in UC in a fully automated manner.

Methods

The ESSM consists of 3 main elements (Fig. 1): 1) a quality algorithm which selects readable frames from a colonoscopy video, 2) a scoring system which assigns an MES to each readable frame (Gutierrez Becker 2020) and 3) a camera localisation algorithm that assigns each frame to an anatomical location within the colon (Yao 2022). The ESSM was trained and tested using 4,306 sigmoidoscopy videos from phase III Etrolizumab clinical trials (Hickory NCT02100696, Laurel NCT02165215, Hibiscus I NCT02163759, Hibiscus II NCT02171429 and Gardenia NCT02136069).

Results

We evaluate the performance of the ESSM by first assessing the agreement of scoring as compared to centrally read MES. The agreement between central reading and the ESSM at the colon section level was high (quadratic-weighted kappa k=0.81; Tab. 1). This was comparable to the agreement between central and local reading (k=0.84), suggesting that the ESSM shows levels of inter-rater variability comparable to experienced readers. Finally, we found correlations between the average ESSM at all anatomical locations and other disease activity markers to be moderate to high: faecal calprotectin rs=0.24, CRP rs=0.29, stool frequency rs=0.49, rectal bleeding rs=0.43 and physician global assessment rs=0.47 (Tab. 1).

Conclusion

Here, we introduced the ESSM, a fully-automated AI-based scoring system that enables accurate, objective and localised assessment of disease severity in UC. In brief, we show that the ESSM compares well with central reading (at the colon section level) and has clinical relevance when compared to other markers of disease activity. This tentatively suggests that the ESSM has the potential to augment the current way of assessing disease severity, both in clinical trials and everyday clinical practice.
P142 Genetic ablation of DNA methyltransferase 3A in myeloid cells impairs M1/M2 polarization and leads to pro-inflammatory macrophages
Authors: Bordoni D , Escudero-Hernández C , Kimmig F , Mishra N , Fazio A , Tran F , Imm S , Hinrichsen F , Weber-Stiehl S , Stengel S , Sommer F , Falk-Paulsen M , Rosenstiel P
Published: January 2024

Background

Genetic variants in the de novo DNA methyltransferase DNMT3A associate with increased risk for inflammatory bowel disease (IBD). While DNMT3A loss-of-function somatic mutations have been attributed to myelodysplasia and acute myeloid leukemia, the exact mechanisms how defective DNMT3A is linked to IBD remain elusive. Since macrophages are key gatekeepers for immune homeostasis, we aimed to characterize how DNMT3A loss affects monocytic lineage differentiation and inflammatory properties.

Methods

We generated mice lacking Dnmt3a expression in myeloid cells (Dnmt3aLysM) using a LysM-driven cre-lox system and confirmed gene deletion by PCR and western blot of gut lamina propria CD11b+ enriched cells and 5-day in vitro differentiated bone marrow-derived macrophages (BMDM, 20ng/mL M-CSF). Four-day polarized BMDMs (resting: 20ng/mL M-CSF; M1: 50ng/mL LPS/IFNγ; M2: 50ng/mL IL-4/IL-13) were functionally characterized using RNA-seq, flow cytometry, and arginase and nitric oxide synthase (NOS) activities. FITC-labelled zymosan served to quantify phagocytosis, and co-culture with intestinal epithelial ModeK wounded monolayers to assess healing. To evaluate Dnmt3a loss during inflammation in vivo, colitis was induced using dextran sodium sulfate (DSS).

Results

M2 polarization induces Dnmt3a expression in wild-type (WT) BMDMs while inflammatory stimuli decrease it. Dnmt3a-deficient BMDMs differentiated with IL4/IL13 in vitro increase ERK1/2 and PI3K signaling, decrease M2 markers and upregulate inflammatory transcripts, e.g. Irg1,Tnf compared to WT cells. IL4/IL13 treated BMDMs confirmed M2 polarization defects as they acquire morphological and functional M1-like features in the absence of Dnmt3a, especially those from aged (>34 weeks old) mice, e.g. failed spindle M2 morphology, increased phagocytosis, decreased arginase and increased NOS activities. In co-cultures, IL4/IL13 treated Dnmt3aLysM BMDMs inhibited intestinal epithelial regeneration after a scratch lesion compared to M2 WT BMDMs. Increased NOS activity was also detected in M1-polarized BMDMs from aged Dnmt3aLysM mice. In vivo, young Dnmt3aLysM mice display mildly increased splenic T-cell and decreased monocyte numbers. Aged Dnmt3aLysM mice (>34 weeks) exhibit altered bone marrow morphology and hepatosplenomegaly linked to increased myeloproliferation. Dnmt3aLysM mice already at young age displayed increased sensitivity to both acute and chronic DSS colitis.

