Disruption of CerS6-mediated sphingolipid metabolism by FTO deficiency aggravates ulcerative colitis.

BACKGROUND AND AIMS: Deregulation of RNA N6-methyladenosine (m6A) modification in intestinal epithelial cells (IECs) influences intestinal immune cells and leads to intestinal inflammation. We studied the function of fat mass-and obesity-associated protein (FTO), one of the m6A demethylases, in patients with ulcerative colitis (UC). METHODS: We analysed colon tissues of Ftoflox/flox; Villin-cre mice and their Ftoflox/flox littermates with dextran sulfate sodium (DSS) using real-time PCR and 16s rRNA sequencing. RNA and methylated RNA immunoprecipitation sequencing were used to analyse immunocytes and IECs. Macrophages were treated with conditioned medium of FTO-knockdown MODE-K cells or sphingosine-1-phosphate (S1P) and analysed for gene expression. Liquid chromatograph mass spectrometry identified C16-ceramide. RESULTS: FTO downregulation was identified in our in-house cohort and external cohorts of UC patients. Dysbiosis of gut microbiota, increased infiltration of proinflammatory macrophages, and enhanced differentiation of Th17 cells were observed in Ftoflox/flox;Villin-cre mice under DSS treatment. FTO deficiency resulted in an increase in m6A modification and a decrease in mRNA stability of CerS6, the gene encoding ceramide synthetase, leading to the downregulation of CerS6 and the accumulation of S1P in IECs. Subsequentially, the secretion of S1P by IECs triggered proinflammatory macrophages to secrete serum amyloid A protein 1/3, ultimately inducing Th17 cell differentiation. In addition, through bioinformatic analysis and experimental validation, we identified UC patients with lower FTO expression might respond better to vedolizumab treatment. CONCLUSIONS: FTO downregulation promoted UC by decreasing CerS6 expression, leading to increased S1P accumulation in IECs and aggravating colitis via m6A-dependent mechanisms. Lower FTO expression in UC patients may enhance their response to vedolizumab treatment.

Gut virome in inflammatory bowel disease and beyond.

OBJECTIVE: The gut virome is a dense community of viruses inhabiting the gastrointestinal tract and an integral part of the microbiota. The virome coexists with the other components of the microbiota and with the host in a dynamic equilibrium, serving as a key contributor to the maintenance of intestinal homeostasis and functions. However, this equilibrium can be interrupted in certain pathological states, including inflammatory bowel disease, causing dysbiosis that may participate in disease pathogenesis. Nevertheless, whether virome dysbiosis is a causal or bystander event requires further clarification. DESIGN: This review seeks to summarise the latest advancements in the study of the gut virome, highlighting its cross-talk with the mucosal microenvironment. It explores how cutting-edge technologies may build upon current knowledge to advance research in this field. An overview of virome transplantation in diseased gastrointestinal tracts is provided along with insights into the development of innovative virome-based therapeutics to improve clinical management. RESULTS: Gut virome dysbiosis, primarily driven by the expansion of Caudovirales, has been shown to impact intestinal immunity and barrier functions, influencing overall intestinal homeostasis. Although emerging innovative technologies still need further implementation, they display the unprecedented potential to better characterise virome composition and delineate its role in intestinal diseases. CONCLUSIONS: The field of gut virome is progressively expanding, thanks to the advancements of sequencing technologies and bioinformatic pipelines. These have contributed to a better understanding of how virome dysbiosis is linked to intestinal disease pathogenesis and how the modulation of virome composition may help the clinical intervention to ameliorate gut disease management.

Alzheimer's disease-related presenilins are key to intestinal epithelial cell function and gut immune homoeostasis.

OBJECTIVE: Mutations in presenilin genes are the major cause of Alzheimer's disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis. DESIGN: Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis. RESULTS: Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation. CONCLUSION: Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.

TiO2 nanoparticles abrogate the protective effect of the Crohn's disease-associated variation within the PTPN22 gene locus.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

MCPIP1 restrains mucosal inflammation by orchestrating the intestinal monocyte to macrophage maturation via an ATF3-AP1S2 axis.

