Faecal Volatile Organic Compound Analysis in De Novo Paediatric Inflammatory Bowel Disease by Gas Chromatography-Ion Mobility Spectrometry: A Case-Control Study.

The gut microbiota and its related metabolites differ between inflammatory bowel disease (IBD) patients and healthy controls. In this study, we compared faecal volatile organic compound (VOC) patterns of paediatric IBD patients and controls with gastrointestinal symptoms (CGIs). Additionally, we aimed to assess if baseline VOC profiles could predict treatment response in paediatric IBD patients. We collected faecal samples from a cohort of de novo therapy-naïve paediatric IBD patients and CGIs. VOCs were analysed using gas chromatography-ion mobility spectrometry (GC-IMS). Response was defined as a combination of clinical response based on disease activity scores, without requiring treatment escalation. We included 109 paediatric IBD patients and 75 CGIs, aged 4 to 17 years. Faecal VOC profiles of paediatric IBD patients were distinguishable from those of CGIs (AUC ± 95% CI, p-values: 0.71 (0.64-0.79), <0.001). This discrimination was observed in both Crohn's disease (CD) (0.75 (0.67-0.84), <0.001) and ulcerative colitis (UC) (0.67 (0.56-0.78), 0.01) patients. VOC profiles between CD and UC patients were not distinguishable (0.57 (0.45-0.69), 0.87). Baseline VOC profiles of responders did not differ from non-responders (0.70 (0.58-0.83), 0.1). In conclusion, faecal VOC profiles of paediatric IBD patients differ significantly from those of CGIs.

MicroRNAs in inflammatory bowel disease: What do we know and what can we expect?

MicroRNAs (miRNAs), small non-coding RNAs composed of 18-24 nucleotides, are potent regulators of gene expression, contributing to the regulation of more than 30% of protein-coding genes. Considering that miRNAs are regulators of inflammatory pathways and the differentiation of intestinal epithelial cells, there is an interest in exploring their importance in inflammatory bowel disease (IBD). IBD is a chronic and multifactorial disease of the gastrointestinal tract; the main forms are Crohn's disease and ulcerative colitis. Several studies have investigated the dysregulated expression of miRNAs in IBD, demonstrating their important roles as regulators and potential biomarkers of this disease. This editorial presents what is known and what is expected regarding miRNAs in IBD. Although the important regulatory roles of miRNAs in IBD are clearly established, biomarkers for IBD that can be applied in clinical practice are lacking, emphasizing the importance of further studies. Discoveries regarding the influence of miRNAs on the inflammatory process and the exploration of their role in gene regulation are expected to provide a basis for the use of miRNAs not only as potent biomarkers in IBD but also as therapeutic targets for the control of inflammatory processes in personalized medicine.

Fecal proteolytic profiling of pediatric inflammatory bowel disease: A pilot study.

Colonoscopy is the gold standard for diagnosing inflammatory bowel disease (IBD). However, this invasive procedure has a high burden for pediatric patients. Previous research has shown elevated fecal amino acid concentrations in children with IBD versus controls. We hypothesized that this finding could result from increased proteolytic activity. Therefore, the aim of this study was to investigate whether fecal protease-based profiling was able to discriminate between IBD and controls. Protease activity was measured in fecal samples from patients with IBD (Crohn's disease (CD) n = 19; ulcerative colitis (UC) n = 19) and non-IBD controls (n = 19) using a fluorescence resonance energy transfer (FRET)-peptide library. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each FRET-peptide substrate. Screening the FRET-peptide library revealed an increased total proteolytic activity (TPA), as well as degradation of specific FRET-peptides specifically in fecal samples from IBD patients. Based on level of significance (p < .001) and ROC curve analysis (AUC > 0.85), the fluorogenic substrates W-W, A-A, a-a, F-h, and H-y showed diagnostic potential for CD. The substrates W-W, a-a, T-t, G-v, and H-y showed diagnostic potential for UC based on significance (p < .001) and ROC analysis (AUC > 0.90). None of the FRET-peptide substrates used was able to differentiate between protease activity in fecal samples from CD versus UC. This study showed an increased fecal proteolytic activity in children with newly diagnosed, treatment-naïve, IBD. This could lead to the development of novel, noninvasive biomarkers for screening and diagnostic purposes.

[Upregulating KLF11 ameliorates intestinal inflammation in mice with 2, 4, 6-trinitrobenesulfonic acid-induced colitis by inhibiting the JAK2/STAT3 signaling pathway].

