bluuebunny/crossref_metadata_2025_split huggingface dataset:
P018 Myeloid diversity in inflammatory bowel disease revealed by single-cell transcriptomics
Authors: Garrido Trigo A , Corraliza A M , Melón-Ardanaz E , Veny M , Ordás I , Ricart E , Masamunt M C , Panès J , Hemberg M , Mereu E , Heyn H , Azucena S 
Published: May 2021

Abstract

Background
Recent studies have applied single-cell RNA sequencing (sc-RNAseq) of the human intestine to resolve the cellular complexity and heterogeneity characteristic of inflammatory bowel disease (IBD). Nonetheless, an in-depth analysis of the myeloid mucosal compartment, a crucial player in gut immune responses, has not yet been conducted.

Methods
Colonic biopsies from healthy controls (HC, n=6), endoscopically active Crohn’s disease (CD, n=5) or ulcerative colitis (UC, n=6) patients were collected and immediately disaggregated. Single-cell suspensions were processed using the 10x Genomics Next-GEM Single Cell kit assays (Chromium 3’ v3.0) and the resulting libraries sequenced (NovaSeq 6000 S1, Illumina). Data from 3452 myeloid cells was separated in silico and analyzed using Cell Ranger v3.1.0 and the R package Seurat.

Results
In HC, mast cells, CD1c+ dendritic cells (DCs) and resident macrophages (RMΦ) in different transcriptional states were identified. All RMΦ clusters were HLA-IIhi and expressed C1Q genes, SELENOP, CD74 and LYZ. Within RMΦ, a distinct cluster of M2-like cells characterized by high expression of CD209, CD163L1, LILRB5, AXL and MRC1 formed an independent cluster. IBD showed a marked increase in both the number of cells and heterogeneity within the myeloid compartment compared to HC. While RMΦ and M2 macrophages were decreased, we observed an infiltration of inflammatory monocytes and M1-like macrophages expressing the monocyte markers VCAN and CD300E. A cluster of TNIP3-expressing M1 macrophages that co-expressed ACOD1, KYNU, TRAF1, MMP19, IRAK2, TNF and IL1B was present in most UC and CD samples. Additional M1-like macrophage subsets, including a cluster characterized by high expression of CXCL5, HTRA1, SPP1, INHBA, MMP9, MMP14 and MMP10, or a cluster of NRG1, RETN, EREG, HBEGF, TGFB1 and VEGFA-expressing macrophages were identified in UC. Furthermore, IBD samples presented an additional cluster of mature DCs expressing CCL22, CCL17, CCL19 and CCR7. Importantly, our study is the first to capture infiltrating granulocytes in IBD patients by scRNA-seq; specifically, eosinophils expressing CLC, IL4 and IL13, and 3 neutrophil states, expressing FCGR2B, CMTM2, S100A8 and CXCL8.

Conclusion
We describe an unbiased approach for the identification of intestinal myeloid cells that revealed previously undescribed macrophage subpopulations. In addition, we discovered a marked heterogeneity within intestinal inflammatory macrophages that showed high patient-to-patient variability. We suggest that the diverse macrophage states in mucosal lesions could explain important differences in disease pathophysiology and contribute to the observed diversity in patient behavior.
P023 SZN-1326, a Wnt agonist, improved epithelial healing and ameliorated colitis in a chronic DSS model, in stark contrast to anti-TNF and anti-IL-12/23p40 antibodies
Authors: Xie L , Handa P , Tun C , Liu A , Lu C , Baribault H , Newman M , Le L , Shah D , Deshmukh S , Mutha D , Le T , Fletcher R , Chen H , Ye J , Tibbitts J , Srinivasan U , Li Y , Vanhove G , Yeh W C 
Published: May 2021

Abstract

Background
All currently approved treatments for Inflammatory bowel disease (IBD) and the majority of drugs in the pipeline are anti-inflammatory agents. Clinical remission rates have reached a plateau, and there is an unmet need for agents that directly repair and regenerate the intestinal epithelial barrier as mucosal healing has been associated with improved clinical outcomes. Wnt/β-catenin signaling promotes intestinal stem cell renewal and epithelial regeneration. We have engineered a Wnt activator, SZN-1326, which increased Wnt target gene expression in the colon of mice treated with Dextran Sulfate Sodium (DSS), an animal model of IBD and demonstrated efficacy in the acute and chronic forms of this model. The goal of this study was to compare the efficacy of SZN-1326 to the anti-inflammatory agents anti-TNFα or anti-IL-12/23p40 antibodies in a chronic DSS model.

