Cystathionine Gamma-Lyase Regulates TNF-α-Mediated Injury Response in Human Colonic Epithelial Cells and Colonoids.

Cystathionine gamma-lyase (CSE) and TNF-α are now recognized as key regulators of intestinal homeostasis, inflammation, and wound healing. In colonic epithelial cells, both molecules have been shown to influence a variety of biological processes, but the specific interactions between intracellular signaling pathways regulated by CSE and TNF-α are poorly understood. In the present study, we investigated these interactions in normal colonocytes and an organoid model of the healthy human colon using CSE-specific pharmacological inhibitors and siRNA-mediated transient gene silencing in analytical and functional assays in vitro. We demonstrated that CSE and TNF-α mutually regulated each other's functions in colonic epithelial cells. TNF-α treatment stimulated CSE activity within minutes and upregulated CSE expression after 24 h, increasing endogenous CSE-derived H2S production. In turn, CSE activity promoted TNF-α-induced NF-ĸB and ERK1/2 activation but did not affect the p38 MAPK signaling pathway. Inhibition of CSE activity completely abolished the TNF-α-induced increase in transepithelial permeability and wound healing. Our data suggest that CSE activity may be essential for effective TNF-α-mediated intestinal injury response. Furthermore, CSE regulation of TNF-α-controlled intracellular signaling pathways could provide new therapeutic targets in diseases of the colon associated with impaired epithelial wound healing.

Identification of differentially expressed genes and splicing events in early-onset colorectal cancer.

Background: The incidence of colorectal cancer (CRC) has been steadily increasing in younger individuals over the past several decades for reasons that are incompletely defined. Identifying differences in gene expression profiles, or transcriptomes, in early-onset colorectal cancer (EOCRC, < 50 years old) patients versus later-onset colorectal cancer (LOCRC, > 50 years old) patients is one approach to understanding molecular and genetic features that distinguish EOCRC. Methods: We performed RNA-sequencing (RNA-seq) to characterize the transcriptomes of patient-matched tumors and adjacent, uninvolved (normal) colonic segments from EOCRC (n=21) and LOCRC (n=22) patients. The EOCRC and LOCRC cohorts were matched for demographic and clinical characteristics. We used The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) database for validation. We used a series of computational and bioinformatic tools to identify EOCRC-specific differentially expressed genes, molecular pathways, predicted cell populations, differential gene splicing events, and predicted neoantigens. Results: We identified an eight-gene signature in EOCRC comprised of ALDOB, FBXL16, IL1RN, MSLN, RAC3, SLC38A11, WBSCR27 and WNT11, from which we developed a score predictive of overall CRC patient survival. On the entire set of genes identified in normal tissues and tumors, cell type deconvolution analysis predicted a differential abundance of immune and non-immune populations in EOCRC versus LOCRC. Gene set enrichment analysis identified increased expression of splicing machinery in EOCRC. We further found differences in alternative splicing (AS) events, including one within the long non-coding RNA, HOTAIRM1. Additional analysis of AS found seven events specific to EOCRC that encode potential neoantigens. Conclusion: Our transcriptome analyses identified genetic and molecular features specific to EOCRC which may inform future screening, development of prognostic indicators, and novel drug targets.

Increased Activity of MAPKAPK2 within Mesenchymal Cells as a Target for Inflammation-Associated Fibrosis in Crohn's Disease.

