bluuebunny/crossref_metadata_2025 huggingface dataset:
COLITIS-ASSOCIATED COLON CANCER: EXPLORING THE DCLK1-S-MMP13-P53 AXIS
Authors: Yusuf Kafayat , Roy Badal , Umar Shahid
Published: January 2023

Chronic intestinal inflammation in patients with inflammatory bowel diseases (IBD) significantly increases the risk of colorectal cancer (CRC). Tuft cells in the intestine marked by doublecortin-like kinase-1 (DCLK1), are long-lived chemosensory cells with newly identified roles in colitis as epithelial ablation of DCLK1 worsens disease outcome. DCLK1 exists in long and short isoforms (DCLK1-L and DCLK1-S). Hypermethylation of DCLK1 alpha promoter encoding DCLK1-L particularly in CRC, allows switching to the DCLK1-S isoform that confers a more invasive tumor phenotype. In an infectious colitis model, we recently discovered that DCLK1-S is predominantly expressed in Ly6G+;MHCII- neutrophils that coincides with elevated levels of CXCL1/KC and MMP13 expression in the colon. In an unbiased molecular docking study, DCLK1’s kinase domain was found to interact with the catalytic domain of MMP13. MMP13 plays a crucial role in the modulation of extracellular matrix (ECM) degradation and cell-matrix interactions and wild type and mutant p53 differentially regulate MMP13 gene expression. Mutations in TP53 (tumor suppressor p53) are crucial for neoplasia initiation in IBD patients and are observed in 50-85% of colitis-associated colon cancers (CACCs). We further observed that p53 mutation status dictates DCLK1-S expression in colon cancer cells and in mice mutant for p53, expression of DCLK1-S and MMP13 correlated with neutrophilic inflammation and crypt hyperplasia. We will expand on these novel discoveries by employing high fidelity proteomic and phosphoproteomic studies and decipher how DCLK1-S-MMP13-p53 axes stimulate fibrosis, tumorigenesis, and tumor progression. These studies are expected to lead to therapeutic avenues to target CACCs.
METABOLOMICS ANALYSIS IDENTIFIED AN ORALLY ACTIVE METABOLITE FOR TREATING CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS
Authors: Koon Hon Wai
Published: January 2023

BACKGROUND

Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Surgical resection is used to treat intestinal strictures, but this approach may adversely affect the patients' quality of life. Based on the findings of our groups and others, we hypothesize that intestinal metabolites are associated with intestinal fibrosis. We aimed to discover novel anti-fibrogenic metabolites.

METHODS

We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in an inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic CSA13 treatment to identify mouse intestinal fibrosis-related metabolites.

RESULTS

Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of inosine. Compared to normal mice, TNBS-treated mice had reduced fecal levels of inosine. CSA13-exposed TNBS-treated mice had increased fecal inosine levels. Oral administration of inosine (10 mg/kg/day for 14 days) effectively reduced ileal fibrosis in fibrotic 40-week-old SAMP1/YitFc mice with 96-100% reduction of intestinal collagen mRNA expression and 87% reduction of overall ileal disease activity. Although inosine did not directly inhibit collagen expression in primary human ileal fibroblasts from stricturing CD patients (CD-HIF), it reduced collagen mRNA expression in fresh ileal tissues from stricturing CD patients. RNA-seq and proteomics identified that peroxiredoxin 2 (PRDX2) is negatively associated with intestinal strictures in CD patients. PRDX2 is a thiol-specific antioxidant gene that reduces peroxides to alcohols. Inosine increased the secretion of peroxiredoxin 2 (PRDX2) in primary human intestinal epithelial cells (HPEC). The inosine-mediated PRDX2 secretion was inhibited by the pretreatment of adenosine 2A receptor (A2AR) inhibitor ZM241385. PRDX2 reduced cell stress, induced cell stress-related protein (cyclooxygenase 2/COX-2), and inhibited collagen expression in the CD-HIF. Inhibition of COX2 by indomethacin reversed the anti-fibrogenic effect of PRDX2 in the CD-HIF. Inhibition of PRDX2 caused increased collagen expression in the CD-HIF. Lentiviral overexpression of Prdx2 ameliorated ileal fibrosis in the fibrotic SAMP1/YitFc mice. Fibrotic SAMP1/YitFc mice had reduced circulating free thiol, reflecting increased systemic oxidative stress. Inosine restored circulating free thiol levels in SAMP1/YitFc mice.

CONCLUSIONS

Inosine is an orally active anti-fibrogenic metabolite that promotes PRDX2 expression from intestinal epithelial cells. PRDX2 is a newly identified anti-fibrogenic protein, which may inhibit collagen expression by reducing cell stress.
SELECTION OF DIETARY FIBERS FOR GUT HEALTH BASED ON THEIR PHYSICOCHEMICAL PROPERTIES
Authors: Moncada Edward , Bulut Nuseybe , Li Shiyu , Hamaker Bruce , Reddivari Lavanya
Published: January 2023

Fiber under-consumption in the American population has been associated with a rise in several gastrointestinal diseases. In this study, we focused on Ulcerative Colitis (UC) which currently affects 1 million Americans needing effective preventive and treatment strategies. Short-chain fatty acid (SCFA) production by fiber fermentation is linked to an improved gut barrier function. However, physicochemical properties of fibers such as rate of fermentability, solubility, and structure complexity can influence SCFA levels and fiber tolerance by UC patients. The goal is to determine which fiber characteristics are associated with health benefits in UC patients.

