Raja Atreya

Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo.

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1–5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130–Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6–sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6–sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-kappaB, and bcl-x(L) was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

NF-κB in inflammatory bowel disease

Apart from genetic and environmental factors, the mucosal immune system of the gut plays a central role in the pathogenesis of inflammatory bowel disease (IBD). In the healthy gut, the mucosal immune system ensures the balance between pro‐ and anti‐inflammatory mediators and thereby allows an effective defence against luminal pathogens but at the same time prevents an overwhelming immune reaction directed against the huge amount of harmless luminal antigens (for example, components of food or nonpathological bacteria). In both entities of IBD (Crohn’s disease and ulcerative colitis) this immunological balance is severely impaired and shifted towards the pro‐inflammatory side. The chronic mucosal inflammation in IBD is caused by hyperactivation of effector immune cells, which produce high levels of pro‐inflammatory cytokines like tumour necrosis factor‐α, interleukin‐6 and interferon‐γ, resulting in colonic tissue damage. The nuclear transcription factor kappaB (NF‐κB) was identified as one of the key regulators in this immunological setting. Its activation is markedly induced in IBD patients and through its ability to promote the expression of various pro‐inflammatory genes, NF‐κB strongly influences the course of mucosal inflammation. Considering the different cell‐type specific effects which are mediated by NF‐κB, this review aims at describing the complex role of NF‐κB in IBD and discusses existing pharmacological attempts to block the activation of NF‐κB to develop new therapeutic strategies in IBD.

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.).

Exacerbation of ulcerative colitis after rituximab salvage therapy

Background: B‐cells are considered to play a pathogenic role in human ulcerative colitis (UC) by producing autoantibodies that cause epithelial cell damage. Here we report on a patient with intractable UC who suffered from a severe exacerbation of UC after salvage therapy with rituximab, a B‐cell‐depleting anti‐CD20‐antibody. Methods: A 58‐year‐old patient with active long‐standing UC and unresponsiveness or adverse events to mesalamine, corticosteroids, azathioprine, methotrexate, infliximab, leukapheresis, mycophenolate mofetil, and adalimumab received 375 mg/m2 rituximab. Results: A severe exacerbation of UC activity was noted upon therapy that required hospitalization. Subsequent studies showed a complete depletion of CD20‐positive mucosal B‐cells associated with a suppression of local IL‐10 production. Conclusions: In contrast to rheumatoid arthritis patients, rituximab had deleterious effects in our UC patient by blocking IL‐10 producing B‐cells. Our data suggest an important anti‐ rather than proinflammatory role of B‐cells in UC.

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohns disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohns disease led to detection of intestinal mTNF+ immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF+ cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF+ cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohns disease and autoimmune or inflammatory disorders.

Involvement of IL-6 in the pathogenesis of inflammatory bowel disease and colon cancer

Inflammatory bowel disease (IBD), which consists of Crohn’s disease and ulcerative colitis, is defined as a chronic inflammation of the gastrointestinbal tract. The etiopathogenetic mechanisms underlying the development of IBD are still not completely understood, and the therapeutic strategies used thus far have been limited to mostly evidence-based principles. There is growing evidence that the pro-inflammatory cytokine interleukin (IL)-6 plays a crucial part in the uncontrolled intestinal inflammatory process, which is a main characteristic of IBD. There is elevated production of IL-6 and its soluble receptor (sIL-6R) by intestinal macrophages and CD4+ T-cells. The increased formation of IL-6-sIL-6R complexes that interact with gp130 on the membrane of CD4+ T-cells (trans-signaling) lead to an increased expression and nuclear translocation of STAT3, which causes the induction of anti-apoptotic genes, such as Bcl-xl. This leads to an augmented resistance of lamina propria T-cells to apoptosis. The ensuing T-cell expansion contributes to the perpetuation of chronic intestinal inflammation. This understanding concerning the predominant pathogenic role of an IL-6-dependent inflammatory cascade may lead to the development of new therapeutic strategies in the treatment of this disease. Recent studies have also suggested a potential role of IL-6-sIL-6R in the pathogenesis of colon cancer and, therefore, imply a possible novel therapeutic strategy targeting the sIL-6R and ensuing IL-6 trans-signaling.

TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells.

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.

Antibodies Against Tumor Necrosis Factor (TNF) Induce T-Cell Apoptosis in Patients With Inflammatory Bowel Diseases via TNF Receptor 2 and Intestinal CD14+ Macrophages

BACKGROUND & AIMS The anti-tumor necrosis factor (TNF) antibodies infliximab, adalimumab, and certolizumab pegol have proven clinical efficacy in Crohns disease. Here, we assessed the effects of anti-TNF antibodies on apoptosis in inflammatory bowel disease (IBD). METHODS CD14(+) macrophages and CD4(+) T cells were isolated from peripheral blood and lamina propria mononuclear cells from patients with IBD and control patients. Cell surface markers and apoptosis were assessed by immunohistology and fluorescence-activated cell sorting techniques. RESULTS Lamina propria CD14(+) macrophages showed significantly more frequent and higher membrane-bound TNF (mTNF) expression than CD4(+) T cells in IBD, whereas mTNF-dependent signaling proteins such as TNF receptor (TNFR) 2, TNFR-associated factor (TRAF) 2, and nuclear factor κB were induced in IBD mucosal CD4(+) T cells. Most anti-TNF antibodies did not induce T-cell apoptosis in purified peripheral or mucosal CD4(+) T cells. However, in contrast to etanercept, administration of all clinically effective anti-TNF antibodies resulted in a significant induction of T-cell apoptosis in IBD when lamina propria CD4(+) T cells expressing TNFR2(+) were cocultured with mTNF(+) CD14(+) intestinal macrophages. In contrast, no effects in control patients were noted. T-cell apoptosis in IBD occurred in vivo after treatment with adalimumab and infliximab, was critically dependent on TNFR2 signaling, and could be prevented via interleukin-6 signal transduction. Blockade of interleukin-6R signaling augmented anti-TNF-induced T-cell apoptosis in IBD. CONCLUSIONS Clinically effective anti-TNF antibodies are able to induce T-cell apoptosis in IBD only when mucosal TNFR2(+) T cells are cocultured with mTNF-expressing CD14(+) macrophages. The finding that anti-TNF antibodies induce apoptosis indirectly by targeting the mTNF/TNFR2 pathway may have important implications for the development of new therapeutic strategies in IBD.

The transcription factor IFN regulatory factor–4 controls experimental colitis in mice via T cell–derived IL-6

The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor-4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transplantation with CD4+CD45RB(hi) T cells, adoptive transfer of wild-type but not IRF4-deficient T cells resulted in severe colitis. Furthermore, IRF4-deficient mice were protected from T cell-dependent chronic intestinal inflammation in trinitrobenzene sulfonic acid- and oxazolone-induced colitis. In addition, IRF4-deficient mice with induced colitis had reduced mucosal IL-6 production, and IRF4 was required for IL-6 production by mucosal CD90+ T cells, which it protected from apoptosis. Finally, the protective effect of IRF4 deficiency could be abrogated by systemic administration of either recombinant IL-6 or a combination of soluble IL-6 receptor (sIL-6R) plus IL-6 (hyper-IL-6). Taken together, our data identify IRF4 as a key regulator of mucosal IL-6 production in T cell-dependent experimental colitis and suggest that IRF4 might provide a therapeutic target for IBDs.