BACKGROUND: Mesenchymal stromal cells are suggested to play a critical role in Crohn's disease [CD]-associated fibrosis. MAPKAPK2 [MK2] has emerged as a potential therapeutic target to reduce inflammation in CD. However, the cell-specific pattern of phospho-MK2 activation and its role in CD-associated fibrosis are unknown. The objectives of this study were to evaluate cell-specific changes in MK2 activity between predominantly inflammatory CD vs CD with fibrotic complications and define the role of stromal cell-specific MK2 activation in CD-associated fibrosis. METHODS: CD tissue, CD tissue-derived mesenchymal stromal cells known as myo-/fibroblasts [CD-MFs], and fibroblast-specific MK2 conditional knockout [KO] mice were used. RESULTS: In the inflamed area of predominantly inflammatory CD, high MK2 activity was equally distributed between mesenchymal and haematopoietic cells. By contrast, in CD with fibrotic complications, high MK2 activity was mostly associated with mesenchymal stromal cells. Using ex vivo CD tissue explants and an IL-10KO murine colitis model, we demonstrated that pro-fibrotic responses are significantly reduced by treatment with the MK2 inhibitor PF-3644022. Inhibition of MK2 activity in primary cultures of CD-MFs significantly reduced basal and TGF-ß1-induced profibrotic responses. Using fibroblast-specific MK2 knockout mice in chronic dextran saline sulphate colitis, we demonstrated that fibroblast intrinsic MK2 signalling is among the key processes involved in the chronic inflammation-induced profibrotic responses. CONCLUSIONS: Our data suggest that activation of MK2 within fibroblasts contributes to the chronic inflammation-induced fibrosis in CD and that targeting MK2 has potential for the development of novel therapeutic approaches for fibrosis in CD.

Loss of STIM2 in colorectal cancer drives growth and metastasis through metabolic reprogramming and PERK-ATF4 endoplasmic reticulum stress pathway.

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis.

Pyoderma Gangrenosum Is Associated With Increased Risk of Inflammatory Pouch-Related Complications: A Retrospective Cohort Study.

Background: Pyoderma gangrenosum (PG) is a rare, neutrophilic dermatosis that is a well-established extraintestinal manifestation (EIM) of inflammatory bowel disease. The clinical implications of developing PG in patients with ulcerative colitis (UC) who undergo total proctocolectomy colectomy and ileal pouch anal anastomosis (TPC-IPAA) surgery remain unknown. Methods: Study participants were selected from patients enrolled in the Carlino Family Inflammatory Bowel and Colorectal Disease Biobank between 1998 and 2021 with a pre-colectomy diagnosis of UC and who underwent TPC-IPAA surgery. A retrospective study comparing patients with PG and those without PG was performed. The outcomes measured included the development of pouchitis, pouchitis classification, presence of pouch fistula, anal fistula, anal stenosis, and pouch failure. Results: In this study, 357 IPAA patients were included, 10 of whom suffered PG. Patients with PG and without PG had similar demographics and clinical characteristics. Both groups had similar rates of pouchitis (80% in PG patients and 64% in patients without PG, P = .504). However, IPAA patients with PG had a higher risk of developing pouch fistula (50% vs 10%, P = .002), anal fistula (40% vs 12%, P = .031), and Crohn's-like disease of the pouch (70% vs 15%, P = .003) compared to patients without PG. Patients who developed PG prior to their first episode of pouchitis were more likely to eventually experience pouch failure (odds ratio: 20.7, 95% confidence interval: 3.9, 110.7, q = 0.003 after false discovery rate adjustment). Conclusions: Among UC patients who undergo TPC-IPAA surgery, the development of PG portends poor pouch outcomes and is predictive of pouch failure.

Control of Paneth cell function by HuR regulates gut mucosal growth by altering stem cell activity.

Rapid self-renewal of the intestinal epithelium requires the activity of intestinal stem cells (ISCs) that are intermingled with Paneth cells (PCs) at the crypt base. PCs provide multiple secreted and surface-bound niche signals and play an important role in the regulation of ISC proliferation. Here, we show that control of PC function by RNA-binding protein HuR via mitochondria affects intestinal mucosal growth by altering ISC activity. Targeted deletion of HuR in mice disrupted PC gene expression profiles, reduced PC-derived niche factors, and impaired ISC function, leading to inhibited renewal of the intestinal epithelium. Human intestinal mucosa from patients with critical surgical disorders exhibited decreased levels of tissue HuR and PC/ISC niche dysfunction, along with disrupted mucosal growth. HuR deletion led to mitochondrial impairment by decreasing the levels of several mitochondrial-associated proteins including prohibitin 1 (PHB1) in the intestinal epithelium, whereas HuR enhanced PHB1 expression by preventing microRNA-195 binding to the Phb1 mRNA. These results indicate that HuR is essential for maintaining the integrity of the PC/ISC niche and highlight a novel role for a defective PC/ISC niche in the pathogenesis of intestinal mucosa atrophy.