Sixteen dietary fibers were selected based on different levels of solubility, rate of fermentation and structure complexity. Fibers were subjected to an in-vitro simulation of the upper gastrointestinal digestion before in-vitro fermentation using fecal samples from a healthy individual. Gas production, pH, and SCFA production were measured at different time points during the fermentation. Resistant Maltodextrins, Pectin, Inulin, and Wheat Bran were selected as high and low tolerable and efficient fibers for a second in-vitro fermentation using IBD patient fecal sample and an in-vivo study with IL 10-KO germ-free mice transplanted with fecal microbiome from a healthy individual. DNA was extracted from the in-vitro fecal samples and the 16S rRNA genes of the V3-V4 region were amplified to assess gut bacterial dysbiosis and modulation.

Results showed a relationship between fiber physicochemical properties and their roles in gut health. The fermentability rate is one of the major characteristics to be considered, since how fast fiber is fermented is correlated with gas production. SCFA and gas production levels differed significantly in in-vitro fermentation between healthy and IBD fecal samples within each fiber. Bacteria sequencing results showed that the α-and β-diversity were significantly different between IBD and healthy groups. Furthermore, α- diversity was not different between the donors within the fiber treatment, while β-diversity only differed among the fibers within the IBD donor. An increase in the relative abundance of bacteria from the Enterobacteriaceae family in the IBD group may explain the SCFA reduction and the role gut bacterial dysbiosis play in the utilization of these fibers and their potential health benefits. Initial data from the in-vivo study showed differential role of these fibers.

Understanding the connection between fiber properties, gut bacterial composition and fiber efficacy in the gut, can significantly aid in the design of clinical trials and future dietary recommendations. Positive impact of our findings will be evidence-based verification of fibers that are well tolerated by healthy individuals and UC patients for the prevention/protection of this disease.
DIFFERENTIAL ABUNDANCE OF THE FUNGAL MYCOBIOME IN PATIENTS WITH ULCERATIVE COLITIS
Authors: Hsia Katie , Kouhsari Laleh Montaser , Algarrahi Khalid , Fu May , Zhao Naisi , Chen Hannah , Michaud Dominique , Jangi Sushrut
Published: January 2023

BACKGROUND

The fungal mycobiome has been increasingly implicated in the pathogenesis of inflammatory bowel disease (IBD). Circulating antibodies to Saccharomyces antibodies are detected in 60-70% of Crohn’s disease and in 10-15% of patients with ulcerative colitis (UC). Specific fungal species, including Candida albicans, are increased in active IBD compared to healthy controls. Recently, UC patients with high fecal Candida prior to fecal microbial transplantation (FMT) were more likely to demonstrate robust treatment responses to transplant, with low Candida levels post-FMT predicting ameliorated inflammation in the recipient. Despite the accumulation of findings supporting the role of fungi in IBD, the mycobiome remains poorly characterized in UC. Few studies have examined changes in the UC mycobiome with respect to validated endoscopic indices or histologic parameters; furthermore, the effects of treatment exposure, including biologic medications, on fungal communities in UC are unknown.

AIMS

In this study, we aim to characterize the fungal mycobiome in UC patients with varying levels of endoscopic activity, endo-histologic activity, and treatment exposure.

METHODS

We performed a secondary analysis using the data extracted from the Crohn’s and Colitis Foundation’s Study of a Prospective Adult Research Cohort with IBD, which contains clinical, endoscopic, histologic, and metagenomic data. Using Internal Transcribed Spacer based deep sequencing of fungal rDNA from fecal samples, we classified sequences utilizing the UNITE fungal database and a fitted classifier. The R package DESeq2 was used to calculate the log-2 fold change of differential abundance.

RESULTS

We identified 504 unique fungal amplicon sequence variants across the cohort of 98 patients, belonging to phylum Ascomycota (71.5%), Basidiomycota (11%), Mucoromycota (0.16%), or unidentified (17.2%). Compared to endoscopic remission, patients with endoscopic activity (n=25) were enriched with Saccharomyces (4.31, p< 5 x 10-5) and Candida (2.53, p< 1 x 10-3). Similarly, compared to endo-histologic remission, patients with endo-histologic activity (n=19) were also enriched for Saccharomyces (2.71, p<0.05) and Candida (2.07, p=0.05). Biologic exposure favored the growth of Candida (p-adj < 1 x10-4). After adjusting for age, gender, and biologic exposure among patients with endoscopic activity, Saccharomyces (p-adj < 5 x 10-12) and Candida (p-adj< 1 x 10-8) remained enriched.

CONCLUSION

These data demonstrate that endoscopic and histologic inflammation in UC is associated with expansion of Saccharomyces and Candida compared to remission, even after controlling for exposure to biologic exposure. These findings are recapitulated during endo-histologic activity. The role of these fungal taxa as potential biomarkers and targets for personalized approaches to therapeutics in UC should be evaluated.
INNATE TL1A SIGNALING PROMOTES INTESTINAL NEUTROPHIL ACTIVATION AND COLITIS ASSOCIATED CANCER
Authors: Pires Silvia , Yang Wei , Longman Randy
Published: January 2023

