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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.
Mass spectrometry-based Proteome analysis of porcine cells infected with African swine fever virus
(2023)
ASFV, a highly contagious, pathogenic and lethal pathogen of swine, poses a major threat to domestic and wild suids worldwide as neither vaccines nor treatments are available. Compared to other well-characterized similarly complex viruses like herpesviruses or adenoviruses, the understanding of ASFV biology is poor.
To improve the understanding of ASFV biology, following the establishment of a robust protocol for the isolation of primary monocyte-derived porcine macrophages (moMΦ) and their infection with ASFV for mass spectrometry (MS)-based proteome analysis was performed.
Under both conditions, naïve and infected, the isolated cells showed cell type-specific characteristics like phagocytosis and antigen presentation and protein expression patterns, including the expression of swine leucocyte antigens and CD markers. Furthermore, moMΦ could be reproducibly infected with ASFV isolates of different genotypes and pathogenicity.
The ASFV protein expression patterns in moMΦ correlate well with those observed in established cell lines at transcript and protein level. The expression of 27 ASFV proteins was confirmed at the protein level. Among them, 9 members of multi-gene families (MGF) and 12 novel open reading frames (nORFs) were recently predicted based on transcription start site mapping.
The direct comparison of closely related ASFV genotype II isolates revealed no virulence-associated protein expression patterns beyond those expected based on the genome sequences of the isolates.
Using different MS quantification strategies, it was shown that ASFV affects both static protein expression levels and protein synthesis. These changes in protein expression impact proteins and pathways known to be targeted by ASFV, including CD-markers, ER-stress and cell death pathways, and cellular antiviral responses. Beyond these observations that further validated the moMΦ infection model, novel effects of the ASFV infection on the cellular proteome were noticed.
These effects include the decreased expression levels of cathepsins, especially cathepsins D (CTSD), H (CTSH) and L (CTSL) as well as the transient activation of MAPK14/p38 prior to its strong downregulation. In addition to MAPK14/p38 further members of the MAPK14/p38 signaling pathway, like MAPKAPK2, were affected by ASFV infection.
As these modulations of the cellular proteome would in general result in decreased pro-inflammatory responses, it did stand out that the synthesis of interferon-response related genes including MX1 and ISG15 evaded the ASFV-induced global reduction of protein synthesis. In contrast, the synthesis of genes involved in RNA processing and splicing was significantly impaired. In total, the regulations of individual host proteins assessed in the context of the whole cellular proteome integrate well with each other and other cellular responses to ASFV infection and may help to improve the understanding of host-virus interactions.
Overall, this thesis provides novel insights into the expression of ASFV-encoded ORFs of different isolates and the host response to ASFV infection. It points out that the current knowledge of the ASFV coding capacity, temporal protein expression patterns, protein functionality, post-translational modifications and host interactions is still sketchy as many aspects of ASFV replication have yet to be understood. The established moMΦ-model to study ASFV infections in vitro provides a powerful tool for future applications to increase the understanding of ASFV biology.
Macrophages are cells of immune system and distributed throughout the body. They provide the first line of defense against microbial pathogen infections. Using bone marrow macrophages (BMMs) which derived from mice of strain BALB/c and strain C57BL/6, this study aimed to identify the changes in proteome of the macrophages due to IFN gamma stimulation and S. aureus infection. Two quantitative proteomic techniques, two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied in this study. The analysis results indicated that many proteins which play important roles in immunological functions of macrophages were changed due to IFN gamma stimulation and S. aureus infection. This study also identified the differences in proteome of macrophages derived from mice of strain BALB/c in comparing to macrophages of strain C57BL/6.
The success of pregnancy depends on precisely adjusted, local immune mechanisms. In early pregnancy, fetal trophoblast cells implant into the endometrium to build and anchor the placenta. Simultaneously, they mediate fetal tolerance and defense against infections. To cover these versatile requirements, local immune factors must be in balance. A too tolerogenic milieu can lead to an inadequate placentation; while a too inflammatory milieu can cause rejection of the semi-allogenic fetus. Bacterial infections can provoke these inflammatory pregnancy complications as well. Therefore, the pregnant uterus was long thought to be sterile. Descriptions of a placental microbiome opened a scientific discourse, which is unsolved due to contrary studies. The colonization of the non-pregnant endometrium is, however, confirmed. It is supposed to affect both, uterine pathologies and fertility. Precise data are lacking. Aim of this work was to assess if and under which circumstances a bacterial colonization would be tolerable.
One of the described species in placental and endometrial samples is Fusobacterium nucleatum. It is an opportunistic bacterium, which is known from the human oral cavity and associated with the development of colon carcinomas. F. nucleatum supports tumorigenesis by the induction of epithelial proliferation, survival, migration and invasion as well as angiogenesis and tumor tolerance. Since similar processes are required for implantation and placentation, F. nucleatum might support these as well. In this work, the effects of F. nucleatum on leukocyte-trophoblast-interactions, especially of macrophages and innate lymphoid cells type 3 (ILC3), were assessed.
The monocytic cells (THP-1) were differentiated into inflammatory M1 (IFN-γ) or tissue-repairing and tolerogenic M2a (IL-4) and M2c (TGF-β) macrophages. Inactivated F. nucleatum, LPS or E. coli was added. Only small concentrations of inactivated bacteria were used (bacteria:leukocyte ratio of 0.1 or 1), since it was not the aim to analyze infections. Conditioned medium of treated leukocytes was added to trophoblastic cells (HTR-8/SVneo). Migratory, invasive and tube formation behavior of trophoblastic cells was quantified.
Treated M1 macrophages impaired trophoblast function, whereas M2a macrophages induced trophoblast invasion. M2c macrophages supported trophoblast migration and tube formation if treated with the smaller, but not with the higher concentration of F. nucleatum. This treatment induced the accumulation of HIF-1α and the secretion of VEGF-A in M2c macrophages as well. Moreover, the higher concentration of F. nucleatum caused rather inflammatory responses (NF-κB activation and cytokine expression). The activation of the HIF-1α-VEGF-A axis under the influence of TGF-β might serve as a mild immune stimulation by low abundant commensal bacteria supporting placentation.
In contrast to macrophages, the function of ILC3s during pregnancy is still unknown. In general, ILC3s are located in mucosal tissue, such as the gut. They participate in tolerance mechanisms and form the local micromilieu by the secretion of cytokines and the presentation of antigens. In order to characterize local, uterine ILC3s, murine ILC3s were compared to peripheral, splenic ILC3s. Uterine ILC3s were more activated and produced higher levels of IL-17 compared to splenic ILC3s. However, uterine ILC3s barely expressed MHCII on their surface. A reduced antigen presentation potential was confirmed in human ILC3s differentiated from cord blood stem cells by the addition of TGF-β or hCG. The treatment with bacteria increased MHCII expression, but not to the initial level. The higher bacterial concentration induced IL-8 secretion and led to an increased trophoblast invasion. ILC3s were less sensitive to bacterial stimulation than macrophages.
Recent studies on the uterine or placental presence of bacteria during pregnancy are discrepant. The results of this project indicate that bacteria or bacterial residues might serve as a mild stimulus under certain circumstances to support implantation without negative effects. The current discussion must therefore not only be expanded by additional studies, but especially include differentiated local conditions. In this context, the sheer presence of bacteria or bacterial components must not be equated with an infection representing a known hazard.