Conclusion

Normal Dnmt3a function is required for physiological macrophage polarization. Deletion of Dnmt3a in mice leads to a pro-inflammatory skewing particularly after M2-inducing cytokines IL4 and IL13. Our findings provide insights on how DNMT3A – via its specific function in myeloid cells – contribute to IBD pathogenesis.
P143 Efficacy of the oral tyrosine kinase 2 (TYK2) inhibitor TAK-279 in two preclinical mouse models of colitis
Authors: Kong K F , Khan M A W , Singh N , Nguyen V A , Wilson E , Tang J , Marusic S , Xia G , Petrakis I , Nguyen D G
Published: January 2024

Background

Tyrosine kinase 2 (TYK2), a member of the Janus kinase (JAK) family, is required for the downstream signalling of interleukin (IL) 12 and 23, and interferons (IFN) α/β, which are involved in the pathogenesis of several autoimmune diseases, including inflammatory bowel disease (IBD).1 JAK inhibitors are approved for treatment of IBD, but their dosing levels may be limited due to adverse effects related to JAK1–3 inhibition. TAK-279 is a highly-selective, oral, allosteric TYK2 inhibitor, which binds to the JAK homology 2 pseudokinase domain of TYK2, but not JAK1–3.1,2 This study assessed the efficacy of TAK-279 in two IL-23-dependent preclinical mouse models of colitis induced by (i) adoptive T-cell transfer (TCT) and (ii) anti-CD40 monoclonal antibody (α-CD40 mAb).

Methods

In the TCT colitis model, CD4+CD45RBhi T cells were transferred to severe combined immunodeficient mice at Day 0, and disease progression followed for 48 days. In the α-CD40 colitis model, T- and B-cell-deficient Rag2-/- mice were challenged with agonistic α-CD40 mAb at Day 0, and disease severity analyzed on Day 7. Mice were divided into four groups per model (n = 12–16 and n = 10 per group in the TCT and α-CD40 models, respectively) to receive vehicle (negative control) or TAK-279, orally, twice-daily, at doses intended to provide either 24hr IC90 (high dose) or IC50 (low dose) coverage, or once-weekly intraperitoneal α-IL-12/23 p40 mAb (positive control). At the end of the treatment period, colonic tissue was obtained; colon weight:length (CWL) ratio, total histology score (THS) and transcriptomic analysis were used to assess efficacy and mechanism of action. Statistical significance of differences between groups was determined using analysis of variance with Tukey’s post hoc test.

Results

In both models, TAK-279 was maximally efficacious at the IC90 dose; CWL ratio and THS were significantly reduced relative to vehicle and the response was comparable with α-IL-12/23 p40. IC50 dosing was not sufficient in the α-CD40 model and IC90 dosing was required for significant efficacy (Figure). Transcriptomic analysis showed TAK-279-dependent downregulation of genes associated with cytokine signalling, including those related to T-helper 17 cell phenotypes and the IFN α/β pathway.

Conclusion

TAK-279 was efficacious in two preclinical mouse models of colitis and induced relevant cytokine signalling changes; IC90 coverage provided maximal efficacy in both models. These results support the potential role of potent and selective TYK2 inhibition for the treatment of IBD, where TAK-279 is expected to achieve IC90 coverage in humans.

1. Leit S et al. J Med Chem 2023;66:10473–96.

2. Gangolli EA et al. STAT 2022;1:5.
P145 Phagocyte responses to gut-derived C. albicans are modified in inflammatory bowel disease
Authors: Mathew L , Carlson S , Savage M , Pardieu C , Sze N , Munro C , Naglik J , Stagg A , Kok K , Lindsay J O , McCarthy N
Published: January 2024

Background

Fungi are a key component of the human gut microbiota and have been strongly implicated in the pathogenesis of inflammatory bowel disease (IBD). Candida albicans typically exists as a commensal yeast within healthy hosts but can form tissue-invasive filaments that secrete Candidalysin (CLYS) toxin to disrupt epithelial barriers. Dynamics of this morphological switch are influenced by genotypic and phenotypic variation of gut-resident C. albicans strains, which may impact host response and extent of mucosal inflammation.

