bluuebunny/crossref_metadata_2025_split huggingface dataset:
A MODEL TO STUDY INTESTINAL FIBROSIS IN IPSC-DERIVED HUMAN EPITHELIAL AND MESENCHYMAL CELLS FROM CROHN’S DISEASE PATIENTS
Authors: Valencia Christian Wong , Bay Olivia , Patel Shachi , Estrada Hannah , Casero David , Targan Stephan , Barrett Robert 
Published: January 2024

Abstract

INTRODUCTION
Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix (ECM) proteins. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. To overcome this, we have previously developed a human model of intestinal fibrosis using iPSC-derived epithelial and mesenchymal cells generated from both control and CD patients. Using this model, we aimed to further characterize the transcriptomic signature associated with the fibrotic response as well as other functional hallmarks of fibrosis.

METHODS
iPSCs from 19 CD patients with (10) and without (9) fibrotic complications were directed to form HIOs. As iPSC-derived HIOs contain epithelial and mesenchymal cells, the organoids were dissociated to generate purified cultures of epithelial only-HIOs (eHIOs) and intestinal mesenchymal cells (MSCs). eHIO and MSC cultures were treated with TNFα and TGFβ, alone or in combination. We evaluated the transcriptomic profile of both cell types via RNA-seq and carried out additional studies via ELISA, ECM staining and scratch assay.

RESULTS
RNAseq analysis on the effects of cytokine induction in eHIOs lead to an enrichment in pathways associated with inflammation and regulation of the immune response. Furthermore, eHIOs from patients with fibrotic complications showed a significant upregulation of ORM1 regardless of culture conditions, which was subsequently confirmed at the protein level indicating an intrinsic difference between both patient groups. RNAseq analysis on MSC cultures found differentially expressed genes clustered in pathways associated with ECM production and remodeling, and cell migration and proliferation. Cell migration capacity of MSCs was assessed by measuring the reduction in scratch area and under basal conditions, MSCs from the fibrotic cohort had a lower rate of cell migration (40.81%) compared to the non-fibrotic cohort (20.97%); notably, the addition of TNFα appeared to accelerate the migration capacity of MSCs in both fibrotic (14.54%) and non-fibrotic (6.56%) samples. Finally, compared to untreated cultures, MSCs treated with TNFα/TGFβ exhibited a significant increase in ECM deposition (2.64- and 2.29-fold increase for non-fibrotic and fibrotic, respectively).

CONCLUSION
We have demonstrated the functionality of our model of intestinal fibrosis generated from iPSC-derived epithelial and mesenchymal cells from CD patients. Furthermore, we have identified both generalized and intrinsic differences in our cohort of patients with fibrotic complications. Future experiments on the role of ORM1 in intestinal fibrosis and its effects on mesenchymal cells could provide a deeper understanding of the events leading the development of intestinal fibrosis in CD patients.
CREEPING FAT-DERIVED FIBROBLASTS PARTICIPATE IN INTESTINAL FIBROSIS IN A NOVEL MOUSE MODEL OF INTESTINAL STRICTURES
Authors: Bauer-Rowe Khristian , Kim Alexia , Pham Benjamin , Griffin Michelle , Norton Jeffrey , Hyun Jeong , Longaker Michael 
Published: January 2024

Abstract

INTRODUCTION
An intestinal stricture is a common morbidity in Crohn's disease, but its pathogenesis is poorly understood. CF forms adjacent to strictures and is associated with stricture recurrence, but whether it promotes stricture formation is unclear. Adipocytes display remarkable plasticity, and adipocyte-derived fibroblasts (ADFs) play important roles in tissue wound healing and fibrosis. Here, we show that CF ADFs may promote intestinal stricture formation.

METHODS
We developed a novel mouse model of CF and intestinal fibrosis by creating anti-mesenteric colotomies. We then created colotomies in Adipoq-Cre; mTmG mice and harvested the mice at post-operative day (POD) 14. This system exclusively labels adipocytes and their derivatives GFP+, whereas all other cells are TdTomato+ (Figure 1A-B). We immunostained sham and colotomy bowel for mature adipocyte marker PLIN1. We quantified GFP+ lineage-negative ADFs (CD45-CD31-Terr119-EPCAM-ECadherin-) isolated from either sham or colotomy bowel at POD 14. We then isolated ADFs (GFP+) and non-ADFs (nADFs) (TdTomato+) by flow cytometry and performed single-cell RNA-seq (scRNA-seq) (Figure 1F).

