Regulation and pathophysiological role of epithelial turnover in the gut.

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Review

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Regulation and pathophysiological role of epithelial turnover in the gut夽

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Claudia Günther, Barbara Buchen, Markus F. Neurath, Christoph Becker ∗ Department of Medicine 1, University of Erlangen-Nuremberg, Erlangen, Germany

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Article history: Available online xxx

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Keywords: Necroptosis Inflammation Ripoptosome

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Contents

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Cell death in the intestinal epithelium has to be tightly controlled. Excessive or misplaced epithelial cell death can result in barrier dysfunction and, as a consequence thereof, uncontrolled translocation of components of the microbial flora from the lumen into the bowel wall. Susceptibility to gastrointestinal infections or chronic inflammation of the gut, as observed in patients with inflammatory bowel disease, can be the result of such dysregulation. Conversely, defects in cell death initiation might lead to an irregular accumulation of epithelial cells and cause intestinal cancer development. Until recently, activation of caspases in the intestinal epithelium was considered as a potential contributor to barrier dysfunction and as a pathogenic factor in the development of intestinal inflammation. Thus blocking of caspases appeared to be a potential therapeutic option for patients with inflammatory bowel disease. Recent studies on necroptosis however demonstrated that also inhibition of caspases can cause barrier dysfunction and intestinal inflammation. Caspase-8 on top of its functions in the extrinsic apoptosis pathway also controls necroptosis and turns out to be an essential molecule in regulating tissue homeostasis in the gut. Epithelial caspase-8 therefore emerges as a checkpoint not only of cell survival and cell death, but also as a regulator of the mode of cell death. According to this model, both excessive activity as well as a lack of activity of caspase-8 results in epithelial cell death and intestinal inflammation and caspase-8 needs to be tightly controlled to warrant tissue homeostasis in the gut. © 2014 Elsevier Ltd. All rights reserved.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of cell death in the intestinal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Necroptosis: a new mode of cell death with implications for gut homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The signalling pathways towards necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Tnf-R complex 2a (cell survival) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Tnf-R complex 2b (apoptosis). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Tnf-R complex 2c (necroptosis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Triggering factors for apoptosis and necroptosis in the intestinal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Death receptor mediated epithelial cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. PAMP mediated cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. TLR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Cell death induced by interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Q1 夽 Grant support: This work has been supported by the Deutsche Forschungs-gemeinschaft (BE3686/2-1, SFB 796, KFO 257 CEDER, SPP1656), the European Community’s Q2 Innovative Medicines Initiative (IMI), acronym BTCure (115142) and the Interdisciplinary Center for Clinical Research (IZKF) of the University Erlangen-Nuremberg. ∗ Corresponding author at: Department of Medicine 1, University of Erlangen-Nuremberg, Hartmannstrasse 14, 91054 Erlangen, Germany. Tel.: +49 9131 8535886; fax: +49 9131 8535959. E-mail address: [email protected] (C. Becker). http://dx.doi.org/10.1016/j.semcdb.2014.06.004 1084-9521/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Günther C, et al. Regulation and pathophysiological role of epithelial turnover in the gut. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.004

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1. Introduction

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The gut harbours the largest part of our individual immune system. The reason for this high number of immune cells is seen in the fact that the gut is permanently exposed to a plethora of antigens and potential pathogens which are present in the food or the microbial flora [1]. To maintain intestinal homeostasis and prevent an excessive activation of immune cells within the gut wall, it is necessary that the microbiota is strictly separated from the underlying immune system. It has been reported that excessive infiltration of bacteria or bacterial products leads to a deregulated intestinal immune response resulting in the development of gastrointestinal disorders, including inflammatory bowel diseases (IBD) or infectious colitis [2]. The first line of defence of the intestinal mucosa is represented by a single cell layer of intestinal epithelial cells (IECs). These epithelial cells are of paramount importance in host defence by providing on the one hand a physical barrier and on the other hand highly specialized innate immune functions [3]. The physical barrier is established by the close and firm contact of neighbouring intestinal epithelial cells, which relies on the formation of tight junctions. Innate immune functions are carried out mainly by goblet cells and paneth cells, highly specialized epithelial cells with secretory functions [3]. Goblet cells release mucins, which give rise to a viscous mucous layer on the gut wall, hampering the access of bacteria to the epithelial surface [4]. Paneth cells in contrast release granules containing antimicrobial peptides [5]. These antimicrobial peptides are thought not only to kill bacteria; their diversity and regulated expression patterns are believed to actively shape the microbial communities present within the gut lumen. Antimicrobial peptides and mucins together form a thick bactericidal mucous layer which hampers access and survival of bacteria adjacent to the epithelium [5]. Besides its important functions in generating a barrier against the external environment, the intestinal epithelium is also the most vigorously self-renewing tissue of adult individuals [3]. Intestinal epithelial cells are generated by proliferating stem cells at the bottom of the Crypts of Lieberkühn [6]. During their short lifetime of 4–5 days, intestinal epithelial cells move up towards the intestinal surface [3]. During this process undifferentiated cells differentiate to functionally active absorptive enterocytes or to other epithelial cell types. Once the cells reach the luminal surface, they die and are released from the epithelial cell layer. Disturbances within this highly regulated system can cause serious diseases. Excessive or misplaced cell death has been shown to cause barrier disruption and chronic inflammation. Conversely, deficient cell death can lead to dysplasia and cancer [7]. It is therefore obvious, that cell death has to be tightly controlled in order to maintain tissue homeostasis in the gut. Historically, cell death was differentiated into apoptosis, a programmed form of cell death initiated by intracellular or extracellular triggers, and necrosis, an unregulated form of injury-like cell lysis [8]. Studies of the past years have made it very clear, that this simplistic discrimination might be an oversimplification of cell death regulation. The discovery of alternative forms of cell death including autophagy and necroptosis has dissolved the strict classification into necrosis and apoptosis. In this review we will revise the differences between apoptotic and necrotic cell death in the intestinal epithelium and will then explore the mechanisms and triggering factors which induce and regulate apoptosis and programmed necrosis.