Methods

C. albicans isolates were derived from healthy control (HC) and IBD gut biopsies. Phenotypic, genomic, and proteomic analyses were conducted on these isolates. To further understand host interplay with intestinal C. albicans under physiologically relevant conditions, a human ‘gut-on-a-chip’ system was developed to assess tissue invasion potential in vitro.

Results

IBD-derived isolates were more readily able to form filaments, displayed altered cell wall composition, and modulated expression of adhesion-associated genes including IHD1. Subsequent innate leukocyte responses to these strain-dependent profiles were investigated to understand impact on host immunity. When exposed to IBD-derived C. albicans strains, blood neutrophils from healthy donors displayed increased swarming behaviour and NETosis induction, whereas monocyte-derived macrophages released greater amounts of pro-inflammatory IL-1b. These responses were substantially diminished in neutrophils and macrophages from IBD patients, suggesting altered reactivity to fungal stimuli in disease settings. The gut-on-a-chip model system confirmed that IBD-derived strains display increased adhesion and translocation in a dynamic human gut environment.

Conclusion

Gut-adapted C. albicans strains from HC and IBD donors exhibit distinct biological profiles that impact on host immune responses and shape interactions with the gut barrier in a novel organ chip model.
P147 IBD relevant pathways are upregulated in Paneth cells in a murine model of dietary induced intestinal inflammation
Authors: Grabherr F , Meyer M , Christina P , Marteau V , Schwärzler J , Mayr L , Enrich B , Jukic A , Grander C , Trajanoski Z , Tilg H , Adolph T E
Published: January 2024

Background

Dietary constituents are believed to play a role in the development of Crohn’s disease (CD) (1). Recently we were able to demonstrate, that polyunsaturated fatty acids (PUFA), induce a CD like enteritis phenotype in genetically susceptible mice (2,3). Furthermore, we could show, that PUFA intake correlated negatively with disease course in CD patients (3). In this project we tried to untangle the role of Paneth cells (PC) in the development of PUFA induced enteritis.

Methods

scRNA sequencing was performed on cells isolated from mice lacking one allele of Gpx4 (GPX4+/-IEC) in their intestinal epithelial cells (IEC) after feeding them a PUFA enriched western style diet (PUFA-WD). Furthermore, mice lacking both alleles of Gpx4 in their PC (GPX4ΔPC) where fed a PUFA-WD for four weeks and enteritis was assessed based on a histological enteritis score and immunhistochemical stainings.

Results

scRNAseq of murine intestinal cells revealed an upregulation of IBD relevant pathways (such as JAK or TNFα) in Gpx4 deficient IECs and PC. GPX4ΔPC mice developed a CD like enteritis phenotype, characterized by a mucosal and submucosal infiltration of neutrophils, macrophages and other leucocytes, after only four weeks of PUFA enriched diet, indicating that GPX4 dependent intestinal inflammation is controlled by PC.

Conclusion

The upregulation of IBD relevant pathways in our model of dietary induced intestinal inflammation renders this model a valuable tool to investigate mechanistical understanding in the role of dietary components in IBD. Our results further link PC metabolism to the development of intestinal inflammation.

References

1. Adolph TE, Meyer M, et al Nat Rev Gastroenterol Hepatol 2022

2. Mayr L, Grabherr F, et al Nat Com 2020

3. Schwaerzler J, Mayr L, et al Gastroenterology 2022
P150 Ulcerative Colitis Patients’ Luminal Zinc Induces Clostridioides difficile Virulence and Exacerbates Inflammatory Responses
Authors: Wu T , Zhou H , Li X , Lin J , Wang F , Da Y , Liang J
Published: January 2024

Background

The refractory acute severe UC patients are often complicated by C. difficile infection (CDI), resulting in higher surgery and mortality rates. However, few studies have observed C. difficile’s pathogenicity in real UC patients’ gastrointestinal microenvironments, and the actual role of C. difficile in UC development remains unknown. We found zinc concentrations among UC patients’ feces were significantly higher than those among Crohn’s disease patients and healthy subjects. Additionally, we previously reported that excessive zinc would promote the growth and virulence of C. difficile. Therefore, we hypothesized that the increased UC patients’ luminal zinc may up-regulate C. difficile virulence and promote intestinal inflammatory responses, leading to an exacerbated UC progression.