OBJECTIVE: To investigate the expression level of Kruppel-like transcription factor family member KLF11 in intestinal mucosal tissues of Crohn's disease (CD) and its regulatory effect on intestinal inflammation in CD-like colitis. METHODS: We examined KLF11 expression levels in diseased and normal colon mucosal tissues from 12 CD patients and 12 patients with colorectal cancer using immunofluorescence staining. KLF11 expression was also detected in the colon mucosal tissues of a mouse model of 2, 4, 6-trinitrobenesulfonic acid (TNBS)-induced colitis. A recombinant adenoviral vector was used to upregulate KLF11 expression in the mouse models and the changes in intestinal inflammation was observed. A Caco-2 cell model with stable KLF11 overexpression was constructed by lentiviral infection. The effect of KLF11 overexpression on expressions of JAK2/STAT3 signaling pathway proteins was investigated using immunoblotting in both the mouse and cell models. The mouse models were treated with coumermycin A1, a JAK2/STAT3 signaling pathway agonist, and the changes in intestinal inflammatory responses were observed. RESULTS: The expression level of KLF11 was significantly lowered in both the clinical specimens of diseased colon mucosal tissues and the colon tissues of mice with TNBS-induced colitis (P < 0.05). Adenovirus-mediated upregulation of KLF11 significantly improved intestinal inflammation and reduced the expression levels of inflammatory factors in the intestinal mucosa of the colitis mouse models (P < 0.05). Overexpression of KLF11 significantly inhibited the expression levels of p-JAK2 and p-STAT3 in intestinal mucosal tissues of the mouse models and in Caco-2 cells (P < 0.05). Treatment with coumermycin A1 obviously inhibited the effect of KLF11 upregulation for improving colitis and significantly increased the expression levels of inflammatory factors in the intestinal mucosa of the mouse models (P < 0.05). CONCLUSION: KLF11 is downregulated in the intestinal mucosa in CD, and upregulation of KLF11 can improve intestinal inflammation and reduce the production of inflammatory factors probably by inhibiting the JAK2/STAT3 signaling pathway.

Gut metabolome and microbiota signatures predict response to treatment with exclusive enteral nutrition in a prospective study in children with active Crohn's disease.

BACKGROUND: Predicting response to exclusive enteral nutrition (EEN) in active Crohn's disease (CD) could lead to therapy personalization and pretreatment optimization. OBJECTIVES: This study aimed to explore the ability of pretreatment parameters to predict fecal calprotectin (FCal) levels at EEN completion in a prospective study in children with CD. METHODS: In children with active CD, clinical parameters, dietary intake, cytokines, inflammation-related blood proteomics, and diet-related metabolites, metabolomics and microbiota in feces, were measured before initiation of 8 wk of EEN. Prediction of FCal levels at EEN completion was performed using machine learning. Data are presented with medians (IQR). RESULTS: Of 37 patients recruited, 15 responded (FCal < 250 µg/g) to EEN (responders) and 22 did not (nonresponders). Clinical and immunological parameters were not associated with response to EEN. Responders had lesser (µmol/g) butyrate [responders: 13.2 (8.63-18.4) compared with nonresponders: 22.3 (12.0-32.0); P = 0.03], acetate [responders: 49.9 (46.4-68.4) compared with nonresponders: 70.4 (57.0-95.5); P = 0.027], phenylacetate [responders: 0.175 (0.013-0.611) compared with nonresponders: 0.943 (0.438-1.35); P = 0.021], and a higher microbiota richness [315 (269-347) compared with nonresponders: 243 (205-297); P = 0.015] in feces than nonresponders. Responders consumed (portions/1000 kcal/d) more confectionery products [responders: 0.55 (0.38-0.72) compared with nonresponders: 0.19 (0.01-0.38); P = 0.045]. A multicomponent model using fecal parameters, dietary data, and clinical and immunological parameters predicted response to EEN with 78% accuracy (sensitivity: 80%; specificity: 77%; positive predictive value: 71%; negative predictive value: 85%). Higher taxon abundance from Ruminococcaceae, Lachnospiraceae, and Bacteroides and phenylacetate, butyrate, and acetate were the most influential variables in predicting lack of response to EEN. CONCLUSIONS: We identify microbial signals and diet-related metabolites in feces, which could comprise targets for pretreatment optimization and personalized nutritional therapy in pediatric CD.

Inflammation and Organic Cation Transporters Novel (OCTNs).

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.

Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohn's disease.

Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected 13 co-regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

hUC-MSCs therapy for Crohn's disease: efficacy in TNBS-induced colitis in rats and pilot clinical study.

