Statistical Analysis of Chao’s Immune System Simulation

Modeling the immune system (IS) means putting together a set of assumptions about its components (cells and organs) and their interactions. Simulations of a model show joint behavior of the components, which for complex realistic models is often impossible to find analytically. Simulations allow us to experiment on how initial concentrations and properties of the immune cells and viruses impact the IS behavior, and gain better quantitative and qualitative insight into how the IS works and why different behavior patterns occur. A simulation, once it has been created, must be reviewed both statistically and analytically as well as validated from the biological point of view. We analyzed Chao’s immune system simulation [1][2] from a statistical and analytical point. We explicited both the Markov chain which was simulated and the underlying process on which Chao’s stage-structured approach was built. Furthermore, we established a test protocol for timestep validation which Chao’s simulator passed. We evaluated Chao’s simulator’s dependence on the random number generator, which was shown to be negligible. Finally, we evaluated the simulator output and our major result is the discovery of a secondary response to a primary infection, an occurrence is not shown in Chao’s dissertation. A tertiary response to the infection is never possible due to the size of the secondary response caused by memory cells.

New immunomodulatory roles of lymphatic endothelium and implications for immunotherapy

Efthymia Vokali

The lymphatic system serves a critical role in fluid homeostasis, lipid metabolism and immune surveillance. The growing appreciation of its implication in various diseases challenges the conventional view of lymphatics as a passive transport system. Traditionally, the lymphatic endothelium has been perceived as a structural scaffold with certain immunological functions but no active involvement in immunomodulation. In the lymph nodes (LNs), the main sites of immune regulation in the periphery, lymphatic endothelial cells (LECs) come to close contact with immune cells, suggesting potential interactions. Indeed, LECs have been recently shown to suppress dendritic cell maturation and present peripheral tissue and tumor antigen for CD8+ T cell deletion. While LECs have only begun to be acknowledged as active regulators of immunity, their function and relative contribution in shaping immune responses is as yet poorly understood. This thesis aimed to elucidate the direct role of LECs in the induction of CD8+ T cell immunity and tolerance. First, we demonstrated that murine LECs can actively scavenge and cross-present exogenous antigen to cognate CD8+ T cells under non-inflamed conditions. By utilizing an in vitro coculture system and the model antigen ovalbumin, we investigated the antigen-specific interactions between LECs and CD8+ T cells. LEC-educated CD8+ T cells proliferated, exhibiting an activated phenotype, however, they displayed early-generation apoptosis and failed to produce effector cytokines. Our findings establish LECs as antigen-presenting cells and suggest that they may assist in the maintenance of peripheral tolerance during homeostasis. The particular differentiation state of LEC-educated CD8+ T cells prompted us to investigate whether they are terminally tolerized or they could escape the dysfunctional state. We demonstrated that LEC-educated CD8+ T cells adopted a distinct phenotype with central memory-like characteristics and shared multiple functional properties with memory cells. Upon antigen re-encounter, LEC-educated CD8+ T cells mounted proliferative responses and generated cytotoxic effector cells. More importantly, they participated in anti-infectious immunity while preserving a secondary-memory persistent population. Our findings reveal a unique differentiation state of antigen-experienced CD8+ T cells, generated under steady-state conditions, which remain inactive but can be functionally reactivated upon antigenic inflammatory challenge. This previously unanticipated feature of LECs triggered questions for their antigen-presenting function in an inflammatory setting. We asked whether the previously observed extensive proliferation of LECs in the LN during inflammation might directly influence the induction of immunity. We employed an anti-VEGFR3 blocking antibody to inhibit LEC proliferation and thus, reduce the number of LECs following vaccine immunization. Alternatively, we generated the Prox1-Cre-DTR mouse model, allowing for specific ablation of LECs following administration of diphtheria toxin in vivo. Our findings advance our perception of the relative contribution of LECs in the establishment of adaptive immunity. This thesis elucidates the multifaceted immunological role of LECs and strongly suggests the importance of harnessing their immunomodulatory function to enhance current vaccines and immunotherapeutic strategies. Looking forward, our work will contribute to future advances in the clinic.

EPFL2016

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

Object-oriented approach for dynamic system modelling and simulation

Adel Besrour

Software engineering always cares to provide solutions for building applications as close as possible to what they should be, according to the requirements and the final users needs. Systems behavior simulation is a very common application to virtually reproduce and often predict the real-world behavior. Simulation is one of the most operational research tool in a large variety of engineering and scientific domains: Transport, telecommunication, medicine, chemical processes, physics, etc. The complexity of such application is relative to the increasing complexity of the systems. In this context, it is relevant to bring together different tools and formalisms such as markovian chain, Petri nets, etc., to improve the existent approaches and so to answer the simulations performances needs. The principle objective of this thesis is to bring together techniques from software engineering and safety engineering in order to improve the state of the art of modeling and simulation of dynamic systems in the industrial context. In addressing this objective, this work initially involves defining the essential limitations of the used formalisms, methods and tools regarding from one hand the software engineering modeling and simulation techniques and from the other hand the existent risk analysis methodologies. This work is conducted with respect to the problem of danger identification, considering the context of the complex systems behavior and their interaction with the human operator. In software engineering, it is well known that Petri/high-level nets have attractive characteristics to be used in systems simulation and behavior prediction such as the natural graphical representation, and their well-defined semantic. They are well-suited for the description of complex situations with concurrency (interleaving and true concurrency depending on the underlying semantics), conflict and confusion. However, the absence of structuring capabilities has been one of the main criticisms raised against Petri nets/high-level nets. Thus, there have been many attempts to introduce structuring principles in nets of this kind [BCM88] [Kie89] [JR91]. The attractive characteristics of Petri/high-level nets have prompted researchers to enrich these formalisms with object-oriented features. CO-OPN (Concurrent Object-Oriented Petri Net) approach, brings together the power of both Petri/high-level nets and object-orientation techniques, it has been devised so as to offer an adequate framework for the specification and design of large scale concurrent system [BG91]. CO-OPN, as a powerful modeling tool, has been used in a limited way to simulate systems. This work aims to provide a CO-OPN extension to allow a more realistic systems' simulation. Actually, its simulator semantic uses to be a suitable approach for modeling near closed systems and software components, because they need to loose coupling with external world. But, when we model more realistic problems like industrial processes, where human interaction is a relevant event, this approach is not sufficient to catch all system activity attributes. Moreover, the CO-OPN interpretation process does not allow interaction with the object internal states. This work provides a new solution to overcome CO-OPN simulation limitations and a set of prototypes to assist dynamic systems simulations. Furthermore, this work has been conducted in a Risk Analysis (RA) context, a domain where computer-based simulations research are of utmost interest. Actually, classical approaches used to address complex workplace hazard in a limited way, using checklists or sequence models. Moreover, the use of single oriented methods, such as AEA (man-oriented), FMEA (machine oriented) or HAZOP (process oriented), is not satisfactory to overcome the increasing sophistication of industrial processes. The automation of a part of the analysis process as well as the multiple-oriented approach allowed by dynamic modeling may indeed enhance significantly the analysis completeness and reduce the time analyzing time. This work, based on Object Oriented Petri net formalism (CO-OPN), propose an alternative multi-oriented approach where existent methods limitations have been criticized to develop a dynamic model, MORM (Man-machine Occupational Risk Modeling). A real industrial system (metal wire making process) has been specified to implement the different approach steps (system identification, model application, system simulation, system analysis).

EPFL2005