genomewide characterization of host-pathogen interactions by ...

Maren Depke
Discussion and Conclusions
Only few genomewide transcriptome profiling studies on IFN-γ and macrophages are available
in literature. Kota et al. analyzed the reaction of murine RAW 264.7 cells to IFN-γ exposition for
4 h (Kota et al. 2006). Despite the difference of macrophage cell line to BMM and of treatment
time and IFN-γ dose (4 h and 1000 U/ml vs. 24 h and 300 U/ml) between the published study and
the study described in this thesis, a very good agreement in the differential regulation of several
genes was observed (e. g. Oas, GTPases, Socs1, Socs3, Nos2, Cxcl9, Cxcl10, Irf1, Cxcr4,
immunoproteasome and associated genes, MHC molecules, Il18bp, Il10ra, Ctsc, Ptgs2 and
others). Ehrt et al. analyzed BMM after 72 h of IFN-γ treatment (100 U/ml), infection for 24 h
with Mycobacterium tuberculosis, or both (Ehrt et al. 2001). The authors observed – in contrast
to the results of this study – slightly more repressed than induced genes after IFN-γ treatment
alone. The infection setting resulted in an additional, specific set of differentially expressed
genes. Such additional set of genes is also expected in case of future inclusion of further infected
samples in the settings of the study described in this thesis. The authors defined a set of
approximately 1300 genes to be regulated upon treatment of BMM with IFN-γ (Ehrt et al. 2001).
This high number is not directly comparable with the results of this study, because Ehrt and
colleagues treated each probe set of the array as if it represented a single gene. This
simplification was necessary because the analysis was performed before the complete
sequencing of the mouse genome was finished. Thus, at that time, the partially overlapping EST
and cDNA sequences were difficult to assign to gene information. Furthermore, the authors
explain the result of the high number of regulated genes with the long IFN-γ stimulation time of
72 h. But also between the study by Ehrt et al. and the study described in this thesis accordance
was observed (e. g. MHC molecules, Gbp2 and other GTPases, Irg1, Nos2, Cxcl10, Cxcr4).
Interestingly, the authors monitored a strong influence of Nos2 deficiency on the gene
expression profile after IFN-γ treatment leading to the conclusion that Nos2 directly or indirectly
influences the cellular reaction to IFN-γ stimulus. Zocco and coworkers focused their analyses on
rat hepatic macrophages, i. e. Kupffer cells, stimulated with 1000 U/ml IFN-α or IFN-γ for 8 h
(Zocco et al. 2006). Using an Affymetrix array with about 8800 probe sets, they observed 70
induced and 72 repressed genes by IFN-γ, the relation of which resembles more that observed by
Ehrt et al. than the relation determined in the study described in this thesis. Nevertheless, also
here a certain concordance of cellular reaction became visible (e. g. Mx, Gbp2, Nos2, Irf1, Stat1,
Oas, immunoproteasome subunits, MHC molecules). In the study of Pereira et al. BMM of A/J or
BALB/c mice were treated for 18 h with 50 U/ml of IFN-γ and analyzed on a 1536 feature cDNA
array from a fetal thymus library (Pereira et al. 2004). For BALB/c BMM, the induction of 297
genes and repression of 58 genes was recorded. Here, the relation of induction to repression is
similar to that observed in the study described in this thesis although the comparison of results is
difficult because of the very different arrays, which were used in both studies. The comparison
with similar transcriptome studies from literature leads to the conclusion that the results of this
study are well supported by knowledge on cellular reactions to IFN-γ. In addition, similar to
comparisons of other studies, also in this study specific gene expression changes were observed
which might be explained with experimental differences. Nevertheless, for a comprehensive
knowledge of IFN-γ effects the study of different experimental settings is necessary to which this
study contributes.
During data analysis using the Ingenuity Pathway Analysis tool (IPA, www.ingenuity.com), it
became clear that only a fraction of the IFN-γ related, differentially expressed genes were linked
to macrophages or RAW cells in the IPA database. Of about 180 genes, approximately 130 were
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