Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection

ABSTRACT: BACKGROUND: Gut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, Erwinia carotovora carotovora 15, induces a massive increase in stem cell proliferation in the gut of Drosophila. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal. RESULTS: To understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult Drosophila undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection. CONCLUSIONS: This study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

Hes1 as a Mediator of Notch Signalling in T-cell Development and Notch1- Induced T-ALL

Agnieszka Anna Wendorff

The evolutionarily conserved Notch signalling pathway controls a broad spectrum of cell fate decisions in organisms as diverse as fruit flies and mice. Whithin the murine haematopoietic system, Notch1 is indispensable for T cell development as conditional inactivation of Notch1 in bone marrow (BM) precursors results in a complete block in T-cell development and ectopic B-cell development in the thymus. Conversely, constitutive activation of Notch1 signalling plays a causative role in the development of acute T-cell lymphoblastic leukaemia (T-ALL). Numerous mutations within the Notch1 gene, which result in aberrent activation of Notch1 signalling, have been identified in > 50% of human T-ALL patients. Therefore, gain-of-function studies in murine models of T-ALL have been employed by many laboratories to elucidate the molecular mechanisms acting either cooperatively or downstream of oncogenic Notch1 signalling in development and progression of T-ALL. The best-characterized Notch1 target gene to date is a member of the bHLH family of transcriptional repressors, Hes1. This study focuses on the role of Hes1 as a mediator of Notch1 signalling in adult murine haematopoietic development, as well as the requirement for Hes1 in T-ALL development and maintenance. Conditional inactivation of Hes1 in adult murine BM cells results in severe thymic hypoplasia, while myeloid and B cell development remain unperturbed. Upon transplantation in congenic recipients, Hes1-/- progenitors give rise to only a small number of mature T cells. Hes1-inactivation does not however cause ectopic B-cell development in the thymus, and therefore does not phenocopy the loss of Notch1. Thus, while Notch1 signalling is indispensable for T versus B-cell fate specification, this function is not mediated exclusively via Hes1. Strikingly, in a competitive BM chimera setting, Hes1-/- progenitors completely fail to generate T cells. The mechanism by which Hes1 inactivation results in depleted T cell numbers remains to be elucidated, although we have shown that loss of Hes1 does not result in enhanced cell death. In addition, preliminary data are indicative of cell-cycle perturbation in Hes1-deficient immature T cells. Finally, we have demonstrated that upon IT transplantation, Hes1-deficient progenitors successfully give rise to T-cells at all stages of development. This data strongly indicates that Hes1 deficiency does not result in an intrinsic developmental defect but rather in the inability of T cell progenitors to efficiently home to the thymus from the BM. Upregulation of Hes1 expression has been observed in both human T-ALL cell lines as well as murine primary Notch1-induced T-ALL samples. However, the function of Hes1 as an effector of oncogenic Notch1 signalling in modulating disease onset or progression remained to be investigated. The requirement for Hes1 as a mediator of Notch1-induced T-ALL was addressed by inducing simultaneous deletion of Hes1 and expression of a dominant active form of Notch1 (NICD) in a murine model of T-ALL. Our data demonstrate that Hes1 is essential for the development of NICD-induced T-ALL. Loss of Hes1 prevents ectopic development of DP (CD4+CD8+) leukemic T cells in the BM and the subsequent accumulation of DP T cells in peripheral tissues, a hallmark of T-ALL, and consequently results in 10-fold increased life expectancy of experimental animals. We have further determined that Hes1 is required for the maintenance of T-ALL by inactivating Hes1 in a retrovirally pre-induced T-ALL model. Deletion of Hes1 in leukemic DP cells results in rapid cell death and enhanced survival. These data demonstrate that Hes1 is required as a mediator of Notch1 signalling in normal T cell development, and is indispensable to mediate the oncogenic effect of constitutive Notch1 activation in the induction of T-ALL.

EPFL2010

Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila

Nichole Broderick, Sveta Chakrabarti, Bruno Lemaitre

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.