Intestinal inflammation associated with chronic inflammatory bowel disease (IBD) increases the risk of developing colitis-associated cancer (CAC), but the cellular mechanisms driving CAC are not well defined. Polymorphisms in TNFSF15 (called TNF-like cytokine 1a or TL1A) are highly associated with IBD and the aim of this work is to evaluate a potential mechanistic role of TL1A in regulating CAC. Data from the human atlas protein database revealed that TL1A protein is present in 50% of colorectal cancer tissue samples and that high expression of TNFSF15 correlated with reduced survival. To test the functional role for TL1A in tumorigenesis, we used the well-established AOM/DSS model of CAC in mice deficient for the TL1A receptor (called death receptor 3 or DR3) and mice deficient for myeloid derived TL1A (CD11C cre+TNFSF15fl/fl). We found that both DR3-deficient mice and myeloid derived TL1A deficient mice had a significant reduction in tumor number compared to heterozygous littermate controls. To evaluate the contribution of innate lymphocytes, we used DR3-deficient mice on a RAG-deficient background in our AOM/DSS model. Even in the absence of B and T cells, DR3-deficient mice still showed significant reduction in tumor burden, suggesting innate lymphoid cell (ILC) contribution. DR3-deficient mice also revealed a significant reduction in neutrophil infiltration. Neutrophil depletion with α-Ly6G similarly resulted in a significant reduction of tumor numbers in our CAC model in a DR3 dependent manner. To test the direct regulation of neutrophils by ILCs, we establish a co-culture model. TL1A activated ILCs triggered neutrophil activation (CD177) and maturation (CD11B) which required GM-CSF. In vivo activation with a DR3 agonist confirmed intestinal ILC3 production of GM-CSF but also impacted bone marrow granulopoiesis by expanding both mature neutrophils and granulocyte-macrophage progenitors (GMPs).

CONCLUSION: Our data reveals a new link between intestinal ILC production of GM-CSF and neutrophil activation that promotes CAC and bone marrow granulopoiesis.
REPOGRAMING OF THE INTESTINAL IMMUNE CELL COMPARTMENT DEFINES THE RESPONSE TO AUTOLOGOUS STEM CELL TRANSPLANTATION IN CROHNS DISEASE
Authors: Guisado Daniela , Talware Sayali , Singh Shishir , Gold Stephanie , Mansi Moutasem , Fozilov Elbek , Etra Aaron , Cho Judy , Cohen Louis
Published: January 2023

INTRODUCTION

For the treatment of refractory Crohn’s disease (CD) autologous stem cell transplant (auto-SCT) is unparalleled in its ability to induce clinical and endoscopic remission.(1, 2) Auto-SCT is unique as a cellular therapy aimed to reset immune pathophysiology to a pre-disease state using hematopoietic stem cells. As such, the study of how the immune system responds to auto-SCT will provide unique insight into CD pathogenesis and treatment. To date, no studies in any cohort have defined the mucosal and peripheral immune response to auto-SCT. Here we report initial studies of high dimensional immune phenotyping of patients with CD during auto-SCT.

METHODS

Patients with CD were enrolled in a Phase IIa study of auto-SCT (NCT03219359). 14 patients were transplanted (2018-2022). Paired blood and intestinal samples were taken prior to transplant and 6 months post-transplant. Fresh leukocytes were isolated and analyzed by mass cytometry (CyTOF). Supervised clustering of immune cell populations using canonical markers was performed in parallel with unsupervised clustering by FlowSOM.(3)

RESULTS

After 6 months post-transplant,12/13 patients had an endoscopic response (↓SES-CD by 50%) and 10/13 patients were in endoscopic remission (SES-CD<4). Supervised clustering of major immune cell subsets demonstrated distinct site-specific responses to transplant in myeloid and lymphoid cell populations (Fig 1). Naïve CD4+ and CD8+ T cells universally decrease in number at 6 months post-transplant whereas all other B and T cell populations have discordant changes in the blood and intestine, especially naïve and transitional CD27- B cell populations which significantly increase in blood and decrease in the intestine. CD14+ populations are universally increased in number post-transplant with a specific increase in CD14+ CD206+ macrophages in the intestine. Unsupervised clustering of blood and intestinal immune cells resolve 100 cell clusters that correlate with canonical immune cell markers (Fig 2). Unsupervised analysis further highlights a significant increase in multiple intestinal CD14+CD206+ immune cell populations reflecting newly arrived and/or differentiating and mature monocyte derived macrophages. Principal component and hierarchical clustering analyses of immune cell clusters suggest a reprograming of the intestinal immune compartment post-transplant whereas changes in circulating immune cells populations fail to separate the pre and post-transplant states.

CONCLUSION

We demonstrate for the first time differences in the intestinal and peripheral immune response to auto-SCT. These studies highlight the changes in intestinal immune cell networks that define the transplant response perhaps through CD14+ cells.
NEW ONSET OF INFLAMMATORY BOWEL DISEASE IN A PATIENT WITH EOSINOPHILIC ESOPHAGITIS: 10-YEAR CONSTELLATION OF AUTOIMMUNE GASTROINTESTINAL DISORDERS
Authors: Guzman Adalberto , Ibarra Maria , Mishagina Irina , Ravichander Benjamin , Sangha Lillian
Published: January 2023

INTRODUCTION

Inflammatory bowel disease (IBD) is well known for concurrent immune-mediated disorders such as eosinophilic esophagitis, psoriasis, and rheumatoid arthritis.(1) For the past two decades, we are seeing an increased occurrence of eosinophilic esophagitis (EoE) in long standing IBD patients (2) (3) (4), but the question has arisen as to new onset IBD in chronic EoE patients. One study that involved 4814 patients demonstrated that the prevalence of eosinophilic esophagitis in IBD was 5 times higher compared to general population and EoE in IBD is 2 times higher than expected.(5) Multiple factors have been identified that may trigger the exact inflammatory response in IBD and EoE patients. Both diseases are associated with an exaggerated response to interleukins, especially IL-13 sharing cytokines and helper T cell mediated pathophysiology that is linked to disruption of the gastrointestinal tract barrier. (6) (7)

