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Background & Aims Interleukin (IL)33/IL1F11 is an important mediator for the development of type 2 T-helper cell (Th2)–driven inflammatory disorders and has also been implicated in the pathogenesis of gastrointestinal (GI)-related cancers, including gastric carcinoma. We therefore sought to mechanistically determine IL33's potential role as a critical factor linking chronic inflammation and gastric carcinogenesis using gastritis-prone SAMP1/YitFc (SAMP) mice. Methods SAMP and (parental control) AKR mice were assessed for baseline gastritis and progression to metaplasia. Expression/localization of IL33 and its receptor, ST2/IL1R4, were characterized in corpus tissues, and activation and neutralization studies were both performed targeting the IL33/ST2 axis. Dissection of immune pathways leading to metaplasia was evaluated, including eosinophil depletion studies using anti-IL5/anti-CCR3 treatment. Results Progressive gastritis and, ultimately, intestinalized spasmolytic polypeptide-expressing metaplasia (SPEM) was detected in SAMP stomachs, which was absent in AKR but could be moderately induced with exogenous, recombinant IL33. Robust peripheral (bone marrow) expansion of eosinophils and local recruitment of both eosinophils and IL33-expressing M2 macrophages into corpus tissues were evident in SAMP. Interestingly, IL33 blockade did not affect bone marrow-derived expansion and local infiltration of eosinophils, but markedly decreased M2 macrophages and SPEM features, while eosinophil depletion caused a significant reduction in both local IL33-producing M2 macrophages and SPEM in SAMP. Conclusions IL33 promotes metaplasia and the sequelae of eosinophil-dependent downstream infiltration of IL33-producing M2 macrophages leading to intestinalized SPEM in SAMP, suggesting that IL33 represents a critical link between chronic gastritis and intestinalizing metaplasia that may serve as a potential therapeutic target for preneoplastic conditions of the GI tract.

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.

Th17 cells, which express the cytokine IL-17A, and master regulator RORγt, are important in the inflammatory response to fungal and bacterial pathogens, but also have a pathogenic role in many inflammatory disorders. In contrast, regulatory T cells (Treg), expressing the Foxp3 transcription factor, have a suppressive function and can dampen an immune response. The appropriate balance of these distinct effector functions is critical for an effective immune response and autoimmunity can arise if this process goes awry. In this issue, Wang et al. demonstrate a critical role for the transcription factor CREB (cyclic AMP-responsive element binding protein) in regulating the balance between inflammatory Th17 and suppressive Treg cells with implications for autoimmunity.

The notion of "immune privilege" of the brain has been revised to accommodate its infiltration, at steady state, by immune cells that participate in normal neurophysiology. However, the immune mechanisms that regulate learning and memory remain poorly understood. Here, we show that noninflammatory interleukin-17 (IL-17) derived from a previously unknown fetal-derived meningeal-resident γδ T cell subset promotes cognition. When tested in classical spatial learning paradigms, mice lacking γδ T cells or IL-17 displayed deficient short-term memory while retaining long-term memory. The plasticity of glutamatergic synapses was reduced in the absence of IL-17, resulting in impaired long-term potentiation in the hippocampus. Conversely, IL-17 enhanced glial cell production of brain-derived neurotropic factor, whose exogenous provision rescued the synaptic and behavioral phenotypes of IL-17-deficient animals. Together, our work provides previously unknown clues on the mechanisms that regulate short-term versus long-term memory and on the evolutionary and functional link between the immune and nervous systems.

