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Objective By interfering with multiple cytokines, human Janus kinase inhibitors (JAKis) are of growing importance in the treatment of malignant and inflammatory conditions. Although tofacitinib has demonstrated efficacy as the first-in-class JAKi in ulcerative colitis many aspects concerning its mode of action and pharmacokinetics remain unresolved. Design We studied tofacitinib’s impact on various primary human innate and adaptive immune cells. In-depth in vivo studies were performed in dextran sodium sulfate–induced colitis in mice. Immune populations were characterized by flow cytometry and critical transcription factors and effector cytokines were analyzed. Pharmacokinetics of tofacitinib was studied by liquid chromatography–tandem mass spectrometry. Results Tofacitinib inhibited proliferation in CD4+ and CD8+ T cells along with Th1 and Th17 differentiation, while Th2 and regulatory T cell lineages were largely unaffected. Monocytes and macrophages were directed toward an anti-inflammatory phenotype and cytokine production was suppressed in intestinal epithelial cells. These findings were largely reproducible in murine cells of the inflamed mucosa in dextran sulfate sodium colitis. Short-term treatment with tofacitinib had little impact on the mouse microbiota. Strikingly, the degree of inflammation and circulating tofacitinib levels showed a strong positive correlation. Finally, we identified inflammation-induced equilibrative nucleoside transporters as regulators of tofacitinib uptake into leukocytes. Conclusions We provide a detailed analysis of the cell-specific immune-suppressive effects of the JAKis tofacitinib on innate and adaptive immunity and reveal that intestinal inflammation critically impacts tofacitinib’s pharmacokinetics in mice. Furthermore, we describe an unappreciated mechanism—namely induction of equilibrative nucleoside transporters—enhancing baseline cellular uptake that can be inhibited pharmaceutically., Graphical abstract

Background & Aims We previously showed that histamine suppressed inflammation-associated colonic tumorigenesis through histamine type 2 receptor (H2R) signaling in mice. This study aimed to precisely elucidate the downstream effects of H2R activation in innate immune cells. Methods Analyses using online databases of single-cell RNA sequencing of intestinal epithelial cells in mice and RNA sequencing of mouse immune cells were performed to determine the relative abundances of 4 histamine receptors among different cell types. Mouse neutrophils, which expressed greater amounts of H2R, were collected from the peritoneum of wild-type and H2R-deficient mice, of which low-density and high-density neutrophils were extracted by centrifugation and were subjected to RNA sequencing. The effects of H2R activation on neutrophil differentiation and its functions in colitis and inflammation-associated colon tumors were investigated in a mouse model of dextran sulfate sodium–induced colitis. Results Data analysis of RNA sequencing and quantitative reverse-transcription polymerase chain reaction showed that Hrh2 is highly expressed in neutrophils, but barely detectable in intestinal epithelial cells. In mice, the absence of H2R activation promoted infiltration of neutrophils into both sites of inflammation and colonic tumors. H2R-deficient high-density neutrophils yielded proinflammatory features via nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and suppressed T-cell proliferation. On the other hand, low-density neutrophils, which totally lack H2R activation, showed an immature phenotype compared with wild-type low-density neutrophils, with enhanced MYC pathway signaling and reduced expression of the maturation marker Toll-like receptor 4. Conclusions Blocking H2R signaling enhanced proinflammatory responses of mature neutrophils and suppressed neutrophil maturation, leading to accelerated progression of inflammation-associated colonic tumorigenesis., Graphical abstract

Background & Aims Mucosal-associated invariant T (MAIT) cells are innate-like T cells restricted by major histocompatibility complex-related molecule 1 (MR1) and express a semi-invariant T cell receptor. Previously, we reported the activation status of circulating MAIT cells in patients with ulcerative colitis (UC) was associated with disease activity and that these cells had infiltrated the inflamed colonic mucosa. These findings suggest MAIT cells are involved in the pathogenesis of inflammatory bowel disease. We investigated the role of MAIT cells in the pathogenesis of colitis by using MR1−/− mice lacking MAIT cells and a synthetic antagonistic MR1 ligand. Methods Oxazolone colitis was induced in MR1−/− mice (C57BL/6 background), their littermate wild-type controls, and C57BL/6 mice orally administered an antagonistic MR1 ligand, isobutyl 6-formyl pterin (i6-FP). Cytokine production of splenocytes and colonic lamina propria lymphocytes from mice receiving i6-FP was analyzed. Intestinal permeability was assessed in MR1−/− and i6-FP-treated mice and their controls. The effect of i6-FP on cytokine production by MAIT cells from patients with UC was assessed. Results MR1 deficiency or i6-FP treatment reduced the severity of oxazolone colitis. i6-FP treatment reduced cytokine production in MAIT cells from mice and patients with UC. Although MR1 deficiency increased the intestinal permeability, i6-FP administration did not affect gut integrity in mice. Conclusions These results indicate MAIT cells have a pathogenic role in colitis and suppression of MAIT cell activation might reduce the severity of colitis without affecting gut integrity. Thus, MAIT cells are potential therapeutic targets for inflammatory bowel disease including UC., Graphical abstract

Background & Aims One of the features of ulcerative colitis (UC) is a defect in the protective mucus layer. This has been attributed to a reduced number of goblet cells (GCs). However, it is not known whether abnormal GC mucus secretion also contributes to the reduced mucus layer. Our aims were to investigate whether GC secretion was abnormal in UC and exists as a long-term effect of chronic inflammation. Methods Colonoids were established from intestinal stem cells of healthy subjects (HS) and patients with UC. Colonoids were maintained as undifferentiated (UD) or induced to differentiate (DF) and studied as three-dimensional or monolayers on Transwell filters. Total RNA was extracted for quantitative real-time polymerase chain reaction analysis. Carbachol and prostaglandin E2 mediated mucin stimulation was examined by MUC2 IF/confocal microscopy and transmission electron microscopy. Results Colonoids from UC patients can be propagated over many passages; however, they exhibit a reduced rate of growth and transepithelial electrical resistance compared with HS. Differentiated UC colonoid monolayers form a thin and non-continuous mucus layer. UC colonoids have increased expression of secretory lineage markers ATOH1 and SPDEF, along with MUC2 positive GCs, but failed to secrete mucin in response to the cholinergic agonist carbachol and prostaglandin E2, which caused increased secretion in HS. Exposure to tumor necrosis factor α (5 days) reduced the number of GCs, with a greater percentage decrease in UC colonoids compared with HS. Conclusions Chronic inflammation in UC causes long-term changes in GCs, leading to abnormal mucus secretion. This continued defect in GC mucus secretion may contribute to the recurrence in UC., Graphical abstract

Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb Lonicera japonica Thunb., here we report its therapeutic effect on intestinal inflammation by binding directly to enhancer of zeste homolog 2 (EZH2) histone methyltransferase. EZH2-mediated modification of H3K27me3 promotes the expression of autophagy-related protein 5, which in turn leads to enhanced autophagy and accelerates autolysosome-mediated NLRP3 degradation. Mutations of EZH2 residues (His129 and Arg685) indicated by the dynamic simulation study have found to greatly diminish the protective effect of lonicerin. More importantly, in vivo studies verify that lonicerin dose-dependently disrupts the NLRP3–ASC–pro-caspase-1 complex assembly and alleviates colitis, which is compromised by administration of EZH2 overexpression plasmid. Thus, these findings together put forth the stage for further considering lonicerin as an anti-inflammatory epigenetic agent and suggesting EZH2/ATG5/NLRP3 axis may serve as a novel strategy to prevent ulcerative colitis as well as other inflammatory diseases., Graphical abstract Lonicerin can be considered as an anti-inflammatory epigenetic agent and EZH2/ATG5/NLRP3 axis may serve as a novel strategy to prevent ulcerative colitis as well as other inflammatory diseases.Image 1

Proteins and peptides (PPs) have gradually become more attractive therapeutic molecules than small molecular drugs due to their high selectivity and efficacy, but fewer side effects. Owing to the poor stability and limited permeability through gastrointestinal (GI) tract and epithelia, the therapeutic PPs are usually administered by parenteral route. Given the big demand for oral administration in clinical use, a variety of researches focused on developing new technologies to overcome GI barriers of PPs, such as enteric coating, enzyme inhibitors, permeation enhancers, nanoparticles, as well as intestinal microdevices. Some new technologies have been developed under clinical trials and even on the market. This review summarizes the history, the physiological barriers and the overcoming approaches, current clinical and preclinical technologies, and future prospects of oral delivery of PPs., Graphical abstract The current strategies for improving oral absorption of proteins and peptides (PPs) include stabilization, absorption enhancement and mucus-related technologies. Most of marketed oral PP products employ two or more strategies.Image 1

Background & Aims We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn’s disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. Methods RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. Results We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. Conclusions PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes., Graphical abstract

Background & Aims Exclusive enteral nutrition (EEN) is used to treat pediatric Crohn’s disease (CD), but therapeutic benefits are variable, and EEN can lead to microbial dysbiosis. Because of reported lower efficacy EEN is not routinely used to treat pediatric ulcerative colitis (UC). Inulin-type fructans (IN) beneficially modulate the gut microbiome and promote expansion of anti-inflammatory immune cells. We hypothesized that enriching EEN with IN (EEN IN) would enhance treatment efficacy. To test this, we examined the effects of EEN IN on colitis development, the gut microbiome, and CD4+ T cells using an adoptive T-cell transfer model of colitis. Methods TCR-β deficient (-/-) mice were randomized to 1 of 4 groups: (1) Control, (2) Chow, (3) EEN, and (4) EEN IN, and naive CD4+ T cells were adoptively transferred into groups 2–4, after which mice were monitored for 5 weeks before experimental endpoint. Results Mice fed EEN IN showed greater colitis protection, with colonic shortening, goblet cell, and crypt density loss reduced compared with EEN fed mice and reduced disease activity and immune cell infiltration compared with chow fed mice, and less crypt hyperplasia and higher survival compared with both groups. EEN IN mice had less deterioration in the colonic mucus layer and had increased levels of Foxp3+IL-10+ and Rorγt+IL-22+ and reduced levels of Tbet+IFNγ+ and Tbet+TNF+ CD4+ T cells. EEN IN also led to higher butyrate concentrations, Bifidobacterium spp. and Anaerostipes caccae relative abundance, and lower [Clostridium] innocuum group spp. and Escherichia-Shigella spp. relative abundance. Conclusions The EEN IN group showed reduced colitis development as compared with the chow and EEN groups. This highlights the potential benefits of EEN IN as a novel induction therapy for pediatric CD and UC patients., Graphical abstract

Clinical trials investigating immune checkpoint inhibitors (ICIs) largely excluded solid organ transplant recipients (SOTRs), as immunostimulation by these drugs may subvert host tolerance to the transplanted tissue, potentially leading to allograft rejection.1 Consequently, data concerning the safety and efficacy of ICIs in these patients remain limited. ICI trials also routinely excluded patients with preexisting autoimmune disease given the concern for possible disease exacerbations, resulting in similarly limited data in this population.2 We report a unique case of a SOTR with ulcerative colitis (UC) and metastatic cutaneous squamous cell carcinoma (SCC) who was successfully treated with anti-programmed cell death protein 1 ICI therapy, initially with nivolumab, then with cemiplimab, which is approved by the US Food and Drug Administration for advanced cutaneous SCC.3

Previously, we found that rosmarinic acid (RA) exerted anti-inflammatory activities in a dextran sulfate sodium (DSS)-induced colitis model. Here, we investigated the anti-tumor effects of RA on colitis-associated colon cancer (CAC) and the underlying molecular mechanisms. We established an azoxymethane (AOM)/DSS-induced CAC murine model for in vivo studies and used a conditioned media (CM) culture system in vitro. H&E staining, immunohistochemistry, western blot assay, enzyme-linked immunosorbent assay, molecular docking, co-immunoprecipitation, and immunofluorescence assay were utilized to investigate how RA prevented colorectal cancer. In the AOM/DSS-induced CAC murine model, RA significantly reduced colitis severity, inflammation-related protein expression, tumor incidence, and colorectal adenoma development. It significantly modulated toll-like receptor-4 (TLR4)-mediated nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) activation, thus attenuating the expression of anti-apoptotic factors, which mediate transcription factor-dependent tumor growth. In vitro, RA inhibited CM-induced TLR4 overexpression and competitively inhibited TLR4-myeloid differentiation factor 2 complex in an inflammatory microenvironment. Thus, RA suppressed NF-κB and STAT3 activation in colon cancer cells in an inflammatory microenvironment. Therefore, RA suppressed colitis-associated tumorigenesis in the AOM/DSS-induced CAC murine model and abrogated human colon cancer progression in an inflammatory microenvironment by propitiating TLR4-mediated NF-κB and STAT3 activation, pleiotropically.