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2. Characteristics of cell death in the intestinal epithelium

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Cell death with its different characteristics is an elementary process for tissue development and homeostasis to eliminate

superfluous, damaged or aged cells. This is of major importance for the GI-tract, since the intestinal epithelium undergoes continuous and rapid self-renewal. The majority of intestinal epithelial cells have a very short life span and are renewed every 4–5 days [3,6]. The small intestinal epithelium can be divided into villi, which contain terminal differentiated cells and the crypts of Lieberkühn which contain a small number of stem cells [6]. Intestinal stem cells at the crypt bottom proliferate and therefore are critical to renew the intestinal epithelial cell layer. Few stem cells at the crypt bottom give rise to a larger number of so called transient amplifying cells, undifferentiated cells that still have mitotic potential [6]. Newly generated cells within the crypts migrate upward towards the villous tip. During this migration period, the cells differentiate to specialized epithelial cells, including absorptive enterocytes, goblet cells or enteroendocrine cells [3]. The continuous proliferation at the crypt bottom is thought to be the driver of this upward cellular movement. Once the cells reach the tip of the villous, they are shed into the gut lumen in a process, which is associated with epithelial cell death [9]. The continuous self-renewal therefore critically depends on proliferation of stem cells within the crypts and cell loss at the villous tip. Epithelial cell renewal and cell death needs to be tightly regulated as irregularities might cause pathologies, like inflammation and cancer [10–13]. As such, excessive cell death of IECs on the one hand is thought to cause barrier defects, invasion of bacteria and subsequent inflammation [10]. On the other hand, defective cell death activation has been associated with the development of colorectal cancer [14]. Historically, cell death has been divided into a regulated or programmed form and an unregulated more injury-like form. The most common form of programmed cell death has been described in the early 1970s as apoptosis [8]. Apoptosis can be initiated by a wide variety of stimuli including DNA damage, nutrient deficiency, endoplasmic reticulum (ER) stress, growth factor withdrawal, heat shock, developmental cues and ligation of death-receptors on the cell surface [15]. Depending on the origin of the death inducing stimuli, apoptosis is mediated through either the intrinsic or extrinsic pathway. Both apoptotic pathways depend on enzymatic cascades of caspases [16]. Morphological characteristics of apoptosis include membrane blebbing, apoptotic body formation, cell shrinkage, chromatin condensation and DNA fragmentation [16,17]. Apoptotic cells can be recognized by neighbouring immune cells, like macrophages, leading to the phagocytosis of dying cells, a mechanism that might protect the host from cell death associated inflammatory processes [Taylor, 2008, #241, 18]. Under steady state conditions cell death is rarely observed in the intestinal epithelium [11]. However, it has been described that it can occur at one hot spot along the crypt-villus axis, represented by the villus tip, where aged epithelial cells are replaced by neighbouring cells [18,19]. This process has been referred to as “homeostatic cell shedding” [20]. Although cells activate caspases during the process of shedding shortly before they are expelled into the gut lumen, accumulating evidence indicates that homeostatic cell shedding occurs at least functionally independent of apoptotic cell death initiation [9,21,22]. The fact that apoptosis might not be required for epithelial turnover in the gut, at least under steady state conditions, is supported by studies demonstrating that mice deficient for central molecules of the apoptosis pathway show only little structural changes [19,23]. For example mice deficient for caspase-3 were described to show no major morphological differences in the development of the gastrointestinal tract, suggesting that the effector caspase-3 might be dispensable for tissue homeostasis in the gut [24–26]. This was further supported by murine knockout studies of other cell death related genes like Bcl-2 (B-cell CLL/lymphoma 2) and Bax (BCL2-associated X protein) [27]. It has been demonstrated that homozygous Bcl2-null mice show equal levels of spontaneous


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apoptosis in small intestinal crypts compared to their wild-type counterparts [28,29]. In addition, also Bax deficient mice do not show any alterations in the occurrence of spontaneous apoptosis compared to their wild-type counterparts in the small intestine, suggesting that Bax expression appears to have little effect on homeostasis in normal intestinal epithelium [30]. In a recent study, it has been demonstrated that predominantly caspase-negative cells extrude from the surfaces of the intestinal epithelium [22]. The authors concluded that shedding, under steady state conditions, is initiated by the rearrangement of tight junction proteins and the recruitment of kinases like the myosin light chain kinase and rho kinase to the membrane. While a lack of apoptosis does not compromise the structural development of the gut, excessive apoptosis can lead to severe gut pathology. Several studies, using mouse models with elevated levels of apoptosis in the intestinal epithelium either described the spontaneous development of inflammatory intestinal lesions or an increased susceptibility towards experimentally induced gut inflammation in mice [31–34]. A well characterized mouse model to demonstrate the contribution of increased apoptosis to intestinal inflammation carries an intestinal epithelial cell specific deletion of the gene NEMO (nuclear factor kappa B [NF-␬B] essential modulator), which is an important component of the NF-␬B pathway These mice spontaneously develop a chronic colitis shortly after birth [31], which was shown to develop as a consequence of excessive Tnf-␣ induced apoptosis of epithelial cells. Death of IECs led to a breakdown of the intestinal barrier, resulting in the translocation of bacteria into subepithelial areas and consequently to mucosal inflammation. This study was important, as it clearly demonstrated that a dysregulation within the epithelium is sufficient to drive chronic intestinal inflammation. Keeping in mind that many susceptibility genes linked to inflammatory bowel disease in humans are genes regulating IEC homeostasis, it is tempting to speculate, that barrier dysfunction due to defective epithelial cell function might be causative for the development of these diseases. To support the role of cell death regulation in intestinal inflammation, deficiency of other members of the NF-␬B pathway, like RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A), TAK1 (TGFbeta activated kinase 1) or both IKK1 (IkappaB kinase 1) and IKK2 (IkappaB kinase 2), resulted in an increased susceptibility to colitis [31,32,34–36]. In summary these data demonstrate the importance of the NF-␬B pathway for intestinal homeostasis and for controlling apoptosis of IEC. Another form of regulated cell death which occurs in the intestinal epithelium is the autophagy-associated cell death [37]. Autophagy represents a survival strategy in times of stress as it allows cells to adapt their metabolism to starvation mediated by restricted extracellular nutrients or by decreased intracellular metabolite concentrations resulting from the loss of growth-factor signalling. Aside from this cellular adaptation process, autophagy may be involved in immune homeostasis due to its participation in the digestion of intracellular pathogens and in antigen presentation. Interestingly, several autophagy-related genes were identified as IBD susceptibility genes in genome-wide association studies, suggesting that this kind of cell death might be involved in the pathogenesis of IBD [38–40]. Following these observations, a number of studies have investigated the role of autophagy in the gut [41–44]. Mice with an epithelial cell specific deletion of the autophagy genes ATG5 (autophagy related 5 homolog) or ATG7 (autophagy related 7 homolog) showed little defects in the development of a special type of epithelial cells (Paneth cells). However this was dispensable for gut immune homeostasis and had no effect on susceptibility in mouse models of experimentally induced colitis [43,44]. Altogether these data suggest that the autophagy pathway plays a minor role in the structural development of the gut.