Methods
7- to 8-week old female C57BL6/J mice were administered 3% DSS for three 7-day cycles separated by 7-days off, followed by a 3-day 1% DSS wash-out period. SZN-1326 was injected intraperitoneally (I.P.) at 1, 3, or 10 mg/kg for 2, 4 or 6 doses. Anti-TNFa (MP6-XT22, Biolegend) was injected I.P. at 5 or 25 mg/kg for 4 or 7 doses, and the mice were euthanized on day 38. A separate study examined the efficacy of SZN-1326 relative to anti-IL-12/23p40 (C17.8, Bioxcell) in the same model. Anti-IL12/23p40 antibody at 3 or 10 mg/kg for 4 or 8 doses was given on days 23 or 7 respectively.

Results
During each cycle of DSS treatment, animals exhibited a marked increase in disease activity index (DAI), characterized by body weight loss, diarrhea and bleeding. SZN-1326 at various dosing regimen (1 mg/kg for 4 doses to 10 mg/kg for 2, 4 or 6 doses) significantly improved body weights and decreased DAI compared to isotype control, while anti-TNFa or anti-IL12/23p40 had no efficacy on body weight restoration or DAI. The antibodies could be detected in the serum in accordance with their doses. Treatment with SZN-1326, but not with anti-TNFα significantly decreased colon inflammation, mucosal erosion and overall histopathological score. Further, SZN-1326 treatment significantly reduced serum levels of IL-6 and lipocalin-2, while anti-TNFa decreased only lipocalin-2 levels and anti-IL12/23p40 reduced serum IL-6 level.

Conclusion
In a chronic mouse IBD model, SZN-1326 at various dosing regimens 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, treatment with anti-TNFα or anti-IL-12/23p40 antibodies led to reduction only in certain serum inflammatory cytokines but had no efficacy on DAI or colon histology in this model.
P033 Dendritic cells from patients with active IBD imprint pro-inflammatory α4β7+CLA+ T cells with potential for gut and skin homing
Authors: Gordon H , Rodger B , Hoti I , Lindsay J O , Stagg A 
Published: May 2021

Abstract

Background
T cell trafficking contributes to inflammation and is a therapeutic target in IBD. Exposure of T cells to retinoic acid during activation induces expression of the gut trafficking integrin α4β7 and suppresses expression of cutaneous leukocyte antigen (CLA) which is required for entry into the skin. Although α4β7+ (gut homing) and CLA+ (skin homing) T cells are generally non-overlapping populations, we have recently observed a low frequency of α4β7+CLA+ (dual tropic) T cells in human blood; these cells may provide an immunological link between gut and skin. Here we investigate the generation of these population by DC in vitro, analyse the function of cells with different tissue tropism and examine the impact of intestinal and skin inflammation.

Methods
Using flow cytometry, expression of α4β7 and CLA was assessed in whole blood, gut tissues, and on proliferating cells generated by stimulation of naïve CD4+ T cells with monoclonal antibodies (anti-CD3/28/2), or with allogeneic colonic or monocyte-derived DC (moDC). Production of TNFα and IFNγ was measured by intracellular cytokine staining.

Results
α4β7+CLA+ T cells were found at low frequency in ileal lamina propria tissue as well as peripheral blood. Naïve CD4 T cells activated in vitro with antibodies expressed α4β7 but not CLA. In contrast, activation with DC resulted in the expression of both α4β7 and CLA and generated a dual tropic a4b7+CLA+ population. Colonic DC generated significantly more α4β7+ cells (p&lt;0.05) and higher levels of α4β7 (p&lt;0.05) than did moDC; colonic DC generated significantly fewer CLA+ cells (p&lt;0.01). Nonetheless, a similar frequency of dual tropic cells was generated by both types of DC. moDC from healthy controls and patients with active IBD or skin inflammation all generated dual tropic cells. moDC from patients with active IBD, but not from patients with active inflammatory skin disease generated more gut tropic (α4β7+) T cells (p&lt;0.05) compared with moDC from healthy controls. Following activation with moDC, cytokine production varied with homing profile. The frequency of TNFα+ and IFNg+ cells was significantly greater in CLA+ expressing populations (p&lt;0.05 and p&lt;0.01, respectively).

