The policy of our research team is multidisciplinary. It is based on questions resulting from the management of infectious diseases due to intracellular bacteria such as Coxiella burnetii, the agent of Q fever. First, we will analyze the cell tropism of C. burnetii for placental cells such as trophoblasts, macrophages and/or dendritic cells because of the role of pregnancy in C. burnetii persistence and chronic infections. The mechanisms used by C. burnetii to replicate within host cells and/or to inhibit their microbicidal machinery will be studied by cell and molecular approaches (intracellular trafficking of C. burnetii or its factors such as LPS, large-scale transcriptional approach). The natural history of infectious diseases reveals that several endogenous parameters control host response. Indeed, clinical observations indicate that the frequency and the severity of C. burnetii infection differ in men and women. We recently showed that C. burnetii infection induced a transcriptional response largely dependent on gender in mice. This response will be analyzed in humans and the potential role of genes specifically modulated in the sex dimorphism, with a special attention to genes involved in the innate immune response since C. burnetii infects myeloid cells. This study will be extended to infectious diseases known to be gender-dependent (Q fever, endocarditis, sepsis). We will develop original mouse models of infection in collaboration with immunophenomic platform. Hence, we will use humanized mice in which human macrophages are expressed and investigate C. burnetii infection with live imaging methods. Finally, we will use the «Transcriptome» platform that we direct to investigate transcriptional responses of circulating cells from patients with Q fever or other rickettsiosis, especially genes that may be involved in defective protective immune response. Finally, a translational research will be also developed in sepsis and preeclampsia, the fields of clinical investigations of several members of the team.
The project of the team “Infection, Gender and Pregnancy” will be an amplification of the previous researches we developed during the last four years. Our team is known to have international expertise in the field of innate immunity against intracellular bacterial pathogens, more precisely in infectious diseases in which macrophages play an essential role. As example, the intramacrophage survival of Coxiella burnetii is a key of the pathophysiology of Q fever. However, it is doubtful that monocytes/macrophages are the sole natural cells in which resides C. burnetii. Indeed, Q fever may become chronic in some patients: the cell territory in which resides C. burnetii during the latent phase of the disease is unknown. It is well known that placenta is an organ very rich in bacteria in animals suffering from C. burnetii infection but the cells which are targeted by C. burnetii are unknown. Our first aim was to identify placental cell types which are targeted by C. burnetii and to analyze the pathways used by C. burnetii to infect and replicate within these cells. On the other hand, the understanding of human infectious diseases cannot be reduced to in vitro models of cell host-pathogen interaction. For instance, epidemiologic observations show that the ability to mount a protective response to C. burnetii differs between men and women. Convenient animal models of C. burnetii infection may be helpful to dissect the molecular mechanisms possibly involved in Q fever. This aspect of our project will be reinforced by the analysis of immune deficiency in Q fever using a large-scale approach such as microarrays and transcriptomic platform. This translational research will be developed in other fields of infectious diseases and will be opened to collaborative projects.
In the next project, Jean-Louis Mege remains director of the team and Eric Ghigo will be co-director. This co-direction is made to prepare him to be the only director in four years. The size of the team is increasing. The arrival of Christophe Buffat, an Assistant-Professor in Biochemistry, who developed research in placenta biology and that of Andreas Stein, A Professor in Infectious Diseases compensated the movement of B. Desnues toward the Center of Immunology Marseille-Luminy. They will clearly reinforce our projects on patients. Also, the arrival of an Assistant-Ingenior will reinforce the project 3. During the present contract, M. Leone was promoted full Professor (Anesthesiology and Intensive Care). This promotion will irrigate our projects in terms of patient cohorts and medical residents. The project of IHU (Institut Hospitalo-Universitaire) will create favorable conditions to develop a pathophysiological axis with a strong visibility. This will consist of the collaboration of our team with different external structures including the Immunophenomic Center (B. Malissen), the Immunomonitoring Platform (D. Olive) and CIML (J.P. Gorvel). This axis is supported by several common publications and grant proposals. In addition, our team has developed two platforms: one is a transcriptomic platform based on Agilent technologies, the other is a cell biology platform equipped with biphotonic confocal microscope, laser capture microdissection, flow cytometer and cell sorting.