Inflammatory bowel disease (IBD) is associated with dysregulated macrophage responses, such that quiescent macrophages acquire a pro-inflammatory activation state and contribute to chronic intestinal inflammation. The transcriptional events governing macrophage activation and gene expression in the context of chronic inflammation such as IBD remain incompletely understood. Here, we identify Kruppel-like transcription factor-6 (KLF6) as a critical regulator of pathogenic myeloid cell activation in human and experimental IBD. We found that KLF6 was significantly upregulated in myeloid cells and intestinal tissue from IBD patients and experimental models of IBD, particularly in actively inflamed regions of the colon. Using complementary gain- and loss-of-function studies, we observed that KLF6 promotes pro-inflammatory gene expression through enhancement of nuclear factor κB (NFκB) signaling, while simultaneously suppressing anti-inflammatory gene expression through repression of signal transducer and activator of transcription 3 (STAT3) signaling. To study the in vivo role of myeloid KLF6, we treated myeloid-specific KLF6-knockout mice (Mac-KLF6-KO) with dextran sulfate sodium (DSS) and found that Mac-KLF6-KO mice were protected against chemically-induced colitis; this highlights the central role of myeloid KLF6 in promoting intestinal inflammation. Collectively, our results point to a novel gene regulatory program underlying pathogenic, pro-inflammatory macrophage activation in the setting of chronic intestinal inflammation.

Summary T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population., Graphical Abstract, Highlights • Tissue-resident, SFB-elicited Th17 cells are non-inflammatory • Citrobacter-elicited Th17 cells show high plasticity towards inflammatory cytokines • SFB Th17 cells are metabolically similar to resting memory cells • Citrobacter Th17 cells are highly glycolytic effector cells, The distinctions between inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals are unclear. Omenetti et al. show that tissue-resident Th17 cells, in contrast to pathogen-elicited Th17 cells, exhibit little plasticity towards inflammatory cytokines, show muted metabolism, and do not participate in inflammatory reactions. These findings highlight the link between metabolic fitness and functional state.

Purpose of review Innate lymphoid cells (ILCs) are a newly-identified population of immune cells prevalent in, but not limited to, mucosal tissues that not only play a significant role in immune homeostasis and host defense, but also in disease pathogenesis. This review highlights the importance of type 3 ILCs (ILC3s) and their interactions with the intestinal microflora, both in maintaining gut health and in the development of inflammatory bowel disease (IBD). Recent findings Distinct lineages of ILCs are defined based on the presence of cell surface proteins, secretion of effector cytokines and expression of master transcription factors that determine their differentiation and inflammatory behavior. These ILC subgroups mirror corresponding CD4 T-cell subsets, with which they share many phenotypic, morphologic and functional attributes. ILC3s, in particular, through direct and indirect interactions with the gut microbiota, have been identified to promote protection and maintenance of epithelial integrity, as well as to regulate intestinal inflammation and fibrosis, such as that observed in IBD. Summary Gut mucosal ILCs respond to environmental cues, such as diet and microflora composition, which can shape downstream immune function. As such, ILCs represent attractive targets for the development of therapeutic modalities to maintain gut health and to potentially treat IBD.

The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors 1–4, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification5. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling 6, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

T-helper 17 (Th17) and T-regulatory (Treg) cells are frequently found at barrier surfaces, particularly within the intestinal mucosa, where they function to protect the host from pathogenic microorganisms and to restrain excessive effector T-cell responses, respectively. Despite their differing functional properties, Th17 cells and Tregs share similar developmental requirements. In fact, the fate of antigen-naïve T-cells to either Th17 or Treg lineages is finely regulated by key mediators, including TGFβ, IL-6 and all-trans retinoic acid (RA). Importantly, the intestinal microbiome also provides immunostimulatory signals, which can activate innate, and downstream adaptive, immune responses. Specific components of the gut microbiome have been implicated in the production of proinflammatory cytokines by innate immune cells, such as IL-6, IL-23, IL-1β, and the subsequent generation and expansion of Th17 cells. Similarly, commensal bacteria and their metabolites can also promote the generation of intestinal Tregs that can actively induce mucosal tolerance. As such, dysbiosis of the gut microbiome may not solely represent a consequence of gut inflammation, but rather shape the Treg/Th17 commitment and influence susceptibility to inflammatory bowel disease (IBD). In this review, we discuss Treg and Th17 cell plasticity, its dynamic regulation by the microbiome, and highlight its impact on intestinal homeostasis and disease.