OBJECTIVE: Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) is highly expressed in inflamed mucosa of inflammatory bowel disease (IBD) and negatively regulates immune response, while the underlying mechanisms regulating mucosal macrophage functions remain unknown. Here, we investigated the roles of MCPIP1 in modulating the differentiation and functions of intestinal macrophages in the pathogenesis of IBD. DESIGN: ScRNA-seq was used to cluster the monocyte/macrophage lineage from macrophage-specific Mcpip1-deficient (Mcpip1 ∆Mye) mice and Mcpip1 fl/fl littermates. The differentially expressed genes were confirmed by RNA-seq, luciferase assay, CUT&Tag assay and Western blotting. Effects of MCPIP1 and the activating transcription factor 3 (ATF3)-AP1S2 axis were assessed in patients with IBD. RESULTS: Mcpip1 ∆Mye mice developed more severe dextran sulfate sodium (DSS)-induced colitis characterised by an increase in macrophage migratory capacity and M1 macrophage polarisation but a decrease in the monocyte-to-macrophage maturation in gut mucosa compared with their littermates. ScRNA-seq unravelled a proinflammatory population (Ccr2+Il-1ß+Tlr2+Cx3cr1-Cd163-Mrc1-Ly6c+) of the monocyte/macrophage lineage from lamina propria CD11b+ cells and an arrest of Mcpip1 ∆Mye monocyte-to-macrophage maturation in an Atf3-Ap1s2 axis-dependent manner. Silencing of Ap1s2 or Atf3 markedly suppressed Mcpip1 ∆Mye macrophage migration, M1-like polarisation, and production of proinflammatory cytokines and chemokines. Notably, in vivo blockage of Ap1s2 ameliorated DSS-induced colitis in Mcpip1 ΔMye mice through enhancing intestinal macrophage maturation. Furthermore, MCPIP1, ATF3 and AP1S2 were highly expressed in inflamed mucosa of active patients with IBD and blockage of ATF3 or AP1S2 significantly suppressed IBD CD14+-derived M1-like macrophage polarisation and proinflammatory cytokine production. CONCLUSIONS: Macrophage-specific Mcpip1 deficiency polarises macrophages towards M1-like phenotype, arrests macrophage maturation and exacerbates intestinal inflammation in an Atf3-Ap1s2-dependent manner, thus providing novel mechanistic insight into intestinal macrophage functions during IBD.

Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn's disease.

OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

Inflammatory bowel disease and Parkinson's disease: common pathophysiological links.

Inflammatory bowel disease and Parkinson's disease are chronic progressive disorders that mainly affect different organs: the gut and brain, respectively. Accumulating evidence has suggested a bidirectional link between gastrointestinal inflammation and neurodegeneration, in accordance with the concept of the 'gut-brain axis'. Moreover, recent population-based studies have shown that inflammatory bowel disease might increase the risk of Parkinson's disease. Although the precise mechanisms underlying gut-brain interactions remain elusive, some of the latest findings have begun to explain the link. Several genetic loci are shared between both disorders with a similar direction of effect on the risk of both diseases. The most interesting example is LRRK2 (leucine-rich repeat kinase 2), initially identified as a causal gene in Parkinson's disease, and recently also implicated in Crohn's disease. In this review, we highlight recent findings on the link between these seemingly unrelated diseases with shared genetic susceptibility. We discuss supporting and conflicting data obtained from epidemiological and genetic studies along with remaining questions and concerns. In addition, we discuss possible biological links including the gut-brain axis, microbiota, autoimmunity, mitochondrial function and autophagy.

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling.

OBJECTIVE: Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined. DESIGN: We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rbhigh T-cell transfer model in combination with several genetic mouse models. RESULTS: Anti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rbhigh T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF. CONCLUSION: The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

CD177+ neutrophils as functionally activated neutrophils negatively regulate IBD.