This project will be directed by Jean-Louis Mege, Florence Bretelle and Eric Ghigo. It will be subdivided in two parts.
The spreading of infectious diseases and bacterial persistence in chronic infectious diseases are a major problem of public health. Q fever, a zoonosis due to the intracellular bacterium C. burnetii illustrates this question. This disease has obstetrical consequences on pregnancy in humans and animals leading to abortions, low birth rate and infertility. The microorganisms use their ability to infect placenta to spread among humans and animals and to create a favorable environment for their persistence, leading to chronic evolution of the disease. Understanding how microorganisms infect placenta is essential but the main difficulty is linked to the isolation and the characterization of primary trophoblasts and immune cells (lymphocytes, macrophages, NK cells and dendritic cells (DCs)) from human placentas. The studies of interactions between bacterial pathogens and placenta cells are rare and no study has concerned C. burnetii. In addition, we ignored if placenta immune cells are infected by microorganisms and which role they play in defense or pathogenicity. Few teams with expertise in the pathophysiology of Q fever and the cell biology of placenta cells can develop this approach. Our project will have two main focuses. First, we will analyze in normal human pregnancy how C. burnetii is able to infect and persist in placenta cells (trophoblasts and/or macrophages), with a special attention to the mechanisms of uterine/placental immune defenses using isolated T cells, NK cells and DCs. A very similar approach may be used to study the placental infection by other pathogens such as Brucella: a collaborative project (with J. P. Gorvel at the CIML) will be undertaken. Second, we will analyze the mechanisms used by C. burnetii to replicate within host cells and/or circumvent the microbicidal machinery of targeted cells.
1 – 1 - Which types of placenta cells are targeted by C. burnetii?
In the first phase of our project, we will determine which types of placenta cells are able to be infected by C. burnetii. As trophoblasts and some immune cells (macrophages, DCs) are candidates, we will first isolate trophoblasts from human placentas with a method based on Percoll gradients and positive selection. Since the differentiation of trophoblasts would be different during the time of pregnancy, we will also select placentas from each trimester (women are mainly infected at the first trimester). As sensitive tools to characterize trophoblast subsets are lacking, we will develop monoclonal antibodies (mAb) to characterize trophoblast subsets and development stages (collaboration with CIML). In parallel, we will isolate placental immune cells, macrophages, T cells, NK cells and DCs using tissue digestion, Ficoll gradient and positive selection. Trophoblasts and immune cells will be infected in vitro with C. burnetii, and the infection will be measured by microbiological and molecular methods.
The second phase of our project will consist of the characterization of compartments in which C. burnetii survives or replicates. Preliminary results in trophoblast cell lines have shown that C. burnetii inhabits a vacuole that expresses Lamp-1 (a marker of late endosomes and lysosomes) and cathepsin D (a marker of lysosomes). This is clearly distinct from human macrophages in which C. burnetii replicates within a phagosome that expresses Lamp-1 but not cathepsin D, suggesting that the replicative compartment of C. burnetii might be distinct in primary human trophoblasts and placental immune cells.
The third phase of our project will characterize the responses of infected cells to C. burnetii using a global approach. In trophoblasts, we will define common and specific genes and their organization. We have already obtained preliminary results in which C. burnetii elicits a transcriptional program of 340 genes in which IL-6, MIF, IkB, TNF and IL-13R pathways are engaged. We will also characterize the transcriptional profile of placental immune cells in response to C. burnetii, and we will compare this profile with that of circulating cells. The last phase will consist to study cell interactions in the context of placental infection. We will establish co-culture of trophoblasts, macrophages and DCs on one hand, and lymphocytes and NK cells on another hand. NK cell and T cell activation will be monitored by flow cytometry (activation antigens and intracellular cytokines). In addition, the analysis of microarrays will provide specific parameters that will be assessed in co-cultures.
The consequences of the project are to describe for the first time the responses of human trophoblasts and immune placental cells to C. burnetii. The project will also document the cross-talk between trophoblasts and cells from placental immune system, which will provide an integrated analysis of placenta infection. In addition, the project will enable the development of new tools to characterize trophoblasts.
