Tofacitinib Downregulates TNF and Poly(I:C)-Dependent MHC-II Expression in the Colonic Epithelium.

Major Histocompatibility Complex (MHC)-I and -II genes are upregulated in intestinal epithelial cells (IECs) during active inflammatory bowel diseases (IBD), but little is known about how IBD-relevant pro-inflammatory signals and IBD drugs can regulate their expression. We have previously shown that the synthetic analog of double-stranded RNA (dsRNA) Polyinosinic:polycytidylic acid (Poly(I:C)), induces interferon stimulated genes (ISGs) in colon organoids (colonoids). These ISGs may be involved in the induction of antigen presentation. In the present study, we applied colonoids derived from non-IBD controls and ulcerative colitis patients to identify induction and effects of IBD-drugs on antigen presentation in IECs in the context of Tumor Necrosis Factor (TNF)-driven inflammation. By RNA sequencing, we show that a combination of TNF and Poly(I:C) strongly induced antigen-presentation gene signatures in colonoids, including expression of MHC-II genes. MHC-I and -II protein expression was confirmed by immunoblotting and immunofluorescence. TNF+Poly(I:C)-dependent upregulation of MHC-II expression was associated with increased expression of Janus Kinases JAK1/2 as well as increased activation of transcription factor Signal transducer and activator of transcription 1 (STAT1). Accordingly, pre-treatment of colonoids with IBD-approved pan-Janus Kinase (JAK) inhibitor Tofacitinib led to the downregulation of TNF+Poly(I:C)-dependent MHC-II expression associated with the abrogation of STAT1 activation. Pre-treatment with corticosteroid Budesonide, commonly used in IBD, did not alter MHC-II expression. Collectively, our results identify a regulatory role for IBD-relevant pro-inflammatory signals on MHC-II expression that is influenced by Tofacitinib.

Mucosal and faecal neutrophil gelatinase-associated lipocalin as potential biomarkers for collagenous colitis.

BACKGROUND: Collagenous colitis (CC) is an inflammatory bowel disease where chronic diarrhoea is the main symptom. Diagnostic markers distinguishing between CC and other causes of chronic diarrhoea remain elusive. This study explores neutrophil gelatinase-associated lipocalin (NGAL) and its mRNA lipocalin2 (LCN2) as histological and faecal disease markers in CC. METHODS: NGAL/LCN2 were studied in colonic biopsies from CC patients before and during budesonide treatment using RNA sequencing (n = 9/group), in situ hybridization (ISH) (n = 13-22/group) and immunohistochemistry (IHC) (n = 14-25/group). Faecal samples from CC (n = 3-28/group), irritable bowel syndrome diarrhoea (IBS-D) (n = 14) and healthy controls (HC) (n = 15) were assayed for NGAL and calprotectin. RESULTS: NGAL/LCN2 protein and mRNA expression were upregulated in active CC vs HC, and vs paired samples of treated CC in clinical remission. IHC and ISH localized increased NGAL/LCN2 mainly to epithelium of active CC, compared to almost absence in HC and treated CC. In contrast, calprotectin was solely expressed in immune cells. Despite great individual differences, faecal NGAL was significantly increased in active CC compared to HC, IBS-D and treated CC and had high test sensitivity. Faecal calprotectin levels were variably increased in active CC, but the values remained below usual clinical cut-offs. CONCLUSION: NGAL/LCN2 is upregulated in the epithelium of active CC and reduced during budesonide-induced clinical remission to the level of HC and IBD-S. This was reflected in NGAL faecal concentrations. We propose NGAL as an IHC marker for disease activity in CC and a potential faecal biomarker discriminating CC from HC and IBS-D.