RESULTS
The creation of a colotomy is sufficient to induce localized mesenteric adipose tissue (MAT) expansion around the colotomy site, consistent with CF formation (Figure 1C). We observed the presence of GFP+ cells with a fibroblast morphology that lost the expression of adipocyte markers and acquired the expression of fibroblast markers. (Figure 1D). Furthermore, we observed that GFP+ ADFs expanded in colotomy bowel compared to sham (Figure 1E). scRNA-seq of ADFs and nADFs revealed 5 major fibroblast clusters, including a Clu+ pro-fibrotic population and a Pi16+ progenitor population, which were enriched in bowel GFP+ ADFs (Figure 1G-H). Finally, quantification of gene expression revealed that GFP+ ADFs lacked mature adipocyte markers, expressed fibroblast markers, and were enriched for various ECM proteins.

CONCLUSIONS
An anti-mesenteric model of intestinal strictures and CF in combination with immunostaining and scRNA-seq revealed that ADFs from CF infiltrate the bowel wall and expand at the site of fibrosis. ADFs exhibit fibroblast heterogeneity and are enriched for pro-fibrotic and stromal progenitor populations. Finally, ADFs produce collagen and fibronectin, suggesting that they may be pro-fibrotic. Altogether, these data highly suggest that CF contains pro-fibrotic stromal cells that infiltrate the stricture site to promote fibrosis.
EPITHELIAL MITOCHONDRIAL DYSFUNCTION IN CHRONIC POUCHITIS
Authors: Patel Parth H , Elsayed Mohamed , Hassan Syed Adeel , ElSaadani Mohamed A , Kasem Ahmad , Wempe Lesley , Bhogoju Sarayu , Goretsky Tatiana , Lee Goo , Barrett Terrence , Kapur Neeraj 
Published: January 2024

Abstract

BACKGROUND
Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgical procedure of choice for patients with refractory ulcerative colitis (UC). However, patients with IPAA are at risk of pouch-related disorders like “pouchitis” with an incidence rate range from 20-50%. The underlying etiopathogenesis of pouchitis has been unclear, which makes management and treatment of active pouchitis difficult. Mitochondrial (Mito) dysfunction is commonly associated with pathogenesis of inflammatory bowel diseases (IBD). We posit the reduced Mito function contributes to chronic pouchitis in IPAA patients. In this study we performed comparative analysis of Mito complex levels in pouchitis patients compared to normal controls.

METHODS
Biopsies collected from ileal pouches +/- inflammation (n=6), and normal (n=3) and inflamed ileum (CD Ileitis; n=3 mean SES=5.2) were preserved in Allprotect® or formalin. Total RNA was extracted, cDNA synthesized and analyzed by RT-qPCR for genes specific for Mito biogenesis and complexes, cytokines and cellular antioxidant machinery. Formalin-fixed tissues were processed for IHC staining of Mito complex-I (NDUFB6) and complex-IV (MTCO1).

RESULTS
Transcriptomic analysis of active pouchitis tissue showed significant (**p&lt;0.01) reduction of Mito biogenesis genes (PGC1α: 69.5±13.9%, TFAM: 48.7±14.5%, NRF1:48.9±17.7%) compared to normal pouch. Similar mRNA analysis in CD-ileitis showed 90.6±15.2% reduction in PGC1α gene (**p&lt;0.01) and more than 62% reduction in TFAM and NRF1 genes compared normal ileum. We also observed 21.6±9.8% reduction in Mito complex I (Ndufa1, Ndufa4, Ndufb2, Ndufb6), 33.6±8.7% reduction in complex-IV (Cox5a Cox6a1, Cox5e) and 38.8±11.8% reduction in complex-V (ATP5a, ATP5b, ATP5e) in chronic pouchitis compared to normal controls. In contrast, CD-ileitis showed 59.5±10.2% reduction in Mito complexes at mRNA level compared to normal ileum. Altogether, CD-ileitis showed 26.3% and 23.8% reduction in Mito complexes and biogenesis genes, respectively, compared to pouchitis. At protein level, tissues from chronic pouchitis showed significantly reduced expression of NDUFB6 and MTCO1 compared to normal controls. However, the decreased expression of NDUFB6 and MTCO1 was more pronounced in active CD-ileitis patients. Concertedly, suppression of Mito biogenesis and complex genes correlated with upregulation of pro-inflammatory cytokines (IL1β and IL-6) and concomitant reduction of anti-oxidant genes (Sod1, Sod2, Gpx, Prdx-2,3,4,6).