1 – 2 - Mechanisms used by C. burnetii to replicate within host cells
This project will be directed by Eric Ghigo (collaborations with B. Henrissat and P. Soubeyran, Marseille, A. Salvetti, Pisa, Italy and R. Toman, Bratislava, Slovaquia.)
The mechanisms which allow C. burnetii to replicate or survive within tissues, cells and organs are poorly understood. As other pathogens, C. burnetii is able to disturb the microbicidal function of host cells, including macrophages and placental cells. Indeed, different pathogens have evolved distinct strategies to control their intracellular fate and enhance their survival within host cells. One of these strategies is to hijack the phagosome maturation and the immune response.
Several bacterial factors have been identified to be involved in the prevention of the phagolysosome formation and the perturbation of the immune response. Thus, for Mycobacteria spp., Lieshmania spp. and Brucella spp., these activities have been attributed to bacterial membrane components, respectively the lipoarabinomannans (LAM), the LPG (lipophosphoglycan) and lipopolysacharide (LPS). In addition, it appears that micro RNA (miRNA) should be involved in the hijacking of microbicidal function of macrophages by the bacteria. We propose to investigate and elucidate the molecular mechanisms of the alteration of phagosome maturation by LPS using the LPS of Coxiella burnetii and the involvement of micro-RNA in the hijacking of microbicidal functions.
1 - 2 - 1 - Role of the LPS in the prevention of the phagolysosome formation
The intracellular localization of C. burnetii has been determined by our team . We are also able to purify the LPS of several C. burnetii strains, and to chemically modified these LPSs. The structure of these LPSs from C. burnetii is known and has been published by a collaborating team [2-4]. C. burnetii replicates within macrophages through the inhibition of the phagosome-lysosome fusion. Our goal is to investigate the effect of Coxiella LPSs on the intracellular trafficking, to identify the cell effectors that are targeted/altered by these LPSs and to understand the LPS structures involved in the survival of C. burnetii. To date, no study clearly describes the relationship between structure/function of LPS in the strategies used by gram negative bacteria to generate an environment suitable for their replication. In this respect, the elucidation of the trafficking of Coxiella LPSs and the identification of LPSs-containing compartments are necessary to better understand the role of LPSs in the intracellular fate of gram-negative bacteria.
To investigate and understand the effect of LPSs on the endocytic pathway, and identify the mechanisms leading the LPSs to modulate the endocytic pathway and generate an environment suitable for the bacterial replication, we have assigned the three following objectives:
- Study of the intracellular localization of LPSs
- Effect of LPSs on the endocytic machinery
- Role of macrophage activation in the intracellular localization of LPSs
1 - 2 - 2 - Role of miRNA in the hijacking of microbical functions of macrophages
It has been recently described that bacteria might produce small non-coding RNAs (sRNA) [5,6]. sRNAs are known as a large class of versatile gene regulators. Post-transcriptional regulation of gene expression by sRNA molecules has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. It is possible that bacteria are able to control the phagosome conversion and immune response using sRNAs. The microbicidal function interference by bacterial small non-coding RNAs might constitute a new strategy for bacteria to control their intracellular fate. In addition, cells are functionaly regulated through a miRNA-dependent mechanism. It is possible that bacterial infections perturb cell miRNA regulation leading to dysfunction of microbicidal activities. To investigate and understand the effect of the C. burnetii small non-coding RNA and cell miRNA in bacterial infection, we have assigned the tree following objectives:
- In silico prediction of C. burnetii small non-coding RNAs and identification of potential targets
- Expression of C. burnetii small non-coding RNA in macrophages and functional consequences on the microbicidal machinery
- Determination of cellular non-coding RNA expression profile during in vitro infection and in patients with Q fever.
This project will be directed by Jean-Louis Mege, Marc Leone and Eric Ghigo. Its objective is to analyze the cell and molecular factors that govern the susceptibility or resistance of hosts to rickettsial diseases. It will be subdivided in two parts.