BACKGROUND: Neutrophils are accumulated in inflamed mucosa of IBD and play an important role in the pathogenesis. CD177 is expressed in neutrophils specifically and upregulated during inflammation. However, the role of CD177+ neutrophils in pathogenesis of IBD remains elusive. MATERIALS AND METHODS: Expression of CD177 was analysed in peripheral blood and intestinal mucosa from patients with IBD using quantitative RT-PCR, flow cytometry and immunohistochemistry. CD177+ and CD177- neutrophils were isolated to determine gene differences by RNA sequencing. Colitis was established in CD177-/- and wild-type mice in response to dextran sulfate sodium (DSS) insults to determine the role of CD177+ neutrophils in IBD. RESULTS: CD177+ neutrophils were markedly increased in peripheral blood and inflamed mucosa from patients with active IBD compared with healthy controls. RNA sequencing revealed that differential gene expression between CD177+ and CD177- neutrophils from patients with IBD was associated with response to bacterial defence, hydrogen peroxide and reactive oxygen species (ROS). CD177+ neutrophils produced lower levels of proinflammatory cytokines (ie, interferon-γ, interleukin (IL)-6, IL-17A), but higher levels of IL-22 and transforming growth factor-ß, and exhibited increased bactericidal activities (ie, ROS, antimicrobial peptides, neutrophil extracellular trap) compared with CD177- subset. CD177-/- mice developed more severe colitis on DSS insults compared with wild-type mice. Moreover, CD177 deficiency led to compromised intestinal barrier and impaired antibacterial immunity through decreased production of IL-22 by CD177- neutrophils. CONCLUSIONS: CD177+ neutrophils represent functionally activated population and play a protective role in IBD through increased bactericidal activity and IL-22 production. Targeting CD177+ neutrophils may be beneficial for treatment of IBD.

NAD metabolism fuels human and mouse intestinal inflammation.

OBJECTIVE: Nicotinamide phosphoribosyltransferase (NAMPT, also referred to as pre-B cell colony-enhancing factor or visfatin) is critically required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD) supply catalysing the rate-limiting step of the NAD salvage pathway. NAMPT is strongly upregulated in inflammation including IBD and counteracts an increased cellular NAD turnover mediated by NAD-depleting enzymes. These constitute an important mechanistic link between inflammatory, metabolic and transcriptional pathways and NAD metabolism. DESIGN: We investigated the impact of NAMPT inhibition by the small-molecule inhibitor FK866 in the dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer. The impact of NAD depletion on differentiation of mouse and human primary monocytes/macrophages was studied in vitro. Finally, we tested the efficacy of FK866 compared with dexamethasone and infliximab in lamina propria mononuclear cells (LPMNC) isolated from patients with IBD. RESULTS: FK866 ameliorated DSS-induced colitis and suppressed inflammation-associated tumorigenesis in mice. FK866 potently inhibited NAMPT activity as demonstrated by reduced mucosal NAD, resulting in reduced abundances and activities of NAD-dependent enzymes including PARP1, Sirt6 and CD38, reduced nuclear factor kappa B activation, and decreased cellular infiltration by inflammatory monocytes, macrophages and activated T cells. Remarkably, FK866 effectively supressed cytokine release from LPMNCs of patients with IBD. As FK866 was also effective in Rag1-/- mice, we mechanistically linked FK866 treatment with altered monocyte/macrophage biology and skewed macrophage polarisation by reducing CD86, CD38, MHC-II and interleukin (IL)-6 and promoting CD206, Egr2 and IL-10. CONCLUSION: Our data emphasise the importance of NAD immunometabolism for mucosal immunity and highlight FK866-mediated NAMPT blockade as a promising therapeutic approach in acute intestinal inflammation.

Raf kinase inhibitor protein mediates intestinal epithelial cell apoptosis and promotes IBDs in humans and mice.

