Refine
Document Type
- Doctoral Thesis (2)
Language
- English (2) (remove)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Keywords
- Cytokine (2) (remove)
IL-10 drives the re-establishment of peritoneal macrophage populations in bacterial peritonitis
(2011)
The aim of this thesis work was to explore the physiological and functional properties of peritoneal macrophage populations in both the steady state and in inflammatory conditions. In the steady state there are two populations of macrophages in the peritoneum which I refer to as the R1 and R2 populations. The R1 cells are a rapidly turning over population which constitute around 20% of the peritoneal macrophages. I show that these cells have the capacity to efficiently present peptides on MHC-II to CD4+ T cells but that they are poor at phagocytosis. Monocytes transferred into the un-infected peritoneum give rise almost exclusively to this R1 population, suggesting that the R1 fate is the default pathway of monocyte development under steady state conditions. In contrast, the R2 population in the peritoneum turns over very slowly in the steady state and is composed of cells which are poor at the presentation of peptide to T cells but which are efficient at phagocytosis. Both of these populations are lost from the peritoneum within an hour of the induction of a poly-microbial peritonitis. A large fraction of the R2 population relocates from the peritoneal wash fraction to the omentum, the fate of the R1 population is less clear. Over the course of the next three days, the macrophage populations in the peritoneum are re-established. Transfer experiments using genetically marked cell populations demonstrated that neither the R1 nor the R2 populations which “disappeared” one hour after infection contributes to the re-established peritoneal wash fraction macrophage pool at day 3. While the re-established R1 population retains the functional properties and the FACS phenotype of the steady state R1 cells, the re-established R2-like population is clearly not identical to the R2 cells present in the pre-infection environment. In particular, this R2-like population can be split into two sub-populations which have non-identical functional properties. In this inflammatory situation monocytes transferred into the peritoneum now acquire the capacity to differentiate not only into R1-like cells but also into R2-like macrophages. I looked for the molecular basis driving this change of monocyte differentiation in the infected peritoneum by using a solid phase cytometry based ELISA procedure to examine the spectrum of cytokines produced in the peritoneum in response to poly-microbial infection. One of the most prominent cytokines produced early in infection is IL-10. To determine whether IL-10 is directly involved in assigning monocyte fate in the peritoneum I looked at the ability of mice carrying a targeted deficiency of either the IL-10 gene or of the IL-10 receptor gene to form the R2-like cells after infection. Neither mouse strain efficiently generates the R2-like population after infection. Adoptive transfer of genetically marked wild type or mutant monocytes into appropriate hosts demonstrated that the effect of IL-10 is not direct. Rather, the IL-10 responding cell produces a mediator which then directs monocyte fate. Thus, the bystander IL-10R deficient monocytes are driven by the mediator produced by wild type monocytes to generate R2 cells with high efficiency. The crucial role of this IL-10 dependent pathway was underscored by supplementation experiments. Mice carrying a targeted deficiency of the IL-10 gene fail to generate the R2 population during peritonitis. However, injection of IL-10 into these animals rescues the capacity to form the R2 population. In addition the normal default pathway of monocyte development in un-infected animals which leads to the R1 population is modulated by injection of IL-10 so that the monocytes can now differentiate into the R2 population. The work presented in this thesis describes the steady state populations of phagocytes in the un-infected peritoneum and the dynamics of these populations during the induction of peritonitis. It also uncovers an IL-10 dependent pathway which regulates the choice of monocyte developmental fate within the peritoneum.
Introduction: For a successful pregnancy, a set of physiological requirements has to be fulfilled. The mother has to provide enough nutrients and the proper anatomical environment for the developing fetus and protect him and herself against pathogens. The cells of the im-mune system constantly monitor the organism in search for pathogens and mount a response to eradicate the threat. The favourable outcome of an immune response re-lays on the capacity of those cells to recognize structures that shouldn’t be present in the organism and the speed or strength at which the cells react. During pregnancy, however, a fetus is able to establish a firm contact with the endometrium of the mother and then grow for an extended period of time. This “exception to the rule” hides behind a set of fine-tuned regulations of the immune responses which are not completely un-derstood. Though many cell types have been extensively investigated in the past dec-ades, B cells play yet enigmatic roles. The aim of this work is to uncover the events occurring within the B cell development during pregnancy and to study the role of certain subtypes in healthy pregnancy and pregnancy miscarriage. Methods: For all experiments, 8-weeks-old female mice either non-pregnant, having normal preg-nancies or miscarriage were used. Organs were removed and cells isolated using standard protocols. The analysis of the population distribution was performed by Flow Cytometry. For in vitro experiments, specific cell subsets were isolated using MACS Cell Separation. Bio-plex method was used for the assessment of Immunoglobulin isotypes in serum, while CBA Array was the method used to measure cytokine levels in the supernatant of cell cultures. Statistical analysis was done using GraphPad Prism software. Results: Pregnancy had a strong impact on the murine B cell development. The restructuration of the B cell compartment could be appreciated already from the bone marrow progeni-tors, reduced in pregnant mice. Peripheral subsets drastically adapted their develop-mental pathways, with a drift towards the generation of marginal zone B cells. B cells also showed functional adaptations to gravidity, as evidenced by the changes in the immunoglobulin production and immunomodulatory capacity. Conclusions: For the first time a deep investigation of the consequences of pregnancy on the B cell development was performed, covering several aspects of B cell functionality. This work shows that B lymphocyte compartment is remodelled during pregnancy. Aberration of this process may lead to pregnancy complications including miscarriage.