2 – 1 - C. burnetii infection and gender
Social factors such as gender inequity can explain differences in the distribution of infectious diseases between men and women. As shown elsewhere, poor women may be at a disadvantage in their ability to access quality health care. However, biological differences are also responsible for part of the epidemiological variation observed between males and females in infectious diseases due to intra- and extracellular pathogens. Gender-based biological differences also affect host immune responses to pathogens. Women elicit more vigorous humoral and cell-mediated immune responses than men in response to antigenic challenges, while men have frequently been observed to exhibit more aggressive and harmful inflammatory responses to pathogens. Tuberculosis and Legionnaire’s disease are reported to be more prevalent and severe in men than in women. Although biological differences have been largely attributed to sex hormones, the precise nature of the cross-talk between gender and infections remains largely unknown. Q fever is more frequent and severe in men than in women for a similar exposure.
We recently studied the relationship between gender and infection in a model of intact and castrated mice infected by C. burnetii. We showed that the gene expression modulated by C. burnetii infection was mostly gender-dependent. Moreover, most of the modulation occurred in males, and was widely dependent on sexual hormones as castration abolished 60% of gene modulation . A large-scale study (microarrays on the whole mouse genome) reveals that males develop an early anti-inflammatory response and that the circadian rhythm is perturbed in females . It is the first demonstration that circadian rhythm plays a major role in host response to bacterial pathogens, and provides a new basis for elucidating the role of sexual dimorphism in human infections.
The study will be extended to humans suffering from Q fever. We will investigate the expression of genes involved in circadian cycle. Preliminary results show that some circadian genes are differently expressed in men and women. We will screen a large number of genes known to be gender-dependent and test their functional role in sexual dimorphism. For instance, we have shown that macrophage M1/M2 polarization is critical in host defence against infections . We will analyze the contribution of gender in macrophage M1/M2 polarization. This study will be extended to other infectious diseases known to be gender-dependent and for which we have cohorts of patients and large data of transcriptomics.
2 – 2 - Mouse models of C. burnetii and rickettsial infections
The idea is that we have the means to follow rickettsial infections by targeting some cells or tissues and the opportunity to genetically manipulate mice. We have developed murine models with C. burnetii (acute and chronic infection), Rickettsia prowazekii, the agent of epidemic typhus (initial infection and relapses) and Tropheryma whipplei, the agent of Whipple’s disease (acute infection in normal and injured mice). All these studies are based on static studies and do not target specific tissues.
2 - 2 – 1 - Macrophage infections
We studied the interaction of C. burnetii and T. whipplei with human monocytes and macrophages, their usual targets, for several years. We especially described the natural history of C. burnetii infection including the internalization receptors, the type of activation (M1-type in monocytes and atypical M2-type in macrophages [10,11]), the nature of the replication compartment and innate immune response in patients (IL-10 or IL-16 context) [12,13]. More recently, we have developed an approach to investigate placenta macrophages and their infection with Coxiella (and Brucella) since it is responsible of obstetrical complications and bacterial persistence. This project is supported by a strong collaboration between our team and that of J.P. Gorvel (CIML). We also showed that T. whipplei differently interacts with human monocytes (elimination) and macrophages (replication). We provided a dissection of the molecular events related to bacterial replication in macrophages: M2-type of activation and overexpression of IL-16 [13,14]. Unfortunately, the profiles of mouse and human infections are distinct. Therefore, as mice represent suitable models of infections it will be necessary to develop the humanization of macrophage compartment in mice. This strategy of humanized mice expressing human macrophages will be developed using Rag-2-/-gc-/- deficient mice in the Immunophenomic Center directed by B. Malissen (CIML). A procedure has been recently described that creates human-mouse chimera with CD34+ HSC (hematopoietic stem cells) and immunodeficient mice; a such approach generates macrophages expressing CD14 and CD68 that are able to infiltrate the tissues and to induce lesions . This approach will be pertinent to study the role of human macrophages in infectious diseases due to intracellular pathogens.
