genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Gene Expression Pattern of Bone-Marrow Derived Macrophages after Interferon-gamma Treatment
number of genes whose corresponding proteins were accessible by LC-MS/MS analysis, the
overlap of differentially abundant proteins and differentially expressed genes was low in absolute
number (12 at control level and 19 at IFN-γ treated level) but it still held 60 % (control level) and
79 % (IFN-γ treated level) of the genes that were both differentially expressed in the array data
set and accessible by proteome analysis (Table R.3.2 B). Vice versa, for only 5.9 % (control level)
and 6.6 % (IFN-γ treated level) of regulated proteins with accessible microarray data a change in
gene expression level was detected. Hence, the majority of protein abundance differences
between the strains were independent from gene expression level.
A refined comparison between proteome and transcriptome analysis which included the
direction of regulation uncovered that the overlap of proteins and genes higher expressed in
C57BL/6 BMM and the overlap of proteins and genes higher expressed in BALB/c BMM (7 and 4
at control level; 10 and 17 at IFN-γ treated level) did not sum up to the total overlap of 12 at
control level and 19 at IFN-γ treated level (Table R.3.2 B). The explanation for the discrepancy is
that one protein/gene (Scp2) at the control level showed a higher expression in BALB/c BMM in
transcriptome but higher abundance in C57BL/6 BMM in proteome analysis. Correspondingly, at
the IFN-γ treated level two proteins/genes did not possess the same direction of regulation: H2-
AB1 and H2-K1 were higher expressed in C57BL/6 BMM in transcriptome but showed higher
abundance in BALB/c BMM in proteome analysis.
The comparison of strain differences which occurred at the non-treated medium control level
and at the IFN-γ treated level demonstrated similar differences between C57BL/6 BMM and
BALB/c BMM at both treatment levels: 128 genes were expressed differentially at both treatment
conditions (Fig. R.3.3 A), and 183 proteins show significantly different abundance (Fig. R.3.3 C).
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 (Fig. R.3.3 B, D).
Revelation of similar main effects of IFN-γ in BALB/c BMM and C57BL/6 BMM by network
analysis and confirmation of known IFN-γ effects in serum-free BMM cultivation system
Ingenuity Pathway Analysis (IPA, www.ingenuity.com) allows to arrange genes of interest, e. g.
differentially expressed genes, in networks which show the interactions in the selected group of
genes. The two networks with the highest score from the separate analyses for IFN-γ effects in
BALB/c BMM and C57BL/6 BMM exhibited an overlap of 22 out of 35 genes (nodes). Therefore,
they covered similar functional aspects. After merging both networks, the comparison of
differential gene expression after IFN-γ treatment in BALB/c BMM (Fig. R.3.4 A) and in C57BL/6
BMM (Fig. R.3.4 B) showed that many of the genes of this network were subjected to comparable
regulation by IFN-γ in BMM of both strains, sometimes with differences in magnitude.
The network confirms already known IFN-γ effects in the new serum-free BMM cultivation
system. The 26 S proteasome was also known to be changed in response to IFN-γ stimulus. The
proteasome, consisting of a ring-shaped 20 S core complex of several subunits, which exhibits
besides others protease function, and two regulatory complexes of 19 S, is the center of
intracellular ubiquitin-dependent protein degradation which is not only needed for the normal
protein turnover but also for the generation of peptides for the presentation to immune cells via
MHC-I molecules. The change of the normal proteasome into the so-called immunoproteasome is
mediated by the exchange of three β-subunits from the core complex by gene products of
Psmb9, Psmb10, and Psmb8 and is induced by IFN-γ. The exchange of these subunits leads to a
change in the proteolytic activity and specificity (Wang/Maldonado 2006, Strehl et al. 2005). Also
an alternative regulatory complex of 11 S (PA28) exists (Rivett et al. 2001). Here, expression of
Psmb9 (3.6 / 3.4), Psmb10 (3.1 / 3.7), and Psmb8 (2.8 / 3.1), coding for β-subunits of the core
complex, and Psme1 (2.1 / 2.1) and Psme2 (2.7 / 3.1), which encode components of the regulatory
11 S complex, were induced in both BALB/c and C57BL/6 BMM after IFN-γ treatment.
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