In the present study, we have investigated and/or compared the role of glibenclamide, G as cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, and lubiprostone, L as chloride channel-2 (ClC-2) activator in the 2,4-dinitrobenzene sulfonic acid (DNBS)-induced gastrointestinal inflammation. GI inflammation was induced by intrarectal administration of DNBS. Rats were randomly allocated in 5 groups as sham control, distilled water + DNBS, sulfasalazine (S) + DNBS, G + DNBS, and L + DNBS. All the groups were pre-treated successively for five days before the induction of colitis. One day before and the first four days after DNBS administration various parameters were studied. Later, blood chemistry, colon’s gross structure, histology, and the antioxidant load was examined. Pre-treatment with G significantly protected the change induced by DNBS concerning the change in body weight, food intake, diarrhea, occult blood in the feces, wet weight of the colon, and spleen. G because of its anti-inflammatory property down-regulated the neutrophil and WBC count and up-regulated the lymphocyte number. Moreover, G efficiently ameliorates the oxidative stress in the colon and declines the level of myeloperoxidase and malondialdehyde and up-regulated the level of superoxide dismutase and glutathione. Lubiprostone has not shown any promising effects, in fact, it causes an increase in diarrheal frequency. Our findings from this study represent that G has good potential to ameliorate GI inflammation induced by DNBS by its multiple actions including CFTR blockage and reducing the release of inflammatory markers from the MCs, anti-inflammatory and free radical scavenging property.

Background & Aims The pathogenesis of immune checkpoint inhibitor (ICI)–colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. Methods We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. Results We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti–CTLA-4/PD-1 combination therapy and in anti–PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. Conclusions Interferon gamma–producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy., Graphical abstract, We present an analysis of the immunopathology in checkpoint-inhibitor colitis, a common adverse effect of cancer immunotherapy. We used our findings to successfully identify a novel therapy for a case of refractory colitis.

The Inflammatory Bowel Diseases (IBD), Ulcerative Colitis (UC) and Crohn’s Disease (CD) are characterised by chronic non-resolving gut mucosal inflammation involving innate and adaptive immune responses. Neutrophils, usually regarded as first responders in inflammation, are a key presence in the gut mucosal inflammatory milieu in IBD. Here, we review the role of neutrophil extracellular trap (NET) formation as a potential effector disease mechanism. NETs are extracellular webs of chromatin, microbicidal proteins and oxidative enzymes that are released by neutrophils to contain pathogens. NETs contribute to the pathogenesis of several immune-mediated diseases such as systemic lupus erythematosus and rheumatoid arthritis; and recently, as a major tissue damaging process involved in the host response to severe acute respiratory syndrome coronavirus 2 infection. NETs are pertinent as a defence mechanism at the gut mucosal interphase exposed to high levels of bacteria, viruses and fungi. On the other hand, NETs can also potentiate and perpetuate gut inflammation. In this review, we discuss the broad protective vs. pathogenic roles of NETs, explanatory factors that could lead to an increase in NET formation in IBD and how NETs may contribute to gut inflammation and IBD-related complications. Finally, we summarise therapeutic opportunities to target NETs in IBD.

Background & Aims Patients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTαIKO mice). Methods Colonic tissue of CTαIKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate–dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTαIKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate. Results Inducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTαIKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes. Conclusions Maintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603., Graphical abstract

Background & Aims Inflammatory bowel disease (IBD) is a polygenic disorder characterized principally by dysregulated inflammation impacting the gastrointestinal tract. However, there also is increasing evidence for a clinical association with stress and depression. Given the role of the hypothalamus in stress responses and in the pathogenesis of depression, useful insights could be gleaned from understanding its genetic role in IBD. Methods We conducted genetic correlation analyses on publicly available genome-wide association study summary statistics for depression and IBD traits to identify genetic commonalities. We used partitioned linkage disequilibrium score regression, leveraging our ATAC sequencing and promoter-focused Capture C data, to measure enrichment of IBD single-nucleotide polymorphisms within promoter-interacting open chromatin regions of human embryonic stem cell-derived hypothalamic-like neurons (HNs). Using the same data sets, we performed variant-to-gene mapping to implicate putative IBD effector genes in HNs. To contrast these results, we similarly analyzed 3-dimensional genomic data generated in epithelium-derived colonoids from rectal biopsy specimens from donors without pathologic disease noted at the time of colonoscopy. Finally, we conducted enrichment pathway analyses on the implicated genes to identify putative IBD dysfunctional pathways. Results We found significant genetic correlations (rg) of 0.122 with an adjusted P (Padj) = 1.4 × 10-4 for IBD: rg = 0.122; Padj = 2.5 × 10-3 for ulcerative colitis and genetic correlation (rg) = 0.094; Padj = 2.5 × 10-3 for Crohn’s disease, and significant approximately 4-fold (P = .005) and approximately 7-fold (P = .03) enrichment of IBD single-nucleotide polymorphisms in HNs and colonoids, respectively. We implicated 25 associated genes in HNs, among which CREM, CNTF, and RHOA encode key regulators of stress. Seven genes also additionally were implicated in the colonoids. We observed an overall enrichment for immune and hormonal signaling pathways, and a colonoid-specific enrichment for microbiota-relevant terms. Conclusions Our results suggest that the hypothalamus warrants further study in the context of IBD pathogenesis., Graphical abstract

Background & Aims Intestinal macrophages adopt a hyporesponsive phenotype through education by local signals. Lack of proper macrophage maturation in patients with ulcerative colitis (UC) in remission may initiate gut inflammation. The aim, therefore, was to determine the effects of fecal luminal factors derived from healthy donors and UC patients in remission on macrophage phenotype and function. Methods Fecal supernatants (FS) were extracted from fecal samples of healthy subjects and UC patients in remission. Monocytes were matured into macrophages in the presence of granulocyte-macrophage colony-stimulating factor without/with FS, stimulated with lipopolysaccharide, and macrophage phenotype and function were assessed. Fecal metabolomic profiles were analyzed by gas-chromatography/mass-spectrometry. Results Fecal luminal factors derived from healthy donors were effective in down-regulating Toll-like receptor signaling, cytokine signaling, and antigen presentation in macrophages. Fecal luminal factors derived from UC patients in remission were less potent in inducing lipopolysaccharide hyporesponsiveness and modulating expression of genes involved in macrophage cytokine and Toll-like receptor signaling pathways. Although phagocytic and bactericidal abilities of macrophages were not affected by FS treatment, healthy FS-treated macrophages showed a greater ability to suppress cluster of differentiation 4+ T-cell activation and interferon γ secretion compared with UC remission FS-treated counterparts. Furthermore, metabolomic analysis showed differential fecal metabolite composition for healthy donors and UC patients in remission. Conclusions Our data indicate that UC patients in remission lack luminal signals able to condition macrophages toward a hyporesponsive and tolerogenic phenotype, which may contribute to their persistent vulnerability to relapse., Graphical abstract