2 - 2 – 2 - Dendritic cells and infections
The URMITE and CIML teams usually study the interaction of intracellular bacteria with myeloid DCs. Hence, it has been shown that Brucella abortus interfere with the maturation of DCs , C. burnetii do not prevent DC maturation and T. whipplei is unable to activate DCs (manuscripts in preparation). The program will consist in the building of mice expressing human DCs and to follow the infection in these humanized mice. Besides Brucella and C. burnetii, these mice will offer the opportunity to assess immune response to Rickettsia. Indeed, rickettsioses result from tick bites and likely Rickettsia are transferred to skin DCs and thereafter to lymph nodes. The availability of mice expressing human DCs will allow the study of the natural history of rickettsioses and the recruitment of immunocompetent cells.
2 - 2 - 3 - Live imaging and infection.
To follow the infection process in mice, the platform will create mice with modified genes by KI to express several fluorescent proteins. Such approach will enable to identify a given cell type and to define an activation or infection state. It will offer the opportunity to analyze and sort cells and/or bacteria expressing fluorescent proteins. We will target different tissues: skin and placenta.
2 – 3 - Investigation of patients with rickettsial infections
This sub-project will be directed by Jean-Louis Mege and Christian Capo.
The chronic evolution of infectious diseases due to intracellular pathogens is associated with altered immune responses. Indeed, Q fever is characterized by a chronic evolution and an impairment of cell-mediated responses, which is very likely related to an aberrant overproduction of IL-10 . Recent publications renew the central role of IL-10 inchronic infectious diseases due to virus and bacteria. The Whipple’s disease is another example since several features of impaired microbicidal responses are found probably in susceptible subjects. The policy of the URMITE was the building of patient cohorts that make possible such studies.
2 - 3 - 1 - Analysis of phenotypic markers of circulating cells
This project undertaken with the collaboration with D. Olive will analyze the complexity of leukocyte population interaction using circulating mononuclear cells and multicolor cytometry (12 markers). This technology allows to identify simultaneously different cell populations (pDC, mDC, NK, monocytes and T cells) and to analyze their activation state (activation markers: CD69, CD83 and functional markers: IFNb, IFNg, TNF, IL-10, IL-12, CD107, CD163). This multiparametric approach will also allow the study of different pathogens with clinical relevance on different subpopulations of mononuclear cells. In addition, evidence showed that auxiliary molecules of immune response such as PD1/PDL1 are necessary for the overproduction of IL-10 inthe context of chronic HIV infection. We think that similar mechanisms may be elicited in chronic Q fever and sepsis. We have cohorts of patients with Q fever and with sepsis (Pr M. Leone) in which these two hypotheses may be studied.
2 - 3 – 2 – Analysis of the transcriptional responses of circulating cells and infected tissues
The development of a transcriptomic platform by our team offers new perspectives to study host responses to rickettsial pathogens including C. burnetii [9,10,11], T. whipplei [13,14], R. prowazekii [17,18] and O. tsutsugamushi (submitted manuscript). First, it allows the study of transcriptional responses of host cells to rickettsial infections and the determination of molecular parameters possibly involved in bacterial persistence within host cells. Second, it allows the study of circulating cells from patients and the definition of specific transcriptional signatures of rickettsial diseases through collaborative projects performed with physicians of infectious diseases (Whipple’s disease, scrub typhus, mediterranean spotted fever). This strategy has produced fruitful collaborations with cardiologists (infective endocarditis) , and more recently, cell biologists (migration of macrophages and gene expression; macrophage fusion and giant multinuclear cells) . Third, a similar approach, namely cell biology and transcriptomics studies, will be used to analyze the placental response in a cohort of pregnant patients with Q fever to find new pathways for placental infection. The comparison of pathological placenta responses with in vitro infection may be useful to determine signatures of C. burnetii infection, and potentially improve the follow-up and treatment of patients.
This policy will be maintained during the next 4 years with a special attention for two clinical situations, bacterial sepsis and preeclampsia (PE). Bacterial sepsis will be studied since M. Leone is full Professor in Anesthesiology and Intensive Care, and his main clinical concern is sepsis, the major cause of death in intensive care units. PE is a public health problem and can appear in up to 5% of pregnancies. This is still a major risk factor for maternal and neonatal mortality and morbidity. F. Bretelle is full Professor in Obstetrics-Gynecology and PE was the major research domain of C. Buffat, Assistant-Professor in Biochemistry. Consequently, their know-how in this domain may be useful to opening new perspectives for a predictive approach of this pathology that is lacking.