CASE DESCRIPTION

A 25-year-old healthy male presented back in 2012 with a history of three months of worsening dysphagia that resulted in 15-lb weight loss. Upper endoscopy showed esophagus with small caliber lumen, linear furrows, and thin concentric rings with microscopic evidence of eosinophilic esophagitis. Over the years he developed multiple episodes of persistent dysphagia, several endoscopies demonstrated pangastritis and duodenitis with a new pyloric and duodenal bulb stricture. Due to his remitting-relapsing course despite long-term medical treatment, patient underwent additional workup including an abdominal CT and upper endoscopy that showed worsening of the pyloric stricture that improved after balloon dilation. In 2020, the patient developed diarrhea, rectal bleeding, generalized abdominal pain and 21 lb weight loss despite medical treatment. Colonoscopy showed multiple erosions in the ileum and rectum not seen on previous studies. The biopsy indicated ulcerated mucosa with necrosis in the ileum and rectal area with mixed inflammatory infiltration with the presence of a non-caseating granuloma consistent with Chron’s disease.

DISCUSSION

It is unclear which disease manifest first or if it is a clustering and co-occurrence of diseases. The fact that both diseases share the same pathogenetic mechanism with overexpression of mucosal interleukin-13 (IL-13) causing loss of functionality of the gastrointestinal barrier could explain symptoms observed on both. Many studies demonstrated that IBD was diagnosed before EoE in almost 2/3 of the cases. The association of EoE-IBD is found significantly more in males and occurs at a younger age compared to IBD alone. Further studies will be needed in order to confirm the co-occurrence of diseases because of the increased risk of IBD-related complications and higher chance of requiring systemic corticosteroids and anti-TNF therapy in patients who have coexisting EoE-IBD.
A NOVEL DRUG THERAPY INDUCES MITOCHONDRIAL BIOGENESIS AND ATTENUATES COLITIS
Authors: Bhogoju Sarayu , Kapur Neeraj , Chaiswing Luksana , Kim Jong Hyun , Awuah Samuel , Goretsky Tatiana , Barrett Terrence
Published: January 2023

Mitochondrial dysfunction is a characteristic feature of intestinal epithelium cells (IEC) in IBD patients, associated with oxidative stress and impaired ATP production. We posit that disrupted mitochondrial bioenergetics impairs ulcer healing in IBD. In the current study, we tested a novel gold-based compound (AuPhos) that improves mitochondrial function by increasing mitochondrial biogenesis and antioxidant enzymes in IEC and human colonic biopsies.

METHODS: Normal human colonic epithelial cells (NCM460) were used to evaluate the effect of AuPhos on mitochondrial proteins and anti-oxidant proteins by western blotting in the presence or absence of low-dose TNF (1ng/ml). To model mitochondrial deficiency, TFAM (mitochondrial transcriptional factor A) was silenced (shTFAM) in NCM460 cells. These cells were treated with low-dose TNF to mimic inflammatory conditions in the human intestine. The mitochondrial mass was assessed by flow cytometry using mito tracker green. The transcriptional activity of genes associated with mitochondrial biogenesis, mitochondrial complexes, and anti-oxidants was evaluated using RT-qPCR in AuPhos or vehicle-treated (overnight) cells with or without TNF. Human colonic biopsy samples were collected in Allprotect, treated ex-vivo with vehicle or AuPhos (3h, 4oC), and processed for RNA isolation followed by RT-qPCR.

RESULTS: Biochemical and mass spectrometry analyses revealed that AuPhos localizes to the mitochondria. We show here that AuPhos increases mitochondrial mass and mRNA for mitochondrial complexes [complex IV (Cox6A1) and complex V (ATP5A1)] in NCM460 cells in TNF or vehicle-treated cells. Similarly, AuPhos increased protein levels of upstream regulators of mitochondrial biogenesis such as TFAM, PGC-1α, and Nrf2 compared to nontreated cells. Further protein analysis of AuPhos-treated cells showed that AuPhos increases downstream antioxidant markers such as manganese superoxide dismutase (MnSOD), peroxidane 3 (Prd3), glutathione peroxidase 4 (GPX4), thioredoxin reductase2 (Trx2), and catalase expression when compared to the TNF or control samples. Interestingly, expression of genes associated with mitochondria-specific (MnSOD) and cytoplasmic superoxide dismutase (CuZnSOD) were also significantly increased in AuPhos-treated shTFAM NCM cells compared to vehicle or TNF-treated cells. AuPhos substantially upregulates the expression of mtCO1, mtCO2, Cox6A1, ATP5A1, NRF1, MnSOD, GPX, PRDX-2,-4, and -6, in ex vivo treated human colonic biopsies compared to vehicle or TNF-treated samples.

CONCLUSIONS: The current data suggest that the AuPhos compound improves mitochondrial function by promoting the expression of mitochondrial-associated proteins and antioxidant enzymes (Figure 1). We believe AuPhos can be used as a potential therapeutic in treating ulcerative colitis and Crohn’s disease by restoring mitochondrial biogenesis.
TRANSFER RNA MODIFICATION CONTRIBUTE TO PATHOGENESIS OF INTESTINAL INFLAMMATION BY ALTERING EPITHELIAL BARRIERS
Authors: Zhang Jilei , Zhang Yong-guo , Xia Yinglin , Sun Jun
Published: January 2023

BACKGROUNDS

Transfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine (Q)-modification is a fundamental process for fidelity and efficiency of translation from RNA to proteins. In eukaryotes, tRNA-Q-modification relies on the intestinal microbial product queuine. Growing evidence indicates that tRNA modifications play important roles in human diseases, e.g., type 2 diabetes. Q depletion led to endoplasmic reticulum stress in mouse liver. Q-tRNA modifications are dynamic and highly variable depending on the developmental stages and species type and tumorigenesis. However, the health consequences of disturbed availability of queuine and altered Q-tRNA modification remain to be investigated. The effects and mechanisms of Q-tRNA in intestinal bowel diseases (IBD) are unknown.