Background Inflammatory bowel disease (IBD) is a complex disease with variable presentation, progression, and response to therapies. Current disease classification is based on subjective clinical phenotypes. The peripheral blood immunophenome can reflect local inflammation, and thus we measured 39 circulating immune cell types in a large cohort of IBD and control subjects and performed immunotype:phenotype associations. Methods We performed fluorescence-activated cell sorting or CyTOF analysis on blood from 728 Crohn’s disease, 464 ulcerative colitis, and 334 non-IBD patients, with available demographics, endoscopic and clinical examinations and medication use. Results We observed few immune cell types commonly affected in IBD (lowered natural killer cells, B cells, and CD45RA– CD8 T cells). Generally, the immunophenome was distinct between ulcerative colitis and Crohn’s disease. Within disease subtype, there were further distinctions, with specific immune cell types associating with disease duration, behavior, and location. Thiopurine monotherapy altered abundance of many cell types, often in the same direction as disease association, while anti-tumor necrosis factor (anti-TNF) monotherapy demonstrated an opposing pattern. Concomitant use of an anti-TNF and thiopurine was not synergistic, but rather was additive. For example, thiopurine monotherapy use alone or in combination with anti-TNF was associated with a dramatic reduction in major subclasses of B cells. Conclusions We present a peripheral map of immune cell changes in IBD related to disease entity and therapies as a resource for hypothesis generation. We propose the changes in B cell subsets could affect antibody formation and potentially explain the mechanism behind the superiority of combination therapy through the impact of thiopurines on pharmacokinetics of anti-TNFs., Graphical abstract

Although impaired intestinal sodium transport has been described for decades as a ubiquitous feature of inflammatory bowel disease (IBD), whether and how it plays a pivotal role in the ailment has remained uncertain. Our identification of dominant mutations in receptor guanylyl cyclase 2C as a cause of IBD-associated familial diarrhea syndrome brought a shift in the way we envision impaired sodium transport. Is this just a passive collateral effect resulting from intestinal inflammation, or is it a crucial regulator of IBD pathogenesis? This review summarizes the mutational spectrum and underlying mechanisms of monogenic IBD associated with congenital sodium diarrhea. We constructed a model proposing that impaired sodium transport is an upstream pathogenic factor in IBD. The review also synthesized emerging insights from microbiome and animal studies to suggest how sodium malabsorption can serve as a unifying mediator of downstream pathophysiology. Further investigations into the mechanisms underlying salt and water transport in the intestine will provide newer approaches for understanding the ion–microbiome–immune cross-talk that serves as a driver of IBD. Model systems, such as patient-derived enteroids or induced pluripotent stem cell models, are warranted to unravel the role of individual genes regulating sodium transport and to develop more effective epithelial rescue and repair therapies., Graphical abstract

Inflammatory bowel disease (IBD) patients have an increased risk of developing colitis-associated colon cancer (CAC); however, the basis for inflammation-induced genetic damage requisite for neoplasia is unclear. Several studies have shown that IBD patients have signs of increased oxidative damage, which could be a result of genetic and environmental factors such as an excess in oxidant molecules released during chronic inflammation, mitochondrial dysfunction, a failure in antioxidant capacity, or oxidant promoting diets. It has been suggested that chronic oxidative environment in the intestine leads to the DNA lesions that precipitate colon carcinogenesis in IBD patients. Indeed, several preclinical and clinical studies show that different endogenous and exogenous antioxidant molecules are effective at reducing oxidation in the intestine. However, most clinical studies have focused on the short-term effects of antioxidants in IBD patients but not in CAC. This review article examines the role of oxidative DNA damage as a possible precipitating event in CAC in the context of chronic intestinal inflammation and the potential role of exogenous antioxidants to prevent these cancers.

Background and aims: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4β7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. Methods: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. Results: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. Conclusions: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues. Keywords: Chemokine Receptors; Dopaminergic Regulation; G Protein-Coupled Receptors Heteromers; Gut Tropism; Inflammatory Bowel Diseases; Inflammatory Colitis; T Cell Migration.

Background and aims The trigger hypothesis opens the possibility of anti-flare initiation therapies by stating that ulcerative colitis (UC) flares originate from inadequate responses to acute mucosal injuries. However, experimental evidence is restricted by a limited use of suitable human models. We thus aimed to investigate the acute mucosal barrier injury responses in humans with and without UC using an experimental injury model. Methods A standardized mucosal break was inflicted in the sigmoid colon of 19 patients with UC in endoscopic and histological remission and 20 control subjects. Postinjury responses were assessed repeatedly by high-resolution imaging and sampling to perform Geboes scoring, RNA sequencing, and injury niche microbiota 16S ribosomal RNA gene sequencing. Results UC patients had more severe endoscopic postinjury inflammation than did control subjects (P < .01), an elevated modified Geboes score (P < .05), a rapid induction of innate response gene sets (P < .05) and antimicrobial peptides (P < .01), and engagement of neutrophils (P < .01). Innate lymphoid cell type 3 (ILC3) markers were increased preinjury (P < .01), and ILC3 activating cytokines were highly induced postinjury, resulting in an increase in ILC3-type cytokine interleukin-17A. Across groups, the postinjury mucosal microbiome had higher bacterial load (P < .0001) and lower α-diversity (P < .05). Conclusions UC patients in remission respond to mucosal breaks by an innate hyperresponse engaging resident regulatory ILC3s and a subsequent adaptive activation. The postinjury inflammatory bowel disease–like microbiota diversity decrease is irrespective of diagnosis, suggesting that the dysbiosis is secondary to host injury responses. We provide a model for the study of flare initiation in the search for antitrigger-directed therapies., Graphical abstract

Background & Aims p21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/β-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice. Methods IL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT. Results When compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein–coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased β-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation. Conclusions PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1–Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC., Graphical abstract