BACKGROUND: The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. METHODS: The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established in vitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1ß) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. FINDINGS: hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1ß, IL-6, and TNFα, p < 0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1ß, IL-6, and TNFα significantly (p < 0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48 h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. INTERPRETATION: hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. FUNDING: This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08), Key Disciplines Group Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZxq2022-06), Medical discipline Construction Project of Pudong Health Committee of Shanghai (Grant No. PWYgf2021-02) and National Natural Science Foundation of China (Grant No. 82300604).

Targeting AQP9 enhanced the anti-TNF therapy response in Crohn's disease by inhibiting LPA-hippo pathway.

Although anti-TNF antibodies are extensively used to treat Crohn's disease (CD), a significant proportion of patients, up to 40%, exhibit an inadequate response to this therapy. Our objective was to identify potential targets that could improve the effectiveness of anti-TNF therapy in CD. Through the integration and analysis of transcriptomic data from various CD databases, we found that the expression of AQP9 was significantly increased in anti-TNF therapy-resistant specimens. The response to anti-TNF therapy in the CD mouse model was significantly enhanced by specifically inhibiting AQP9. Further experiments found that the blockade of AQP9, which is dominantly expressed in macrophages, decreased inflamed macrophage functions and cytokine expression. Mechanistic studies revealed that AQP9 transported glycerol into macrophages, where it was metabolized to LPA, which was further metabolized to LPA, resulting in the activation of the LPAR2 receptor and downstream hippo pathway, finally promoting the expression of cytokines, especially IL23 and IL1ß⊡ Taken together, the expansion of AQP9+ macrophages is associated with resistance to anti-TNF therapy in Crohn's disease. These findings indicated that AQP9 could be a potential target for enhancing anti-TNF therapy in Crohn's disease.

Sophoricoside improved Crohn's disease-like colitis by inhibiting intestinal epithelial cell apoptosis through PI3K/AKT signaling.

BACKGROUND AND AIMS: Increased apoptosis of intestinal epithelial cells (IECs) is a significant cause of intestinal barrier dysfunction in Crohn's disease (CD). Sophoricoside (SOP) is an isoflavone glycoside known for its anti-apoptotic properties. The aim of this study was to investigate the effects of SOP on mice with CD-like colitis and to understand the underlying mechanisms. METHODS: Mice treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS) were used to examine the therapeutic effect of SOP on CD-like colitis and intestinal barrier damage. To further explore SOP's impact on IECs apoptosis and intestinal barrier protection, an in vitro colonic organoid apoptosis model induced by TNF-α was utilized. Network pharmacology was employed to predict the relevant pathways and molecular processes associated with SOP in the treatment of CD. RESULTS: Treatment with SOP significantly improved colitis symptoms in TNBS mice, as demonstrated by reductions in the Disease Activity Index (DAI), weight loss, colon shortening, macroscopic scores, colonic tissue inflammatory scores, and the expression of pro-inflammatory factors. Our experiments confirmed that SOP protects the intestinal barrier by counteracting IECs apoptosis. Additionally, this study established that SOP reduced IECs apoptosis by inhibiting the PI3K/AKT signaling pathway. CONCLUSIONS: SOP can reduce IECs apoptosis through the inhibition of the PI3K/AKT signaling pathway, thereby protecting the intestinal barrier. This study is the first to illustrate how SOP ameliorates colitis and protects the intestinal barrier, suggesting SOP has potential clinical application in treating CD.

An in vitro model to study immune activation, epithelial disruption and stromal remodelling in inflammatory bowel disease and fistulising Crohn's disease.

At present, preclinical models of inflammatory bowel disease (IBD) are insufficient, limiting translation between research and new therapeutics. This is especially true for fistulising Crohn's disease (CD), as the severe lack of relevant models hinders research progression. To address this, we present in vitro human IBD mucosal models that recapitulate multiple pathological hallmarks of IBD simultaneously in one model system - immune cell infiltration, stromal remodelling and epithelial disruption. Stimulation of models induces epithelial aberrations common in IBD tissue including altered morphology, microvilli abnormalities, claudin gene expression changes and increased permeability. Inflammatory biomarkers are also significantly increased including cytokines and chemokines integral to IBD pathogenesis. Evidence of extracellular matrix remodelling, including upregulated matrix-metalloproteinases and altered basement membrane components, suggests the models simulate pathological stromal remodelling events that closely resemble fistulising CD. Importantly, MMP-9 is the most abundant MMP and mimics the unique localisation observed in IBD tissue. The inflamed models were subsequently used to elucidate the involvement of TNF-α and IFN- γ in intestinal stromal remodelling, in which TNF-α but not IFN- γ induced MMP upregulation, specifically of MMP-3 and MMP-9. Collectively, our results demonstrate the potential of the IBD models for use in preclinical research in IBD, particularly for fistulising CD.