METHODS

We explored the Q-tRNA-related tRNA synthetases and expression of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used several colitis mouse models, organoids, and cultured cells for loss- and gain-of-function studies to investigate the mechanisms of Q-tRNA modifications in intestinal barrier functions, proliferation, and inflammation.

RESULTS

In ulcerative colitis and Crohn’s disease patients, QTRT1 expression was significantly downregulated. Altered QTRT1-related metabolites were found in human IBD. The four Q-tRNA-related tRNA synthetases, asparaginyl-aspartyl-, histidyl-, and tyrosyl-tRNA synthetase, were decreased in IBD patients. This reduction was further confirmed in DSS-induced colitis and IL10-deficient mice. Reduced QTRT1 was significantly correlated with intestinal junctions, including downregulated β-catenin and upregulated Claudin-2. These alterations were confirmed in vitro by deleting QTRT1 from cells. Queuine treatment significantly enhanced cell junctional functions and reduced inflammation in epithelial cells.

CONCLUSION

tRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial barriers. Insights into Q-tRNA modification in regulating barrier functions would facilitate the development of targeted interventions for IBD through queuine and tRNA modifications.
GUT MICROBIAL METABOLISM OF 5-ASA IS PROSPECTIVELY ASSOCIATED WITH TREATMENT FAILURE IN PATIENTS WITH IBD
Authors: Mehta Raaj , Mayers Jared , Zhang Yancong , Bhosle Amrisha , Nguyen Long , Ma Wenjie , Ananthakrishnan Ashwin , Franzosa Eric , Balskus Emily , Chan Andrew , Huttenhower Curtis
Published: January 2023

BACKGROUND

Variation in clinical response to 5-aminosalicylic acid (5-ASA) has been attributed in part to its inactivation by gut microbes. Recently, in the Inflammatory Bowel Disease (IBD) Multi’omics Database (IBDMDB), a multicenter year-long cohort of 100+ participants with IBD, we identified 12 gut microbial enzymes from two protein families that convert 5-ASA to N-acetyl 5-ASA, a compound that lacks anti-inflammatory effects. Within the IBDMDB, we then found that a subset of these enzymes was cross-sectionally linked with greater risk of treatment failure, defined by corticosteroid use. The aim of this study was to prospectively associate these drug-degrading gut microbial enzymes with treatment failure and to confirm in vitro conversion of 5-ASA by these enzymes.

METHODS

We examined the prospective association between gut microbial acetyltransferases with risk of 5-ASA treatment failure in the ongoing “Study of a Prospective Adult Research Cohort with IBD” (SPARC IBD). We included 208 participants who at study entry 1) gave a stool sample at baseline, 2) were on 5-ASA, and 3) were steroid-free. Fecal metagenomic data was processed by the same analysis pipeline (biobakery3) as in the IBDMDB. Exposure was defined identically as in the IBDMDB as metagenomic carriage of 3-4 drug-degrading acetyltransferases compared to 0-2 acetyltransferases. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) of incident steroid use using multivariable generalized estimating equations, adjusting for age and sex. For biochemical characterization, the 12 enzymes identified in the IBDMDB were heterologously expressed by E. coli. A representative enzyme was selected from each protein family for purification, and then incubated with 5-ASA and acetyl CoA for 6 hr at 37C with 1 mM of each substrate and 50 μM enzyme. Using a custom LC–MS assay, we then detected N-acetyl 5-ASA production.

RESULTS

Over a median follow-up of 8 months, we identified 60 cases of corticosteroid use. Consistent with data from the IBDMDB, we found that metagenomic carriage of 3 or more microbial acetyltransferase genes in SPARC IBD (compared to 2 or fewer) was associated with treatment failure (OR 2.77, 95% CI 1.03-7.43). In a random effects meta-analysis of the two cohorts, we observed a three-fold increased risk of drug failure (OR 3.12, 95% CI 1.41-6.89) (Fig 1). In vitro biochemical experiments confirmed the ability of a thiolase and an acyl-CoA N-acetyltransferase to acetylate 5-ASA, generating product at levels consistent with >25% conversion (Fig 2).

CONCLUSIONS

We characterized two gut microbial protein families that are directly involved in 5-ASA metabolism and, in turn, are prospectively associated with 5-ASA treatment failure. These findings advance the possibility of microbiome-based personalized medicine for patients with IBD.
MUCOSAL IMMUNE SIGNATURES OF PERIANAL FISTULIZING CROHN’S DISEASE BY MASS CYTOMETRY
Authors: Cao Siyan , Escudero Guadalupe Oliva , Deepak Parakkal , Cella Marina , Colonna Marco
Published: January 2023

BACKGROUNDS

Perianal fistulas occur in ~30-40% of patients associated with high morbidity and an impaired quality of life. The management of perianal fistulizing CD is a major clinical challenge, where its underlying etiology of is poorly understood.

METHODS

We recruited patients with a history of: 1) CD with perianal fistulas; 2) CD without perianal involvement; 3) idiopathic perianal fistulas. Biopsies were taken from the fistula tracts, external opening of the fistulas, and rectal mucosa during examination under anesthesia or colonoscopy. Isolated mucosal immune cells were analyzed using mass cytometry. Data was analyzed using viSNE for dimensionality reduction.