Inflammatory bowel disease (IBD) is a chronic intestinal disease with painful clinical manifestations and high risks of cancerization. With no curative therapy for IBD at present, the development of effective therapeutics is highly advocated. Drug delivery systems have been extensively studied to transmit therapeutics to inflamed colon sites through the enhanced permeability and retention (EPR) effect caused by the inflammation. However, the drug still could not achieve effective concentration value that merely utilized on EPR effect and display better therapeutic efficacy in the inflamed region because of nontargeted drug release. Substantial researches have shown that some specific receptors and cell adhesion molecules highly expresses on the surface of colonic endothelial and/or immune cells when IBD occurs, ligand-modified drug delivery systems targeting such receptors and cell adhesion molecules can specifically deliver drug into inflamed sites and obtain great curative effects. This review introduces the overexpressed receptors and cell adhesion molecules in inflamed colon sites and retrospects the drug delivery systems functionalized by related ligands. Finally, challenges and future directions in this field are presented to advance the development of the receptor-mediated targeted drug delivery systems for the therapy of IBD., Graphical abstract When inflammatory bowel disease occurs, some specific receptors and cell adhesion molecules, like mannose receptor, CD98, CD44 and ICAM-1, are overexpressed on the colonic epithelial cells and/or immune cells. Drug delivery systems superficially modified with related ligands can specifically bind to receptors on inflamed cells and deliver drug into target area.Image 1

Background & Aims Biotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs. Methods Mice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1β), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB). Results Biotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB. Conclusions Oral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD., Graphical abstract

Background & Aims Bacterial flagellin is an important antigen in inflammatory bowel disease, but the role of flagellin-specific CD4+ T cells in disease pathogenesis remains unclear. Also unknown is how changes in intestinal microbiome intersect with those in microbiota-specific CD4+ T cells. We aimed to quantify and characterize flagellin-specific CD4+ T cells in Crohn’s disease (CD) and ulcerative colitis (UC) patients and study their relationship with intestinal microbiome diversity. Methods Blood was collected from 3 cohorts that included CD patients, UC patients, and healthy controls. Flow cytometry analyzed CD4+ T cells specific for Lachnospiraceae-derived A4-Fla2 and Escherichia coli H18 FliC flagellins, or control vaccine antigens. Serum antiflagellin IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay and stool samples were collected and subjected to 16S ribosomal DNA sequencing. Results Compared with healthy controls, CD and UC patients had lower frequencies of vaccine-antigen–specific CD4+ T cells and, as a proportion of vaccine-specific cells, higher frequencies of flagellin-specific CD4+ T cells. The proportion of flagellin-specific CD4+ T cells that were CXCR3negCCR4+CCR6+ Th17 cells was reduced in CD and UC patients, with increased proportions of CD39+, PD-1+, and integrin β7+ cells. Microbiome analysis showed differentially abundant bacterial species in patient groups that correlated with immune responses to flagellin. Conclusions Both CD and UC patients have relative increases in the proportion of circulating Fla2-specific CD4+ T cells, which may be associated with changes in the intestinal microbiome. Evidence that the phenotype of these cells strongly correlate with disease severity provides insight into the potential roles of flagellin-specific CD4+ T cells in inflammatory bowel disease., Graphical abstract

Inflammatory bowel disease (IBD) consists of two major idiopathic gastrointestinal diseases: ulcerative colitis and Crohn's disease. Although a significant advance has been achieved in the treatment of IBD, there remains a particular population of patients that are refractory to the conventional treatments, including the biologic agents. Studies have revealed the importance of “mucosal healing” in improving the prognosis of those difficult-to-treat patients, which indicates the proper and complete regeneration of the damaged intestinal tissue. In this regard, organoid-based regenerative medicine may have the potential to dramatically promote the achievement of mucosal healing in refractory IBD patients, and thereby improve their long-term prognosis as well. So far, studies have shown that hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) may have some beneficial effect on IBD patients through their transplantation or transfusion. Recent advance in stem cell biology has added intestinal stem cells (ISCs) as a new player in this field. It has been shown that ISCs can be grown in vitro as organoids and that those ex-vivo cultured organoids can be employed as donor cells for transplantation studies. Further studies using mice colitis models have shown that ex-vivo cultured organoids can engraft onto the colitic ulcers and reconstruct the crypt-villus structures. Such transplantation of organoids may not only facilitate the regeneration of the refractory ulcers that may persist in IBD patients but may also reduce the risk of developing colitis-associated cancers. Endoscopy-assisted transplantation of organoids may, therefore, become one of the alternative therapies for refractory IBD patients. Keywords: Intestinal stem cells, Organoids, Ulcerative colitis, Crohn's disease, Organoid medicine

Introduction Neuroendocrine tumors (NETs) of the rectum are rare, with an estimated incidence of 0.1% of all colorectal cancers. However, it is crucial to diagnose NET, particularly in patients with ulcerative colitis, who seem to have a higher risk of cancerization according to recent studies, given the aggressiveness and poor prognosis of these tumors. Presentation of case We report the case of a 54-year-old man who developed multiple rectal NETs (approximately 10), measuring 1–6 mm, only 2 years after a primary diagnosis of ulcerative colitis. Discussion In the literature, all reported cases of NETs present patients with a long history of several years of inflammatory bowel disease; however, very little literature exists on this subject. Herein, we discussed the outcomes and a literature review of the pathology, prognosis, and management of NETs. Conclusion Despite advances in research on rectal NETs, many aspects about the disease remain unclear, partly due to its rarity., Highlights • A 54-year-old man developed NETs 2 years after ulcerative colitis diagnosis. • We discussed the pathology, prognosis, and management of NETs. • Ulcerative colitis seems to predispose patients to NET development. • Many aspects about multiple NETs remain unclear.

Background & Aims We previously showed that abundance of Candida tropicalis is significantly greater in Crohn’s disease patients compared with first-degree relatives without Crohn’s disease. The aim of this study was to determine the effects and mechanisms of action of C tropicalis infection on intestinal inflammation and injury in mice. Methods C57BL/6 mice were inoculated with C tropicalis, and colitis was induced by administration of dextran sodium sulfate in drinking water. Disease severity and intestinal permeability subsequently were evaluated by endoscopy, histology, quantitative reverse-transcription polymerase chain reaction, as well as 16S ribosomal RNA and NanoString analyses (NanoString Technologies, Seattle, WA). Results Infected mice showed more severe colitis, with alterations in gut mucosal helper T cells (Th)1 and Th17 cytokine expression, and an increased frequency of mesenteric lymph node–derived group 2 innate lymphoid cells compared with uninfected controls. Gut microbiome composition, including changes in the mucin-degrading bacteria, Akkermansia muciniphila and Ruminococcus gnavus, was altered significantly, as was expression of several genes affecting intestinal epithelial homeostasis in isolated colonoids, after C tropicalis infection compared with uninfected controls. In line with these findings, fecal microbiome transplantation of germ-free recipient mice using infected vs uninfected donors showed altered expression of several tight-junction proteins and increased susceptibility to dextran sodium sulfate–induced colitis. Conclusions C tropicalis induces dysbiosis that involves changes in the presence of mucin-degrading bacteria, leading to altered tight junction protein expression with increased intestinal permeability and followed by induction of robust Th1/Th17 responses, which ultimately lead to an accelerated proinflammatory phenotype in experimental colitic mice., Graphical abstract

