Ulrike Billmeier

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.

Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus.

Hepatic insulin resistance is a driving force in the pathogenesis of type 2 diabetes mellitus (T2DM) and is tightly coupled with excessive storage of fat and the ensuing inflammation within the liver. There is compelling evidence that activation of the transcription factor nuclear factor-κB (NF-κB) and downstream inflammatory signaling pathways systemically and in the liver are key events in the etiology of hepatic insulin resistance and β-cell dysfunction, although the molecular mechanisms involved are incompletely understood. We here test the hypothesis that receptor activator of NF-κB ligand (RANKL), a prototypic activator of NF-κB, contributes to this process using both an epidemiological and experimental approach. In the prospective population-based Bruneck Study, a high serum concentration of soluble RANKL emerged as a significant (P < 0.001) and independent risk predictor of T2DM manifestation. In close agreement, systemic or hepatic blockage of RANKL signaling in genetic and nutritional mouse models of T2DM resulted in a marked improvement of hepatic insulin sensitivity and amelioration or even normalization of plasma glucose concentrations and glucose tolerance. Overall, this study provides evidence for a role of RANKL signaling in the pathogenesis of T2DM. If so, translation to the clinic may be feasible given current pharmacological strategies to lower RANKL activity to treat osteoporosis.

Interleukin-35 Mediates Mucosal Immune Responses That Protect Against T-Cell–Dependent Colitis

BACKGROUND & AIMS The soluble hematopoietin receptor Epstein-Barr virus-induced protein (EBI)-3 is an immune regulator that has been associated with the pathogenesis of inflammatory bowel disease. However, the concept that EBI3 is part of an interleukin (IL)-27 heterodimer that mediates chronic inflammatory and autoimmune diseases has been challenged by the description of IL-35, a bioactive cytokine comprising EBI3 and IL-12 p35. We investigated the roles of IL-27 and IL-35 in chronic inflammation of the intestine. METHODS We analyzed EBI3-deficient mice and IL-27p28-deficient mice with spontaneous or T-cell transfer-induced colitis and compared outcomes with wild-type mice (controls). We constructed vectors that express EBI3 covalently linked to the IL-12p35 chain (recombinant [r]IL-35). RESULTS Intestines of EBI3-deficient mice had increased pathologic features of colitis, compared with IL-27p28-deficient or control mice; they also had shorter survival times, indicating that IL-35, rather than IL-27, protects the intestine from immune responses in mice. The mucosa of EBI3-deficient mice accumulated subsets of activated CD4+ T cells that produced T-helper (Th)1 and Th17 cytokines. Adoptive transfer of these T cells induced colitis in RAG-deficient mice. The rIL-35 significantly reduced the development of several forms of experimental colitis and reduced levels of markers of Th1 and Th17 cells. CONCLUSIONS IL-35 controls the development of T-cell-dependent colitis in mice. It might be developed as a therapeutic target for patients with chronic intestinal inflammation.

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts.

STAT3 Activation in Th17 and Th22 Cells Controls IL-22–Mediated Epithelial Host Defense during Infectious Colitis

The Citrobacter rodentium model mimics the pathogenesis of infectious colitis and requires sequential contributions from different immune cell populations, including innate lymphoid cells (ILCs) and CD4+ lymphocytes. In this study, we addressed the role of STAT3 activation in CD4+ cells during host defense in mice against C. rodentium. In mice with defective STAT3 in CD4+ cells (Stat3ΔCD4), the course of infection was unchanged during the innate lymphoid cell–dependent early phase, but significantly altered during the lymphocyte-dependent later phase. Stat3ΔCD4 mice exhibited intestinal epithelial barrier defects, including downregulation of antimicrobial peptides, increased systemic distribution of bacteria, and prolonged reduction in the overall burden of C. rodentium infection. Immunomonitoring of lamina propria cells revealed loss of virtually all IL-22–producing CD4+ lymphocytes, suggesting that STAT3 activation was required for IL-22 production not only in Th17 cells, but also in Th22 cells. Notably, the defective host defense against C. rodentium in Stat3∆CD4 mice could be fully restored by specific overexpression of IL-22 through a minicircle vector–based technology. Moreover, expression of a constitutive active STAT3 in CD4+ cells shaped strong intestinal epithelial barrier function in vitro and in vivo through IL-22, and it promoted protection from enteropathogenic bacteria. Thus, our work indicates a critical role of STAT3 activation in Th17 and Th22 cells for control of the IL-22–mediated host defense, and strategies expanding STAT3-activated CD4+ lymphocytes may be considered as future therapeutic options for improving intestinal barrier function in infectious colitis.

From physiology to disease and targeted therapy: interleukin-6 in inflammation and inflammation-associated carcinogenesis

Abstract Since its discovery in 1986, originally as B cell stimulating factor 2, the knowledge on IL-6 for immune homeostasis and its pathophysiological implications has rapidly increased. It is now clear that IL-6, alone or in combination with other cytokines, is an architect for shaping and generating immune responses which exerts profound activities on the induction of acute-phase reactions, the differentiation of B lymphocytes, the modulation of T cell apoptosis, the activation of T helper cells and the balance between regulatory T cells and Th17 cells. In parallel to the identification of these physiologic functions, IL-6 has emerged as a critical mediator for perpetuating chronic inflammation and autoimmunity and is increasingly recognized as a key cytokine for linking chronic inflammation to cancer development. In this review, we begin by briefly summarizing the molecular events of IL-6 regulation and signaling and then describe the role of IL-6 in orchestrating innate and adaptive immune responses and its immunopathological relevance for chronic inflammatory diseases. We further outline how IL-6 links chronic inflammation and cancer development and finally provide an outlook on novel therapeutic strategies targeting IL-6 signaling for the treatment of chronic inflammatory diseases and cancer.

Molecular mechanism of action of anti-tumor necrosis factor antibodies in inflammatory bowel diseases

Anti-tumor necrosis factor (TNF) antibodies are successfully used in the therapy of inflammatory bowel diseases (IBD). However, the molecular mechanism of action of these agents is still a matter of debate. Apart from neutralization of TNF, influence on the intestinal barrier function, induction of apoptosis in mucosal immune cells, formation of regulatory macrophages as well as other immune modulating properties have been discussed as central features. Nevertheless, clinically effective anti-TNF antibodies were shown to differ in their mode-of-action in vivo and in vitro. Furthermore, the anti-TNF agent etanercept is effective in the treatment of rheumatoid arthritis but failed to induce clinical response in Crohn’s disease patients, suggesting different contributions of TNF in the pathogenesis of these inflammatory diseases. In the following, we will review different aspects regarding the mechanism of action of anti-TNF agents in general and analyze comparatively different effects of each anti-TNF agent such as TNF neutralization, modulation of the immune system, reverse signaling and induction of apoptosis. We discuss the relevance of the membrane-bound form of TNF compared to the soluble form for the immunopathogenesis of IBD. Furthermore, we review reports that could lead to personalized medicine approaches regarding treatment with anti-TNF antibodies in chronic intestinal inflammation, by predicting response to therapy.

Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium – a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

Wnt Inhibitory Factor 1 Deficiency Uncouples Cartilage and Bone Destruction in Tumor Necrosis Factor α–Mediated Experimental Arthritis

OBJECTIVE Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. METHODS Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/β-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. RESULTS WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. CONCLUSION These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I-deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.