Tbet Expression in Regulatory T Cells Is Required to Initiate Th1-Mediated Colitis.

In normal conditions gut homeostasis is maintained by the suppressive activity of regulatory T cells (Tregs), characterized by the expression of the transcription factor FoxP3. In human inflammatory bowel disease, which is believed to be the consequence of the loss of tolerance toward antigens normally contained in the gut lumen, Tregs have been found to be increased and functionally active, thus pointing against their possible role in the pathogenesis of this immune-mediated disease. Though, in inflammatory conditions, Tregs have been shown to upregulate the T helper (Th) type 1-related transcription factor Tbet and to express the pro-inflammatory cytokine IFNγ, thus suggesting that at a certain point of the inflammatory process, Tregs might contribute to inflammation rather than suppress it. Starting from the observation that Tregs isolated from the lamina propria of active but not inactive IBD patients or uninflamed controls express Tbet and IFNγ, we investigated the functional role of Th1-like Tregs in the dextran sulfate model of colitis. As observed in human IBD, Th1-like Tregs were upregulated in the inflamed lamina propria of treated mice and the expression of Tbet and IFNγ in Tregs preceded the accumulation of conventional Th1 cells. By using a Treg-specific Tbet conditional knockout, we demonstrated that Tbet expression in Tregs is required for the development of colitis. Indeed, Tbet knockout mice developed milder colitis and showed an impaired Th1 immune response. In these mice not only the Tbet deficient Tregs but also the Tbet proficient conventional T cells showed reduced IFNγ expression. However, Tbet deficiency did not affect the Tregs suppressive capacity in vitro and in vivo in the adoptive transfer model of colitis. In conclusion here we show that Tbet expression by Tregs sustains the early phase of the Th1-mediated inflammatory response in the gut.

Reciprocal Regulation Between Smad7 and Sirt1 in the Gut.

In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-ß1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, ex vivo organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut.

RORγt-Expressing Tregs Drive the Growth of Colitis-Associated Colorectal Cancer by Controlling IL6 in Dendritic Cells.

Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082-92. ©2018 AACR.

Knockdown of Smad7 With a Specific Antisense Oligonucleotide Attenuates Colitis and Colitis-Driven Colonic Fibrosis in Mice.

Background: In Crohn's disease (CD), the pathogenic immune response is associated with high Smad7, an inhibitor of TGF-ß1 signaling. Smad7 knockdown with Mongersen, a specific antisense oligonucleotide-containing compound, restores TGF-ß1 activity leading to inhibition of inflammatory signals and associates with clinical benefit in CD patients. As TGF-ß1 is pro-fibrogenic, it remains unclear whether Mongersen-induced Smad7 inhibition increases the risk of intestinal fibrosis. We assessed the impact of Smad7 inhibition on the course of colitis-driven intestinal fibrosis in mice. Methods: BALB/c mice were rectally treated with increasing doses of trinitrobenzene sulfonic acid (TNBS) for 8 or 12 weeks. The effect of oral Smad7 antisense or control oligonucleotide, administered to mice starting from week 5 or week 8, respectively, on mucosal inflammation and colitis-associated colonic fibrosis was assessed. Mucosal samples were analyzed for Smad7 by immunoblotting and immunohistochemistry, TGF-ß1 by enzyme-linked immunosorbent assay, and collagen by immunohistochemistry. Results: TNBS-induced chronic colitis was associated with colonic deposition of collagen I and fibrosis, which were evident at week 8 and became more pronounced at week 12. TNBS treatment enhanced Smad7 in both colonic epithelial and lamina propria mononuclear cells. Colitic mice treated with Smad7 antisense oligonucleotide exhibited reduced signs of colitis, less collagen deposition, and diminished fibrosis. These findings were associated with diminished synthesis of TGF-ß1 and reduced p-Smad3 protein expression. Conclusion: Attenuation of colitis with Smad7 antisense oligonucleotide limits development of colonic fibrosis.

Interleukin-34 sustains pro-tumorigenic signals in colon cancer tissue.

Interleukin-34 (IL-34), a cytokine produced by a wide range of cells, binds to the macrophage colony-stimulating factor receptor (M-CSFR-1) and receptor-type protein-tyrosine phosphatase zeta (PTP-z) and controls myeloid cell differentiation, proliferation and survival. various types of cancers over-express IL-34 but the role of the cytokine in colorectal cancer (CRC) remains unknown. We here investigated the expression and functional role of IL-34 in CRC. A more pronounced expression of IL-34 was seen in CRC samples as compared to matched normal/benign colonic samples and this occurred at both RNA and protein level. Immunohistochemical analysis of CRC tissue samples showed that both cancer cells and lamina propria mononuclear cells over-expressed IL-34. Additionally, CRC cells expressed both M-CSFR-1 and PTP-z, thus suggesting that CRC cells can be responsive to IL-34. Indeed, stimulation of DLD-1 cancer cells with IL-34, but not with MSCF1, enhanced the cell proliferation and cell invasion without affecting cell survival. Analysis of intracellular signals underlying the mitogenic effect of IL-34 revealed that the cytokine enhanced activation of ERK1/2 and pharmacologic inhibition of ERK1/2 abrogated IL-34-driven cell proliferation. Consistently, IL-34 knockdown in HT-29 cells with a specific IL-34 antisense oligonucleotide reduced ERK1/2 activation, cell proliferation and enhanced the susceptibility of cells to Oxaliplatin-induced death. This is the first study showing up-regulation of IL-34 in CRC and suggesting a role for this cytokine in colon tumorigenesis.