Alopecia areata (AA) is an autoimmune form of hair loss characterized by T-cell-mediated damage to hair follicles.1 Inflammatory bowel disease (IBD) is an autoimmune disease of the bowel characterized by dysregulation of T cells, increased production of proinflammatory cytokines (eg, interleukin [IL] 6, IL-23, IL-12, and IL-21), and intestinal epithelial dysfunction.2 Genome-wide association studies, performed separately in AA and IBD populations, reveal overlapping susceptibility loci in AA and Crohn disease (PRDX5 and IL-2RA) and AA and ulcerative colitis (UC) (IL-2/IL-21).3,4 The incidence of comorbid AA and IBD is unknown. Despite the success of biologics for the treatment of IBD, it may be that some biologics, in particular tumor necrosis factor α inhibitors, precipitate AA. The Janus kinase (JAK) inhibitor tofacitinib is approved for the treatment of UC, and there are several ongoing trials of other JAK inhibitors for IBD. JAK inhibitors are an emerging treatment for AA.1,5 In this study, we describe the results of treatment of patients with comorbid IBD and AA with tofacitinib.

Background & Aims Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. Methods The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning–based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. Results Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. Conclusions The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease., Graphical abstract, Bacterial biofilms were observed by colonoscopy as yellow-green membranous layers on the mucosa of the small and large intestinal junction and are specifically prevalent in irritable bowel syndrome and inflammatory bowel disease.

Background: The disease course of inflammatory bowel disease (IBD) following treatment with glucagon-like peptide (GLP)-1 based therapies is unclear. Objectives: To examine clinical outcomes of IBD in patients with IBD and type 2 diabetes treated with GLP-1 based therapies compared with treatment with other antidiabetics. Methods: Using nationwide Danish registries, we identified patients with IBD and type 2 diabetes who received antidiabetic treatment between 1 January 2007 and 31 March 2019. The primary outcome was a composite of the need for oral corticosteroids, tumour necrosis factor-α inhibitors, IBD-related hospitalisation, or IBD-related surgery. In the setting of a new-user active comparator design, we used Poisson regression to estimate incidence rate ratios (IRRs) comparing treatment with GLP-1 receptor agonists and dipeptidyl peptidase (DPP)-4 inhibitors with other antidiabetic therapies. The analyses were adjusted for age, sex, calendar year, IBD severity, and metformin use. Results: We identified 3751 patients with a diagnosis of IBD and type 2 diabetes and with a prescription of an antidiabetic drug (GLP-1 receptor agonists/DPP-4 inhibitors: 982 patients; other antidiabetic treatment: 2769 patients). The adjusted IRR of the composite outcome was 0.52 (95% CI: 0.42-0.65) for patients exposed to GLP-1 receptor agonists/DPP-4 inhibitors compared with patients exposed to other antidiabetics. Conclusion: In patients with IBD and type 2 diabetes, treatment with GLP-1 receptor agonists/DPP-4 inhibitors are associated with a lower risk of adverse clinical events compared with treatment with other antidiabetics. Funding Information: The study was funded by the Novo Nordisk Foundation [grant number NNF16OC0022586 and NNF17OC0029768]. Declaration of Interests: None of the authors have any conflicts of interest to disclose. Ethics Approval Statement: The study was approved by the Danish Data Protection Agency, and data were analysed on a secure research server at the Danish Health Data Authority. In Denmark, ethical approval is not required for research using pre-existing, routinely collected data.

Background & Aims Inflammatory bowel diseases (IBDs) that encompass both ulcerative colitis and Crohn’s disease are a major public health problem with an etiology that has not been fully elucidated. There is a need to improve disease outcomes and preventive measures by developing new effective and lasting treatments. Although polyunsaturated fatty acid metabolites play an important role in the pathogenesis of several disorders, their contribution to IBD is yet to be understood. Methods Polyunsaturated fatty acids metabolite profiles were established from biopsy samples obtained from Crohn’s disease, ulcerative colitis, or control patients. The impact of a prostaglandin I2 (PGI2) analog on intestinal epithelial permeability was tested in vitro using Caco-2 cells and ex vivo using human or mouse explants. In addition, mice were treated with PGI2 to observe dextran sulfate sodium (DSS)-induced colitis. Tight junction protein expression, subcellular location, and apoptosis were measured in the different models by immunohistochemistry and Western blotting. Results A significant reduction of PGI2 in IBD patient biopsies was identified. PGI2 treatment reduced colonic inflammation, increased occludin expression, decreased caspase-3 cleavage and intestinal permeability, and prevented colitis development in DSS-induced mice. Using colonic explants from mouse and human control subjects, the staurosporine-induced increase in paracellular permeability was prevented by PGI2. PGI2 also induced the membrane location of occludin and reduced the permeability observed in colonic biopsies from IBD patients. Conclusions The present study identified a PGI2 defect in the intestinal mucosa of IBD patients and demonstrated its protective role during colitis., Graphical abstract

Highlights • Enema examination is considered safe, but in rare cases, complications may result. • Iatrogenic bowel perforation can occur without leakage of contrast agent during enema examination through a colostomy. • In the case of an enema examination through a colostomy, clinicians must be aware of the possibility of bowel perforation even if leakage of contrast agent is not observed., Introduction Enema examination is considered safe, but in rare cases, complications may result. Here, we report a rare case of iatrogenic bowel perforation during enema examination through a colostomy without leakage of contrast agent. Presentation of case A 36-year-old man who had undergone a sigmoid loop colostomy was diagnosed with ulcerative colitis. A bowel enema through a colostomy was performed by nurses and radiological technologists. During the procedure, a balloon catheter was inserted into the proximal lumen of the colostomy, and the balloon was inflated. The patient developed severe abdominal pain a few minutes following withdrawal of the catheter. Computed tomography showed intraperitoneal free air, although contrast agent leakage into the intraperitoneal cavity was not observed. The patient underwent emergency laparotomy. Intraoperatively, there was a 3-cm bowel perforation just inside the colostomy where the inflated balloon was pressing. Discussion The perforation site may have been sealed by the inflated balloon during the enema examination. In addition, the patient maintained a supine position during and after the examination. This led to contrast agent accumulating on the dorsal side and not leaking out from the perforation site after the balloon was deflated. Conclusion Iatrogenic bowel perforation can occur without leakage of contrast agent during enema examination through a colostomy, and the examination should be performed under the supervision of an attending doctor. In the case of an enema examination through a colostomy, clinicians must be aware of the possibility of bowel perforation even if leakage of contrast agent is not observed.