3 - 1 - Bacterial sepsis
This project will be directed by Marc Leone.
The incidence of sepsis is rising, partly related to medical progress, which allows patients to survive longer, resulting in increased numbers of older, debilated, or immunocompromised patients passing through intensive care unit. Ten to 15 percent of intensive care unit patients develop septic shock, the form of acute circulatory shock that occurs secondary to severe infection. The mortality rate is 50% to 60%. In clinical studies, lower mortality rates have been reported but this is due to exclusion criteria such as cirrhosis, immunosuppression, or “do not resuscitate” order . The organisms involved in severe sepsis and septic shock are most often bacterial. The gram-negative bacilli are commonly implicated, although there is an increasing part of gram-positive cocci. The lung is the most common source of infection, followed by abdomen, catheter and urine . The pathophysiology of sepsis is complex and remains under investigation. The traditional approach was to consider septic shock as a major pro-inflammatory response. However, there is today some conflicts about this approach. Briefly, this excessive pro-inflammatory phase is probably of very short duration, even nonexistent in old and debilated patients. The second phase consists on a deep and prolonged inhibition of the immune response, favoring the development of secondary nosocomial infection, and leading to a progressive multi-organ failure [23,24]. In the laboratory, we propose to participate to this debate by elaborating murine models and investigating intensive care unit patients. In patients, we will test part of our findings in animal models. We are currently looking at the immune response with on-going studies on the production of interleukin-16 inhuman sepsis. We also will investigate the expression of genes involved in the circadian rhythm in intensive care unit patients, with a special attention to gender and sepsis effects. We will next develop an axis on the immune cell response to stimulation in neutropenic patients, once again with a special focus on gender. In addition, we plan to develop clinical projects on the use of antibiotics in intensive care unit patients. Guidelines recommend to use broad spectrum antibiotics in the first hour after sepsis diagnosis, and to narrow the spectrum of antibiotics after pathogen identification (so-called desescalade) . However, there is no randomized clinical trial testing the impact of such policy on the outcomes. Hence, we propose to build a randomized clinical trial to test the desescalade in real life conditions, by using a network of French intensive care units. We will rely on the laboratory to manage the biological collections in this project, by elaborating for instance a transcriptional profile of treatment responding patients.
3 - 2 - Preeclampsia and pregnancy
This project will be directed by Florence Bretelle and Christophe Buffat.
To predict PE is a major health goal because it can allow to select patients for therapeutics studies and improve papthophysiology knowledge. Because of the central role of placenta in the development of PE, most of the transcriptomic studies were initially focused on the analysis of gene expression in trophoblastic cells of patients. This analysis can only be obtained after the delivery and is therefore useless in predicting the disease. We propose to investigate which genes were differently expressed using a large-scale transcriptomic approach in whole blood obtained in early pregnancy, at time of diagnosis and 2 months after delivery.
Our approach will allow to:
1. Identify prognosis factors. The current classification in 3 stages from low to severe does not reflect the natural evolution of the disease, particularly maternal morbidity and fetal growth intrauterine restriction.
The characterization of powerful diagnosis markers could help to predict the disease evolution.
2. Identify early markers of PE. Whereas the development of the disease occurs early in the first trimester of pregnancy, the diagnosis is made later when maternal and potentially fetal impact are observed. The main objective of the practitioner would be to distinguish in the early course of pregnancy the group of women with a high risk to develop PE. This may guide the counselling and apply preventive treatment.
3. Identify novel therapeutic targets and predictive factors of treatment response to avoid overtreatment of patients as well as an unnecessary risk of toxicity for both the mother and fetus.
4. Tailored treatment of the disease. The transcriptional signature of PE could permit to distinguish patients with a risk probability and allow a better understanding of the disease. The development of a tailored treatment for individual patients could be proposed according this signature.
This project has received a 50 0000 Euros grant (AORc excellence 2010). We aim to test 20 PE women and 20 normal pregnancies. After identification of modulated genes, we will apply microarray results on a cohort of 100 normal pregnancy and 100 PE patients using RT-PCR. The project has received the ethics committee approval.
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