Flavio Caprioli

Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells.

Objective: In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (Treg) cells. Further, it was recently described that the cross-talk between human intestinal epithelial cells (IECs) and DCs helps in maintaining gut immune homeostasis via the induction of non-inflammatory DCs. In this study, an analysis was carried out to determine whether IECs could promote the differentiation of CD103+ tolerogenic DCs, and the function of primary CD103+ DCs isolated from human mesenteric lymph nodes (MLNs) was evaluated. Methods: Monocyte-derived DCs (MoDCs) and circulating CD1c+ DCs were conditioned or not with supernatants from Caco-2 cells or IECs isolated from healthy donors or donors with Crohn’s disease and analysed for their ability to induce Treg cell differentiation. In some cases, transforming growth factor β (TGFβ), retinoic acid (RA) or thymic stromal lymphopoietin (TSLP) were neutralised before conditioning. CD103+ and CD103− DCs were sorted by fluorescence-activated cell sorting (FACS) from MLNs and used in Treg cell differentiation experiments. Results: It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3+ Treg cells. This control was lost in patients with Crohn’s disease and paralleled a reduced expression of tolerogenic factors by primary IECs. MoDCs differentiated with RA or IEC supernatant upregulated the expression of CD103. Consistently, human primary CD103+ DCs isolated from MLNs were endowed with the ability to drive Treg cell differentiation. This subset of DCs expressed CCR7 and probably represents a lamina propria-derived migratory population. Conclusions: A population of tolerogenic CD103+ DCs was identified in the human gut that probably differentiate in response to IEC-derived factors and drive Treg cell development.

Mongersen, an Oral SMAD7 Antisense Oligonucleotide, and Crohn’s Disease

BACKGROUND Crohns disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. METHODS In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohns disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohns Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. RESULTS The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohns disease. CONCLUSIONS We found that study participants with Crohns disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

BACKGROUND & AIMS Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21

Background: T cell-mediated immunity plays a central part in the pathogenesis of tissue damage in inflammatory bowel disease (IBD). The mechanism by which T cells mediate tissue damage during IBD remains unclear, but evidence indicates that T cell-derived cytokines stimulate fibroblasts to synthesise matrix metalloproteinases (MMPs), which then mediate mucosal degradation. We have previously shown that, in IBD, there is high production of interleukin (IL) 21, a T cell-derived cytokine, which enhances Th1 activity. Aim: To investigate whether IL21 controls MMP production by intestinal fibroblasts. Methods: IL21 receptor (IL21R) was evaluated in intestinal fibroblasts by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Fibroblasts were stimulated with IL21 and MMPs were evaluated by RT-PCR and western blotting. The effect of a neutralising IL21R fusion protein (IL21R/Fc) on the induction of MMPs in fibroblasts stimulated with IBD lamina propria mononuclear cell (LPMC) supernatants was also evaluated. Results: Intestinal fibroblasts constitutively express both IL21R and the common γ chain receptor, which are necessary for IL21-driven signalling. IL21 enhances fibroblast production of MMP-1, MMP-2, MMP-3 and MMP-9, but not tissue inhibitors of MMP-1 and MMP-2. Moreover, IL21 synergises with tumour necrosis factor α to increase synthesis of MMP synthesis. IL21 enhances MMP secretion without affecting gene transcription and protein synthesis. IBD LPMC supernatants stimulate MMP secretion by intestinal fibroblasts, and this effect is partly inhibited by IL21R/Fc. Conclusions: These results suggest that fibroblasts are a potential target of IL21 in the gut and that IL21 controls MMP secretion by fibroblasts.

IL‐23‐mediated regulation of IL‐17 production in Helicobacter pylori‐infected gastric mucosa

Helicobacter pylori (Hp) infection is associated with a marked infiltration of the gastric mucosa by inflammatory cells. The molecular pathways that control Hp‐associated inflammatory reaction are complex, but locally induced cytokines seem to contribute to maintaining the ongoing inflammation. We have previously shown that IL‐17 is over‐produced in Hp‐infected gastric mucosa, and that IL‐17 stimulates the synthesis of IL‐8, the major neutrophil chemoattractant. Factors/mechanisms that regulate IL‐17 expression remain, however, unknown. In this study, we initially expanded our previous data, showing that CD4+ and CD8+ T cells are a source of IL‐17 in Hp‐infected samples. Since IL‐23 enhances T cell‐derived IL‐17 during bacterial infections, we then assessed the role of IL‐23 in controlling IL‐17 expression in Hp‐colonized stomach. Using real‐time PCR and ELISA, IL‐23 was detected in all gastric biopsies, but its expression was more pronounced in Hp‐infected samples in comparison to controls. Treatment of normal gastric lamina propria mononuclear cells (LPMC) with IL‐23 enhanced Stat3 activation and IL‐17 secretion, and pharmacological inhibition of Stat3 prevented IL‐23‐driven IL‐17 synthesis. Consistently, blockade of IL‐23 in cultures of LPMC from Hp‐infected patients reduced Stat3 activation and IL‐17 production. Data show that IL‐23 is overexpressed in Hp‐infected gastric mucosa where it could contribute to sustaining IL‐17 production.

Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model

Background and aims Probiotics and their metabolic products, here called postbiotics, have been proposed as food supplements for a healthier intestinal homeostasis, but also as therapeutic aids in inflammatory bowel disease (IBD) with, however, very little clinical benefit. This may be due to the lack of reliable preclinical models for testing the efficacy of different strains. Methods The activity of three probiotic strains of Lactobacillus (or a postbiotic) was analysed and compared with a pathogenic strain of Salmonella on a novel organ culture system of human healthy and IBD intestinal mucosa developed in our laboratory. The system maintains an apical to basolateral polarity during stimulation due to the presence of a glued cave cylinder. The cylinder is detached at the end of the experiment and the tissue is processed for histology and immunohistochemistry. Cytokines released from the basolateral side are analysed. Results The model system provides several physiological characteristics typical of a mucosal microenvironment including the presence of an organised mucus layer and an apical to basolateral polarity. Polarised administration of bacteria is critical to control the ensuing immune response as it mimics the physiological entrance of bacteria. The authors show that probiotics are not always beneficial for the healthy host and can also be detrimental in inflamed IBD. This study shows that a potent postbiotic can protect against the inflammatory properties of invasive Salmonella on healthy tissue and also downregulate ongoing inflammatory processes in IBD tissue. Conclusions Probiotics can have inflammatory activities in both healthy and IBD tissue. Valid preclinical data on proper model systems should therefore be obtained before specific probiotic strains enter the clinics, especially if administered during acute inflammatory responses. Postbiotics may be a safe alternative for the treatment of patients with IBD in the acute inflammatory phase.

Involvement of interleukin-21 in the regulation of colitis-associated colon cancer

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane.

Autocrine Regulation of IL-21 Production in Human T Lymphocytes

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common γ-chain. IL-21 is mostly produced by CD4+ T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4+ T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3+ T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common γ-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3+ lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3+ lamina propria lymphocytes isolated from patients with Crohn’s disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.

Plasma chromogranin a in patients with inflammatory bowel disease.

Background: Circulating chromogranin A (CgA) levels, a marker for neuroendocrine tumors including carcinoids, have recently been found elevated in some patients with inflammatory bowel disease (IBD), although their significance is unclear. Therefore, we aimed to evaluate CgA levels and their possible relationship with clinical and biochemical disease activity indexes in 119 IBD patients. Methods: The study groups comprised 75 patients with ulcerative colitis, 44 with Crohns disease, in both active and quiescent phases, and 85 controls. Results: Mean CgA levels were significantly higher in IBD patients than in controls (20.4 ± 14.0 [SD] versus 11.3 ± 4.3 U/L, P < 0.001), without any statistical significant difference among the IBD subgroups. However, CgA levels were above the normal range (20 U/L) in 25/45 patients with active IBD (55%; 95% confidence interval [CI]: 40%–70%) and in 18/74 patients with remission IBD (24%; 95% CI: 15%–36%) (P < 0.001, Fishers test). Among biochemical parameters, CgA correlated with serum TNF‐&agr; levels (rs = 0.398, P < 0.001). Conclusions: High CgA levels can occur in IBD. The disease activity and TNF‐&agr; levels seem to influence the CgA pattern, which could reflect the neuroendocrine system activation in response to inflammation. From a clinical point of view, the possibility of high CgA levels in IBD should be taken into consideration when a carcinoid is suspected in such patients, since this event seems to be more frequent than previously considered. Indeed, revision of our 83 patients with gastrointestinal carcinoids, studied between 1997 and 2006, showed that 4 patients had IBD, with a prevalence of 4.8%, which is markedly higher than that of the general population.

A gut-vascular barrier controls the systemic dissemination of bacteria

A gut bacterial containment system Trillions of bacteria selectively inhabit our guts, but how do our bodies keep them contained? Spadoni et al. describe a “gut-vascular barrier” that prevents intestinal microbes from accessing the liver and the bloodstream in mice (see the Perspective by Bouziat and Jabri). Studies with human samples and in mice revealed that the cell biology of the gut-vascular barrier shares similarities with the blood-brain barrier of the central nervous system. Pathogenic bacteria such as Salmonella typhimurium could penetrate the gut-vascular barrier in mice, gaining access to the liver and bloodstream, in a manner dependent on the Salmonella pathogenicity island 2–type III secretion system. Science, this issue p. 830; see also p. 742 An endothelial barrier between the gut and the bloodstream helps keep intestinal microbes contained. [Also see Perspective by Bouziat and Jabri] In healthy individuals, the intestinal microbiota cannot access the liver, spleen, or other peripheral tissues. Some pathogenic bacteria can reach these sites, however, and can induce a systemic immune response. How such compartmentalization is achieved is unknown. We identify a gut-vascular barrier (GVB) in mice and humans that controls the translocation of antigens into the blood stream and prohibits entry of the microbiota. Salmonella typhimurium can penetrate the GVB in a manner dependent on its pathogenicity island (Spi) 2–encoded type III secretion system and on decreased β-catenin–dependent signaling in gut endothelial cells. The GVB is modified in celiac disease patients with elevated serum transaminases, which indicates that GVB dismantling may be responsible for liver damage in these patients. Understanding the GVB may provide new insights into the regulation of the gut-liver axis.