genomewide characterization of host-pathogen interactions by ...

Maren Depke
Discussion and Conclusions
proteins different in abundance amounted to approximately 40 % to 60 % in reference to
genes/proteins which were accessible with both methods. Most recently, a study on LPSactivated
RAW 264.7 macrophages and C57BL/6 BMM was published. Applying isotope coded
affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry, 1064
proteins were identified and quantified of which 36 differed in abundance between LPS-treated
and control cells. Comparison to microarray data revealed approximately 75% concordance
(Swearingen et al. 2010). Although the treatments in both studies were different, the total,
regulated and overlap numbers were roughly comparable.
Nevertheless, a different phenomenon was observed when analyzing differences between
BMM of both strains. Expectedly, a high number of differentially expressed genes was observed,
which were not accessible with the proteome approach. But also a high number of regulated
proteins was recorded, for which transcriptome data were mostly available but did not show
differential expression. Thus, BMM in both strains might differ in post-transcriptional,
translational, and protein stability/turnover processes.
Although the regulated genes/proteins were not always identical for the different
comparisons, similar global results were received by both approaches.
1) IFN-γ treatment mainly led to induction of gene expression or an increase in protein
abundance.
2) IFN-γ induced changes were highly similar in BALB/c BMM and C57BL/6 BMM, since many of
them were observed in BMM of both strains or, when observed only in one of them, showed a
highly similar trend in the other in reference to their fold change values.
3) In the comparison between BMM of the two analyzed mouse strains, about 50 % of regulated
genes/proteins exhibited higher expression/abundance in BALB/c BMM whereas the other
50 % showed higher expression/abundance in C57BL/6 BMM.
4) Strain differences were highly similar in the comparison of control level BMM and in the
comparison of IFN-γ treated BMM.
Apart from the comparison to proteome results, the transcriptome data were analyzed for
their biological content. The confirmation of known IFN-γ effects in the data set proved the
relevance of the results. Very prominent, the induction of immunoproteasome and antigen
presentation genes became visible. These included Psmb8, Psmb9, Psmb10, Psme1, and Psme2,
genes of proteasome subunits, Tap1 and Tap2, peptide transporters from cytosol to the
endoplasmatic reticulum (ER), Erap1, ER aminopeptidase, and MHC class I and class II genes,
which were already described to be induced by interferon (Aki et al. 1994, Van den Eynde/Morel
2001, Brucet et al. 2004, Ma W et al. 1997, Schiffer et al. 2002, Chang et al. 2005, Jung et al.
2009, Benoist/Mathis 1990, Boehm et al. 1997, Gobin/van den Elsen 2000). IFN-γ is also in vivo
responsible for the complete exchange of the constitutive to the immunoproteasome since this
exchange was shown to be reduced to 50 % in the liver of IFN-γ -/- BALB/c mice after infection with
lymphocytic choriomeningitis virus. The authors suppose TNF-α to be responsible for the
remaining fraction of exchange (Khan S et al. 2001). Other in vivo experiments using the fungal
pathogen Histoplasma capsulatum in infections of IFN-γ -/- C57BL/6 mice resulted in a complete
inability to induce the immunoproteasome. In that study, the authors could not find hints that
other cytokines can compensate for the loss of IFN-γ in reference to the immunoproteasome
(Barton et al. 2002). The knockout of inducible proteasome β-subunit LMP-7 (Psmb8) led to a
strongly increased susceptibility of mice during Toxoplasma gondii infections, which was
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