Background & Aims Liver inflammation is a common extraintestinal manifestation of inflammatory bowel disease (IBD), but whether liver involvement is a consequence of a primary intestinal defect or results from alternative pathogenic processes remains unclear. Therefore, we sought to determine the potential pathogenic mechanism(s) of concomitant liver inflammation in an established murine model of IBD. Methods Liver inflammation and immune cell subsets were characterized in ileitis-prone SAMP1/YitFc (SAMP) and AKR/J (AKR) control mice, lymphocyte-depleted SAMP (SAMPxRag-1−/−), and immunodeficient SCID recipient mice receiving SAMP or AKR donor CD4+ T cells. Proliferation and suppressive capacity of CD4+ T-effector (Teff) and T-regulatory (Treg) cells from gut-associated lymphoid tissue (GALT) and livers of SAMP and AKR mice were measured. Results Surprisingly, prominent inflammation was detected in 4-week-old SAMP livers before histologic evidence of ileitis, whereas both disease phenotypes were absent in age-matched AKR mice. SAMP liver disease was characterized by abundant infiltration of lymphocytes, required for hepatic inflammation to occur, a TH1-skewed environment, and phenotypically activated CD4+ T cells. SAMP intrahepatic CD4+ T cells also had the ability to induce liver and ileal inflammation when adoptively transferred into SCID recipients, whereas GALT-derived CD4+ T cells produced milder ileitis but not liver inflammation. Interestingly, SAMP intrahepatic CD4+ Teff cells showed increased proliferation compared with both SAMP GALT- and AKR liver-derived CD4+ Teff cells, and SAMP intrahepatic Tregs were decreased among CD4+ T cells and impaired in in vitro suppressive function compared with AKR. Conclusions Activated intrahepatic CD4+ T cells induce liver inflammation and contribute to experimental ileitis via locally impaired hepatic immunosuppressive function.

Summary The epithelium and immune compartment in the intestine are constantly exposed to a fluctuating external environment. Defective communication between these compartments at this barrier surface underlies susceptibility to infections and chronic inflammation. Environmental factors play a significant, but mechanistically poorly understood, role in intestinal homeostasis. We found that regeneration of intestinal epithelial cells (IECs) upon injury through infection or chemical insults was profoundly influenced by the environmental sensor aryl hydrocarbon receptor (AHR). IEC-specific deletion of Ahr resulted in failure to control C. rodentium infection due to unrestricted intestinal stem cell (ISC) proliferation and impaired differentiation, culminating in malignant transformation. AHR activation by dietary ligands restored barrier homeostasis, protected the stem cell niche, and prevented tumorigenesis via transcriptional regulation of of Rnf43 and Znrf3, E3 ubiquitin ligases that inhibit Wnt-β-catenin signaling and restrict ISC proliferation. Thus, activation of the AHR pathway in IECs guards the stem cell niche to maintain intestinal barrier integrity., Graphical Abstract, Highlights • AHR deficiency in IECs promotes inflammation and colon tumorigenesis • AHR activation regulates intestinal crypt stem cell differentiation • AHR controls negative regulators of the Wnt-β-catenin pathway • A diet rich in AHR ligands can prevent or halt tumorigenesis, Metidji et al. show that deletion of Ahr in intestinal epithelial cells results in a defective barrier and unrestricted proliferation of intestinal stem cells (ISCs), culminating in malignant transformation. Activation of AHR by dietary ligands guards the ISC niche and maintains intestinal barrier homeostasis.