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3. Necroptosis: a new mode of cell death with implications for gut homeostasis In contrast to apoptosis and autophagy, necrosis has traditionally been described as an unregulated and uncontrolled process [45]. It is mainly initiated by external factors such as ischaemiareperfusion, viral or bacterial infection, toxins, neurodegenerative processes or physical or chemical traumata and is characterized by a rapid breakdown of the membrane, resulting in the release of intracellular compounds into the extracellular space. Such cellular compounds can damage neighbouring cells which finally leads to the release of proinflammatory cytokines and the activation of the immune system [46]. Recently, an apoptosis-independent mode of necrosis, termed necroptosis, has been identified and described in different organs, including the gut [23,24,47,48]. Strikingly, both apoptosis as well as necroptosis can be induced by receptor ligation and therefore both pathways are genetically determined and regulated by very similar intracellular protein platforms [49]. While apoptosis absolutely requires caspase activation, necroptosis is negatively regulated by caspases and instead is dependent on the kinase activity of Receptor-interacting proteins (RIP) [50–52]. Opposed to apoptotic cells, necroptotic cells show the morphological features of necrosis, with the final cell lysis as described above. Certain proteins that are released from cells during necrotic cell death, including danger-associated molecular patterns (DAMPs), have been shown to activate pattern-recognition receptors (PRRs), e.g. Toll like receptors (TLRs) to further promote an inflammatory response. Thus the current dogma suggests that induction of apoptosis and other forms of non-necrotic cell death is necessary to maintain homeostasis by enabling cellular turnover. Opposed to that, necrotic forms of cellular death alert the host’s immune system which subsequently leads to the induction of an inflammatory response. In 2011 two papers independently described necroptotic cell death in vivo in the intestinal epithelium [23,47]. Caspase-8 is a central component in the programmed cell death pathway because its activation status determines whether a cell undergoes apoptosis or necroptosis. Despite the paradigm that apoptosis is important for regulating epithelial cell numbers, histological and morphometrical analysis of the distal small intestine of mice which specifically lack caspase-8 (Casp8IEC ) or its adapter FADD (FADDIEC ) in IECs revealed no obvious changes of tissue architecture, including villus length or number of dying cells in the villus region [23,47]. Thus these two studies again confirmed that activation of the caspase-cascade is not essential for the development and the overall structural integrity of the gut. However, both mouse strains spontaneously developed inflammatory lesions in the terminal ileum. This seemed surprising as excessive apoptosis of IEC rather than a lack of the apoptotic cell death was considered to drive intestinal inflammation. An interesting feature of both mouse strains is the complete absence of Paneth cells, which contribute to the integrity of the epithelial barrier by secreting antimicrobial peptides and thereby take part in the immune defence of the intestine. Subsequent analysis revealed that these Paneth cells undergo necroptosis which was formally proven by additional deletion of the necroptosis mediator RIP3 [23,47]. Moreover disruption of RIP3 prevented the development of spontaneous ileitis in Casp8IEC - and FADDIEC -mice, indicating that intestinal inflammation was RIP3-dependent. These studies clearly demonstrated that Paneth cells might be specifically sensitive to necroptosis, since these cells are the only type of differentiated epithelial cells in the small intestine which are affected by caspase-8 deficiency. The underlying cause of that specific sensitivity still remains unclear, but one possible explanation could be the constitutive high expression of RIP3 compared to other epithelial cell types [23]. It is tempting to speculate that dysregulated


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necroptosis of Paneth cells might also promote intestinal inflammation in human IBD. Defects in Paneth cell function have been hypothesized to contribute to the development of Crohn’s disease (CD), one major form of IBD [23]. Interestingly dying Paneth cells, showing signs of necrotic cell death, could be identified in biopsies taken from areas of acute inflammation in (CD) patients [23]. Comparable to Casp8IEC mice, human IECs can undergo RIP-mediated necroptosis which further implicates a potential role for necroptosis in the pathological changes that are observed in the small intestine of CD patients [23]. Another example for the potential contribution of necroptosis to human inflammation has recently been described. In this study the authors collected biopsies from children suffering from Crohn’s disease or ulcerative colitis [53]. Interestingly they identified elevated levels of the necroptosis mediators RIP3 and MLKL in inflamed areas, while at the same time expression of caspase-8 was reduced. These findings suggest that necroptosis is strongly associated with the development of intestinal inflammation in children with IBD. However the triggering factors for necroptosis in Casp8IEC or FADDIEC mice and humans still remain unclear. Furthermore it is still not clear whether necroptosis as a pro-inflammatory mode of cell death in general promotes inflammation or if the inflammation actually depends on the specific depletion of Paneth cells.

4. The signalling pathways towards necroptosis The best characterized pathway triggering cell death within the gut (including apoptosis and necroptosis), is the Tnf-Receptor (TnfR) signalling cascade. Ligation of the Tnf-R traditionally results in the assembly of one of the following cytosolic complexes: Tnf-R complex 1, which mediates pro-survival functions, or Tnf-R complex 2 which can either lead to the promotion of cell death in various cell types, including intestinal epithelial cells [54,55,1] or to cell survival (Fig. 1). One major molecule which decides between survival and cell death is the Receptor-interacting protein kinase 1 (RIP1) [56–58]. Activation of Tnf-R complex 1 triggers the polyubiquitination of RIP1 by inhibitor of apoptosis proteins (IAPs), called cIAP1 and cIAP2, which in turn promotes the downstream activation of mitogen-activated protein kinases (MAPKs) or the canonical NF-␬B pathway, resulting in the expression of pro-survival genes [59,60]. If RIP1 is deubiquitinated for example by the ubiquitinediting enzyme deubiquitinase cylindromatosis (CYLD) or A20, it can translocate to the Tnf-R complex 2 and bind to caspase-8 via its RIP homotype interaction motif (RHIM) [58,61,62]. Recent studies indicate that the composition of this complex not only decides on cell death or survival, it also determines what kind of cell death will be activated [63–65].

4.1. Tnf-R complex 2a (cell survival) The cysteine protease caspase-8 can heterodimerize with the long form of its cellular inhibitor cFLIPlong via the Fas-associated protein with Death Domain (FADD) adapter [66]. cFLIP carries a caspase-like domain which is very similar to that of caspase-8, but lacks some active site residues, rendering it catalytically inactive [67–69]. Although cFLIP inhibits the full activation of caspase8 and thereby blocks apoptosis, the heterodimer still possesses some catalytic activity [70–72] which is sufficient to prevent RIPdependent necroptosis presumably by cleaving and inactivating the RIP kinases [73]. With its partial activation, caspase-8 can cleave the deubiquitinase CYLD, which was also shown to be a substrate of caspase-8 [72], and thereby prevents the deubiquitination of RIP1, resulting in the association of RIP1 with the Tnf-R1 complex to mediate cell survival.