OBJECTIVE: Raf kinase inhibitor protein (RKIP) appears to control cancer cell metastasis and its expression in colonic tissue is related to colonic cancer development. We sought to identify the roles of RKIP in maintaining homeostasis of GI tract. DESIGN: The expression of RKIP was determined by immunohistochemistry and western blot analysis. RKIP knockout and wild-type mice were administered dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce experimental colitis, and the mice were assessed based on colitis symptoms and biochemical approaches. The mechanism was analysed using immunoprecipitation and pull-down experiments. RESULTS: The RKIP expression is positively correlated with the severity of IBD. RKIP deficiency protects mice from DSS-induced or TNBS-induced colitis and accelerated recovery from colitis. RKIP deficiency inhibits DSS-induced infiltration of acute-phase immune cells and reduces production of proinflammatory cytokines and chemokines in colon. RKIP deficiency inhibits DSS-induced or TNBS-induced colonic epithelial barrier damage and intestinal epithelial cell (IEC) apoptosis. RKIP deficiency also inhibits tumour necrosis factor-alpha-induced IEC apoptosis and colitis. Mechanistically, RKIP enhances the induction of P53-upregulated modulator of apoptosis by interacting with TGF-ß-activated kinase 1 (TAK1) and promoting TAK1-mediated NF-κB activation. This is supported by the observation that TAK1 activation is positively correlated with the expression of RKIP in human clinical samples and the development of IBD. CONCLUSIONS: RKIP contributes to colitis development by promoting inflammation and mediating IEC apoptosis and might represent a therapeutic target of IBD.

Specific members of the predominant gut microbiota predict pouchitis following colectomy and IPAA in UC.

OBJECTIVE: Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy. DESIGN: Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation. RESULTS: Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively. CONCLUSIONS: Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA.

Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann-Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease.

OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.

Proteomic analysis of ascending colon biopsies from a paediatric inflammatory bowel disease inception cohort identifies protein biomarkers that differentiate Crohn's disease from UC.

OBJECTIVE: Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. DESIGN: Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). RESULTS: In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2. CONCLUSIONS: This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.

Methyl-deficient diet promotes colitis and SIRT1-mediated endoplasmic reticulum stress.

BACKGROUND: Methyl donor deficiency (MDD) aggravates experimental colitis in rats and increases endoplasmic reticulum (ER) stress through decreased sirtuin 1 (SIRT1) in neuronal cells and myocardium. ER stress plays a key role in IBD pathogenesis. AIM: We investigated whether the influence of MDD on colitis resulted from an ER stress response triggered by decreased SIRT1 expression. DESIGN: The unfolded protein response (UPR), chaperones proteins, heat shock factor protein 1 (HSF1) and SIRT1 were examined in rats with MDD and dextran sulfate sodium (DSS)-induced colitis in a Caco-2 cell model with stable expression of transcobalamin-oleosin (TO) chimera, which impairs cellular availability of vitamin B12, and in IBD. The effects of SIRT1 activation were studied both in vitro and in vivo. RESULTS: MDD aggravated DSS-induced colitis clinically, endoscopically and histologically. MDD activated ER stress pathways, with increased phosphorylate-PKR-like ER kinase, P-eiF-2α, P-IRE-1α, activating transcription factor (ATF)6, XBP1-S protein and ATF4 mRNA expression levels in rats. This was accompanied by reduced SIRT1 expression level and greater acetylation of HSF1, in relation with a dramatic decrease of chaperones (binding immunoglobulin protein (BIP), heat shock protein (HSP)27 and HSP90). Adding either vitamin B12, S-adenosylmethionine or an SIRT1 activator (SRT1720) reduced the UPR in vitro. In rats, SIRT1 activation by SRT1720 prevented colitis by reducing HSF1 acetylation and increasing expression of BIP, HSP27 and HSP90. Immunohistochemistry showed impaired expression of SIRT1 in the colonic epithelium of patients with IBD. CONCLUSIONS: SIRT1 is a master regulator of ER stress and severity of experimental colitis in case of MDD. It could deserve further interest as a therapeutic target of IBD.

A TPL2 (MAP3K8) disease-risk polymorphism increases TPL2 expression thereby leading to increased pattern recognition receptor-initiated caspase-1 and caspase-8 activation, signalling and cytokine secretion.