Alpha-tocopherylquinone-mediated activation of the Aryl Hydrocarbon Receptor regulates the production of inflammation-inducing cytokines and ameliorates intestinal inflammation.

This study investigated the role of Alpha-tocopherylquinone (TQ) in regulating the intestinal immune system and the underlying mechanisms. In the experimental dextran sodium sulfate and T cell-mediated colitis models, TQ significantly reduced the mRNA levels of interleukin (IL)-6, IL-1ß, IL-17A, IL-23, and tumor necrosis factor (TNF)-α and the abundance of proinflammatory macrophages, T helper (Th)17 cells, and ILC3s in the colons of wild-type mice. TQ also prevented lipopolysaccharide (LPS)-induced activation of NFκB and signal transducer and activator of transcription (Stat)-3 pathways in the human macrophage U937 cells. Pharmacological inhibition or CRISPR-Cas-9-mediated knockout of Aryl hydrocarbon Receptor (AhR) prevented the anti-inflammatory effects of TQ in the LPS-treated U937 cells. Furthermore, TQ reduced the mRNA levels of the LPS-induced pro-inflammatory cytokines in the WT but not Ahr-/- mice splenocytes. TQ also reduced IL-6R protein levels and IL-6-induced Stat-3 activation in Jurkat cells and in vitro differentiation of Th17 cells from wild-type but not Ahr-/- mice naive T cells. Additionally, TQ prevented the pro-inflammatory effects of LPS on macrophages and stimulation of T cells in human PBMCs and significantly reduced the abundance of tumor necrosis factor-α, IL-1ß, and IL-6hi inflammatory macrophages and Th17 cells in surgically resected Crohn's disease (CD) tissue. Our study shows that TQ is a naturally occurring, non-toxic, and effective immune modulator that activates AhR and suppresses the Stat-3-NFκB signaling.

Creating a Surgical Biobank: The Hershey Medical Center Experience.

BACKGROUND: Tissue harvesting at the time of surgery offers surgeons and scientists a unique opportunity to discover and better understand disease pathophysiology. Tissue biobanking presents challenges in patient consents, specimen collection, preparation, and storage, but the potential for scientific discovery justifies the effort. Although the number of tissue biobanks is increasing worldwide, information regarding necessary infrastructure, process flow, and management of expected obstacles is lacking. OBJECTIVE: To provide a framework and motivation for clinician scientists intending to start an intestinal tissue biobank under their direction. DATA SOURCES: The Carlino Family Inflammatory Bowel and Colorectal Diseases Biobank is housed at the Milton S. Hershey Medical Center. STUDY SELECTION: Review. INTERVENTION: Implementation of a surgical tissue biobank at a large tertiary care institution. MAIN OUTCOME MEASURES: Assess critical challenges and obstacles over the years as well as keys to the success of the program. RESULTS: Over 2 decades, the institutional biobank grew from an IBD biobank to one which now incorporates thousands of surgical specimens representing numerous colorectal diseases. This was done through a process of refinement focusing on patient recruitment and an efficient consenting and specimen management process. The biobank's success is further insured by institutional, external, and philanthropic support; scientific collaborations; and sharing of biological specimens with other groups of dedicated researchers. LIMITATIONS: This is a single-center experience in collecting surgically resected colorectal specimens. CONCLUSIONS: Surgical specimen biobanks are essential in studying disease cause using genomics, transcriptomics, and proteomic technologies. Therefore, surgeons, clinicians, and scientists should build biobanks at their institutions to promote further scientific discovery and improve specimen diversity.

The Microbiome of Complicated Diverticulitis: An Imbalance of Sulfur-Metabolizing Bacteria.

