Stefania Vetrano

The lymphatic system controls intestinal inflammation and inflammation-associated colon cancer through the chemokine decoy receptor D6

Background and aims Inflammatory CC chemokines have long been associated with cancer, but unequivocal evidence of a role in clinically relevant models of carcinogenesis is lacking. D6, a promiscuous decoy receptor that scavenges inflammatory CC chemokines, plays a non-redundant role in reducing the inflammatory response in various organs. As inflammation is a key player in the development of inflammatory bowel disease (IBD) and IBD-associated colorectal cancer, we investigated D6 expression in human colitis and colon cancer, and its role in experimental colitis and inflammation-associated colon cancer. Results In humans, D6 was mainly expressed by lymphatic vessels and leukocytes in the mucosa of individuals with IBD and colon cancer, as well as the mucosa of control individuals. Mice lacking expression of D6 were significantly more susceptible to experimental colitis than wild-type mice and failed to resolve colitis, with significantly higher levels of several pro-inflammatory chemokines. In bone marrow chimeric mice, the ability of D6 to regulate colitis was tracked to the stromal/lymphatic compartment, with no contribution of haemopoietic cells. Finally, after administration of the carcinogen azoxymethane, D6−/− mice showed increased susceptibility to colitis-associated cancer in the distal segment of the colon compared with wild-type mice. Conclusions D6 expressed on lymphatic vessels plays a key role in the control of intestinal inflammation and the development of inflammation-associated colon cancer. Our results reveal a new unexpected role for the lymphatic system in the pathogenesis of IBD and intestinal cancer, and candidate chemokines as novel players in tumour promotion and progression.

Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications

Inflammation and coagulation are closely linked interdependent processes. Under physiologic conditions, the tissue microcirculation functions in anticoagulant and anti-inflammatory fashions. However, when inflammation occurs, coagulation is also set in motion and actively participates in enhancing inflammation. Recently, novel and unexpected roles of hemostasis in the humoral and cellular components of innate immunity have been described. In particular, the protein C system, besides its well-recognized role in anticoagulation, plays a crucial role in inflammation. Indeed, the protein C system is now emerging as a novel participant in the pathogenesis of acute and chronic inflammatory diseases, such as sepsis, asthma, inflammatory bowel disease, atherosclerosis, and lung and heart inflammation, and may emerge as unexpected therapeutic targets for intervention.

Multiple Pathogenic Roles of Microvasculature in Inflammatory Bowel Disease: A Jack of All Trades

The etiology of Crohns disease and ulcerative colitis, the two major forms of inflammatory bowel disease (IBD), is still largely unknown. However, it is now clear that the abnormalities underlying pathogenesis of intestinal inflammation are not restricted to those mediated by classic immune cells but also involve nonimmune cells. In particular, advances in vascular biology have outlined a central and multifaceted pathogenic role for the microcirculation in the initiation and perpetuation of IBD. The microcirculation and its endothelial lining play a crucial role in mucosal immune homeostasis through tight regulation of the nature and magnitude of leukocyte migration from the intravascular to the interstitial space. Chronically inflamed IBD microvessels display significant alterations in microvascular physiology and function compared with vessels from healthy and uninvolved IBD intestine. The investigation into human IBD has demonstrated how endothelial activation present in chronically inflamed IBD microvessels results in a functional phenotype that also includes leakiness, chemokine and cytokine expression, procoagulant activity, and angiogenesis. This review contemplates the newly uncovered contribution of intestinal microcirculation to pathogenesis and maintenance of chronic intestinal inflammation. In particular, we assess the multiple roles of the microvascular endothelium in innate immunity, leukocyte recruitment, coagulation and perfusion, and immune-driven angiogenesis in IBD.

VEGF-C–dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease

Crohns disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) of unknown etiology that are associated with an aberrant mucosal immune response. Neoangiogenesis and vascular injury are observed in IBD along with increased lymphangiogenesis. While the pathogenic role of angiogenesis in IBD is well characterized, it is not clear how or if increased lymphangiogenesis promotes disease. Here, we determined that enhancing lymphangiogenesis and lymphatic function reduces experimental IBD. Specifically, we demonstrated that adenoviral induction of prolymphangiogenic factor VEGF-C provides marked protection against the development of acute and chronic colitis in 2 different animal models. VEGF-C-dependent protection was observed in combination with increased inflammatory cell mobilization and bacterial antigen clearance from the inflamed colon to the draining lymph nodes. Moreover, we found that the VEGF-C/VEGFR3 pathway regulates macrophage (MΦ) plasticity and activation both in cultured MΦs and in vivo, imparting a hybrid M1-M2 phenotype. The protective function of VEGF-C was meditated by the so-called resolving MΦs during chronic experimental colitis in a STAT6-dependent manner. Together, these findings shed light on the contribution of lymphatics to the pathogenesis of gut inflammation and suggest that correction of defective lymphatic function with VEGF-C has potential as a therapeutic strategy for IBD.

Effects of immunosuppression on immune response to pneumococcal vaccine in inflammatory bowel disease: a prospective study.