Transcriptomic Profiling of Collagenous Colitis Identifies Hallmarks of Nondestructive Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The pathophysiology of the inflammatory bowel disease collagenous colitis (CC) is poorly described. Our aim was to use RNA sequencing of mucosal samples from patients with active CC, CC in remission, refractory CC, ulcerative colitis (UC), and control subjects to gain insight into CC pathophysiology, identify genetic signatures linked to CC, and uncover potentially druggable disease pathways. METHODS: We performed whole transcriptome sequencing of CC samples from patients before and during treatment with the corticosteroid drug budesonide, CC steroid-refractory patients, UC patients, and healthy control subjects (n = 9-13). Bulk mucosa and laser-captured microdissected intestinal epithelial cell (IEC) gene expression were analyzed by gene set enrichment and gene set variation analyses to identify significant pathways and cells, respectively, altered in CC. Leading genes and cells were validated using reverse-transcription quantitative polymerase chain reaction or immunohistochemistry. RESULTS: We identified an activation of the adaptive immune response to bacteria and viruses in active CC that could be mediated by dendritic cells. Moreover, IECs display hyperproliferation and increased antigen presentation in active CC. Further analysis revealed that genes related to the immune response (DUOX2, PLA2G2A, CXCL9), DNA transcription (CTR9), protein processing (JOSD1, URI1), and ion transport (SLC9A3) remained dysregulated even after budesonide-induced remission. Budesonide-refractory CC patients fail to restore normal gene expression, and displayed a transcriptomic profile close to UC. CONCLUSIONS: Our study confirmed the implication of innate and adaptive immune responses in CC, governed by IECs and dendritic cells, respectively, and identified ongoing epithelial damage. Refractory CC could share pathomechanisms with UC.

The serotonin reuptake transporter is reduced in the epithelium of active Crohn's disease and ulcerative colitis.

Serotonin is a highly conserved and ubiquitous signaling molecule involved in a vast variety of biological processes. A majority of serotonin is produced in the gastrointestinal epithelium, where it is suggested to act as a prominent regulatory molecule in the inflammatory bowel diseases (IBDs) Crohn's disease (CD) and ulcerative colitis (UC). Extracellular and circulating serotonin levels are thought to be elevated during intestinal inflammation, but the underlying mechanisms have been poorly understood. The data on human material are limited, contradictory, and in need of further investigation and substantiating. In this study, we show a potent and significant downregulation of the dominant serotonin reuptake transporter (SERT) mRNA (SLC6A4) in the epithelium from active CD ileitis, CD colitis, and UC colitis, compared with healthy controls. The mRNA of tryptophan hydroxylase 1, the rate-limiting enzyme in serotonin synthesis, was unregulated. Immunohistochemistry showed expression of the SERT protein in both the epithelium and the lamina propria and localized the downregulation to the epithelial monolayer. Laser capture microdissection followed by RNA sequencing confirmed downregulation of SLC6A4 in the epithelial monolayer during intestinal inflammation. Patient-derived colon epithelial cell lines (colonoids) incubated with the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) reduced SERT expression. In summary, these results show that intestinal inflammation potently reduces the expression of SERT in both CD and UC and that TNF-α alone is sufficient to induce a similar reduction in colonoids. The reduced serotonin reuptake capacity may contribute to the increased interstitial serotonin level associated with intestinal inflammation.NEW & NOTEWORTHY The serotonin reuptake transporter is potently reduced in inflamed areas of Crohn's ileitis, Crohn's colitis, and ulcerative colitis. The changes are localized to the intestinal epithelium and can be induced by TNF-α. The serotonin synthesis through tryptophan hydroxylase 1 is unchanged. This regulation is suggested as a mechanism underlying the increased extracellular serotonin levels associated with intestinal inflammation.

Ulcer-associated cell lineage expresses genes involved in regeneration and is hallmarked by high neutrophil gelatinase-associated lipocalin (NGAL) levels.