Despite growing literature characterizing the fecal microbiome and its association with health and disease, few studies have analyzed the microbiome of the small intestine. Here, we examine what is known about the human small intestinal microbiota in terms of community structure and functional properties. We examine temporal dynamics of select bacterial populations in the small intestine, and the effects of dietary carbohydrates and fats on shaping these populations. We then evaluate dysbiosis in the small intestine in several human disease models, including small intestinal bacterial overgrowth, short-bowel syndrome, pouchitis, environmental enteric dysfunction, and irritable bowel syndrome. What is clear is that the bacterial biology, and mechanisms of bacteria-induced pathophysiology, are enormously broad and elegant in the small intestine. Studying the small intestinal microbiota is challenged by rapidly fluctuating environmental conditions in these intestinal segments, as well as the complexity of sample collection and bioinformatic analysis. Because the functionality of the digestive tract is determined primarily by the small intestine, efforts must be made to better characterize this unique and important microbial ecosystem. Keywords: Microbiota, Microbiome, Small Intestine, Structure, Function, Dysbiosis

Immunoglobulin G4–related diseases (IgG4-RD) are a group of immune-mediated disorders characterized by organ infiltration with lymphocytes and IgG4+ plasma cells. IgG4-RD has multiple clinical manifestations, affecting distant organs ranging from the orbit to the pancreas. Ulcerative colitis (UC) is an inflammatory bowel disease that causes chronic colonic mucosal inflammation, most likely due to a damaged epithelial barrier, an abnormal reaction to intestinal bacteria, and a dysregulated immune response.1 UC is also associated with extraintestinal manifestations including ocular complications. Here we present an unusual case of IgG4-related orbital disease (IgG4-ROD) arising in a patient with ulcerative colitis.

Background & Aims Crohn’s disease (CD) is a debilitating inflammatory disorder that affects more than 1.6 million people in North America alone. Members of the tumor necrosis factor superfamily are key regulators of intestinal inflammation; specifically, tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor–inducible 14 (Fn14), are involved in normal and pathologic tissue remodeling. Our aim was to determine the role of TWEAK/Fn14 in CD and a murine model of CD-like ileitis (ie, SAMP1/YitFc [SAMP] strain). Methods SAMP mice deficient in Fn14 (SAMP × Fn14-/-) were developed and a detailed time-course study was performed evaluating ileal tissues by histology and stereomicroscopy, as well as quantitative polymerase chain reaction and NanoString technology (Seattle, WA). Reciprocal bone marrow chimeras were generated to assess the relevance of Fn14 in hematopoietic vs nonhematopoietic compartments. Surgically resected intestinal tissues and mucosal biopsy specimens from patients with CD, ulcerative colitis, and healthy controls were analyzed for the expression of TWEAK/Fn14 by quantitative polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. Results SAMP × Fn14-/- showed a marked decrease in ileitis severity at 20 weeks of age compared with SAMP WT controls. Bone marrow chimeras showed that Fn14 was required in both hematopoietic and nonhematopoietic compartments for ileitis to develop. Transcriptome data showed multiple cellular pathways regulated by Fn14 signaling. Finally, increased expression of TWEAK and Fn14 was observed in tissue lesions from CD patients compared with ulcerative colitis and healthy controls. Conclusions TWEAK/Fn14 are up-regulated in CD, and also mediate experimental CD-like ileitis, by regulation of multiple innate and adaptive cellular pathways. Therefore, TWEAK/Fn14 may represent a novel therapeutic target for the treatment of small intestinal inflammation in CD., Graphical abstract

Background & Aims Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal Na+/H+ exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in an ulcerative colitis–like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors. Methods We assessed NHE8 expression in human CRCs by immunohistochemistry and studied tumor burden in NHE8 knockout (KO) mice using an azoxymethane/dextran sodium sulfate colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NOD scid gamma (NSG) mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by polymerase chain reaction and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc also were analyzed to evaluate Wnt/β-catenin activation. Results NHE8 was undetectable in human CRC tissues. Although only 9% of NHE8 wild-type mice showed tumorigenesis in the azoxymethane/dextran sodium sulfate colon cancer model, almost 10 times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared with HT29NHE8 wild type cells. Furthermore, NHE8 deficiency resulted in increased Lgr5 expression in the colon, in HT29-derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation. Conclusions NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine., Graphical abstract

Background & Aims Serotonin (5-hydroxytryptamine [5-HT]) is synthesized mainly within enterochromaffin (EC) cells in the gut, and tryptophan hydroxylase 1 (Tph1) is the rate-limiting enzyme for 5-HT synthesis in EC cells. Accumulating evidence suggests the importance of gut microbiota in intestinal inflammation. Considering the close proximity of EC cells and the microbes, we investigated the influence of gut-derived 5-HT on the microbiota and the susceptibility to colitis. Methods Gut microbiota of Tph1-/- and Tph1+/- mice were investigated by deep sequencing. Direct influence of 5-HT on bacteria was assessed by using in vitro system of isolated commensals. The indirect influence of 5-HT on microbiota was assessed by measuring antimicrobial peptides, specifically β-defensins, in the colon of mice and HT-29 colonic epithelial cells. The impact of gut microbiota on the development of dextran sulfate sodium–induced colitis was assessed by transferring gut microbiota from Tph1-/- mice to Tph1+/- littermates and vice versa, as well as in germ-free mice. Results A significant difference in microbial composition between Tph1-/- and Tph1+/- littermates was observed. 5-HT directly stimulated and inhibited the growth of commensal bacteria in vitro, exhibiting a concentration-dependent and species-specific effect. 5-HT also inhibited β-defensin production by HT-29 cells. Microbial transfer from Tph1-/- to Tph1+/- littermates and vice versa altered colitis severity, with microbiota from Tph1-/- mice mediating the protective effects. Furthermore, germ-free mice colonized with microbiota from Tph1-/- mice exhibited less severe dextran sulfate sodium–induced colitis. Conclusions These findings demonstrate a novel role of gut-derived 5-HT in shaping gut microbiota composition in relation to susceptibility to colitis, identifying 5-HT–microbiota axis as a potential new therapeutic target in intestinal inflammatory disorders., Graphical abstract