Conclusion
CLA expression is induced by gut DC even though they potently upregulate α4β7. The dual tropic population generated is enriched for cells that produce inflammatory cytokines and it may provide a link between gut and skin inflammation. Monocyte-derived cells may contribute to the imprinting of T cell homing to the intestine and upregulation of this activity, specifically in active IBD, supports the concept that intestinal inflammation reprogrammes circulating monocytes.
P049 Development of human embryonic stem cell-derived intestinal organoids for in vitro studies on intestinal inflammation and fibrosis
Authors: Kandilogiannakis L , Filidou E , Drygiannakis I , Tarapatzi G , Arvanitidis K , Bamias G , Valatas V , Paspaliaris V , Kolios G 
Published: May 2021

Abstract

Background
Organoids are self-renewing, 3D structures, consisting of different cell types, with histology and physiology features very close to the physiology of the studied organ. Specifically, human Intestinal Organoids (HIOs) develop epithelial crypts consisting of all subtypes of intestinal epithelial cells which are surrounded by mesenchymal cells. Our aim was to develop 3D HIOs from human embryonic stem cells (hESCs) and examine the expression of fibrotic and mesenchymal factors during their maturation process. Additionally, we investigated the effect of the pro-inflammatory cytokines, IL-1α and TNF-α on the expression of fibrotic and inflammatory mediators in HIOs.

Methods
The human ESC line (H1) was cultured and then differentiated towards HIOs using commercially available kit. HIOs were characterized by immunofluorescence in all differentiation stages. In order to examine their maturation process, we compared the mRNA expression of fibrotic and mesenchymal markers from passages 1–10. In order to examine their functionality, HIOs from different passages were stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 12 hours, total RNA was collected and the fibrotic and inflammatory mRNA expression was examined. The mRNA transcripts of CD90, collagen type I, III, fibronectin, CXCL8, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR.

Results
HIOs were successfully developed as they were stained positive for all tested markers throughout their developmental process. Regarding their maturation process, we observed high expression of CD90, collagen type I, type III and fibronectin that was gradually decreased during passages. As for the fibrotic and inflammatory responses from HIOs, we found that the IL-1α and TNF-α stimulation resulted in statistically significant upregulation of the fibrotic factors, fibronectin, collagen type I and type III in culture passages 2 and 4, but had no effect in culture passages 8 and 10. Similarly, IL-1α and TNF-α stimulation led to the statistically significant induction of the inflammatory chemokines CXCL8, CXCL10 and CXCL11 in culture passages 2 and 4, while no effect was observed in culture passages 8 and 10.

Conclusion
Our findings indicate that HIOs contain a functional mesenchymal component that is gradually diminished during passages. Inflammatory and fibrotic responses of HIOs seem to depend on the fitness of their mesenchyme. IBD studies using HIOs as in vitro models should be performed on early passages, when HIO’s mesenchymal component is still functional.
P050 Effect of biological treatments (anti-TNFs) in the creeping fat of Crohn’s disease patients
Authors: Montfort-Ferré D , Serena C , Millan M , Boronat-Toscano A , Maymó-Masip E , Caro A , Espina B , Vendrell J , Fernández-Veledo S 
Published: May 2021

Abstract

Background
Crohn’s disease (CD) is characterized by persistent inflammation and ulcerations at the small or large bowel, provoking chronic low-grade systemic inflammation. Adipose tissue (AT) is believed to play an active role in the pathogenesis of CD, as the expansion of mesenteric fat attached to the inflamed segments of the intestine, also known as “creeping fat,” is a hallmark of the disease that seems to be directly related to disease activity. We demonstrated that adipose-stem cells (ASC) isolated from the creeping fat of CD patients showed a proinflammatory phenotype and increased the proliferation, migration, and phagocytic capacities of these cells. Taking into account the widely described effects of TNFalpha on the biology and functionality of adipocytes, we believe that biological therapies based on anti-TNF agents modify the inflammatory status of creeping fat. In this context, the effect of anti-TNF treatment on mesenteric fat is poorly studied, and the results are divergent.

Methods
Creeping fat biopsies were obtained from active CD patients that underwent surgery for symptomatic complications: 10 patients were on anti-TNF therapy (at least 6-months prior to surgery) and 10 patients never received any biological therapy. The groups were comparable in age, sex, and body mass index. We isolated from AT biopsies: AT explants, ASC, and adipose tissue macrophages (ATM). Adipose tissue was fixed in 10% phosphate-buffered formalin and embedded in paraffin for histological studies. The proliferation of ASC was performed using the CellTraceTM Violet Cell proliferation kit using flow cytometry and the cell migration of ASC was analyzed using a Tranwell system (8 mmpore polycarbonate membrane).