The intestinal microbial metabolite acetyl l-carnitine improves gut inflammation and immune homeostasis via CADM2.

Intestinal symbiotic bacteria play a key role in the regulation of immune tolerance in inflammatory bowel disease (IBD) hosts. However, the bacterial strains directly involved in this regulation and their related metabolites are largely unknown. We sought to investigate the effects of intestinal microbial metabolites on intestinal epithelium and to elucidate their therapeutic potential in regulating intestinal mucosal inflammation and immune homeostasis. Here, we used metagenomic data from Crohn's disease (CD) patients to analyze the composition of intestinal flora and identify metabolite profiles associated with disease behavior, and used the mouse model of dextran sodium sulfate (DSS)-induced colitis to characterize the therapeutic effects of the flora metabolite acetyl l-carnitine (ALC) on DSS-induced colitis. We found that intraperitoneal injection of ALC treatment could significantly alleviate the symptoms of DSS-induced colitis in mice, including prevention of weight loss, reduction in disease activity index (DAI) scores, increasing of colonic length, reduction in histological scores, and improvement in intestinal barrier function. Further, transcriptome sequencing analysis and gene silencing experiments revealed that the absence of CADM2 abolished the inhibitory effect of ALC on the TLR-MyD88 pathway in colonic epithelial cells, thereby reducing the release of inflammatory factors in colon epithelial cells. And we confirmed a significant downregulation of CADM2 expression in intestinal tissues of CD patients compared to healthy people in a population cohort. In addition, we also found that ALC increased the ratio of Treg cells in colon, and decreased the ratio of Th17 cells and macrophages, thereby improving the immune tolerance of the organism. The proposed study could be a potential approach for the treatment of CD.

LncRNA MSC-AS1 regulates SNIP1 SUMOylation-mediated EMT by binding to SENP1 to promote intestinal fibrosis in Crohn's disease.

As a common complication of Crohn's disease (CD), the mechanism underlying CD intestinal fibrosis remains unclear. Studies have shown that epithelial-mesenchymal transition (EMT) is a key step in the development of intestinal fibrosis in CD. It is currently known that the long non-coding RNA (lncRNA) MSC-AS1 plays an important role in regulating the secretion of inflammatory mediators and EMT; however, its role in intestinal fibrosis remains unclear. MSC-AS1 was significantly upregulated in the CD intestinal tissue and intestinal tissue of mice treated with 2,4,6-trinitrobenzenesulfonic acid. Downregulation of its expression can inhibit EMT and alleviates intestinal fibrosis by regulating SNIP1. In addition, MSC-AS1 directly interacted with SENP1, blocking the deSUMOylation of SNIP1 and inhibiting its activity. Furthermore, we found that SENP1 enhanced the expression of SNIP1 and reduced intestinal fibrosis. In summary, MSC-AS1 regulates EMT through the SENP1/SNIP1 axis to promote fibrosis, and may be considered a potential molecular target for the treatment of CD and intestinal fibrosis.

Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4+ Tissue-resident Memory T cells in Crohn's Disease.

BACKGROUND & AIMS: As the most abundant memory T cells and major source of tumor necrosis factor α in the intestinal mucosa of Crohn's disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells. METHODS: CD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction experiments were used to assess cytokines level in CD4+ TRM cells; activation-induced cell apoptosis rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells. RESULTS: Transcriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells up-regulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated nuclear factor kappa B signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake, and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD patients. CONCLUSIONS: CD4+ TRM cells process an accelerated FAO mediated by activated nuclear factor kappa B signaling in CD patients; targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

Recent advances in intestinal fibrosis.

Despite many progresses have been made in the treatment of inflammatory bowel disease, especially due to the increasing number of effective therapies, the development of tissue fibrosis is a very common occurrence along the natural history of this condition. To a certain extent, fibrogenesis is a physiological and necessary process in all those conditions characterised by chronic inflammation. However, the excessive deposition of extracellular matrix within the bowel wall will end up in the formation of strictures, with the consequent need for surgery. A number of mechanisms have been described in this process, but some of them are not yet clear. For sure, the main trigger is the presence of a persistent inflammatory status within the mucosa, which in turn favours the occurrence of a pro-fibrogenic environment. Among the main key players, myofibroblasts, fibroblasts, immune cells, growth factors and cytokines must be mentioned. Although there are no available therapies able to target fibrosis, the only way to prevent it is by controlling inflammation. In this review, we summarize the state of art of the mechanisms involved in gut fibrogenesis, how to diagnose it, and which potential targets could be druggable to tackle fibrosis.