OBJECTIVE: IBD is characterised by dysregulated intestinal immune homeostasis and cytokine secretion. In the intestine, properly regulating pattern recognition receptor (PRR)-mediated signalling and cytokines is crucial given the ongoing host-microbial interactions. TPL2 (MAP3K8, COT) contributes to PRR-initiated pathways, yet the mechanisms for TPL2 signalling contributions in primary human myeloid cells are incompletely understood and its role in intestinal myeloid cells is poorly defined. Furthermore, functional consequences for the IBD-risk locus rs1042058 in TPL2 are unknown. METHODS: We analysed protein, cytokine and RNA expression, and signalling in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR and flow cytometry. RESULTS: PRR-induced cytokine secretion was increased in MDMs from rs1042058 TPL2 GG risk individuals. TPL2 activation by the Crohn's disease-associated PRR nucleotide-oligomerisation domain (NOD)2 required PKC, and IKKß, IKKα and IKKγ signalling. TPL2, in turn, significantly enhanced NOD2-induced ERK, JNK and NFκB signalling. We found that another major mechanism for the TPL2 contribution to NOD2 signalling was through ERK-dependent and JNK-dependent caspase-1 and caspase-8 activation, which in turn, led to early autocrine interleukin (IL)-1ß and IL-18 secretion and amplification of long-term cytokines. Importantly, Salmonella typhimurium-induced cytokines from human intestinal myeloid-derived cells required TPL2 as well as autocrine IL-1ß and IL-18. Finally, rs1042058 GG risk carrier MDMs from healthy individuals and patients with Crohn's disease had increased TPL2 expression and NOD2-initiated TPL2 phosphorylation, ERK, JNK and NFκB activation, and early autocrine IL-1ß and IL-18 secretion. CONCLUSIONS: Taken together, the rs1042058 GG IBD-risk polymorphism in TPL2 results in a gain-of-function by increasing TPL2 expression and signalling, thereby amplifying PRR-initiated outcomes.

miR-301a promotes intestinal mucosal inflammation through induction of IL-17A and TNF-α in IBD.

OBJECTIVE: MicroRNA (miR)-301a is known to be involved in the tumourigenesis and pathogenesis of several autoimmune diseases, but it remains unclear whether miR-301a is associated with the pathogenesis of IBD. METHODS: miR-301a expression was assessed in peripheral blood mononuclear cells (PBMC) and inflamed mucosa of patients with IBD by quantitative real-time-PCR. Peripheral blood CD4+ T cells were transduced with lentivirus-encoding pre-miR-301a (LV-miR-301a) or a reverse complementary sequence of miR-301a (LV-anti-miR-301a), and their differentiation and activation were investigated in vitro. Antisense miR-301a was administered into mice during trinitrobenzene sulphonic acid (TNBS)-induced colitis to determine its role in colitis. RESULTS: miR-301a expression was significantly upregulated in PBMC and inflamed mucosa of patients with IBD compared with healthy controls. Stimulation with tumour necrosis factor-α (TNF-α) significantly enhanced miR-301a expression in IBD CD4+ T cells, which was markedly reversed by anti-TNF-α mAb (Infliximab) treatment. Transduction of LV-miR-301a into CD4+ T cells from patients with IBD promoted the Th17 cell differentiation and TNF-α production compared with the cells with expression of LV-anti-miR-301a. SNIP1 as a functional target of miR-301a was reduced in miR-301a expression but increased in LV-anti-miR-301a expression. Knockdown of SNIP1 could enhance Th17 cell differentiation. Furthermore, intracolonical administration of antisense miR-301a in TNBS-induced mouse colitis model significantly decreased numbers of interleukin (IL)-17A+ cells and amounts of pro-inflammatory cytokines (eg, IL-17A, TNF-α) in inflamed colon. CONCLUSIONS: Our data reveal a novel mechanism in which the elevated miR-301a in PBMC and inflamed mucosa of IBD promotes Th17 cell differentiation through downregulation of SNIP1. Blockade of miR-301a in vivo may serve as a novel therapeutic approach in the treatment of IBD.