Sebastian Zundler

Interleukin-12: Functional activities and implications for disease.

Interleukin-12 (IL-12) was the first member of the IL-12 family of cytokines to be identified and has therefore become its eponym. It is a heterodimeric protein of two subunits (p35, p40) secreted by phagocytic cells in response to pathogens and mainly acts through STAT4 to induce IFN-γ production in T and NK cells. IFN-γ in turn mediates proinflammatory functions and activates T-bet. As IL-12 engages in TH1 development, it is believed to represent an important link between innate and adaptive immunity. Following its identification and the finding of its association to TH1 commitment, great hopes were placed in IL-12 to become a target for therapeutic applications in multiple settings of autoimmunity and cancer. Though, the discovery of the related members of the IL-12 family and several rather disappointing attempts to translate experimental results into clinical practice, have relativized these hopes. Nevertheless, IL-12 remains a cytokine of outstanding importance with lots of unresolved questions. In this review, we will first briefly depict the biochemistry of the cytokine, its receptor and the related signal transduction, before summarizing the regulation of IL-12 production and its biological functions. We will then describe the current knowledge about the implication of IL-12 in different murine disease models as well as in the corresponding human conditions and comment on possible consequences for future clinical applications.

Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo

Objective Gut homing of lymphocytes via adhesion molecules has recently emerged as new target for therapy in IBDs. We aimed to analyse the in vivo homing of effector (Teff) and regulatory (Treg) T cells to the inflamed gut via α4β7 and G protein receptor GPR15. Design We assessed the expression of homing receptors on T cells in peripheral blood and inflamed mucosa. We studied the migration pattern and homing of Teff and Treg cells to the inflamed gut using intravital confocal microscopy and FACS in a humanised mouse model in dextran sodium sulfate-treated NSG (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) mice. Results Expression of GPR15 and α4β7 was significantly increased on Treg rather than Teff cells in peripheral blood of patients with UC as compared with Crohn’s disease and controls. In vivo analysis in a humanised mouse model showed augmented gut homing of UC Treg cells as compared with controls. Moreover, suppression of UC (but not control) Teff and Treg cell homing was noted upon treatment with the α4β7 antibody vedolizumab. In contrast, siRNA blockade of GPR15 had only effects on homing of Teff cells but did not affect Treg homing in UC. Clinical vedolizumab treatment was associated with marked expansion of UC Treg cells in peripheral blood. Conclusions α4β7 rather than GPR15 is crucial for increased colonic homing of UC Treg cells in vivo, while both receptors control UC Teff cell homing. Vedolizumab treatment impairs homing of UC Treg cells leading to their accumulation in peripheral blood with subsequent suppression of systemic Teff cell expansion.

Blockade of αEβ7 integrin suppresses accumulation of CD8+ and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo

Objective Therapeutically targeting lymphocyte adhesion is of increasing relevance in IBD. Yet, central aspects of the action of antiadhesion compounds are incompletely understood. We investigated the role of αEβ7 and α4β7 integrins and their blockade by vedolizumab and etrolizumab for trafficking of IBD T lymphocytes in an in vivo model of homing to and retention in the inflamed gut. Design We explored integrin expression in patients with IBD by flow cytometry and immunohistochemistry, while regulation of integrins was studied in T cell cultures. The functional relevance of integrins was assessed by adhesion assays and a recently established humanised mouse model in dextran sodium sulfate-treated immunodeficient mice. Results High expression of αEβ7 was noted on CD8+ and CD4+ Th9 cells, while α4β7 was expressed on CD8+, Th2 and Th17 cells. T cell receptor stimulation and transforming growth factor β were key inducers of αEβ7 on human T cells, while butyric acid suppressed αEβ7. In comparison to α4β7 blockade via vedolizumab, blockade of β7 via etrolizumab surrogate antibody superiorly reduced colonic numbers of CD8+ and Th9 cells in vivo after 3 hours, while no difference was noted after 0.5 hours. AEβ7 expression was higher on CD8+ T cells from patients with IBD under vedolizumab therapy. Conclusions AEβ7 is of key relevance for gut trafficking of IBD CD8+ T cells and CD4+ Th9 cells in vivo and mainly retention might account for this effect. These findings indicate that blockade of αEβ7 in addition to α4β7 may be particularly effective in intestinal disorders with expansion of CD8+ and Th9 cells such as IBD.

The α4β1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

Background: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohns disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-&agr;4&bgr;7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. Methods: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. Results: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-&agr;4&bgr;7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin &agr;4&bgr;1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of &agr;4 and &agr;4&bgr;1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of &agr;4 and &agr;4&bgr;1 integrins, but not &agr;4&bgr;7 integrins. Conclusions: Our findings suggest that Teff cell homing to the ileum through the axis &agr;4&bgr;1–VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases

BACKGROUND AND AIMS The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. METHODS Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues. RESULTS Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity. CONCLUSIONS The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.

Novel Insights into the Mechanisms of Gut Homing and Antiadhesion Therapies in Inflammatory Bowel Diseases

Therapeutic compounds interfering with T cell trafficking are a new column of inflammatory bowel disease (IBD) treatment. Currently, the anti-α4β7 integrin antibody vedolizumab is successfully used in the clinic and further drugs are likely to follow. Despite these clinical advances, the precise mechanistic background of their action is only gradually elucidated and still a matter of intensive research. Only recently, advances made with the help of new in vivo models and human studies have contributed to shape our concept of T cell trafficking in IBD by deciphering some important and so far unanswered questions. At the same time, basic and clinical data have generated new issues to be addressed on the way toward a clear perception of trafficking mechanisms and toward elucidation of the action of compounds interfering with this process. In this review, we will give a comprehensive outline of all components of T cell trafficking in regard to IBD before discussing the current knowledge concerning targeted interference with integrins in this complex network. Moreover, we will summarize remaining ambiguity and give an outlook on potential future targets.