BACKGROUND: The progression to acute diverticulitis from the relatively benign condition of colonic diverticulosis is not well characterized. A smaller subset may even develop complicated (perforated) diverticulitis resulting in sepsis and/or death. Characterizing the differences between recurrent, uncomplicated diverticulitis, and the more virulent, complicated diverticulitis is necessary to guide clinical decision-making. Alterations to the microbiome offer a possible explanation for local inflammation and the pathophysiology of diverticular disease. OBJECTIVE: This study aimed to characterize the mucosal-associated microbiome in patients with recurrent uncomplicated diverticulitis and complicated (perforated) diverticulitis. DESIGN: Microbial DNA was extracted from full-thickness surgical specimens for 16S rRNA gene sequencing, targeting the V4 hypervariable region. Sequences were analyzed and a quantitative characterization based on taxonomic classification was performed. SETTING: A tertiary care academic medical center. PATIENTS: This study compared 48 patients with recurrent, uncomplicated diverticulitis and 35 patients with radiographically confirmed perforated (complicated) diverticulitis. Tissues were harvested from surgical resection specimens to include both diseased regions and nondiseased (adjacent normal) regions. MAIN OUTCOME MEASURES: We assessed differences in relative abundance and taxonomic classification of mucosal-associated microbes in surgical resection specimens from diverticular disease. RESULTS: When analyzing the tissue of diverticular resection specimens, the complicated diseased segments demonstrated an increased abundance of sulfur-reducing and sulfur-oxidizing bacteria compared to nondiseased, adjacent normal regions. When comparing diseased segments, tissues of patients with complicated diverticulitis had a marked increase in sulfur-reducing microbes. LIMITATIONS: We characterized the mucosal-associated microbiome present at the time of surgical resection, limiting conclusions on its role in pathophysiology. Furthermore, antibiotic usage and bowel preparation before surgery may result in perturbations to microbial flora. CONCLUSIONS: The microbiome of complicated diverticulitis is marked by a localized imbalance of sulfur-metabolizing microbes. The abundance of sulfur-reducing microbes may lead to an excess of hydrogen sulfide and subsequent inflammation. See Video Abstract at http://links.lww.com/DCR/C175 . LA MICROBIOMA DE LA DIVERTICULITIS COMPLICADA UN DESEQUILIBRIO DE LAS BACTERIAS METABOLIZADORAS DE AZUFRE: ANTECEDENTES: La progresión a diverticulitis aguda de la condición relativamente benigna de diverticulosis colónica no está bien caracterizada. Un subgrupo más pequeño puede incluso desarrollar diverticulitis complicada (perforada) que resulta en sepsis y/o muerte. Es necesario caracterizar las diferencias entre la diverticulitis recurrente no complicada y la diverticulitis complicada más virulenta para guiar la toma de decisiones clínicas. Las alteraciones del microbioma ofrecen una posible explicación de la inflamación local y la fisiopatología de la enfermedad diverticular.OBJETIVO: Caracterizar el microbioma asociado a la mucosa en pacientes con diverticulitis no complicada recurrente y diverticulitis complicada (perforada).DISEÑO: El ADN microbiano se extrajo de especímenes quirúrgicos de espesor completo para la secuenciación del gen 16S rRNA, dirigido a la región hipervariable V4. Se analizaron las secuencias y se realizó una caracterización cuantitativa basada en la clasificación taxonómica.AJUSTE: Un centro médico académico de atención terciaria.PACIENTES: Este estudio comparó 48 pacientes con diverticulitis recurrente no complicada y 35 pacientes con diverticulitis perforada (complicada) confirmada radiográficamente. Se recogieron tejidos de especímenes de resección quirúrgica para incluir tanto regiones enfermas como regiones no enfermas (normales adyacentes).PRINCIPALES MEDIDAS DE RESULTADO: Evaluamos las diferencias en la abundancia relativa y la clasificación taxonómica de los microbios asociados a la mucosa en muestras de resección quirúrgica de enfermedad diverticular.RESULTADOS: Al analizar el tejido de las muestras de resección diverticular, los segmentos enfermos complicados demostraron una mayor abundancia de bacterias reductoras de azufre y oxidantes de azufre en comparación con las regiones normales adyacentes no enfermas. Al comparar segmentos enfermos, los tejidos de pacientes complicados tenían un marcado aumento de microbios reductores de azufre.LIMITACIONES: Caracterizamos el microbioma asociado a la mucosa presente en el momento de la resección quirúrgica, lo que limita las conclusiones sobre su papel en la fisiopatología. Además, el uso de antibióticos y la preparación intestinal antes de la cirugía pueden provocar alteraciones en la flora microbiana.CONCLUSIONES: El microbioma de la diverticulitis complicada está marcado por un desequilibrio localizado de microbios metabolizadores de azufre. La abundancia de microbios reductores de azufre puede provocar un exceso de sulfuro de hidrógeno y la consiguiente inflamación. Consulte Video Resumen en http://links.lww.com/DCR/C175 . (Traducción-Dr. Ingrid Melo ).

TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells.

Mutations in components of the Wnt/ß-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.

Characteristics and Outcomes of Right- Versus Left-Sided Early-Onset Colorectal Cancer.

BACKGROUND: Early-onset colorectal cancers are increasing in incidence. Studies reported more left-sided cancers in patients aged <50 years. Some advocate for screening via flexible sigmoidoscopy at age 40 years. OBJECTIVE: The purpose of this study was to investigate characteristics and outcomes in sporadic right- and left-sided early-onset colorectal cancers. DESIGN: This was a retrospective cohort study. SETTINGS: This study was conducted at a single, tertiary care institution. PATIENTS: This study included patients aged <50 years diagnosed with colorectal cancer between 2000 and 2018. MAIN OUTCOME MEASURES: We analyzed patient demographics, tumor characteristics, and survival. RESULTS: A total of 489 patients aged 20 to 49 years were identified from 2000 to 2018. The majority of patients were white (90%) and male (57%). The median age at diagnosis was 44 years, and 75% were diagnosed at age 40-49 years. There was a predominance of left-sided tumors (80%). The majority of patients presented with stage 3 (35%) and stage 4 (35%) disease. Right-sided tumors were more likely to have mucinous (24% vs 7.4%; p < 0.001) and signet-ring cell (4.4% vs 1.7%; p < 0.001) histology. There was no difference in age, sex, race, ethnicity, and stage at presentation. Right-sided tumors were associated with lower 5-year overall survival (44% vs 61%; p < 0.005) with the decrease in survival most prominent in right-sided stage 3 tumors (41% vs 72%; p < 0.0001) and in ages 40 to 49 years (43% vs 61%; p = 0.03). Sex, tumor location, increasing stage, and signet-ring cell histology were independent prognostic factors of overall survival. There was no difference in disease-free survival. LIMITATIONS: This study was a retrospective review at a single institution. CONCLUSIONS: The majority of early-onset colorectal cancers arise from age 40 to 49 years with a left-sided predominance but higher mortality in right-sided tumors. These findings provide further evidence in favor of recommending earlier initial screening colonoscopy for colorectal cancer. See Video Abstract at http://links.lww.com/DCR/B892 . CARACTERSTICAS Y RESULTADOS DEL CNCER COLORRECTAL DE INICIO TEMPRANO DEL LADO DERECHO FRENTE AL IZQUIERDO: ANTECEDENTES:Los cánceres colorrectales de aparición temprana están aumentando en incidencia. Los estudios han informado una preponderancia de cánceres en el lado izquierdo en pacientes <50 años, lo que ha llevado a algunos a abogar por la detección con sigmoidoscopia flexible a los 40 años.OBJETIVO:El propósito de nuestro estudio fue investigar las características del tumor y los resultados de los pacientes en cánceres colorrectales esporádicos del lado derecho e izquierdo de aparición temprana.DISEÑO:Este fue un estudio de cohorte retrospectivo.ENTORNO CLÍNICO:Este estudio se realizó en una única institución de atención terciaria.PACIENTES:Pacientes <50 años diagnosticados de cáncer colorrectal entre 2000 y 2018.RESULTADO PRINCIPAL:Analizamos los datos demográficos de los pacientes, las características del tumor, la supervivencia general y la supervivencia libre de enfermedad.RESULTADOS:Se identificaron un total de 489 pacientes de entre 20 y 49 años entre 2000 y 2018. La mayoría de los pacientes eran blancos (90%) y varones (57%). La mediana de edad en el momento del diagnóstico fue de 44 años y el 75% se diagnosticó entre los 40 y los 49 años. Predominó los tumores del lado izquierdo (80%). La mayoría de los pacientes presentaban enfermedad en estadio 3 (35%) y estadio 4 (35%). Los tumores del lado derecho tenían más probabilidades de tener histología mucinosa (24% frente a 7,4%, p < 0,001) y de células en anillo de sello (4,4% frente a 1,7%, p < 0,001). No hubo diferencia en edad, sexo, raza, etnia, estadio AJCC en la presentación. Los tumores del lado derecho se asociaron con una menor supervivencia general a 5 años (44% frente al 61%, p < 0,005) con la disminución de la supervivencia más prominente en los tumores del lado derecho en estadio 3 (41% frente al 72%, p < 0,0001) y en edades 40-49 (43% vs 61%, p = 0.03). El sexo, la ubicación del tumor, el estadio AJCC en aumento y la histología de las células en anillo de sello fueron factores pronósticos independientes de la supervivencia general. No hubo diferencias significativas en la supervivencia libre de enfermedad.LIMITACIONES:Este estudio fue una revisión retrospectiva en una sola institución.CONCLUSIONES:La mayoría de los cánceres colorrectales de aparición temprana surgen entre los 40 y los 49 años con un predominio en el lado izquierdo pero una mayor mortalidad en los tumores del lado derecho. Estos hallazgos proporcionan evidencia adicional a favor de recomendar una colonoscopia de detección inicial más temprana para el cáncer colorrectal. Consulte Video Resumen en http://links.lww.com/DCR/B892 . (Traducción-Dr. Ingrid Melo ).

Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux.

BACKGROUND AND AIMS: Functional loss of the gut epithelium's paracellular tight junction [TJ] barrier and defective autophagy are factors potentiating inflammatory bowel disease [IBD]. Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier-forming proteins remains unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. RESULTS: Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier-enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In vivo, autophagy induction by rapamycin enhanced occludin levels in wild-type mouse intestines and protected against lipopolysaccharide- and tumour necrosis factor-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. CONCLUSION: Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 mitogen-activated protein kinase-dependent mechanism.

Th17 Cell-Derived Amphiregulin Promotes Colitis-Associated Intestinal Fibrosis Through Activation of mTOR and MEK in Intestinal Myofibroblasts.

BACKGROUND & AIMS: Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS: In this study, T-cell transfer model with wild-type (WT) and Areg-/- Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg-/- mice were used. CD4+ T-cell expression of AREG was determined by quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of AREG on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. AREG expression was assessed in healthy controls and patients with CD with or without intestinal fibrosis. RESULTS: Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrßxδ-/- mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of AREG than Th1 cells. Areg-/- mice developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg-/- Th17 cells induced less severe fibrosis in Tcrßxδ-/- mice compared with WT Th17 cells. Interleukin (IL)6 and IL21 promoted AREG expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. AREG promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mammalian target of rapamycin and MEK. AREG expression was increased in intestinal CD4+ T cells in fibrotic sites compared with nonfibrotic sites from patients with CD. CONCLUSIONS: These findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

Transcriptome Analyses Identify Deregulated MYC in Early Onset Colorectal Cancer.