CONCLUSION
These data indicate decreased levels of Mito complexes in pouchitis, although reductions are less prominent in active CD. We postulate that Mito dysfunction plays a role in the pathogenesis of chronic pouchitis after IPAA surgery in IBD patients. Furthermore, we posit mitochondria-targeted therapy may deliver therapeutic benefits in refractory pouchitis.
GRANZYME A-PRODUCING CD4 T CELLS ARE DRIVERS OF IMMUNE CHECKPOINT BLOCKADE-INDUCED COLITIS
Authors: Park Sungtae , Wang Mengbo , Anderson Nicole , Kazemian Majid , Olson Matthew 
Published: January 2024

Abstract
Immune checkpoint blockade (ICB) (i.e. anti-CTLA4 and/or anti-PD-1) is a type of cancer immunotherapy that is broadly and effectively used across multiple cancer types. Despite this efficacy, ~60% of individuals of ICB recipients develop autoimmune-like inflammation in the skin, lungs and intestines. Of these barrier sites, an inflamed intestine is the most common form of severe disease that requires cessation of immunotherapy and potential tumor rebound. ICB-induced colitis therefore represents a significant health concern in an increasing population of cancer patients receiving this otherwise effective immunotherapy. Previous work has shown evidence of a dysregulated intestinal CD4 T cell response in ICB colitis patients, however, how CD4 T cells might contribute to disease has remained unclear. Here, we identify a unique subset of T helper (Th) 1-like CD4 T cells that express the serine protease granzyme A (GZMA) that are specifically elevated in ICB-induced colitis patients, suggesting a pathogenic role for GZMA-expressing Th cells in disease.
As mouse models for ICB-induced colitis are virtually non-existent, we have profiled existing mouse models of intestinal inflammation for the presence of colonic GZMA-expressing CD4 T cells for more in-depth functional studies. Using this approach, we have identified the T cell transfer model of colitis as a model that mimics the GZMA CD4 T cell phenotype observed in human ICB colitis patients. Within this model, GZMA-expressing CD4 T cells arise early in the intestines after transfer and only minimally express other CD4 T helper (Th) cell lineage cytokines (i.e. IFN-γ or IL-17) indicating that these cells may represent a unique Th cell subtype that are associated with intestinal disease. Indeed, transfer of GZMA-deficient CD4 T cells into colitis-prone mice resulted in reduced intestinal inflammation and damage, despite efficient trafficking of these cells to the intestines. Mechanistically, CD4 T cell-derived GZMA promoted an exaggerated intestinal epithelial cell response to the microbiota, but was not required for immune-editing of the microbiota in this setting. Taken together, our data indicate that GZMA-producing Th cells are likely major contributors to ICB-induced colitis and may represent a valuable therapeutic target for limiting intestinal disease during ICB therapy.
IRE1A-XBP1 CONTROLS GROUP 2 INNATE LYMPHOID CELLS IN INTESTINAL INFLAMMATION AND FIBROSIS
Authors: Cao Siyan , Colonna Marco 
Published: January 2024

Abstract

BACKGROUNDS
Group 2 innate lymphoid cells (ILC2s) are increased in peripheral blood and intestinal samples from patients with ulcerative colitis or Crohn’s disease and murine models of IBD. ILC2s produce a large amount of type 2 cytokines including IL-5 and IL-13 and growth factor amphiregulin (AREG) that induce goblet cell hyperplasia and mitigate acute inflammation in the gut. On the other hand, prolonged or uncontrolled ILC2 activation may contribute to chronic inflammation and tissue fibrosis. However, the regulatory mechanisms and functional impact of ILC2s in IBD remains elusive. The IRE1a-XBP1 pathway orchestrates cellular stress response that plays a key role in intestinal inflammation.