Anti-Adhesion Therapies in Inflammatory Bowel Disease—Molecular and Clinical Aspects

The number of biologicals for the therapy of immunologically mediated diseases is constantly growing. In contrast to other agents that were previously introduced in rheumatologic or dermatologic diseases and only later adopted for the treatment of inflammatory bowel diseases (IBDs), the field of IBD was ground breaking for the concept of anti-adhesion blockade. Anti-adhesion antibodies selectively target integrins controlling cell homing to the intestine, which leads to reduction of inflammatory infiltration to the gut in chronic intestinal inflammation. Currently, the anti-α4β7-antibody vedolizumab is successfully used for both Crohn’s disease and ulcerative colitis worldwide. In this mini-review, we will summarize the fundamental basis of intestinal T cell homing and explain the molecular groundwork underlying current and potential future anti-adhesion therapies. Finally, we will comment on noteworthy clinical aspects of anti-adhesion therapy and give an outlook to the future of anti-integrin antibodies and inhibitors.

Clinical Response to Vedolizumab in Ulcerative Colitis Patients Is Associated with Changes in Integrin Expression Profiles

Background Despite large clinical success, deeper insights into the immunological effects of vedolizumab therapy for inflammatory bowel diseases are scarce. In particular, the reasons for differential clinical response in individual patients, the precise impact on the equilibrium of integrin-expressing T cell subsets, and possible associations between these issues are not clear. Methods Blood samples from patients receiving clinical vedolizumab therapy were sequentially collected and analyzed for expression of integrins and chemokine receptors on T cells. Moreover, clinical and laboratory data from the patients were collected, and changes between homing marker expression and clinical parameters were analyzed for possible correlations. Results While no significant correlation of changes in integrin expression and changes in outcome parameters were identified in Crohn’s disease (CD), increasing α4β7 levels in ulcerative colitis (UC) seemed to be associated with favorable clinical development, whereas increasing α4β1 and αEβ7 correlated with negative changes in outcome parameters. Changes in α4β1 integrin expression after 6 weeks were significantly different in responders and non-responders to vedolizumab therapy as assessed after 16 weeks with a cutoff of +4.2% yielding 100% sensitivity and 100% specificity in receiver-operator-characteristic analysis. Discussion Our data show that clinical response to vedolizumab therapy in UC but not in CD is associated with specific changes in integrin expression profiles opening novel avenues for mechanistic research and possibly prediction of response to therapy.

Integrating Immunologic Signaling Networks: The JAK/STAT Pathway in Colitis and Colitis-Associated Cancer

Cytokines are believed to be crucial mediators of chronic intestinal inflammation in inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC). Many of these cytokines trigger cellular effects and functions through signaling via janus kinase (JAK) and signal transducer and activator of transcription (STAT) molecules. In this way, JAK/STAT signaling controls important events like cell differentiation, secretion of cytokines or proliferation and apoptosis in IBD in both adaptive and innate immune cells. Moreover, JAK/STAT signaling, especially via the IL-6/STAT3 axis, is believed to be involved in the transition of inflammatory lesions to tumors leading to colitis-associated cancer (CAC). In this review, we will introduce the main cellular players and cytokines that contribute to pathogenesis of IBD by JAK/STAT signaling, and will highlight the integrative function that JAK/STATs exert in this context as well as their divergent role in different cells and processes. Moreover, we will explain current concepts of the implication of JAK/STAT signaling in CAC and finally discuss present and future therapies for IBD that interfere with JAK/STAT signaling.

K+ Channel Inhibition Differentially Regulates Migration of Intestinal Epithelial Cells in Inflamed vs. Non-Inflamed Conditions in a PI3K/Akt-Mediated Manner.

Background Potassium channels have been shown to determine wound healing in different tissues, but their role in intestinal epithelial restitution–the rapid closure of superficial wounds by intestinal epithelial cells (IEC)–remains unclear. Methods In this study, the regulation of IEC migration by potassium channel modulation was explored with and without additional epidermal growth factor (EGF) under baseline and interferon-γ (IFN-γ)-pretreated conditions in scratch assays and Boyden chamber assays using the intestinal epithelial cell lines IEC-18 and HT-29. To identify possibly involved subcellular pathways, Western Blot (WB)-analysis of ERK and Akt phosphorylation was conducted and PI3K and ERK inhibitors were used in scratch assays. Furthermore, mRNA-levels of the potassium channel KCNN4 were determined in IEC from patients suffering from inflammatory bowel diseases (IBD). Results Inhibition of Ca2+-dependent potassium channels significantly increased intestinal epithelial restitution, which could not be further promoted by additional EGF. In contrast, inhibition of KCNN4 after pretreatment with IFN-γ led to decreased or unaffected migration. This effect was abolished by EGF. Changes in Akt, but not in ERK phosphorylation strongly correlated with these findings and PI3K but not ERK inhibition abrogated the effect of KCNN4 inhibition. Levels of KCNN4 mRNA were higher in samples from IBD patients compared with controls. Conclusions Taken together, we demonstrate that inhibition of KCNN4 differentially regulates IEC migration in IFN-γ-pretreated vs. non pretreated conditions. Moreover, our data propose that the PI3K signaling cascade is responsible for this differential regulation. Therefore, we present a cellular model that contributes new aspects to epithelial barrier dysfunction in chronic intestinal inflammation, resulting in propagation of inflammation and symptoms like ulcers or diarrhea.