4.2. Tnf-R complex 2b (apoptosis) If expression of cFLIP is decreased, under pro-apoptotic conditions, caspase-8 can form a homodimer [74]. This close location of two caspase molecules initiates an autocatalytic cleavage and results in the release of two fully activated caspase subunits from the death receptor complex, which forms the mature active enzyme. This initial cleavage step represents the first step in the caspase-activating cascade that finally causes the cleavage of the effector caspase-3, resulting in apoptotic cell death. This pathway can be divided into two different types. In type 1 cells, cleavage of the initiator caspase alone is sufficient to induce apoptosis by activating the effector caspases [75]. In type 2 cells, receptor signalling alone is not potent enough to mediate apoptosis, death receptor signalling additionally requires to be amplified by the mitochondrial apoptosis pathway or the downregulation of prosurvival genes, like cFLIP [75]. Notably, in apoptotic cells caspase-8 also controls the RIP1 and RIP3 activity by proteolytic cleavage, thereby blocking necroptosis. 4.3. Tnf-R complex 2c (necroptosis) If the caspase-8 activity is impaired due to genetic deletion or pharmacological inhibition, proteolytic cleavage of the RIP kinases cannot longer be maintained, resulting in the autophosphorylation of these kinases. RIP3 then recruits and activates the mixed lineage kinase domain-like (MLKL) protein, a crucial downstream substrate of RIP3 in the necroptotic pathway [76–82]. Furthermore, MLKL recruits phosphoglycerate mutase 5 (PGAM5), a resident mitochondrial protein that may play a role in the execution of cellular necrosis [83,84]. It is believed that RIP3-MLKL dependent phosphorylation of PGAM5 results in the dephosphorylation and activation of the mitochondrial fusion protein Drp1 [83,85,1]. Excessive activity of Drp1 causes the production of reactive oxygen species (ROS), which disrupt the mitochondrial function and promote other organelles and membrane damage ending up in necroptosis [85,86]. As a result, the composition of this Tnf-R complex has direct implications for tissue homeostasis, as necroptosis in contrast to apoptosis might trigger immune cell activation. As described in this chapter, Tnf-␣ plays a major role in cell homeostasis, as it is capable not only to control cell survival versus death, but also to trigger both types of programmed cell death, apoptosis as well as necroptosis. Interestingly Tnf-␣ is considered as an important contributor to the pathogenesis of intestinal inflammation because patients suffering from Crohn’s disease or ulcerative colitis have elevated mucosal levels of this cytokine [87–90]. Additionally, genetic studies identified a chromosomal region containing the Tnf-␣ gene as an IBD susceptibility locus [1,91–93]. The contribution of this cytokine to the development of CD is moreover emphasized by the fact that Tnf-␣ blocking antibodies, like Infliximab or Adalimumab, are successfully used in the therapy of CD patients [94–98]. In fact, randomized controlled trials unveiled that the anti-Tnf antibodies were effective for the induction and maintenance of remission in both CD and UC [96]. Recent studies have elucidated the potential mechanisms of action of antiTnf therapy among the different cell types in the gut. Several lines of evidence suggest that the apoptotic destruction of activated effector cells is one key mechanism of action of anti-Tnf antibodies. This is supported by studies demonstrating that T-cell apoptosis in the intestinal mucosa occurs within the first days after initiation of blocking therapy [98]. Thus, one key effect of anti-Tnf therapy is the disappearance of inflammatory cells through activation of apoptosis from the previously inflamed mucosa [99]. While this effect is beneficial for the immune cell compartment of the gut, initiation of apoptosis in the intestinal epithelium would support inflammation, since breakdown of the intestinal barrier is one major factor in


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Fig. 1. Model summarizing the signalling pathway to trigger different forms of cell death. (1) Extrinsic apoptosis occurs when death receptor ligands, like Tnf, activate their receptors and if at the same time Nf␬B signalling is impaired, like seen in NEMOIEC and IKK1/2IEC mice. This results in an affected expression of anti-apoptotic genes, which cannot longer inhibit caspase-8 activation. This drives excessive extrinsic stimulated caspase-8 activation, resulting in intestinal inflammation. (2) Extrinsic apoptosis is also observed in mice with an epithelial cell specific deficiency for the caspase-8 regulator cFLIP (cFLIPIEC ). This deletion leads to extrinsic apoptosis due to uncontrolled caspase-8 activation, which further activates downstream caspases. This increased apoptosis frequency promotes the inflammation. (3) A heterodimer of caspase-8 and its cellular inhibitor cFLIP mediates epithelial cell survival and intestinal homeostasis. The partially activated caspase-8 in this heterodimer is not sufficient to induce apoptosis, but is active enough to cleave RIP kinases. Thus apoptosis and necroptosis are blocked. Moreover Tnf receptor activation results in the expression of anti-apoptotic genes, like cFLIP, which further promotes cell survival. (4) Deficiency of FADD or caspase-8 in epithelial cells (Casp8IEC or FADDIEC mice) drives RIP1/RIP3 assembly and phosphorylation, resulting in their activation. RIP3 further activates MLKL and PGAM5, which promotes necroptosis and small intestinal inflammation.

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the development of inflammatory bowel diseases [100–102]. Thus an additional effect of anti-Tnf therapy could be the inhibition of epithelial cell death by trapping Tnf-␣, which would support the restitution of the intestinal barrier. Altogether these data demonstrate that the development of neutralizing antibodies against Tnf has been a crucial milestone for IBD therapy. Noteworthy, a subgroup of patients does not respond to anti-Tnf therapy [94,97]. The potential reason for the failure of anti-Tnf antibody therapy in these patients in still not known. Thus, it is of interest to understand the exact mechanism of action of anti-Tnf antibodies in IBD to identify reasons for therapy failure. 5. Triggering factors for apoptosis and necroptosis in the intestinal epithelium

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As mentioned above, apoptosis does not seem to be required for gut development and epithelial turnover in the steady state gut. However, under pathophysiological conditions, inflammatory stimuli might dysregulate cell death causing excessive epithelial turnover. Among the triggering factors of cell death in the gut, death receptor ligands, cytokines and pattern recognition receptors have been shown to regulate epithelial homeostasis.

