genomewide characterization of host-pathogen interactions by ...

Maren Depke
Summary of Dissertation
function, macrophages take part in regulation of the adaptive immune response. The preparation
of bone marrow stem cells and the in vitro differentiation of the stem cells into so-called bonemarrow
derived macrophages (BMM) is a model to study reactions of macrophages. The
advantage of this approach is that these macrophages have never been under any immunological
influence which might result from the immune status of the animal even under standardized
laboratory conditions. Until recently, BMM experiments were standardized only to a limited
extent because serum-supplemented culture medium was used. To overcome sources of
experimental variation resulting from undefined and varying substances in serum, Eske et al.
introduced serum-free culture conditions for BMM (Eske et al. 2009; J Immunol Methods. 342(1-
2):13) which were now applied to study the reaction of BMM as third part of this thesis. BMM of
different mouse strains were treated with IFN-γ, a modulator of macrophage function, which is
one of the first signals during initiation of the immune response in vivo. Host-pathogen
interaction experiments have revealed differences in reactions of BMM derived from BALB/c and
C57BL/6 mice when confronted with Burkholderia pseudomallei especially after IFN-γ stimulation
and at higher multiplicities of infection (Breitbach et al. 2006; Infect Immun. 74(11):6300). Also
other infection studies uncovered differences between these two mouse strains in vivo and
in vitro. Against the background of genetic influences on the BMM reactions, BALB/c and C57BL/6
BMM were stimulated with IFN-γ to specify on a molecular level the reaction to the priming
signal IFN-γ as basic principle. Furthermore, the study aimed to profile potential differences of
reactions between the BMM of both mouse strains.
Gene expression profiling revealed mainly induction of gene expression after treatment of
BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like the induction of
immunoproteasome, antigen presentation and associated genes, interferon signaling related
genes, GTPase/GTP binding protein genes, inducible nitric oxide synthase and others. IFN-γ
dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Even for
genes, which were differentially expressed only in BMM of one strain, a similar trend was
observed in the other strain’s BMM. Gene expression differences between BMM of both strains
were analyzed on the level of untreated controls as well as after IFN-γ treatment. Here,
approximately 55 % to 60 % of the differentially expressed genes exhibited higher expression in
BALB/c BMM, whereas the remaining genes featured higher expression in C57BL/6 BMM.
Equivalent to the IFN-γ effects, a similar expression trend in the strain comparisons was visible at
both treatment levels, even when regulation was significant only in one. Differentially expressed
genes between BMM of both strains included immune-relevant genes as well as genes linked to
cell death, but the coverage of functional groups was limited. The phenotypical differences
between the reaction of BALB/c and C57BL/6 BMM were often determined in the presence of
IFN-γ and a second stimulus like LPS or infection. To elucidate molecular reasons for the observed
differences in killing of pathogens or cytokine production, the inclusion of samples subjected to a
second stimulus in addition to IFN-γ is recommended.
Not only immune cells or specially phagocytes get in touch with pathogens, but also cells
responsible for functional and structural integrity of host organs and tissue, like epithelial and
endothelial cells. Such cells are actually part of the first line of recognition and reaction to a
pathogenic invasion into the host. While S. aureus colonizes humans in the anterior nares it can
be a cause of pneumonia when transferred to the lung e. g. by aspiration or medical devices. The
bronchial epithelial cell line S9 was used as an in vitro model system for the infection with
staphylococci. A new experimental system permits the study of host-pathogen interactions in the
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