METHODS
We generated conditional knockout Ire1aflo/floxIl5-Cre (Ire1aΔIl5) with Ire1a deleted in ILC2s. Rag-/-Ire1aΔIl5 mice were also generated to exclude possible contribution of T cells during chronic inflammation. Dextran sodium sulfate (DSS) was given in drinking water to induce acute/chronic colitis and intestinal fibrosis. We also collected colonic biopsies from healthy individuals and sort-purified ILC2s for flow cytometry analysis.

RESULTS
Mouse intestinal ILC2s express high level of Ire1a. The number of ILC2s increased in small intestine (SI) and colon of Ire1aΔIl5 mice compared to Il5-Cre controls. Additionally, SI and colonic Ire1aΔIl5 ILC2s produced more IL-5, IL-13, and AREG after ex vivo stimulation with IL-33 and IL-25. Upon DSS challenge, Ire1aΔIl5 mice exhibited milder signs of disease including weight loss, clinical scores as well as colon shortening and histology (Figure 1), which was accompanied by elevations in ILC2-derived cytokines and AREG as well as heightened goblet cell differentiation and mucin production. In response to 3 cycles of DSS, Rag-/-Ire1aΔIl5 mice developed worsened chronic colitis, fibrosis, and increased fibrosis markers including collagens and vimentin. Intraperitoneal injection of neutralizing antibody against IL-13 or AREG reversed the phenotype. In sort-purified human ILC2s, selective IRE1 inhibitor 4u8C enhanced production of IL-13, while selective IRE1 activator IXA4 suppressed IL-13 in response to IL-2, IL-7, and IL-33 ex vivo (Figure 2).

CONCLUSIONS
We demonstrate that IRE1a-XBP1 constrains cytokine production by intestinal ILC2s during inflammation. IRE1a deficiency in ILC2s protected against acute colitis with heightened type 2 cytokines and AREG; while loss of IRE1a exacerbated chronic colitis and fibrosis. We identified IRE1a as a potential therapeutic target for fibrostenotic CD.

Figure 1 Ire1aΔIl5 mice harbor elevated ILC2s (A) and produce increased IL-5, IL-13, and AREG by (B) SI and (C) colonic ILC2s ex vivo. (D-H) Ire1aΔIl5 mice are protected from acute DSS colitis. *P&lt;0.001.

Figure 2 IRE1 modulators control IL-13 production by human ILC2s. **P&lt;0.01.
NON-CASEATING GRANULOMAS ARE ASSOCIATED WITH STRICTURING CROHN’S DISEASE AND THEIR NUMBER AND SIZE ARE INCREASED IN THE MUSCULARIS PROPRIA OF STRICTURED COMPARED TO NON-STRICTURED INTESTINGAL SEGMENTS
Authors: Massey William , Li Jiannan , Goren Idan , Gordon Ilyssa , Rieder Florian 
Published: January 2024

Abstract

BACKGROUND
Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract that progressively leads to fibrosis-induced narrowing of the intestinal lumen, bowel obstruction, and need for surgery. CD exhibits transmural inflammation manifested by immune cell infiltrates composed of multiple immune cell types spanning the entire gut wall. Non-caseating granulomas are a typical hallmark of up to 60% of CD patients. Scarce evidence indicates an association of granulomas with post-operative recurrence suggesting a role of granulomas in disease pathogenesis. Strikingly, while evidence exists from other granulomatous diseases, granulomas have not been linked with stricturing CD.

METHODS
A total of 348 H&E stained sections from segments of small bowel CD surgical resection specimens were evaluated for non-caseating granulomas. Granulomas were defined by a trained inflammatory bowel disease pathologist as a cluster of ≥ 5 epithelioid histiocytes with or without the presence of multinucleated giant cells. CD segments were classified as non-involved (CDni), inflamed non-strictured (CDi), and strictured (CDs) to confirm whether histopathological presence of granulomas correlates with stricturing CD. Paired sets were available in 150 segments of 50 CD patients and analyzed further to determine whether granuloma number, location within the intestinal wall, or size of granulomas correlates with stricturing CD.