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5.1. Death receptor mediated epithelial cell death

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Death receptors, including Tnf-R and FAS have been demonstrated on the surface of the intestinal epithelium. When Tnf-␣

is administered to wildtype mice, intestinal epithelial cells at the villous tip become positive for activated caspases and cellular turnover is largely increased [103–105]. Caspase activation was shown to be functionally important for Tnf-␣ induced epithelial turnover, since perfusion of Tnf-treated mice with a caspase inhibitor blocked more than 90% of shedding events [104]. A major downstream component of TNFR signalling is NF-␬B. Interestingly administration of TNF-␣ to NF-␬B1−/− mice has been reported to result in high sensitivity of these mice towards epithelial cell shedding [103]. The latter mice exhibited a significant reduction in villus height due to increased IEC apoptosis when compared to TNF-␣ treated wildtype animals. The increased sensitivity of NF-␬B1−/− mice to respond to Tnf-␣ is not surprising since TnfR1, besides initiating apoptosis, also triggers the expression of anti-apoptotic genes via the canonical NF-␬B1 pathway [106]. Thus, if the canonical NF-␬B1 pathway is blocked, the expression of prosurvival genes is inhibited, resulting in activation of the cell death machinery after Tnf-R1 ligation. This is supported by data investigating mice with an epithelial cell specific deletion of Nemo (NemoIEC mice), another component of the canonical NF␬B pathway [31]. NemoIEC mice showed increased epithelial cell death after injection of Tnf-␣, demonstrating that NEMO-deficient intestinal epithelial cells are more sensitive to Tnf-induced killing in vivo than wildtype cells. Interestingly this increased sensitivity is not reported for NF-␬B2−/− mice, which signal via an alternative NF-␬B pathway [107]. These mice were resistant to Tnf-induced shedding and villus apoptosis. The literature suggests two potential models that may explain the protection of NF-␬B2−/− mice from Tnf-␣ induced cell death. First it has been mentioned that the NF␬B2


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precursor acts as an inhibitor of NF-␬B activity [108,109]. Thus, deficiency of this inhibitor results in an increased expression of NF-␬B1 induced anti-apoptotic genes. Alternatively, NF-␬B2 may directly inhibit the expression of anti-apoptotic genes or promotes transcription of proapoptotic genes. Collectively these data suggest that Tnf-induced turnover is suppressed by NF-␬B1 but promoted by NF-␬B2. In summary previous studies indicate that villus epithelial cell elimination under inflammatory conditions can be triggered by the proinflammatory cytokine Tnf and that this form of cell loss is mediated via caspase dependent cell death, identifying it as extrinsic apoptosis. Interestingly, administration of Tnf-␣ to mice with a conditional deletion of caspase-8 in the intestinal epithelium results in high lethality, due to massive epithelial cell death [23]. In case of Tnf-␣ this type of cell death has been identified as Rip-mediated necroptosis [23]. Cell loss, which is mediated via caspase-8, has been referred to as “inflammatory shedding” and was thought to be an unfavourable way to die for epithelial cells. However the recent studies have demonstrated an essential role for caspase-8 in maintaining the gut barrier in response to Tnf-␣ by permitting inflammatory cell loss and preventing necroptosis [23]. Although caspase-8 mediated cell loss interrupts the intestinal epithelium at some locations, the barrier function is largely preserved [23]. In sharp contrast, Rip-mediated epithelial necroptosis results in a complete breakdown of the intestinal barrier and a systemic spread of the intestinal microbiota that finally causes a systemic toxic shock [23]. In summary caspase-8 activation in the intestinal epithelium seems to be required to control Tnf-␣ induced cell elimination. This is supported by the constitutive expression of caspase-8 in intestinal epithelial cells [33]. However caspase-8 activation appears to be tightly controlled. This has been identified in mice with a conditional deletion of the cellular caspase-8 inhibitor cFLIP in intestinal epithelial cells [33]. These mice are embryonically lethal, demonstrating that inhibition of caspase-8 activation in the intestinal epithelium is required for embryonic development. Notably, cFLIP deletion in the intestinal epithelium of adult mice results in lethality within a few days [33]. Death was mediated by severe gut tissue destruction, ligand induced epithelial cell death and intestinal inflammation. Although excessive epithelial cell death of caspase8 deficient or sufficient cells can be triggered by the same ligands (e.g. Tnf-␣), the death mediating pathways are different [23,33]. While caspase-8 deficient cells undergo RIP-mediated necroptosis, cell death in a cFLIP deficient epithelium occurs independently of RIP3 and is instead associated with a strong upregulation of caspase-8 and caspase-3, suggesting that this cell death is triggered by extrinsic apoptosis. In striking contrast to the lethality of the mice in vivo, deletion of cFLIP or caspase-8 in epithelial cells of gut organoid cultures in vitro did not result in intestinal epithelial cell death. These results indicate that cell death triggering factors are present in the steady-state gut and that death of cFLIP or caspase-8 deficient epithelial cells in vivo is regulated extrinsically, requiring the presence of death receptor ligands, such as Tnf-␣. The capacity of this cytokine to induce cell death identifies it as a potential triggering factor for the induction of apoptosis or necroptosis in mice and humans. However, Tnf-␣ alone does not seem to be the main trigger for caspase-8 dependent or independent cell death, since neither general deletion of the Tnf-R1 signalling pathway in Casp8IEC or FADDIEC mice, nor depletion of Tnf-␣ in cFLIP deficient mice is sufficient to inhibit cell death as well as inflammation and Paneth cell necroptosis [23,33,47]. These data suggest that caspase-8 in vivo is either activated by a Tnf-R1 independent pathway or that redundant receptor signals are constantly present in the steady state gut. This is further supported by the fact that cFLIP deficient mice already died in utero independently of inflammation [33].