Notch signaling is activated in the intestinal epithelial cells (IECs) of patients with inflammatory bowel disease (IBD), and contributes to mucosal regeneration. Our previous study indicated that TNF-α and Notch signaling may synergistically promote the expression of the intestinal stem cell (ISC) marker OLFM4 in human IECs. In the present study, we investigated the gene regulation and function of OLFM4 in human IEC lines. We confirmed that TNF-α and Notch synergistically upregulate the mRNA expression of OLFM4. Luciferase reporter assay showed that OLFM4 transcription is regulated by the synergy of TNF-α and Notch. At the protein level, synergy between TNF-α and Notch promoted cytoplasmic accumulation of OLFM4, which has potential anti-apoptotic properties in human IECs. Analysis of patient-derived tissues and organoids consistently showed cytoplasmic accumulation of OLFM4 in response to NF-κB and Notch activation. Cytoplasmic accumulation of OLFM4 in human IECs is tightly regulated by Notch and TNF-α in synergy. Such cytoplasmic accumulation of OLFM4 may have a cell-protective role in the inflamed mucosa of patients with IBD., Highlights • Notch and TNF-α signaling is important in IECs of patients with IBD. • Notch and TNF-α signaling promotes the cytoplasmic accumulation of OLFM4. • OLFM4 accumulation may have anti-apoptotic properties. • OLFM4 could protect against mucosal inflammation in IBD.

Background & Aims Data on the clinical characteristics of patients with inflammatory bowel diseases (IBDs) with coronavirus disease 2019 (COVID-19) are scarce. The aim of our systematic review was to investigate symptoms and diagnostic–therapeutic management of IBD patients with COVID-19. Methods We searched PubMed, Embase, Web of Science, and MedRxiv up to July 29, 2020, to identify all studies reporting clinical information on adult and pediatric IBD patients with confirmed COVID-19. Results Twenty-three studies met our inclusion criteria, including 243,760 IBD patients. COVID-19 was diagnosed in 1028 patients (509 with Crohn’s disease [49.5%], 428 with ulcerative colitis [41.6%], 49 with indeterminate colitis [4.8%], and 42 with missing data [4.1%]), accounting for a cumulative prevalence of 0.4%. Viral infection occurred more frequently in males than in females (56.5% vs 39.7%), and the mean age ranged from 14 to 85 years. The most common symptoms were fever (48.3%), cough (46.5%), and diarrhea (20.5%), and a COVID-19 diagnosis was achieved mainly through polymerase chain reaction analysis of nasopharyngeal swabs (94.4%) and chest computed tomography scans (38.9%). Hydroxychloroquine (23.9%), lopinavir/ritonavir (8.2%), steroids (3.2%), and antibiotics (3.1%) were the most used drugs. Overall, approximately a third of patients were hospitalized (30.6%), and 11.4% of them required admission to the intensive care unit. In total, 29 COVID-19–related deaths were reported (3.8%), and increasing age and the presence of comorbidities were recognized as risk factors for COVID-19 and negative outcomes. Conclusions Diarrhea occurs more frequently in IBD patients with COVID-19 than in the non-IBD population. Further studies are needed to define the optimal diagnostic–therapeutic approach in IBD patients with COVID-19.

Background & Aims Genome-wide association studies (GWAS) and transcriptome analyses have been performed to better understand the pathogenesis of ulcerative colitis (UC). However, current studies mainly focus on European ancestry, highlighting a great need to identify the key genes, pathways and cell types in colonic mucosal cells of adult UC patients from other ancestries. Here we aimed to identify key genes and cell types in colonic mucosal of UC. Methods We performed Single-cell RNA sequencing (scRNA-seq) analysis of 12 colon biopsies of UC patients and healthy controls from Chinese Han ancestry. Results Two novel plasma subsets were identified. Five epithelial/stromal and three immune cell subsets show significant difference in abundance between inflamed and non-inflamed samples. In general, UC risk genes show consistent expression alteration in both Immune cells of inflamed and non-inflamed tissues. As one of the exceptions, IgA defection, marking the signal of immune dysfunction, is specific to the inflamed area. Moreover, Th17 derived activation was observed in both epithelial cell lineage and immune cell lineage of UC patients as compared to controls , suggesting a systemic change of immune activities driven by Th17. The UC risk genes show enrichment in progenitors, glial cells and immune cells, and drug-target genes are differentially expressed in antigen presenting cells. Conclusions Our work identifies novel population-specific plasma cell molecular signatures of UC. The transcriptional signature of UC is shared in immune cells from both inflamed and non-inflamed tissues, whereas the transcriptional response to disease is a local effect only in inflamed epithelial/stromal cells., Graphical abstract

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., Graphical abstract

Background & Aims Inflammatory bowel disease is commonly treated by administration of glucocorticoids. While the importance of intestinal epithelial cells for the pathogenesis of this disorder is widely accepted, their role as target cells for glucocorticoids has not been explored. To address this issue, we induced colonic inflammation in GRvillin mice, which carry an inducible deletion of the glucocorticoid receptor in intestinal epithelial cells. Methods Colitis and colitis-associated colorectal cancer were induced by administration of dextran sulfate sodium and azoxymethane in mice. Clinical parameters, epithelial permeability and tumor development were monitored during disease progression. Colon tissue, lamina propria cells and intestinal epithelial cells were examined by gene expression analyses, flow cytometry, histopathology, and immunohistochemistry. Results The absence of the intestinal epithelial glucocorticoid receptor aggravated clinical symptoms and tissue damage, and compromised epithelial barrier integrity during colitis. Gene expression of chemokines, pattern recognition receptors and molecules controlling epithelial permeability was dysregulated in intestinal epithelial cells of GRvillin mice, leading to a reduced recruitment and a hyperactivation of leukocytes in the lamina propria of the colon. Importantly, the exaggerated inflammatory response in GRvillin mice also enhanced associated tumorigenesis, resulting in a higher number and larger size of tumors in the colon. Conclusions Our results reveal an important role of intestinal epithelial cells as targets of glucocorticoid action in inflammatory bowel disease and suggest that the efficacy with which colitis is kept at bay directly affects the progression of colorectal cancer., Graphical abstract

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world’s population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell–based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases., Graphical abstract Image 1, Highlights • The depletion of NAMPT in macrophages promotes susceptibility to colitis. • NAMPT-mediated phagocytic activity is required for engulfing dead cells, leading to tissue repair. • NMN administration mitigates colitis severity. • NAMPT-dependent NAD+ biosynthetic pathway activation may treat inflammatory disease.