RESULTS
Granulomas were present in 20% of all CD tissue segments. 28% of CDs, 9% of CDi, and 5% of CDni segments were found to have granulomas and the fraction of segments containing granulomas was significantly higher in CDs compared to CDni and CDi (P&lt;0.0001). The paired patient sections resembled the larger dataset with 16% of patients having granulomas in at least one of the three segments (16% of CDs, 8% of CDi, and 0% CDni). Further comparing the CDi and CDs sections in this subset of patients, we found that neither the total number nor the average size of granulomas were significantly different between CDs versus CDi tissue sections. Analysis of the layers of the intestinal wall revealed that the number (P&lt;0.05) and average size (P&lt;0.01) of granulomas were significantly increased in the muscularis propria of CDs versus CDi sections. There were no significant changes in number or average size in the mucosal or submucosal layers of the intestinal wall.

CONCLUSION
Together these data indicate that non-caseating granulomas are associated with stricturing CD and these granulomas are increased in number and size in the muscularis propria of strictured versus inflamed tissues from the same patient, but not other intestinal layers. This suggests a potential link between granulomas and the pathogenesis of stricturing CD.
UPREGULATION OF SERPINE1/PAI-1 INDUCED BY IL-33 CONTRIBUTES TO INTESTINAL FIBROSIS IN A CROHN’S DISEASE-LIKE ILEITIS MODEL
Authors: De Santis Stefania , Vidmar Kaylynn , Williams Joseph , Mattioli Benedetta , Chieppa Marcello , Del Pinto Rita , Pietropaoli Davide , Corridoni Daniele , Cominelli Fabio , De Salvo Carlo , Pizarro Theresa 
Published: January 2024

Abstract
IL-33 is a pleiotropic cytokine known to possess dichotomous roles during gut health and disease. We previously described that IL-33 promotes epithelial restitution/repair in otherwise healthy (C57BL/6) mice that have been challenged with dextran sodium sulphate (DSS) to induce acute colitis, with the end result of efficient resolution of inflammation. However, in SAMP1/YitFc (SAMP) mice that spontaneously develop Crohn’s disease (CD)-like ileitis, elevated IL-33 levels persist as disease progresses, perpetuating chronic gut inflammation and severe fibrosis. While IL-33 has been implicated in the development of inflammation-associated fibrosis, the precise mechanism(s) by which this occurs remains unclear. The aim of this study was to determine IL-33-dependent events leading to intestinal fibrosis in ileitis-prone SAMP mice. Our results show IHC co-localization of IL-33 with fibrotic lesions, specifically in cells morphologically-consistent with both macrophages and subepithelial myofibroblasts (SEMFs). Bulk RNA-Seq analysis of the human SEMF cell line, CCD-18Co, stimulated +/- IL-33 identifies one of the highest-expressing transcripts as SERPINE1, encoding for plasminogen activator inhibitor-1 (PAI-1), which has previously been reported as highly-enriched in IBD patients with active disease that do not respond to anti-TNF therapy. Analysis of publicly-available scRNA-Seq data confirms the increased expression of SERPINE1 in IBD patients that localizes to mesenchymal cell populations. Of note, robust upregulation of SERPINE1 is detected in activated fibroblasts from involved vs. non-involved areas of CD patients. Furthermore, spatial transcriptomics of SAMP ilea reveal a progressive and concomitant increase in both Il33 and Serpine1, with strong clusterization compared to healthy AKR controls, particularly during later time points when fibrosis is evident. Currently, results are pending on the treatment of SAMP mice with the PAI-1 inhibitor, MDI-2268, to determine its direct effect(s) on the development of inflammation-associated intestinal fibrosis. Taken together, these findings suggest IL-33-dependent regulation of Serpine1/PAI-1 that promotes intestinal fibrosis, commonly observed in CD patients, and may provide a novel target to treat IBD patients with fibrostenoic disease.
A CONSORTIA OF CLINICAL E. COLI STRAINS WITH DISTINCT IN-VITRO ADHERENT/INVASIVE PROPERTIES ESTABLISH THEIR OWN CO-COLONIZATION NICHE AND SHAPE THE INTESTINAL MICROBIOTA IN INFLAMMATION-SUSCEPTIBLE MICE
Authors: Li Chuang , Bleich Rachel , Sun Shan , Ahn Ju-Hyun , Dogan Belgin , Barlogio Cassandra , Broberg Christopher , Bulik-Sullivan Emily , Carroll Ian , Simpson Kenneth , Fodor Anthony , Arthur Janelle 
Published: January 2024

Abstract
Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation and associated inflammatory bowel disease (IBD). However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner based on histological analysis and 16S sequencing. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.