Despite a global decrease in colorectal cancer (CRC) incidence, the prevalence of early-onset colorectal cancer (EOCRC), or those occurring in individuals before the age of 50, has steadily increased over the past several decades. When compared to later onset colorectal cancer (LOCRC) in individuals over 50, our understanding of the genetic and molecular underpinnings of EOCRCs is limited. Here, we conducted transcriptomic analyses of patient-matched normal colonic segments and tumors to identify gene expression programs involved in carcinogenesis. Amongst differentially expressed genes, we found increased expression of the c-MYC proto-oncogene (MYC) and its downstream targets in tumor samples. We identified tumors with high and low differential MYC expression and found patients with high-MYC tumors were older and overweight or obese. We also detected elevated expression of the PVT1 long-non-coding RNA (lncRNA) in most tumors and found gains in copy number for both MYC and PVT1 gene loci in 35% of tumors evaluated. Our transcriptome analyses indicate that EOCRC can be sub-classified into groups based on differential MYC expression and suggest that deregulated MYC contributes to CRCs that develop in younger patients.

Symptoms associated with healthcare resource utilization in the setting of inflammatory bowel disease.

Several symptoms have been connected to increased healthcare resource utilization (HRU) in the context of inflammatory bowel disease (IBD), including both Crohn's disease (CD) and ulcerative colitis (UC). This study was designed to investigate the prevalence of IBD-associated symptoms and to determine whether any are independently associated with HRU. We undertook a retrospective analysis of data related to consecutive IBD patient encounters from a tertiary care referral center between 1/1/2015 and 8/31/2019. Demographics, clinical activity, endoscopic severity, IBD-related symptom scores, anxiety and depression scores, and other key clinical data were abstracted. Four hundred sixty-seven IBD patients [247f.: 220 m; 315 CD, 142 UC and 11 indeterminate colitis] were included in this study. The most common symptoms were fatigue (83.6%), fecal urgency (68.2%) and abdominal pain (63.5%). Fatigue, abdominal pain, anxiety or depression, corticosteroids, and opioids were each positively associated with HRU, while NSAID and mesalamine use were inversely associated on bivariate analysis. The only factor that demonstrated a statistically significant association with HRU in the whole cohort on multivariable analysis was abdominal pain. Abdominal pain is independently associated with HRU and should be specifically screened for in IBD patients to identify individuals at risk of undergoing expensive interventions. This study also reinforces the importance of optimizing diagnostic and therapeutic management of abdominal pain in IBD.

Special Considerations of Anastomotic Leaks in Crohn's Disease.

Rates of anastomotic leak following intestinal resections in the setting of inflammatory bowel disease are significantly influenced by clinical characteristics. While the literature can be contradictory due to significant heterogeneity in the published data, several common themes appear to consistently arise. With respect to Crohn's disease, low serum albumin, preoperative abscess, reoperative abdominal surgery, and steroid use are associated with an increased risk of postoperative intra-abdominal septic complications. On the contrary, biologic therapy, immunomodulator use, and method of anastomosis appear not to confer increased anastomotic-related complications. Undoubtedly, a low rate of anastomotic leakage is inherent to procedures within colorectal surgery but diligent attention must be paid to identify, optimize, and, therefore, reduce known risks.

Polysubstance use in inflammatory bowel disease.

OBJECTIVES: We aimed to evaluate the incidence, predisposing factors and impacts of polysubstance use (PSU) (ie, the concurrent use or abuse of two or more drugs or substances) in inflammatory bowel disease (IBD). METHODS: Data of patients enrolled between 1 January 2015 and 31 August 2019 from a single tertiary care referral center were retrospectively collected. Patients' baseline and clinical characteristics and their antidepressant and/or anxiolytic medications were abstracted. Associations between PSU and patients' characteristics were analyzed. Multivariate logistic regression models were fit, incorporating significant clinical factors. RESULTS: Altogether 315 patients with IBD (166 women, 149 men; 214 with Crohn's disease and 101 ulcerative colitis) were enrolled. Of them, 66 (21.0%) exhibited PSU (CD 21.5%, UC 19.8%); 37.5% had moderate to severe disease activity, 34.3% with extraintestinal manifestations (EIM), 41.6% with an anxious or depressed state and 69.8% had used healthcare resources in the prior 12 months. Moreover, 71.2% used two substances, while 27.3% used three substances. In the total cohort, EIM (odds ratio [OR] 1.97, 95% confidence interval [CI] 1.14-3.34, P = 0.019) and antidepressant or anxiolytic use (OR 2.51, 95% CI 1.45-4.39, P < 0.001) were positively associated with PSU on multivariate analysis. PSU was associated with increased rate of IBD-associated imaging (57.6% vs 47.0%, P < 0.05). CONCLUSIONS: PSU is common in IBD. EIM, antidepressant and/or anxiolytic use and imaging studies were independently associated with PSU. This study reinforces the importance of screening patients with IBD for substance use, particularly those with EIM and using antidepressants and/or anxiolytics.