Results
Histological studies revealed that AT of patients treated with anti-TNF therapy recovered adipocytes morphology and showed lower infiltration of immune cells. Interestingly, we found a significant decrease in the gene expression of pro-inflammatory cytokines (IL1B, IL6, TNFA) in the creeping fat of CD patients treated with anti-TNF (Figure 1A). Furthermore, ATMs isolated from patients treated with anti-TNF showed a significant decrease in the gene expression of antigen-presenting markers (CD74, CIITa, HLA-DPB and HLA-DM) (Figure 1B). To note, ASC isolated from patients with anti-TNF therapy has significantly decreased their proliferation and migration capacities as well as the pro-inflammatory gene expression. Moreover, the anti-inflammatory gene expression and secretion were significantly increased in these cells (Figure 1C).

Conclusion
Anti-TNF therapies impact on the creeping fat of CD patients improving the phenotype of this tissue and this may cause a beneficial effect on CD.
P084 C-Reactive Protein Correlates with Tumor Necrosis Factor-α in Ulcerative Colitis
Authors: McDonagh P , O’Connell F , O’Connell J , Argue R , Corcoran R , Kevans D , O’Sullivan J 
Published: May 2021

Abstract

Background
Monitoring disease activity in Ulcerative colitis (UC) is essential. Inflammatory markers like C-Reactive Protein (CRP) and faecal calprotectin have been shown to correlate with clinical and endoscopic disease activity. To date the relationship between CRP and the inflammatory analytes within the colonic microenvironment have not been analysed.

Methods
Our primary aim was to evaluate the relationship between CRP and key inflammatory cytokine, TNF-α, in UC patients. We also sought to investigate whether CRP correlates with clinical parameters. Ethical approval was granted by our Research Ethics Committee. Patients over the age of 17 with a confirmed diagnosis of UC presenting for colonoscopy were offered the opportunity to participate. Basic patient demographics and Mayo score were recorded. At endoscopy colonic biopsies were taken and cultured in dimethyl sulfoxide (DMSO). Multiplex inflammatory and angiogenic enzyme-linked immunosorbent assay (ELISAs) were performed to evaluate the colonic microenvironment and assess real time secretion of TNF-α. Correlations were carried out on SPSS 24 and plotted using R Studio corrplot.

Results
26 patients with UC participated in the study. The mean Mayo score was 4 (range 0–10). At endoscopy 15% had severe colitis (Mayo endoscopy sub-score 3), 31% had moderate disease, 54% had mild disease. 35% of patients were receiving biologic therapy. The mean CRP was 6.19mg/L (range 1–48.35mg/L), albumin 43.5g/L (range 31-49g/L). CRP had a negative correlation with albumin (r=-0.585, p-value 0.002). CRP had a statistically significant positive correlation with clinical, endoscopic and total Mayo scores (r= 0.469, 0.543, 0.526. p-values 0.016, 0.004, 0.006, respectively). Bleeding at colonoscopy also correlated very strongly with an elevated CRP (r=0.859, p-value=0.00). CRP had a moderate positive correlation with TNF-α (r=0.603, p-value=0.001) and VEGF receptor (r=0.492, p-value=0.011) levels.

Conclusion
CRP displayed a statistically significant positive correlation with the Mayo score. There was a significant correlation between the CRP and TNF-α expression within the colonic micro-environment. TNF-α is a key cytokine and therapeutic target in UC, its relationship with CRP has not been previously assessed. CRP is produced primarily by hepatocytes and is activated by TNF-α, interleukin 6 and interleukin 1β. It therefore stands to reason that there would be correlation between the two. The importance of TNF-α in UC is well known, this study supports and provides new evidence for the use of CRP as a non-invasive marker of disease activity.
P085 Expression profiling of Wnt pathway genes in colon biopsies of patients with Ulcerative Colitis
Authors: Lu C , Shah D , Wijnands A , Oldenburg B , Yeh W C , Vanhove G , Li Y 
Published: May 2021

Abstract

Background
There is an increasing demand of agents that can promote mucosal healing in Inflammatory Bowel Disease (IBD). Wnt/β-catenin signaling plays a critical role in epithelial regeneration and repair, and stimulating regeneration in the damaged epithelium by modulating Wnt signaling has been suggested as a potential treatment option for IBD. To guide development of Wnt modulating therapeutic molecules for IBD, an understanding of how Wnt signaling may be altered in IBD tissues is required. While earlier work showed altered Wnt pathway gene expression in UC tissues, these studies failed to consider disease conditions (moderate vs severe) and patient treatment history on expression of the Wnt family genes. These previous studies utilized RT-qPCR or microarray and did not reveal how Wnt pathway gene expression might be affected specifically in the epithelium and in the adjacent stromal stem cell niche. Here we report our work investigating expression patterns of Wnt pathway genes in UC biopsies from 12 patients with moderate and severe disease. Patients had either received no anti-TNF treatment or had gone through anti-TNF treatment and partially responded to the treatment.