Chronic colitis is accompanied by extensive myelopoiesis and accumulation of CD11b+Gr-1+ cells in spleens and secondary lymphoid tissues. Although cells with similar phenotype have been described in cancer, chronic infection, or autoimmunity, where they were associated with suppression of T cell responses, little is known regarding how these cells affect CD4 T cell responses in the context of chronic intestinal inflammation. Therefore, we undertook this study to characterize the interplay between colitis-induced myeloid cells and CD4 T cell. Within the CD11b+Gr-1+ population, only monocytes (Ly6GnegLy6Chigh) but not other myeloid cell subsets suppressed proliferation and production of cytokines by CD4 T cells. Suppression was mediated by cell-contact, NO and partially by IFN-γ and PGs. Interestingly, Ly6Chigh MDCs, isolated from colitic colons, showed up-regulation of iNOS and arginase-1 and were more potent suppressors than those isolated from spleen. On a single-cell level, MDCs inhibited Th1 responses but enhanced generation of foxp3+ T cells. MDCs, cocultured with activated/Teffs, isolated from inflamed colons under hypoxic (1% O2) conditions typical for the inflamed intestine, suppressed proliferation but not their production of proinflammatory cytokines and chemokines. Taken together, expansion of monocytes and MDCs and activation of their suppressive properties may represent a homeostatic mechanism aimed at restraining excessive T cell activation during chronic inflammatory settings. The contribution of immunosuppressive monocytes/MDCs to chronic colitis and their role in shaping T cell responses in vivo require further investigation.

Several actin-binding proteins have been shown to be altered in metastatic cell lines and tumours and, in particular, Myristoylated Alanine-Rich protein Kinase C substrate (MARCKS) has been implicated in the pathogenesis of various highly metastatic epithelial malignancies. Considering that a large percentage of deaths due to colorectal cancer (CRC) are consequent to hepatic metastasization, aim of this study was to elucidate the involvement and mechanism of MARCKS in CRC by employing in vitro and in vivo approaches. Loss-of and-gain-on function approaches of MARCKS were employed in two human CRC cell lines: Clone A cells expressing MARCKS and LoVo cells known to have a frameshift mutation of MARCKS i.e. typically for MSI-H CRC. The data unveiled that altering MARCKS expression suppresses cell motility and invasion in human colon carcinoma cells when conditioned medium of liver-specific stromal cells (hepatic stellate cells) was used as chemoattractant. Depletion or re-expression of MARCKS inhibited proliferation with a reduction in expression of the mitotic regulator Aurora B kinase (AURKB), whereas AURKB-depletion did not modify MARCKS expression. In murine colon carcinoma CT26 cells, shRNA MARCKS-depletion reduced motility and invasion, and induced an aberrant, prolonged mitotic process. Significantly less metastases were produced in a syngeneic model of colon metastasis by MARCKS-depleted CT26 in comparison to CT26-tumour challenged mice. In conclusion, MARCKS plays an articulated role in the progression of colorectal cancer and might represent a suitable target to interfere and overcome the invasive behaviour of colon carcinoma cells at primary and distant sites.

P286 EFFECTS OF LIVER-SPECIFIC NO-DONOR, V-PYRRO/NO ON LIVER STEATOSIS IN MICE FED HIGH FAT DIET E. Maślak, A. Jasztal, K. Kochan, B. Sitek, P. Zabielski, A. Chabowski, K. Kuś, M. Walczak, B. Proniewski, M. Baranska, L. Keefer, S. Chlopicki. Jagiellonian Centre for Experimental Therapeutics (JCET), Faculty of Chemistry, Jagiellonian University, Krakow, Department of Physiology, Medical University of Bialystok, Bialystok, Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University, Krakow, Poland; Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD, United States; Department of Experimental Pharmacology, Jagiellonian University, Krakow, Poland E-mail: edyta.maslak@jcet.eu

Liver inflammation, a common extraintestinal manifestation of inflammatory bowel disease (IBD), was reported to unexpectedly precede ileitis onset in SAMP1/YitFc (SAMP) mice, a spontaneous model of IBD. We hypothesized that since SAMP possess an inherent epithelial barrier defect that can facilitate bacterial translocation, ultimately into the liver, toll-like receptor (TLR) activation of resident immune cells may occur, leading to liver inflammation. To test this, SAMP X MyD88KO mice were generated and liver inflammation assessed. TLRs were measured by qPCR and TLR9+ cells identified by FACS in isolated non-parenchymal liver cells (NPLs). Cytokines were measured by ELISA from NPLs +/- a TLR9 agonist, and SAMP treated with a TLR9 agonist or antagonist evaluated. Deletion of MyD88 resulted in complete amelioration of SAMP liver inflammation as early as 4, and up to 40, wks vs age-matched WT SAMP (both p