5.2. PAMP mediated cell death Caspase-8 mediated cell death might also be influenced and regulated by the intestinal microbiota, since epithelial cell elimination can be induced by viral and microbial products which bind to Pattern recognition receptors (PRRs) [103,105]. PRRs, such as Toll like receptors (TLRs) and nucleotide-binding oligomerization domain protein receptors (NOD), provide a critical function in facilitating extracellular and intracellular microbial recognition, including commensal and pathogenic bacteria or viruses. Thus they play a key role in the induction of pro/anti-inflammatory gene expression and the control of adaptive immune responses. Under steady state conditions, IECs express low levels of TLRs, which may contribute to the tolerance of bacteria in the gut, since bacteria represent a major part of the mucosal immune system [110,111]. In contrast to the induction of immune tolerance by TLRs on IECs, stimulation of TLR ligands can also induce a transient increase in epithelial cell turnover, a mechanism that promotes the elimination of intracellular pathogens by pushing damaged or infected cells into the gut lumen [103,105]. Although activation of the extrinsic apoptosis pathway has been described in response to microbial stimulation, its functional involvement in TLR mediated cell elimination remains still unclear. 5.3. TLR4 Toll like receptor 4 is one of the best characterized PRR. It mainly recognizes lipopolysaccharides (LPS) which are present in the outer membrane of gram-negative bacteria. Previous studies have shown, that in the normal intestine, TLR4 is only little expressed on IECs and is preferentially present at the apical side of differentiated enterocytes [112–117]. Besides IECs, TLR4 is expressed on lamina propria mononuclear cells where it has a well-characterized cytoprotective role as TLR4 ligation results in NF-␬B activation and cytokine release [114]. The inflammatory response includes the transcription of several proinflammatory cytokines, chemokines, type I interferons and immune response genes. In contrast to this predominant beneficial effect in pathogen recognition and clearance, emerging evidence suggests that TLR4 signalling in the setting of inflammation may have deviating consequences for the epithelium. It is believed that under inflammatory conditions, the presence of different factors, e.g. hypoxia and high concentrations of endotoxins, results in the upregulation of TLR4 expression on IECs which may alter the extent and nature of TLR4 signalling in the intestinal epithelium. This is supported by an expression analysis which demonstrated that TLR4 mRNA is strongly upregulated in IECs of patients with IBD [112,117–119]. It can be assumed that such altered TLR4 expression promotes a greater sensitivity of these patients to the intestinal microflora, which may induce cell death. This hypothesis is also supported by mouse studies. For instance it has been shown that TLR4 ligation on IECs under inflammatory conditions (e.g. necrotizing enterocolitis; NEC) led to increased enterocyte apoptosis and inhibition of enterocyte migration and proliferation, suggesting a potential role for TLR4 signalling in promoting barrier defects of the intestinal epithelium [120–122]. This conclusion is supported by studies demonstrating that TLR4 signalling in enterocytes results in epithelial cell death, while deficiency of TLR4 in the newborn intestinal epithelium prevented experimental NEC development and attenuated enterocyte apoptosis [120]. Although TLR4 signalling is believed to activate the extrinsic apoptosis pathway, the functional involvement of caspases, as central cell death mediators, has not been clearly documented for LPS induced intestinal cell elimination. Recently, Williams and coworkers have focused their studies on LPS induced TLR4 mediated intestinal epithelial cell death [103]. The authors investigated IEC turnover after systemic LPS administration in


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wildtype animals and could demonstrate that TLR4 ligation causes rapid epithelial cell elimination, resulting in significant villus shortening, fluid exudation into the gut lumen and diarrhoea. TLR4 mediated tissue injury in the gut was independent of the mode of LPS delivery, since either intraperitoneal, intravenous or subcutaneous administration resulted in inflammatory cell loss with a peak at least after 90 min. Interestingly, all parts of the small intestine responded in a similar manner to LPS and in all cases cell elimination was restricted to the upper part of the villus epithelium. In contrast to these findings, LPS induced injury could not be observed in the stomach or colon. The reason behind the specific sensitivity of the small intestinal epithelium to respond to LPS is not entirely understood. It can be assumed that the absence of tissue injury in the colon after LPS administration is based on the fact that the colon is constantly exposed to a higher concentration of bacterial products, resulting in a diminished sensitivity of this cell layer to respond to these triggers. A major downstream component that is expressed upon TLR4 ligation is Tnf-␣, a well-known trigger for intestinal epithelial cell death. Notably, Tnf-␣ mRNA level and plasma concentration were enriched after LPS administration, suggesting a potential contribution of this cytokine in LPS mediated gut damage [103]. Indeed Tnf-R1 deficient mice were protected from LPS-induced cell elimination, suggesting that Tnf-␣ is required to drive LPS-induced apoptosis in the intestinal epithelium via the Tnf-R1 signalling pathway [103]. In contrast to the dependency of TLR4 signalling on Tnf in IECs, one recent publication has shown that the bacterial product LPS is able to activate necroptosis in FADD-deficient dendritic cells in gut-associated lymphoid tissues directly via the TLR-MyD88 pathway independent of Tnf [123]. Similar to LPS induced cell death of dendritic cells, TLR4 mediated apoptosis of macrophages is also independent of Tnf-R1 signalling [124]. A major regulator, acting downstream of TLR4 and Tnf-R1 signalling is the transcription factor NF-␬B. Indeed, it has been shown that LPS-induced intestinal injury is dependent on NF-␬B2, whereas NF-␬B1 might be necessary to suppress IEC apoptosis [107]. This is further supported by a study in which it has been shown that NF-␬B inhibition renders macrophages susceptible to LPS induced, caspase-8 mediated cell death [124]. The additional inhibition of caspase-8 in these macrophages further promotes LPS triggered necroptosis, directly mediated by the TLR-MyD88 pathway. These observations demonstrate that after TLR4 ligation, NF-␬B is critical to control the life or death decision in macrophages, whereas caspase-8 determines the form of cell death. By using mice with an IEC specific deletion of the TLR signal transducer Myd88 it has been shown that TLR4 signalling peripheral to the IECs was required for LPS induced cell elimination [103]. Therefore, initial recognition of LPS, associated with cytokine secretion (e.g. Tnf-␣) occurs via TLR ligation in lamina propria cells rather than in epithelial cells themselves. As it is known that IECs are constantly exposed to TLR ligands, it would be perilous if intestinal microbes could directly trigger epithelial cell loss via TLR4 on epithelial cells. At least in vitro, TLR4 is found to reside in the Golgi apparatus, rather than at the cell membrane of IECs [125]. Therefore LPS signalling in IECs depends on cellular internalization and cytoplasmic traffic of LPS to the Golgi apparatus. In summary, this indirect effect of LPS may represent a host protective mechanism during steady state conditions. Under inflammatory conditions, an increased intestinal permeability results in an enhanced paracellular flux of intestinal pathogens. This may result in the activation of innate immune cells via TLR4, promoting the secretion of cytokines like Tnf-␣, which subsequently induces caspase-3 dependent cell death via Tnf-R1 on epithelial cells [103]. Additionally, our own yet unpublished data demonstrate that LPS induced cell death is mediated via caspase-8, identifying this form of elimination as caspase-dependent apoptosis (Günther et al., unpublished). Caspase activation in the intestinal