RESULTS

We have revealed previously unknown mucosal immune landscapes in perianal CD using mass cytometry. Key cell populations with significant difference in frequencies are summarize in Table 1 (data of external fistula opening not shown due to limited space): 1) CD45RO+ T cells including Tregs are elevated in CD fistulas compared to those from idiopathic fistulas, which is consistent with a prior histological study. The expression of co-inhibitory receptor TIGIT and co-stimulatory receptor CD226 are increased in Tregs in CD fistulas, although their function in IBD and especially perianal CD remains unclear. 2) Perianal CD is associated with increased Th17 cells in the fistula tracts and elevated IL-17-producing CD8 T cells (Tc17) in the rectum, which highlight the importance of IL-17 signaling in perianal CD. 3) CD fistulas show increased CD39 and decreased CD127 (IL-7Ra) in both CD4 and CD8 T cells. CD39 and CD127 are markers of T cell exhaustion, which may control the pathogenesis of perianal CD by regulating cytokine production. 4) Total B cells are elevated in CD fistulas compared to idiopathic fistulas. In contrast, regulatory B cells (Bregs), with immunomodulatory function in the gut, are dramatically diminished in CD fistulas compared to idiopathic fistulas. 5) Besides adaptive cells, CD3-CD19- innate immune cells including innate lymphoid cells (ILCs), macrophages, and NK cells are also skewed in perianal CD. Fistula tracts, external fistula opening (data not shown), and rectum of perianal CD all show dramatically elevated CD172+TREM1+ macrophages, which were previously found to promote chronic inflammation in luminal CD.

CONCLUSIONS

The immune responses in perianal CD demonstrate unique features that are not present in luminal CD (summarized in Figure 1). This indicates that perianal CD represents an immune identity distinct from luminal CD in the intestine. Importantly, altered T cell exhaustion markers (CD39 and CD127) and increased CD172+TREM1+ pathogenic macrophages are present in all three locations (fistula tracts, external opening, and rectum) of perianal CD, suggesting their roles in the pathogenesis of perianal CD and may become novel therapeutic targets.
PRO-INFLAMMATORY SIRPα-EXPRESSING MONOCYTES AND MACROPHAGES MEDIATE ANTI-TNF REFRACTORY CROHN'S DISEASE IN CHILDREN
Authors: Geem Duke , Maddipatla Sushma , Pelia Ranjit , Venkateswaran Suresh , Matthews Jason , Anbazhagan Murugadas , Kolachala Vasantha , Dodd Anne , Kugathasan Subra
Published: January 2023

BACKGROUND

Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract with detrimental consequences on the pediatric population. Anti-tumor necrosis factor (aTNF) therapies are the only FDA-approved biologics for pediatric CD but one-third fail anti-TNF induction, and of those who initially respond, approximately 15% relapse every year. Consequently, there is a pressing need to elucidate the underlying immunogenetic mechanisms of anti-TNF refractory CD.

METHODS

A prospective cohort study was employed of treatment-naïve (TN-CD; n= 12), aTNF refractory (REF-CD; n=11), and endoscopic remission CD (REM-CD; n=16) along with non-IBD control (n=13) children. High dimensional flow cytometric analyses (HD FACS) of the blood, ileal and rectal biopsies were performed and analyzed by t-distributed stochastic neighbor embedding (t-SNE) clustering. Single-cell RNA sequencing (scRNA-seq) was conducted using the 10X Chromium platform with a 3’ reagent kit and sequenced on Illumina’s Novaseq600. The Seurat v4.1 workflow was used for the UMAP cell clustering algorithm. Effects of conditioned media (CM) from SIRPα cells on ileal organoids from non-IBD controls was assessed by enriching blood SIRPα+ cells from REF-CD via magnetic activated cell-sorting and stimulating with LPS (10 ng/mL) and flagellin (FLG; 10 ng/mL) for 96 hrs in culture. CM was collected and ileal organoids were treated for 72 hrs and re-stimulated with LPS+FLG followed by viability and intracellular cytokine staining. Descriptive statistics, one-way ANOVA with multiple comparisons testing, and univariate linear regression were utilized.

RESULTS

HD FACS revealed significant enrichment of SIRPα+CD11c+HLA-DR+CD64+ (SIRPα) cells in the ileum, rectum, and blood of REF-CD (P< 0.05). These cells expressed pro-inflammatory cytokines, IL-1β, IL-6, and TNFα. Deeper characterization with scRNA-seq revealed SIRPα cells to be macrophages based on expression of: CD14, FCGR3A, CD68, CD163, FCGR1A, and MRC1. In addition to pro-inflammatory cytokines detected by HD FACS, SIRPα macrophages expressed TGFB1 and CXCL8. There were significant positive correlations of intestinal SIRPα macrophages with fecal calprotectin (P<0.05) and CRP (P<0.05). CM from SIRPα+ cells of REF-CD induced significant cell death and intracellular cytokine expression of TNFα and IL-1β from non-IBD ileal organoids.

CONCLUSION

SIRPα cells are a distinct pro-inflammatory macrophage population enriched in the blood and intestinal lamina propria of REF-CD that may mediate resistance to anti-TNF therapy via secretion of pro-inflammatory cytokines and disruption of the intestinal epithelial barrier. These findings provide mechanistic insight into pathogenesis of anti-TNF refractory CD in children and highlight these cells to be a potential therapeutic target.
GROWTH OUTCOMES OF IV ANTI-TNF BIOSIMILARS IN PEDIATRIC INFLAMMATORY BOWEL DISEASE
Authors: McClinchie Madeline , Lakhani Alyshah , Abdel-Rasoul Mahmoud , McNicol Megan , Shaikhkhalil Ala , Boyle Brendan , Maltz Ross
Published: January 2023

BACKGROUND

Biosimilars are biologic products similar in safety and efficacy to the originator product. Tumor necrosis factor-alpha inhibitors (anti-TNFs) are used in the treatment of inflammatory bowel diseases (IBD). There are limited studies evaluating the long-term outcomes of anti-TNF biosimilars in children with IBD. Growth is an important clinical outcome, especially in childhood-onset IBD. However, no study to date has compared growth outcomes in patients initiated on the IV anti-TNF originator as compared to a biosimilar.

