Function of constitutive IκB Kinase 2 activation in the intestinal epithelium of mice

Die NF-κB Signalkaskade stellt eine Verbindung zwischen Entzündungsprozessen und Krebs dar. Der NF-κB Signalweg wird durch Stress-assoziierte Reize wie Pathogen-assoziierte Molekulare Muster (PAMPs), inflammatorische Zytokine und UV-Licht initiiert. Da die Aktivierung dieses Signalweges zur Expression von antiapoptotischen Molekülen, inflammatorischen Signalkomponenten, sowie antibakteriellen Peptiden führt, stellt NF-κB einen zentralen Regulator von entzündlichen Prozessen dar. Darüber hinaus weisen verschiedene Krebsarten eine erhöhte NF-κB Aktivität auf, die sich nicht nur auf Tumor-infiltrierende Immunzellen beschränkt,sondern auch in den Krebszellen selber aufzufinden ist. Es wird vermutet, dass die Wirkung von NF-κB in Tumorzellen den Zelltod verhindert und die Zellproliferation fördert. Dahingegen induziert NF-κB in Zellen des Immunsystems die Sekretion von Zytokinen und Wachstumsfaktoren, wodurch Tumorwachstum und Angiogenese gefördert werden. Allerdings ist bisher nicht geklärt worden, ob die Aktivierung von NF-κB die Entstehung von Tumoren durch zellinterne Tumor-fördernde Wirkungen in nicht transformierten Zellen zu initiieren vermag. In dieser Arbeit wird gezeigt, dass die Expression einer konstitutiv aktiven Form der zur Aktivierung des kanonischen NF-κB Signalwegs notwendigen zentralen Kinase IKK2 (IKK2ca) in intestinalen Epithelzellen von Mäusen zu einer spontanen Entzündung sowohl des Dick- als auch des Dünndarms führte. Die konstitutive Aktivierung von IKK2/NF-κB im Darmepithel resultierte in einer Rekrutierung und Aktivierung von Immunzellen und Fibroblasten. Darüber hinaus erhöhte die Expression von IKK2ca im Darmepithel die Sensitivität gegenüber DSS-induzierter Colitis und steigerte die Tumorrate in chemisch- sowie Apc-Mutation-initiiertem Darmkrebs. Intestinale Epithelzellen, die konstitutiv aktive IKK2 exprimierten, zeigten eine erhöhte Proliferationsrate, eine Stabilisierung von β-catenin und eine verstärkte Transkription von Stammzell-assoziierten Faktoren. Hierbei ist zu betonen, dass die Expression von IKK2ca im Darmepithel von Mäusen zur spontanen Entwicklung von Colon- und Dünndarmtumoren führte. Die in dieser Dissertation vorgelegten Daten implizieren, dass die konstitutive Aktivierung von IKK2/NF-κB im Darmepithel ausreichend ist um sowohl durch zellinterne Faktoren, als auch durch die Erzeugung eines Tumor-förderlichen, entzündlichen Milieus, eine Tumorgenese nicht nur zu unterstützten, sondern auch zu induzieren

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-kappaB-Dependent and -Independent Functions

Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor kappaB (NF-kappaB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-kappaB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD

The interplay of IKK, NF-kappa B and RIPK1 signaling in the regulation of cell death, tissue homeostasis and inflammation

Regulated cell death pathways have important functions in host defense and tissue homeostasis. Studies in genetic mouse models provided evidence that cell death could cause inflammation in different tissues. Inhibition of RIPK3-MLKL-dependent necroptosis by FADD and caspase-8 was identified as a key mechanism preventing inflammation in epithelial barriers. Moreover, the interplay between IKK/NF-kappa B and RIPK1 signaling was recognized as a critical determinant of tissue homeostasis and inflammation. NEMO was shown to regulate RIPK1 kinase activity-mediated apoptosis by NF-kappa B-dependent and -independent functions, which are critical for averting chronic tissue injury and inflammation in the intestine and the liver. In addition, RIPK1 was shown to exhibit kinase activity-independent functions that are essential for preventing cell death, maintaining tissue architecture and inhibiting inflammation. In the intestine, RIPK1 acts as a scaffold to prevent epithelial cell apoptosis and preserve tissue integrity. In the skin, RIPK1 functions via its RHIM to counteract ZBP1/DAI-dependent activation of RIPK3-MLKL-dependent necroptosis and inflammation. Collectively, these studies provided evidence that the regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis, and suggested that cell death could be causally involved in the pathogenesis of inflammatory diseases

Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo

Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch

TLR-independent anti-inflammatory function of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice

Objective: The gut microbiota modulates host susceptibility to intestinal inflammation, but the cell types and the signalling pathways orchestrating this bacterial regulation of intestinal homeostasis remain poorly understood. Here, we investigated the function of intestinal epithelial toll-like receptor (TLR) responses in the dextran sodium sulfate (DSS)-induced mouse model of colitis. Design: We applied an in vivo genetic approach allowing intestinal epithelial cell (IEC)-specific deletion of the critical TLR signalling adaptors, MyD88 and/or TIR-domain-containing adapter-inducing interferon-β (TRIF), as well as the downstream ubiquitin ligase TRAF6 in order to reveal the IEC-intrinsic function of these TLR signalling molecules during DSS colitis. Results: Mice lacking TRAF6 in IECs showed exacerbated DSS-induced inflammatory responses that ensued in the development of chronic colon inflammation. Antibiotic pretreatment abolished the increased DSS susceptibility of these mice, showing that epithelial TRAF6 signalling pathways prevent the gut microbiota from driving excessive colitis. However, in contrast to epithelial TRAF6 deletion, blocking epithelial TLR signalling by simultaneous deletion of MyD88 and TRIF specifically in IECs did not affect DSS-induced colitis severity. This in vivo functional comparison between TRAF6 and MyD88/TRIF deletion in IECs shows that the colitis-protecting effects of epithelial TRAF6 signalling are not triggered by TLRs. Conclusions: Intestinal epithelial TRAF6-dependent but MyD88/TRIF-independent and, thus, TLR-independent signalling pathways are critical for preventing propagation of DSS-induced colon inflammation by the gut microbiota. Moreover, our experiments using mice with dual MyD88/TRIF deletion in IECs unequivocally show that the gut microbiota trigger non-epithelial TLRs rather than epithelial TLRs to restrict DSS colitis severity

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs)(1-4). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood(1,5). Here we show that mice with IEC-specific knockout of FADD (FADD(IEC-KO)), an adaptor protein required for death-receptor-induced apoptosis(6), spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis(7-9), prevented the development of spontaneous pathology in both the small intestine and colon of FADD(IEC-KO) mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis(10), prevented colitis development in FADD(IEC-KO) but not in NEMOIEC-KO mice(11), showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADD(IEC-KO) mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADD(IEC-KO) mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation

Constitutive IKK2 activation in intestinal epithelial cells induces intestinal tumors in mice

Many cancers display increased NF-κB activity, and NF-κB inhibition is known to diminish tumor development in multiple mouse models, supporting an important role of NF-κB in carcinogenesis. NF-κB activation in premalignant or cancer cells is believed to promote tumor development mainly by protecting these cells from apoptosis. However, it remains unclear to what extent NF-κB activation exhibits additional protumorigenic functions in premalignant cells that could be sufficient to induce spontaneous tumor development. Here we show that expression of constitutively active IκB kinase 2 (IKK2ca) in mouse intestinal epithelial cells (IECs) induced spontaneous tumors in aged mice and also strongly enhanced chemical- and Apc mutation–mediated carcinogenesis. IECs expressing IKK2ca displayed altered Wnt signaling and increased proliferation and elevated expression of genes encoding intestinal stem cell–associated factors including Ascl2, Olfm4, DLK1, and Bmi-1, indicating that increased IKK2/NF-κB activation synergized with Wnt signaling to drive intestinal tumorigenesis. Moreover, IECs expressing IKK2ca produced cytokines and chemokines that induced the recruitment of myeloid cells and activated stromal fibroblasts to become myofibroblasts, thus creating a tumor-promoting microenvironment. Taken together, our results show that constitutively increased activation of IKK2/NF-κB signaling in the intestinal epithelium is sufficient to induce the full spectrum of cell-intrinsic and stromal alterations required for intestinal tumorigenesis

Temporally Distinct Functions of the Cytokines IL-12 and IL-23 Drive Chronic Colon Inflammation in Response to Intestinal Barrier Impairment

Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-gamma. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects