Transcriptional expression of Interferon-γ and immunoproteasomal subunits in liver and brain of LCMV-infected mice

To investigate the molecular basis of the observed failure in proper immunoproteasome formation, we analysed the transcriptional expression of Interferon-gamma (IFN-γ) and immunoproteasomal subunits by real-time PCR:

Immunocompetent 8- to 12- week-old mice were infected either with 200 pfu LCMV-WE intravenously, 30 pfu intracranially or to attenuate disease progression and to prolong life with 10⁵ pfu intraperitoneally and 30 pfu intracranially. At different time points, RNA was isolated from liver and brain, transcribed into cDNA, and gene induction analysed by real-time PCR using the Light Cycler^® instrument and a SYBR-Green I - based assay. For the detection of IFN-γ expression an IFN-γ-specific probe was employed. All samples were normalized to HPRT. Relative gene expression was evaluated using the Pfaffl-method (Pfaffl 2001) and the Excel-based software tool REST^® (Pfaffl et al. 2002).

Following intravenous infection, we observed an up to 60-fold induction of IFN-γ in the liver on day 6 post infection, which decreased quite rapidly to a less than 20-fold induction on day 8 (Figure 8). Immunoproteasomal subunits were up-regulated with a 4- to 10- fold induction on day 4. This level remained stable till day 6 and then decreased to a lower magnitude on day 8.

Surprisingly, this induction was already observable on day 4, when there was still no IFN-γ expression detectable. Probably, this can be attributed to other pro-inflammatory cytokines such as TNF-α, and type I interferons (IFN-α, IFN-β), which are known to induce immunoproteasome expression (Groettrup et al. 2001) (Shin et al. 2006)

as well, and whose expression levels have not been analysed in this study. Another possibility would be that it results from a technically-based, lower sensitivity due to the employment of an IFN-γ specific probe.

Intracranial infection as well as the co-infection showed a similar kinetic in the liver, whereas - except from IFN-γ and MECL-1 in the co-infection model - the absolute values were significantly decreased in both applications compared to intravenous infection.

Immunoproteasome assembly in the brain of LCMV-infected mice Results

Figure 8: mRNA levels of immunoproteasomal subunits in the liver of naive and LCMV-WE infected BALB/c mice. Mice were either left untreated, or infected with 200 pfu LCMV-WE intravenously, 30 pfu intracranially, or 30 pfu intracranially and 10⁵ pfu intraperitoneally (Coinfection). At indicated time points post infection, RNA was isolated from the liver, transcribed into cDNA and the gene induction of IFN-γ, MECL-1, LMP2, and LMP7 was analyzed by real-time PCR. All samples were normalized to HPRT expression. Relative gene expression was evaluated using the Excel-based software tool REST^®.

Immunoproteasome assembly in the brain of LCMV-infected mice Results

As expected, intravenous LCMV infection did not lead to an inflammation of the brain.

Although we could observe an up to 100-fold induction of Interferon-γ in the brain on day 6 post intravenous infection, this up-regulation was not capable of inducing immunoproteasome expression (Figure 9).

In contrast, following co- and intracranial infection we saw a fulminate 200- to 400- fold up-regulation of IFN-γ in the brain, which can be exclusively associated with infiltrating mononuclear cells (Campbell et al. 1994). While Interferon-γ expression in the intracranial infection model was strongly induced on day 6 and further increased till day 7, up-regulation of this cytokine was only a short-time phenomenon in the brain of co-infected mice, where it also started at day 6 with a comparable kinetic, but was again strongly decreased on day 7. For the co-infection model this observation indicates a faster clearance of the virus and a shorter period of ongoing inflammation that finally results in a reduced CTL-driven immunopathology and the survival of the mice.

Concerning the immunoproteasome formation in the brain of intracranially LCMV-infected mice we saw a constant increase in the expression levels of inducible subunits, which were culminating on day 7 with an up to 6-fold induction for MECL-1, up to 8-fold induction for LMP7, and up to 120-fold induction for LMP2.

In correlation with IFN-γ the absolute numbers of immunoproteasomal induction were slightly decreased in the co-infection model culminating at day 6 with an up to 100-fold induction for LMP2, and a 3- to 6-100-fold induction for MECL-1 and LMP7. Following ongoing inflammation in the co-infection model, the expression levels of immunoproteasomal subunits again decreased on day 7 to some extent, but still remained up-regulated until day 10 p.i. .

Taken together, these data indicate that following intracranial LCMV infection the observed failure to properly form immunoproteasomes cannot be associated with a time-dependent limitation of transcriptional induction. Even in the co-infection model, where the attenuated disease progression led to a prolonged life span and mostly resulted in a complete recovery, the ongoing, transcriptional up-regulation of immunoproteasomal subunits in the brain (Figure 9) could not recover proper immunoproteasome formation (Figure 7) that was observed in other organs upon inflammation (Figure 6).

Immunoproteasome assembly in the brain of LCMV-infected mice Results

Figure 9: mRNA levels of immunoproteasomal subunits in the brain of naive and LCMV-WE infected BALB/c mice. Mice were either left untreated, or infected with 200 pfu LCMV-WE intravenously, 30 pfu intracranially, or 30 pfu intracranially and 10⁵ pfu intraperitoneally. At indicated time points post infection, total RNA was isolated from the brain, transcribed into cDNA and the gene induction of IFN-γ, MECL-1, LMP2, and LMP7 analyzed by real-time PCR. All samples were normalized to HPRT expression. Relative gene expression was evaluated using the Excel-based software tool REST^®.

Immunoproteasome assembly in the brain of LCMV-infected mice Results

3.1.4 Translational expression of immunoproteasomal subunits in liver and