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epithelium could be detected already 60 min after LPS administration, whereas cell loss peaked after 90 min [103] (Günther et al., unpublished). These data clearly point to an involvement of the extrinsic apoptosis pathway in an early step of LPS induced epithelial cell loss. However, it has not yet been formally proven if caspase-8 is required to mediate LPS induced epithelial turnover. Moreover the role of LPS in RIP mediated intestinal epithelial necroptosis is still not investigated. Since it has been demonstrated that deficiency of caspase-8 in the intestinal epithelium causes Rip-mediated necroptosis after treating the mice with Tnf-␣, as described above, it is tempting to assume that necroptosis can also be triggered by LPS indirect through Tnf-␣[23]. Although deletion of Tnf-R signalling in Casp8IEC or FADDIEC mice could not inhibit necroptosis, it is not surprising that neither genetic deletion of the TLR4 adapter MyD88 nor antibiotic treatment could protect FADDIEC mice from necroptosis and spontaneous small intestinal inflammation [33,47]. These data suggest that bacterial pathogens via Tnf-␣ secretion are sufficient, but not required for the induction of epithelial necroptosis, since other pathways are also capable to activate this form of cell death (Fig. 2). 5.4. TLR3 Besides microbial pathogens, the intestinal epithelium has the capacity to detect and respond to viral products, like double stranded RNA (dsRNA) via TLR3. TLR3 is expressed at low levels in the epithelium of suckling mice but the expression strongly increases during the postnatal development [126]. A protective role for TLR3 signalling in the pathogenesis of intestinal inflammation has been suggested, as administration of the TLR3 ligand poly(I:C), to mimic dsRNA, prior to the induction of experimental colitis, protected animals against the severity of the disease [127]. Poly(I:C) has a well-known ability to activate type I IFNs. Furthermore it has been demonstrated that type III IFNs are the predominant IFNs produced by the airway epithelium after TLR3 activation by poly(I:C) [128,129], also suggesting a potential expression by the intestinal epithelium. Additionally a very recent study demonstrated that type III IFNs are strongly upregulated after experimentally induced colitis and that this upregulation fosters intestinal regeneration by promoting intestinal epithelial regeneration via the IFN-Stat1 pathway (Dornhoff et al., JEM accepted for publication). Thus it can be assumed that poly(I:C) protects against experimental colitis via its ability to activate IFN signalling pathways to further promote tissue repair. While these studies suggest that TLR3 ligation may have a therapeutic value for inflammatory diseases, it has also been shown that administration of poly(I:C) results in a dramatic but reversible remodelling of the small intestinal mucosa with significant villus shortening [105]. This shortening was mediated via increased cell loss which was again restricted to the upper part of the villus. In contrast to the dependency of TLR4 mediated cell loss on Tnf-␣, dsRNA induced cell elimination was independent of Tnf-R signalling and other cytokines like IL-6 and IL-1 [105]. As mentioned above, TLR3 ligation results in the secretion of type I IFNs, suggesting that these cytokines might support cell loss via the IRF3-BAX dependent pathway. However poly(I:C) induced cell elimination was independent of the type I IFN and IRF3 signalling pathway [105]. The fact that reconstitution of TLR3 deficient mice with wildtype bone marrow did not induce poly(I:C) mediated cell loss indicated, that TLR3 signalling in cells of non-hematopoietic origin is required for IEC shedding [105]. On a molecular level, it has been shown that recognition of dsRNA by TLR3 triggers the recruitment of the TRIF-adaptor (Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-␤), which interacts with RIP1 via the RHIM domain. RIP1 is further known to interact with caspase-8, suggesting the possible activation of caspase-8 and downstream caspases via the direct


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Fig. 2. Model summarizing the potential mechanism by which LPS or poly(I:C) induce necroptosis in the villus epithelium. LEFT: bacterial components: LPS is first recognized by mononuclear cells expressing TLR4, resulting in the production of Tnf. Tnf activates the TNFR1 on epithelial cells which leads to the phosphorylation of RIP1 and RIP3, if the caspase-8 activity is impaired. This further drives necroptosis. RIGHT: viral components: poly(I:C) is directly able to induce necroptosis in the caspase-8 deficient intestinal epithelium, by binding to TLR3 on epithelial cells, which promotes the assembly and phosphorylation of RIP1 and RIP3 via the TRIF adapter. This mechanism seems to be independent of mononuclear cells of the lamina propria.

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TLR3-TRIF pathway [61,130,131]. Indeed, shed IECs are positive for activated caspase-3 after dsRNA administration [105]. Interestingly as already described for LPS, activation of caspases occurred before villus shortening became apparent, suggesting that caspase activation represents an early event during TLR3 mediated cell elimination. However deletion of caspase-8 from the epithelium does not protect epithelial cells against dsRNA induced cell death, but instead causes tissue damage comparable to administration of Tnf-␣ to Casp8IEC mice [23,105]. While poly(I:C) treated wildtype mice quickly recover tissue injury, Casp8IEC mice could not compensate cell death, resulting in death of the animals within a few hours. It is tempting to speculate that poly(I:C) induces epithelial necroptosis in caspase-8 deficient mice. At least it has been shown in vitro that poly(I:C) stimulation results in the recruitment of an intracellular complex containing all necessary components to mediate either apoptosis or necroptosis via the TRIF adapter [132] (Fig. 2).

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As discussed above, IFN secretion is a well-known consequence of TLR3 activation. While it has been shown that TLR3 induced cell death does not require, at least type I IFN signalling, it has recently been demonstrated that these IFNs can induce necroptosis in macrophages during infection with Salmonella typhimurium [133]. In this study the authors showed that infection of wildtype mice with S. typhimurium leads to a downregulation of caspase8 in macrophages, resulting in an enhanced susceptibility of these macrophages towards cell death. This fact could not be observed for macrophages isolated from infected Ifnar1−/− mice, which show an impaired type I IFN signalling, suggesting that S. typhimurium

induces the production of type I IFNs, which then promote cell death [133]. To further prove if this cell death was RIP dependent, the authors also infected RIP3 deficient mice with S. typhimurium and observed that deficiency of this kinase could block the death of macrophages. In this case however it seems likely that necroptosis of macrophages represents a host protective mechanism that promotes survival. Since the late stage during infection, S. typhimurium is confined mainly to the intracellular compartment, an elimination of macrophages by necroptosis, may be beneficial to reduce the number of pathogens. Delivery of S. typhimurium to the extracellular space also facilitates the detection of the pathogens by the immune system, leading to an inflammatory response. In addition to the studies regarding type I IFNs, it has recently been shown that IFN-␥, a type II IFN which is strongly implicated in the pathogenesis of IBD, induces necroptosis in vitro in a human colon cancer cell line HT29 [134]. Exposure of these cells to IFN␥, after incubation with a caspase inhibitor, leads to increased cell death. This cell death could be successfully blocked by coincubation with necrostatin-1 (nec-1), an inhibitor of RIP1 kinases, identifying this form of cell death as necroptosis. Interestingly this study also demonstrated that HT29 cells, while undergoing necroptosis, showed increased mRNA levels of the proinflammatory cytokines Il-8 and IL-1␤ in comparison to control cells or cells additionally treated with the necroptosis inhibitor nec-1 [53]. These data suggest that necroptosis may actively contribute to mucosal inflammation, by the release of proinflammatory cytokines, since increased expression of IL-8 was also observed in inflamed biopsy specimens from human CD patients [53]. It has been demonstrated in vitro, that both type I and type II IFNs transcriptionally activate the RNA-responsive protein kinase PKR which is able to interact with RIP1 and RIP3, resulting in the