CD163L1+CXCL10+ Macrophages are Enriched Within Colonic Lamina Propria of Diverticulitis Patients.

BACKGROUND: Inflammation of diverticula, which are outpouchings of the colonic bowl wall, causes diverticulitis. Severe cases of diverticulitis require surgical intervention. Through RNA-seq analysis of intestinal tissues, we previously found that the innate immune response was deregulated in surgical diverticulitis patients. In that study, pro-inflammatory and macrophage markers were differentially expressed in the colons of diverticulitis versus control patients. Here we investigate CD163L1+ macrophages and the pro-inflammatory chemokine, CXCL10, in diverticulitis. MATERIALS AND METHODS: We assessed tissue from an uninvolved area adjacent to a region of the sigmoid colon chronically affected by diverticulitis and performed Spearman's correlation on transcripts associated with macrophage signaling. We identified altered CD163L1 and CXCL10 gene expression levels that we confirmed by RT-qPCR analysis on an independent cohort of diverticulitis patients and controls. We used immunofluorescence microscopy to localize CD163L1+ macrophages and CXCL10 levels in intestinal tissue and ELISA to measure CXCL10 levels in patient serum. RESULTS: We found a positive correlation between intestinal CD163L1 and CXCL10 gene expression and an increased number of CD163L1+ macrophages in the sigmoid colons of diverticulitis patients relative to controls (P = 0.036). Macrophages at the apices of colonic crypts expressed the chemokine CXCL10. Correspondingly, these diverticulitis patients also displayed heightened CXCL10 levels in their serum (P = 0.007). CONCLUSIONS: We identified a novel population of CD163L1+CXCL10+ macrophages in the colonic crypts of diverticulitis patients and demonstrated increased expression of serum CXCL10 in these patients. CXCL10 may serve as a prognostic biomarker to aid in clinical decision making for diverticulitis patients.

Impairment of Tissue-Resident Mesenchymal Stem Cells in Chronic Ulcerative Colitis and Crohn's Disease.

BACKGROUND AND AIMS: Little is known about the presence and function of tissue-resident mesenchymal stem cells [MtSCs] within the gastrointestinal mucosa in health and inflammatory bowel disease [IBD]. The contribution of MtSCs to the generation of inflammatory fibroblasts during IBD is also poorly understood. We hypothesized that IBD-MtSCs are impaired and contribute to the generation of the pathological myofibroblasts in IBD. METHODS: In a cohort of clinically and endoscopically active IBD patients and normal controls, we used quantitative RT-PCR and stem cell differentiation assays, as well as confocal microscopy, to characterize MtSCs. RESULTS: Expression of two stem cell markers, Oct4 and ALDH1A, was increased in the inflamed IBD colonic mucosa and correlated with an increase of the mesenchymal lineage marker Grem1 in ulcerative colitis [UC], but not Crohn's disease [CD]. Increased proliferation and aberrant differentiation of Oct4+Grem1+ MtSC-like cells was observed in UC, but not in CD colonic mucosa. In contrast to normal and UC-derived MtSCs, CD-MtSCs lose their clonogenic and most of their differentiation capacities. Our data also suggest that severe damage to these cells in CD may account for the pathological PD-L1low phenotype of CD myofibroblasts. In contrast, aberrant differentiation of MtSCs appears to be involved in the appearance of pathological partially differentiated PD-L1high myofibroblasts within the inflammed colonic mucosa in UC. CONCLUSION: Our data show, for the first time, that the progenitor functions of MtSCs are differentially impaired in CD vs UC, providing a scientific rationale for the use of allogeneic MSC therapy in IBD, and particularly in CD.