CCR2-dependent monocyte-derived macrophages resolve inflammation and restore gut motility in postoperative ileus.

OBJECTIVE: Postoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinal muscularis externa (ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of infiltrating monocytes in a murine model of intestinal manipulation (IM)-induced POI in order to clarify whether monocytes mediate tissue damage and intestinal dysfunction or they are rather involved in the recovery of gastrointestinal (GI) motility. DESIGN: IM was performed in mice with defective monocyte migration to tissues (C-C motif chemokine receptor 2, Ccr2-/ - mice) and wild-type (WT) mice to study the role of monocytes and monocyte-derived macrophages (MΦs) during onset and resolution of ME inflammation. RESULTS: At early time points, IM-induced GI transit delay and inflammation were equal in WT and Ccr2 -/- mice. However, GI transit recovery after IM was significantly delayed in Ccr2 -/- mice compared with WT mice, associated with increased neutrophil-mediated immunopathology and persistent impaired neuromuscular function. During recovery, monocyte-derived MΦs acquire pro-resolving features that aided in the resolution of inflammation. In line, bone marrow reconstitution and treatment with MΦ colony-stimulating factor 1 enhanced monocyte recruitment and MΦ differentiation and ameliorated GI transit in Ccr2 -/- mice. CONCLUSION: Our study reveals a critical role for monocyte-derived MΦs in restoring intestinal homeostasis after surgical trauma. From a therapeutic point of view, our data indicate that inappropriate targeting of monocytes may increase neutrophil-mediated immunopathology and prolong the clinical outcome of POI, while future therapies should be aimed at enhancing MΦ physiological repair functions.

Vagotomy affects the development of oral tolerance and increases susceptibility to develop colitis independently of the alpha-7 nicotinic receptor.

Vagotomy (VGX) increases the susceptibility to develop colitis suggesting a crucial role for the cholinergic anti-inflammatory pathway in the regulation of the immune responses. Since oral tolerance and the generation of regulatory T cells (Tregs) are crucial to preserve mucosal immune homeostasis, we studied the effect of vagotomy and the involvement of α7 nicotinic receptors (α7nAChR) at the steady state and during colitis. Therefore, the development of both oral tolerance and colitis (induced by dextran sulfate sodium (DSS) or via T cell transfer) was studied in vagotomized mice and in α7nAChR-/- mice. VGX, but not α7nAChR deficiency, prevented oral tolerance establishment. This effect was associated with reduced Treg conversion in the lamina propria and mesenteric lymphnodes. To the same extent, vagotomized mice, but not α7nAChR-/- mice, developed a more severe DSS colitis compared with control mice treated with DSS, associated with a decreased number of colonic Tregs. However, neither VGX nor absence of α7nAChR in recipient mice affected colitis development in the T cell transfer model. In line, deficiency of α7nAChR exclusively in T cells did not influence the development of colitis induced by T cell transfer. Our results indicate a key role for the vagal intestinal innervation in the development of oral tolerance and colitis, most likely by modulating induction of Tregs independently of α7nAChR.

The Neuromodulation of the Intestinal Immune System and Its Relevance in Inflammatory Bowel Disease.

One of the main tasks of the immune system is to discriminate and appropriately react to "danger" or "non-danger" signals. This is crucial in the gastrointestinal tract, where the immune system is confronted with a myriad of food antigens and symbiotic microflora that are in constant contact with the mucosa, in addition to any potential pathogens. This large number of antigens and commensal microflora, which are essential for providing vital nutrients, must be tolerated by the intestinal immune system to prevent aberrant inflammation. Hence, the balance between immune activation versus tolerance should be tightly regulated to maintain intestinal homeostasis and to prevent immune activation indiscriminately against all luminal antigens. Loss of this delicate equilibrium can lead to chronic activation of the intestinal immune response resulting in intestinal disorders, such as inflammatory bowel diseases (IBD). In order to maintain homeostasis, the immune system has evolved diverse regulatory strategies including additional non-immunological actors able to control the immune response. Accumulating evidence strongly indicates a bidirectional link between the two systems in which the brain modulates the immune response via the detection of circulating cytokines and via direct afferent input from sensory fibers and from enteric neurons. In the current review, we will highlight the most recent findings regarding the cross-talk between the nervous system and the mucosal immune system and will discuss the potential use of these neuronal circuits and neuromediators as novel therapeutic tools to reestablish immune tolerance and treat intestinal chronic inflammation.