Background: Since immunomodulators and antitumor necrosis factor (TNF) agents are increasingly used to treat inflammatory bowel disease (IBD), it is recommended to administer antipneumococcal vaccination to prevent opportunistic pneumonia. There is some evidence that concomitant immunosuppression may impair the immune response to vaccination. We aimed to evaluate the response rates to pneumococcal vaccination in four different treatment groups (mesalamine, azathioprine, infliximab, infliximab plus azathioprine). Methods: In all, 96 patients with IBD (54 with Crohns disease; 42 with ulcerative colitis) were administered a 23‐valent polysaccharide pneumococcal vaccine (PSV‐23). The levels of antipneumococcal antibodies were measured prior to and at least 3 weeks after vaccination. Response rates and risk factors for impaired immunosuppression were investigated. Patients on mesalamine were used as a control group. Results: Patients administered infliximab or the combination immunosuppressive therapy had significantly lower response rates to vaccination (57.6% and 62.5%, respectively) compared with the group on mesalamine (88.6%; P < 0.05 for both comparisons). Azathioprine alone did not influence the response rate to vaccination (78.9%; P = 0.43 vs. mesalamine group). Mean antibody titers after vaccination were significantly lower in patients under infliximab or combined immunosuppression than controls (P < 0.05). Immunosuppression with infliximab or combination therapy significantly decreased the likelihood of responding to vaccination (odds ratio [OR] = 0.17, 95% confidence interval [CI] 0.04–0.64, P = 0.009, and OR = 0.21, 95% CI 0.05–0.91, P = 0.038, respectively). Pneumococcal vaccination was generally safe and well tolerated. Conclusions: Anti‐TNF therapy alone or in combination with azathioprine impairs the response to pneumococcal vaccination in patients with IBD. All patients with IBD should therefore be vaccinated before starting anti‐TNF therapy. (Inflamm Bowel Dis 2012;)

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine

BACKGROUND & AIMS Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. METHODS MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. RESULTS Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. CONCLUSIONS BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

Efficacy of a potent and safe vitamin D receptor agonist for the treatment of inflammatory bowel disease

Deficiency in 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) has been suggested as an important environmental factor for immuno-mediated disorders including inflammatory bowel diseases (IBD), comprising Crohns disease and ulcerative colitis, both characterized by chronic intestinal inflammation. Administration of vitamin D receptor (VDR) agonists can ameliorate spontaneous and induced animal models of colitis, but hypercalcemia is a dose-limiting adverse event. Previous work in our laboratory has identified 1alpha,25(OH)(2)-16-ene-20-cyclopropyl-vitamin D(3) (BXL-62) as a potent anti-inflammatory VDR agonist with a low calcemic activity. In the present study, we confirm the marked anti-inflammatory properties of BXL-62 and show its capacity to induce VDR primary response genes, like CYP24A1 and CAMP, at lower concentrations than 1,25D(3), in PBMCs from IBD patients. Its higher anti-inflammatory potency compared to 1,25D(3) was demonstrated by the significantly more potent inhibition in PBMCs and in lymphocyte-enriched lamina propria mononuclear cells of the pro-inflammatory cytokines TNF-alpha, IL-12/23p40, IL-6 and IFN-gamma, both at mRNA and protein level. The therapeutic efficacy of intra-rectal administration of BXL-62 in experimental IBD is shown by its beneficial effects, significantly higher than 1,25D(3), to induce recovery of clinical symptoms of colitis at normocalcemic doses in mice undergoing dextran sodium sulfate-induced colitis. These results confirm the therapeutic efficacy of VDR agonists in experimental colitis, and suggest BXL-62 as a promising compound for IBD treatment.

Infliximab Therapy Inhibits Inflammation-Induced Angiogenesis in the Mucosa of Patients With Crohn's Disease

OBJECTIVES:Inflammation-driven angiogenesis contributes to the pathogenesis of inflammatory bowel disease (IBD). In line with this, the efficacy of inhibitors of angiogenesis has been demonstrated in experimental models of colitis. Currently, the ability of infliximab, an anti-tumor necrosis factor-α (TNF-α) agent that is highly beneficial in patients with IBD, to affect mucosal angiogenesis in patients with Crohns disease (CD) and ulcerative colitis (UC) is unknown.METHODS:Patients with active CD (n=14) were treated with infliximab for 1 year, and peripheral blood and intestinal mucosa samples were collected before and after treatment. Mucosal angiogenesis was evaluated by CD31 and Ki-67 staining in endoscopic biopsies at baseline (week 0) and at week 54. The release of vascular endothelial growth factor-A (VEGF-A) by cultured mucosal extracts was measured by enzyme-linked immunosorbent assay (ELISA), before and after administration of infliximab, as well as in cultures of human intestinal fibroblasts (HIFs) stimulated with TNF-α in the presence or absence of infliximab. Migration of human intestinal microvascular endothelial cells (HIMECs) was investigated by migration assays.RESULTS:Microvessel density was significantly higher in the mucosa from patients with CD compared with tissue from healthy control individuals. Of the 14 patients, 8 (57%) showed a clinical remission in response to infliximab, which was associated with a significant reduction of microvascular density. Morphometric vessel analysis further confirmed the significant reduction of the area of vascular section after administration of infliximab. Furthermore, the expression levels of the proliferation marker Ki-67 in endothelial cells were significantly reduced after treatment. The mucosal concentration of VEGF-A was also significantly decreased, whereas in vitro exposure of HIF to infliximab virtually abolished TNF-α-induced VEGF-A production. These phenomena did not occur in patients who showed no clinical response to infliximab.CONCLUSIONS:Administration of infliximab downregulates mucosal angiogenesis in patients with CD and restrains production of VEGF-A by mucosal fibroblasts. It is proposed that this ameliorates inflammation-driven angiogenesis in the gut mucosa and contributes to the therapeutic efficacy of blockade of TNF-α.

Unexpected role of anticoagulant protein C in controlling epithelial barrier integrity and intestinal inflammation

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC−/−/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.