Neutrophil gelatinase-associated lipocalin (NGAL), also known as Lipocalin 2, is an antimicrobial protein, encoded by the gene LCN2, strongly upregulated in inflammatory bowel disease (IBD) and a promising biomarker for IBD. Here we demonstrate that NGAL is highly expressed in all parts of pyloric metaplasia, also known as the ulcer-associated cell lineage (UACL), a metaplastic cell lineage suggested to play a role in wound healing in Crohn's disease (CD). We further show NGAL expression in regenerative intestinal crypts and in undifferentiated patient-derived colonoids. This indicates that NGAL is important in the tissue regeneration process. The remarkable overexpression of NGAL in UACL led us to explore the pathobiology of these cells by transcriptome-wide RNA sequencing. This study is, to our knowledge, the first to characterize the UACL at this level. Biopsies with UACL and inflamed non-UACL epithelium from the terminal ileum of CD patients and epithelium from healthy controls were laser capture microdissected for RNA sequencing. Among the 180 genes differentially expressed between UACL and control epithelium, the ten most-upregulated genes specific for UACL were MUC5AC, PGC, MUC6, MUC5B, LCN2, POU2AF1, MUC1, SDC3, IGFBP5, and SLC7A5. PDX1 was among the most upregulated in both UACL and inflamed non-UACL epithelium. Immunohistochemistry and iDisco 3D visualization was used to characterize UACL histo-morphologically, and to validate protein expression of 11 selected differentially expressed genes. Among these genes, LCN2, NOTCH2, PHLDA1, IGFBP5, SDC3, BPIFB1, and RCN1 have previously not been linked to UACL. Gene expression results were analyzed for functional implications using MetaCore, showing that differentially expressed genes are enriched for genes involved in cell migration and motility, and for biomarkers of gastrointestinal neoplasia. These results support a role for UACL as part of the reepithelialization process during and after destructive intestinal inflammation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in the gut in Crohn's disease.

The antimicrobial glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) is strongly expressed in several infectious, inflammatory and malignant disorders, among these inflammatory bowel disease (IBD). Fecal and serum NGAL is elevated during active IBD and we have recently shown that fecal NGAL is a novel biomarker for IBD with a test performance comparable to the established fecal biomarker calprotectin. This study examines expression of NGAL in the healthy gut and in Crohn's disease (CD), with emphasis on the previously unexplored small intestine. Pinch biopsies were taken from active and inactive CD in jejunum, ileum and colon and from the same sites in healthy controls. Microarray gene expression showed that the NGAL gene, LCN2, was the second most upregulated among 1820 differentially expressed genes in terminal ileum comparing active CD and controls (FC 5.86, p = 0.027). Based on immunohistochemistry and in situ hybridization findings, this upregulation most likely represented increased expression in epithelial cells. Double immunofluorescence showed NGAL expression in 49% (range 19-70) of Paneth cells (PCs) in control ileum with no change during inflammation. In healthy jejunum, the NGAL expression in PCs was weak to none but markedly increased during active CD. We further found NGAL also in metaplastic PCs in colon. Finally, we show for the first time that NGAL is expressed in enteroendocrine cells in small intestine as well as in colon.

Intestinal barrier homeostasis in inflammatory bowel disease.

The single-cell thick intestinal epithelial cell (IEC) lining with its protective layer of mucus is the primary barrier protecting the organism from the harsh environment of the intestinal lumen. Today it is clear that the balancing act necessary to maintain intestinal homeostasis is dependent on the coordinated action of all cell types of the IEC, and that there are no passive bystanders to gut immunity solely acting as absorptive or regenerative cells: Mucin and antimicrobial peptides on the epithelial surface are continually being replenished by goblet and Paneth's cells. Luminal antigens are being sensed by pattern recognition receptors on the enterocytes. The enteroendocrine cells sense the environment and coordinate the intestinal function by releasing neuropeptides acting both on IEC and inflammatory cells. All this while cells are continuously and rapidly being regenerated from a limited number of stem cells close to the intestinal crypt base. This review seeks to describe the cell types and structures of the intestinal epithelial barrier supporting intestinal homeostasis, and how disturbance in these systems might relate to inflammatory bowel disease.

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation.

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.

Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and Transcriptomic [corrected] characterization and correlation to IBD.

BACKGROUND: Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1ß, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. CONCLUSIONS/SIGNIFICANCE: Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.