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formation of the so called “PKR necrosome” [135]. This further induces the phosphorylation of RIP kinases to finally execute necroptosis. Since IFNs are typical cytokines secreted after viral infection, these data may support a model in which viral pathogens promote necroptosis. Additional studies are necessary to clarify, if IFN signalling contributes to Paneth cell necroptosis or small intestinal inflammation, as observed in Casp8IEC mice or FADDIEC mice. References [1] O’Hara AM, Shanahn F. The gut flora as a forgotten organ. EMBO Rep 2006;7:688–93. [2] Kamada N, Seo SU, Chen GY, Nunez G. Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol 2013;13:321–35. [3] van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 2009;71:241–60. [4] Specian RD, Oliver MG. Functional biology of intestinal goblet cells. Am J Physiol 1991;260:C183–93. [5] Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol 2011;9:356–68. [6] Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 2006;7:349–59. [7] Gunther C, Neumann H, Neurath MF, Becker C. Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium. Gut 2012. [8] Mallat Z, Tedgui A. Apoptosis in the vasculature: mechanisms and functional importance. Br J Pharmacol 2000;130:947–62. [9] Bullen TF, Forrest S, Campbell F, Dodson AR, Hershman MJ, Pritchard DM, et al. Characterization of epithelial cell shedding from human small intestine. Lab Invest 2006;86:1052–63. [10] Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011;474:298–306. [11] Edelblum KL, Yan F, Yamaoka T, Polk DB. Regulation of apoptosis during homeostasis and disease in the intestinal epithelium. Inflamm Bowel Dis 2006;12:413–24. [12] Mehlen P, Puisieux A. Metastasis: a question of life or death. Nat Rev Cancer 2006;6:449–58. [13] Meddings J. The significance of the gut barrier in disease. Gut 2008;57:438–40. [14] Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010;140:883–99. [15] Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 2010;11:700–14. [16] Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 2008;9:231–41. [17] Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell 2011;147:742–58. [18] Watson AJ, Duckworth CA, Guan Y, Montrose MH. Mechanisms of epithelial cell shedding in the Mammalian intestine and maintenance of barrier function. Ann N Y Acad Sci 2009;1165:135–42. [19] Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Genes Dev 2010;24:2592–602. [20] Becker C, Watson AJ, Neurath MF. Complex roles of caspases in the pathogenesis of inflammatory bowel disease. Gastroenterology 2013;144:283–93. [21] Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990;110:1001–20. [22] Eisenhoffer GT, Loftus PD, Yoshigi M, Otsuna H, Chien CB, Morcos PA, et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 2012;484:546–9. [23] Gunther C, Martini E, Wittkopf N, Amann K, Weigmann B, Neumann H, et al. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature 2011;477:335–9. [24] Duprez L, Takahashi N, Van Hauwermeiren F, Vandendriessche B, Goossens V, Vanden Berghe T, et al. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity 2011;35:908–18. [25] Brinkman BM, Hildebrand F, Kubica M, Goosens D, Del Favero J, Declercq W, et al. Caspase deficiency alters the murine gut microbiome. Cell Death Dis 2011;2:e220. [26] Colussi PA, Kumar S. Targeted disruption of caspase genes in mice: what they tell us about the functions of individual caspases in apoptosis. Immunol Cell Biol 1999;77:58–63. [27] Watson AJ, Pritchard DM. Lessons from genetically engineered animal models. VII. Apoptosis in intestinal epithelium: lessons from transgenic and knockout mice. Am J Physiol Gastrointest Liver Physiol 2000;278:G1–5. [28] Nakayama K, Negishi I, Kuida K, Sawa H, Loh DY. Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci USA 1994;91:3700–4. [29] Nakayama K, Negishi I, Kuida K, Shinkai Y, Louie MC, Fields LE, et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993;261:1584–8. [30] Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Baxdeficient mice with lymphoid hyperplasia and male germ cell death. Science 1995;270:96–9.

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Obesity and the gut microbiome: pathophysiological aspects.

Physiological and pathophysiological signalling between the gut and the kidney: role in diabetic kidney disease.

On the role of actomyosin ATPases in regulation of ATP turnover rates during intense exercise.

A novel role for p115RhoGEF in regulation of epithelial plasticity.

Role of AE2 for pHi regulation in biliary epithelial cells.

Paracrine Regulation of Adrenal Steroid Secretion in Physiological and Pathophysiological Conditions.

Th17 Cells in Type 1 Diabetes: Role in the Pathogenesis and Regulation by Gut Microbiome.

Microbiota-host interactions in irritable bowel syndrome: epithelial barrier, immune regulation and brain-gut interactions.

The role of the adaptive immune system in regulation of gut microbiota.

The regulation of muscle-protein turnover in growth and development.

The Regulation of Synaptic Protein Turnover.

Pathophysiological role of host microbiota in the development of obesity.

The pathophysiological role of bacterial biofilms in chronic sinusitis.

The role of transcriptional factor p63 in regulation of epithelial barrier and ciliogenesis of human nasal epithelial cells.

The pathophysiological role of dendritic cell subsets in psoriasis.

Roles and regulation of the mucus barrier in the gut.

Pathophysiological Role of Neuroinflammation in Neurodegenerative Diseases and Psychiatric Disorders.

The Role of MicroRNAs in the Regulation of K(+) Channels in Epithelial Tissue.

Mucosal cell turnover in the upper gut of the mouse and its modification by carbenoxolone [proceedings].

Regulation of bradykinin-induced phosphoinositide turnover in cultured cerebellar astrocytes: possible role of protein kinase C.

Promotion of Intestinal Epithelial Cell Turnover by Commensal Bacteria: Role of Short-Chain Fatty Acids.

Role of protein kinase C in the regulation of histamine and bradykinin stimulated inositol polyphosphate turnover in adrenal chromaffin cells.

FtsZ2 Turnover in Chloroplasts and the Role of ARC3.

Role of galanin in the gut.