METHODS

This was a single-center retrospective review of patients with IBD, younger than 16 years old, and initiated on the IV anti-TNF originator or a biosimilar between April 2016 and February 2021 at Nationwide Children’s Hospital. To be eligible for the study and evaluate the primary outcome, patients must remain on the IV anti-TNF therapy for at least 12-months. We used propensity score methodology to identify 37 pairs of matched patients based on baseline characteristics (age of anti-TNF initiation, disease (CD vs UC/IC), behavior, location, perianal type, race). Demographic data, laboratory values, disease activity scores, and growth values (z-scores were recorded for weight, height, and BMI) were collected at baseline (prior to anti-TNF initiation) and 6-months and 12-months post initiation. Linear mixed models with random intercepts accounting for within patient correlation of measurements were fit to test differences in measures over time and between study groups. Hypothesis testing was conducted at an alpha of 0.05 to be used for all statistical analyses.

RESULTS

There were 113 patients on the originator and 39 patients on a biosimilar who met our eligibility criteria. Nineteen percent of patients on the originator and 16% of patients on a biosimilar were excluded from analysis due to discontinuing the IV anti-TNF agent prior to 12 months. Weight, height, and BMI z-scores increased over time (from baseline to 12-months) for both groups (p<0.05). There was not a significant difference in the rate of change for weight, height, or BMI z-scores between study groups. Clinical outcomes of lab values (albumin, c-reactive protein, and hemoglobin) were not statistically different in the rate of change from baseline to 12-months between patients initiated on the originator or biosimilar. There also was not a significant difference in the pediatric Crohn’s disease activity index, pediatric ulcerative colitis activity index, or the physician global assessment between patients initiated on the biosimilar or originator from baseline to 12-months.

CONCLUSIONS

There were similar improvements in growth and clinical outcomes in patients initiated on the IV anti-TNF originator compared to a biosimilar agent. This is the first study to evaluate whether anti-TNF biosimilars have similar growth outcomes in children with IBD.
PATIENT PERSPECTIVE OF BOWEL URGENCY AND BOWEL URGENCY-RELATED ACCIDENTS IN CROHN’S DISEASE
Authors: Jairath Vipul , Hunter Theresa , Moses Richard , Bleakman Alison Potts , Chatterton Kaitlin , Medrano Paolo , Walker Marisa , McLafferty Megan , Klooster Brittany , Delbecque Laure
Published: January 2023

OBJECTIVE

This study aimed to explore the Crohn’s disease (CD) patient experience of Bowel Urgency (BU) and BU-related accidents to identify what is most relevant and important to patients.

METHODS

Patients in the US were eligible to participate in this qualitative study if they were ≥18 years of age, had a diagnosis of moderate to severe CD for ≥6 months and had experienced BU in the last 6 months. Interviews were conducted to explore the signs, symptoms, and impacts related to BU and BU-related accidents. Specifically, patients were asked to describe BU and BU-related accident experiences of CD in their own words. Transcripts were coded and analyzed in ATLAS.ti 9 using a systematic thematic analysis.

RESULTS

A total of 15 patients participated; majority were female (53%), white (53%), receiving biologic and/or conventional therapy (87%), and mean age of 50 yrs. A majority of patients (80%) experienced BU at least once in the last week and 20% had experienced ≥1 BU-related accident in the last month. The time-sensitive need to use or be near the bathroom, uncertainty of BU, and fear of experiencing an accident were reported as the most bothersome aspects of the BU experience.

Patients reported 16 signs and symptoms that co-occur with BU, with abdominal pain (87%), fatigue (87%), and abdominal cramping (53%) being most frequently reported (Table 1). Patients reported 36 BU-related impact concepts across 16 domains (Table 1). Impact domains most frequently reported were adaptive behaviors (93%), household chores (87%), and social activities (87%). Impact concepts most frequently reported were dietary changes (87%) and inability to participate in social activities (87%). Inability to participate in family activities, interrupted work activities, and inability to participate in social activities were reported as the most bothersome impacts to patients.

All 15 patients reported ever experiencing BU-related accidents. Patients reported 12 signs and symptoms that co-occur with BU-related accidents, with abdominal pain (67%), fatigue (67%), and abdominal cramping (60%) being most frequently reported (Table 2). Patients reported 44 impacts of BU-related accidents across 16 domains (Table 2). Impact domains most frequently reported were adaptive behaviors (100%) and emotional functioning (87%). Impact concepts most frequently reported were dietary changes (80%) and embarrassment (60%).

CONCLUSION

This study revealed that the experience of BU and BU-related accidents is bothersome to patients with CD, impactful on their daily lives, and occurs despite the use of biologic and/or conventional therapy.
PATIENT PERSPECTIVES OF BOWEL URGENCY AND BOWEL URGENCY-RELATED ACCIDENTS IN ULCERATIVE COLITIS
Authors: Jairath Vipul , Hunter Theresa , Moses Richard , Bleakman Alison Potts , Chatterton Kaitlin , Medrano Paolo , Hoffman Ryan , Walker Marisa , McLafferty Megan , Klooster Brittany , Delbecque Laure
Published: January 2023

OBJECTIVE

This study aimed to explore the Ulcerative Colitis (UC) patient experience of Bowel Urgency (BU) and BU-related accidents to identify what is most relevant and important to patients.

METHODS

Patients in the US were eligible to participate in this qualitative study if they were ≥18 years of age, had a diagnosis of moderate to severe UC for ≥6 months and had experienced BU in the last 6 months. Interviews were conducted to explore the signs, symptoms, and impacts related to BU and BU-related accidents. Transcripts were coded and analyzed in ATLAS.ti 9 using a systematic thematic analysis.

RESULTS

15 patients with moderate to severe UC participated; majority were female (53%), white (80%), receiving biologic and/or conventional therapy (80%), and had a mean age of 51.6 years. A majority of patients (80%) indicated that they had experienced BU at least once in the last week and 33% indicated that they had experienced at least 1 BU-related accident in the last month.

Participants reported a total of 13 sign and symptom concepts that co-occur with BU, with abdominal pain (87%), fatigue (80%), abdominal cramping (67%), and diarrhea (33%) being the most frequently reported (Table 1). Abdominal pain (40%) and abdominal cramping (40%) were frequently reported as the most bothersome symptoms of BU to participants. A total of 32 BU-related impact concepts were reported by participants across 16 impact domains (Table 1). The impact domains most frequently reported by participants were adaptive behaviors (100%), emotional functioning (87%), and social activities (67%). The most frequently reported impact concepts were dietary changes (73%), inability to participate in social activities (67%), and difficulty running errands (60%). Inability to participate in social activities (27%) and bathroom mapping (20%) were frequently reported as the most bothersome impacts to participants.

A total of thirteen (87%) participants reported experiencing BU-related accidents. Of the 7 signs and symptoms reported by participants to co-occur with BU-related accidents, the most frequently reported were abdominal pain (77%), gas (77%), and abdominal cramping (69%) (Table 2). A total of 30 impact concepts of BU-related accidents were reported by participants across 13 impact domains (Table 2). The domains most frequently reported by participants were adaptive behaviors (100%), emotional functioning (69), and social activities (62%). The most frequently reported impact concepts were bathroom mapping (62%), inability to participate in social activities (62%), and embarrassment (54%).

CONCLUSION

This qualitative study revealed that BU, BU-related accidents, and BU co-occurring symptoms are bothersome, impactful on participants’ daily lives, and occur despite the use of biologic and/or conventional therapy.
BUTYRATE-INDUCED MITOCHONDRIAL FUNCTION IMPROVES BARRIER FUNCTION IN INFLAMMATORY BOWEL DISEASE (IBD)
Authors: Conder Evan , Shay Henry C , Vekaria Hemendra , Erinkitola Iyabo , Bhogoju Sarayu , Goretsky Tatiana , Sullivan Patrick , Barrett Terrence , Kapur Neeraj
Published: January 2023

BACKGROUND

Short-chain fatty acids (SCFAs), end-product of gut microbial metabolism, play an important role in colonic homeostasis. Butyrate is a prime SCFA that fuels intestinal epithelial cells (IECs). However, this energy source is deficient in inflammatory bowel disease (IBD) due to dysbiotic microbiota. In this study, we posit that butyrate supplementation drives mitochondrial respiration induced barrier function in IBD.

METHODS

Normal human Colon mucosal (NCM460) epithelial cells were treated overnight with different concentration of sodium butyrate (SB; 0.5-5.0mM) with or without TNF (1ng/ml). To model mitochondrial deficiency, TFAM (mitochondrial transcriptional factor A), was silenced (shTFAM) in NCM460 cells. For mitochondrial respiration assessment, NCM cells were maintained and treated with SB (-/+TNF) under glucose-free medium and Oxygen consumption rate (OCR) was measured using high-throughput Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies). Gene expression analysis was determined by RT-qPCR from total RNA isolated from untreated and treated NCM cells as well as in mitochondria-deficient shTFAM NCMs compared to shControl cells. All assays were performed in triplicate and fold changes were calculated using the ΔΔCT method.

RESULT

Seahorse analysis showed significant increase in Maximal respiratory capacity, spare respiratory capacity and ATP production at sub-optimal (2mM) and optimal (5mM) SB concentrations, suggesting a SB induced mitochondrial respiration and capacity to overcome oxidative stress. At molecular level, SB significantly increased the expression of genes associated with mitochondrial biogenesis (TFAM), complex I (Ndufa1, Ndufa4, Ndufa6), complex IV (Cox6A1) and complex V (ATP5E, ATP8), at optimal 5mM concentration. The effect of SB was more pronounced under inflammatory condition when NCM cells were treated in the presence of low dose TNF. Further transcriptomic analysis of TFAM-deficient NCMs demonstrated significant decrease in mRNA of major butyrate transporters (MCT1, MCT4, ABCG2) compared to TFAM-proficient shControl cells, irrespective of any treatment. Similar decreased expression pattern was observed for tight junction genes (ZO1 and Occludin) in shTFAM compared to shControl cells. However, butyrate transporters and ZO1 mRNA increased significantly in both shTFAM and shControl cells after SB treatment at sub-optimal concentration (2mM) in the presence of TNF. Interestingly, only shTFAM cells showed increase in transcript level of Occludin after SB+TNF treatment, with no effect in shControl.

CONCLUSION

These data suggest that butyrate metabolism is a critical player in promoting oxidative health of mitochondria, which further drives transporter-mediated butyrate utilization and promotes barrier integrity under inflammatory conditions as in IBD.