Absence of intestinal inflammation and postoperative ileus in a mouse model of laparoscopic surgery.

BACKGROUND: Postoperative ileus (POI) is characterized by impaired gastrointestinal motility resulting from intestinal handling-associated inflammation. The introduction of laparoscopic surgery has dramatically reduced the duration of POI. However, it remains unclear to what extent this results in a reduction of intestinal inflammation. The aim of the present study is to compare the degree of intestinal inflammation and gastrointestinal transit following laparoscopic surgery and open abdominal surgery. METHODS: Mice were subjected to laparoscopic surgery or laparotomy alone or, in combination with standardized intestinal manipulation of the small bowel (IM). Gastrointestinal transit and intestinal inflammation were assessed 24 h after surgery by the number of myeloperoxidase (MPO) positive cells and the level of cytokine expression. The recovery time and the degree of inflammation were also analyzed in patients subjected to colectomy under open conditions (laparotomy) or laparoscopic conditions. KEY RESULTS: Mice undergoing IM by laparotomy (open IM), but not by laparoscopy (Lap IM) developed a significant delay in gastrointestinal transit compared to laparotomy or laparoscopy alone. In addition, there was significant intestinal inflammation only after open IM. Similarly, cytokine levels in peritoneal lavage fluid were lower while recovery time was faster in patients subjected to colectomy under laparoscopic conditions compared to open colectomy. CONCLUSIONS & INFERENCES: Our data confirms that intestinal inflammation is underlying the delayed gastrointestinal transit observed after open surgery. Most importantly, we demonstrate that intestinal inflammation under laparoscopic conditions is significantly lower compared to open surgery, most likely explaining the faster recovery following laparoscopic surgery.

Neuro-anatomical evidence indicating indirect modulation of macrophages by vagal efferents in the intestine but not in the spleen.

BACKGROUND: Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine. METHODS: Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages. RESULTS: Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers. CONCLUSION: In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.

Mast cells play no role in the pathogenesis of postoperative ileus induced by intestinal manipulation.

INTRODUCTION: Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling. DESIGN: The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit(W-sh/W-sh) and Cpa3(Cre/+) mice, and by use of the mast cell stabilizer cromolyn. RESULTS: Kit(W-sh/W-sh) mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3(Cre/+) mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3(Cre/+) mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3(+/+) ). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. CONCLUSIONS: Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.

A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca(2+) imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca(2+) response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

Inhibition of spleen tyrosine kinase as treatment of postoperative ileus.

OBJECTIVE: Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. DESIGN: In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. RESULTS: In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4 ± 0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2 ± 0.7; GSK143 (1 mg/kg): 7.6 ± 0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0-90 µM) and trinitrophenyl (0-4 µg/ml) induced a concentration-dependent release of ß-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03-10 µM) concentration dependently blocked substance P and trinitrophenyl induced ß-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. CONCLUSIONS: The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.

Novel method for studying postoperative ileus in mice.

INTRODUCTION: Postoperative ileus (POI) is characterized by a transient inhibition of coordinated motility of the gastrointestinal (GI) tract after abdominal surgery and leads to increased morbidity and prolonged hospitalization. Currently, intestinal manipulation of the intestine is widely used as a preclinical model of POI. The technique used to manipulate the intestine is however highly variable and difficult to standardize, leading to large variations and inconsistent findings between different investigators. Therefore, we developed a device by which a fixed and adjustable pressure can be applied during intestinal manipulation. METHODS: The standardized pressure manipulation method was developed using the purpose-designed device. First, the effect of graded manipulation was examined on postoperative GI transit. Next, this new technique was compared to the conventional manipulation technique used in previous studies. GI transit was measured by evaluating the intestinal distribution of orally gavaged fluorescein isothiocyanate (FITC)-labeled dextran. Infiltration of myeloperoxidase positive cells and cytokine production (ELISA) in the muscularis externa of the intestine were assessed. RESULTS: Increasing pressures resulted in a graded reduction of intestinal transit and was associated with intestinal inflammation as demonstrated by influx of leukocytes and increased levels of IL-6, IL-1ß and MCP-1 compared to control mice. With an applied pressure of 9 grams a similar delay in intestinal transit could be obtained with a smaller standard deviation, leading to a reduced intra-individual variation. CONCLUSIONS: This method provides a reproducible model with small variation to study the pathophysiology of POI and to evaluate new anti-inflammatory strategies.