Treatment regimens and disease activity could alter salivary myeloperoxidase levels in patients with inflammatory bowel diseases.

INTRODUCTION: Inflammatory bowel diseases (IBDs) present with alternating periods of exacerbation and remission; therefore, it is necessary to develop noninvasive diagnostic tools to control the disease activity and improve therapeutic effectiveness. Recently, we have found that patients with ulcerative colitis (UC) who qualified for biologic therapy had significantly lower salivary myeloperoxidase (MPO) levels. OBJECTIVES: This cross­sectional study aimed to assess the impact of IBD activity and applied treatment on salivary antioxidant system as reflected by the levels of catalase, total antioxidant status, and MPO. PATIENTS AND METHODS: The study group comprised 99 patients diagnosed with Crohn disease (CD) and 61 patients with UC. The Crohn Disease Activity Index and modified Mayo scale were used to estimate the clinical activity of CD and UC, respectively. Unstimulated whole mixed saliva was collected. Salivary levels of selected markers were measured with enzyme­linked immunosorbent assays and colorimetric assays. RESULTS: The patients with clinically active UC showed significantly decreased median (interquartile range) salivary MPO levels (79.4 [30.1-157.5] vs 94.8 [58.2-274.7] ng/ml) with significant correlations with the endoscopic stage on the Mayo scale (R = 0.423; P = 0.02). Receiver operating characteristic analysis confirmed a potential usefulness of MPO concentrations in predicting clinically active UC (area under the curve = 0.654; P = 0.03; cutoff <210.4 ng/ml). Moreover, in the patients treated with biologics and without steroid therapy, salivary MPO concentrations negatively correlated with neutrophil counts in the individuals with UC and positively with C­reactive protein level in the patients with CD. CONCLUSIONS: Salivary MPO levels changed depending on the disease activity in the patients with UC. Decreased MPO concentration in the saliva could be a predictor of clinically active UC.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn's Disease.

BACKGROUND & AIMS: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. METHODS: We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat.

The gut-brain axis: Correlation of choroid plexus volume and permeability with inflammatory biomarkers in Crohn's disease.

BACKGROUND: The dysregulation of the gut-brain axis in chronic inflammatory bowel diseases can cause neuro-psychological disturbances, but the underlying mechanisms are still not fully understood. The choroid plexus (CP) maintains brain homeostasis and nourishment through the secretion and clearance of cerebrospinal fluid. Recent research has demonstrated the existence of a CP vascular barrier in mice which is modulated during intestinal inflammation. This study investigates possible correlations between CP modifications and inflammatory activity in patients with Crohn's disease (CD). METHODS: In this prospective study, 17 patients with CD underwent concomitant abdominal and brain 3 T MRI. The volume and permeability of CP were compared with levels of C-reactive protein (CRP), fecal calprotectin (FC), sMARIA and SES-CD scores. RESULTS: The CP volume was negatively correlated with CRP levels (R = -0.643, p-value = 0.024) and FC (R = -0.571, p-value = 0.050). DCE metrics normalized by CP volume were positively correlated with CRP (K-trans: R = 0.587, p-value = 0.045; Vp: R = 0.706, p-value = 0.010; T1: R = 0.699, p-value = 0.011), and FC (Vp: R = 0.606, p-value = 0.037). CONCLUSIONS: Inflammatory activity in patients with CD is associated with changes in CP volume and permeability, thus supporting the hypothesis that intestinal inflammation could affect the brain through the modulation of CP vascular barrier also in humans.

Interleukin 21 Drives a Hypermetabolic State and CD4+ T-Cell-Associated Pathogenicity in Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease; however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODS: Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing the Seahorse XF analyzer. We used a Crohn's disease single-cell RNA sequencing dataset to infer the therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically modified Tregs in CD4+ T-cell-induced murine colitis models. RESULTS: Mitochondria-endoplasmic reticulum appositions, known to mediate pyruvate entry into mitochondria via voltage-dependent anion channel 1 (VDAC1), are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate supplementation. Notably, interleukin (IL) 21 diminished mitochondria-endoplasmic reticulum appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 ß, a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. Methyl pyruvate and glycogen synthase kinase 3 ß pharmacologic inhibitor (LY2090314) reversed IL21-induced metabolic rewiring and inflammatory state. Moreover, IL21-induced metabolic genes in Tregs in vitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONS: IL21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL21-induced metabolism in Tregs may mitigate CD4+ T-cell-driven chronic intestinal inflammation.

Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. METHODS: Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. RESULTS: Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. CONCLUSIONS: In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.