Methods
Expression of a set of Wnt pathway genes was assessed in UC colon and rectum biopsies by RNAscope in situ hybridization and compared to expression patterns in normal control colon. The genes included the Wnt target genes AXIN2, LGR5 and RNF43, Wnt ligands and the FZD5 and LRP6 receptors enriched in the intestinal epithelium as well as key Wnt signal modulators RSPO1-4.

Results
Expression of Wnt target genes were mildly reduced in the UC colon epithelium, while their expression in some crypts appeared much lower. Overall expression levels of Wnt pathway genes did not differ between moderate and severe UC colon and Wnt target gene expression was more affected in the anti-TNF treated colons, which may reflect more refractory disease. Expression of FZD5, LRP6 and the key niche factor RSPO2, was reduced in the UC colon. RSPOs are normally expressed in the stromal cells next to the crypt bottom stem cell compartment but this expression pattern was disrupted in the UC colon as a result of immune cell infiltration. Although expression of Wnts was induced in the UC colon tissues, the location of expression was altered due to tissue damage, potentially making the Wnts less accessible to the intestinal stem cells.

Conclusion
Reduced expression of Wnt receptors, RSPOs and Wnt target genes indicate insufficient Wnt signal induction in the damaged colon epithelium of UC patients. This suggests that repair of the damaged epithelium by Wnt agonist treatment may constitute a new mechanism of action and benefit patients with UC.
P093 COVID-19 lockdown and mental health burden in Inflammatory Bowel Disease paediatric patients: a case-control study
Authors: Milo F , Romeo E F , Rea F , Grimaldi Capitello T , De Angelis P , Tabarini P 
Published: May 2021

Abstract

Background
From March 10 to May 3, 2020, Coronavirus disease 2019 (COVID-19) and subsequent enforced lockdown in Italy has led to an increased general psychological distress1. In particular, people with both compromised immune function and pre-existing physical or psychiatric problems are at increased risk of adverse psychosocial outcomes2. The purpose of this study was to compare mental health burden in Inflammatory Bowel Disease (IBD) paediatric patients with and without COVID-19 lockdown exposure.

Methods
We conducted a retrospective case-control study comparing mental health outcomes of two matched 1:1 IBD paediatric groups: Group 1 - before the exposure (January 2019-September 2019) and Group 2 - after COVID-19 pandemic lockdown (April 2020-December 2020). Matching criteria included gender, age, disease duration, IBD subtype, Body Mass Index and disease activity (remission or active). This study included patients with Crohn Disease (CD) and Ulcerative Colitis (UC), &gt;12 years, attending outpatient visits at Bambino Gesù Children Hospital. Data were collected using the Generalised Anxiety Disorder (GAD-7) for anxiety and the Patient Health Questionnaire (PHQ-9) for depression. We have estimated the prevalence of anxiety and depression between the two groups. Both groups were also compared with respect to the average score of anxiety and depression symptoms. Sociodemographic and clinical data were recorded from patients’ medical chart.

Results
A total of 108 IBD paediatric patients (62 males, 54 with CD and 54 with UC, mean age=16±2,5 years) was enrolled in this study. Out of these, 54 patients were enrolled in Group 1 and 54 in Group 2. Using a cut-off score ≥10, we found similar prevalence of anxiety (13.0% vs 22.2%, P=.206) and depression (13.0% vs 18.5%, P=.428) among the groups. Average scores were quite similar among the two groups, both for anxiety (5.78 vs 5.96, P=.924) and depression (5.89 vs 5.87, P=. 704). Symptom severity levels were mild (≃6) in both groups, according to standardized instrument cut-offs. No significant associations were found between disease activity and symptoms of anxiety and depression in both groups.

Conclusion
IBD-related symptoms (i.e. weight loss, persistent diarrhoea, abdominal pain and fatigue) lead to severe psychosocial impairment in paediatric IBD patients. COVID-19 pandemic outbreak and enforced isolation (i.e., lockdown, long distance learning, and social restrictions) limited patients’ stressful social situations engagement and paradoxically prevented a worsening of their mental health.

References