Methods

UC patients’ stool samples (n=40) were collected and analyzed by qPCR and ICP-MS to determine the presence of C. difficile and the fecal zinc concentrations. C. difficile was cultured in BHIs with or without zinc (ZnSO4) in an anaerobic chamber. Expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. Human colonic organoids and Caco-2 cells were co-cultured with C. difficile or zinc-treated C. difficile, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. TLR5 knock-out Caco-2 cells were constructed using sgRNA-guided Cas9 nuclease and co-cultured with C. difficile or zinc-treated C. difficile to verify whether zinc-induced C. difficile’s pathogenicity was mainly through flagellar formation.

Results

C. difficile was detected in ~35% of UC patients’ fecal samples. Luminal zinc concentrations (~915μM, p<0.01) were significantly higher among UC patients than CD patients or healthy subjects. The UC patients’ luminal concentration of zinc promoted C. difficile growth, suppressed its exotoxins release, and induced higher expression of its flagella-related genes, resulting in higher cytotoxicity and inflammatory responses in human colonic organoids and Caco-2 cells. However, zinc-treated C. difficile did not induce higher inflammation and pathogenicity in TLR5 knock-out Caco-2 cells.

Conclusion

The association between C. difficile and UC aggravation may partially stem from UC patients’ excess luminal zinc promoting C. difficile flagellin expression, inducing inflammatory responses, and worsening UC progression.
P152 Longitudinal immune profiling of peripheral blood in patients with Ulcerative Colitis
Authors: Wider-Eberspächer E , Garza A P , Albrecht T , Rosania R , Dunay I R
Published: January 2024

Background

Recent literature has established the communication and mutual influence between the gut and the brain, referred to as the gut-brain axis. While inflammatory bowel disease (IBD) poses a risk for neuroinflammation and brain dysfunction, the molecular mechanisms are not fully understood. This study aims to investigate longitudinal changes during Ulcerative Colitis (UC), focusing first on the characterization of peripheral immune cell subsets, circulating cytokines, and extracellular vesicles (EVs).

Methods

In our study, we examined 19 UC patients in both active (mean age in years: 45,63) and remission phases (mean age in years: 46,16). A gastroenterologist at the Gastroenterology Department from the Otto-von-Guericke University in Magdeburg, Germany assessed all participants, categorizing them into stages using Mayo scoring and calprotectin levels. We analysed peripheral whole blood and plasma samples using different flow cytometry-based approaches.

Results

Our findings indicate an upregulation of the neutrophil (CD16+, CD15+) numbers during the active stage followed by a significant reduction during remission, indicating an increase in innate inflammation as a first line of defence and a balanced state during remission. Classical (CD14+, CD16-) monocytes were decreased, along with similar results in the intermediate (CD14+, CD16+) subset. Nonclassical monocytes (CD14+, CD16++) increased from the active to the remission stage. Surface expression of activation marker CCR2 was downregulated for all monocyte subsets, whereas CX3CR1 was upregulated specifically in intermediate monocytes during remission. Compared to the active stage peripheral dendritic cell (CD14-, CD16-) numbers increase during remission suggesting less colonic infiltration. Peripheral regulatory T cells (Tregs) (CD127 low/-, CD25+), which are important for the reestablishment of homeostasis are upregulated during remission. Characterization of cytokine levels in plasma revealed an upregulation of the proinflammatory milieu during the active stage, especially with elevated IL-18 and TGF- β1 levels. Finally, those changes were accompanied by alterations of the surface markers expressed by isolated circulating EVs.

Conclusion

Our initial results indicate the feasibility of evaluating the gut-brain axis through liquid biopsy. We unravel the composition of the peripheral inflammatory milieu during the active phase. Currently we are focusing on the assessment of the CNS-derived metabolites and their bidirectional communication with the gastrointestinal system.