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Clinical Trial
. 2014 Aug 6;9(8):e104048.
doi: 10.1371/journal.pone.0104048. eCollection 2014.

Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ

Khetam Ghannam  1 , Lorena Martinez-Gamboa  1 , Lydia Spengler  1 , Sabine Krause  2 , Biljana Smiljanovic  1 , Marc Bonin  1 , Salyan Bhattarai  1 , Andreas Grützkau  3 , Gerd-R Burmester  1 , Thomas Häupl  1 , Eugen Feist  1
Affiliations
Clinical Trial

Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ

Khetam Ghannam et al. PLoS One. .

Abstract

Objective: In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers.

Methods: Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses.

Results: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood.

Conclusions: Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of immunoproteasomal subunits in immune cells:
Gene expression of immunoproteasomal subunits (PSMB8–10) in CD4+, CD8+, CD19+, CD14+ and DCs of patients with myopathies (PM, DM, OM, NIM) and controls (HD). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**). Significantly higher expression of PSMB8 was observed in CD14+ cells and DC of PM patients compared to HD or DM, NIM and HDs, respectively. PSMB9 was increased in CD8+ and CD14+ of DM and in DCs of PM and DM patients compared to NIM. PSMB10 was found increased in PM patients compared to DM, NIM and HD in CD8+, compared to HD in CD19+ and CD14+ cells and compared to OM, NIM, and HD in DCs.
Figure 2
Figure 2. Expression of catalytic proteasomal subunits in muscle:
Gene expression analysis of constitutive and immunoproteasomal subunits (PSMB5–10) in muscle biopsies of patients with inflammatory myopathies (IM) and patients with non inflammatory myopathies (NIM). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*), p<0.01 (**) and p<0.001 (***). Comparing to NIM, mean relative expression levels in IM revealed 4-fold for PSMB8 [0.302±0.139 and 0.075±0.041] and about 5-fold increase for PSMB9 [0.049±0.029 and 0.009±0.002] but less than 2-fold for PSMB10 [0.065±0.064 and 0.036±0.022].
Figure 3
Figure 3. Comparison of immunoproteosomal subunit expression between paired samples from isolated cells and muscle tissue:
Gene expression analysis of immunoproteasomal subunits (PSMB8–10) in muscle biopsies vs. CD4+, CD8+, CD14+, CD19+, and DCs from patients with inflammatory myopathies (IM) and patients with non-inflammatory myopathies (NIM). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**).
Figure 4
Figure 4. Protein expression of the immunoproteasome subunits PSMB8 and PSMB9 relative to beta actin in IM and NIM.
The protein expression of the two subunits was analyzed by western blot in muscle biopsies from four non-inflammatory myopathies (NIM) and four inflammatory myopathies (IM). Intensity of chemiluminescent signals for PSMB8 and PSMB9 was normalized to corresponding signals of the housekeeping protein beta actin, which was detected in a second staining procedure on the same membrane. Only IM samples revealed PSMB8 and -9 protein expression.
Figure 5
Figure 5. Correlation of proteasome subunit expression with myositis signatures and overlap with immune cell infiltration:
Expression of all proteasome units were investigated in transcriptome data of muscle biopsies referenced in table S2 and were correlated with genes increased in myositis muscle tissue. The heatmap of the probeset signal intensities (A) is opposed to the heatmap of the gene by gene correlation matrix (B) in 133P array experiments. Identical analysis in 133A arrays are shown in D and E. Expression of the 1209 probesets in reference transcriptomes of purified immune cells from the blood of healthy donors served as basis for clustering by probesets and cell types (C). Identification of cell type specific transcriptional patterns in the 1209 probesets demonstrates the extent of overlap of these probesets with immune cell related transcripts. Myositis samples include IBM (red), PM (orange), DM (yellow), NM (necrotizing myopathy)/IM (inflammatory myopathy) (light green) and HD (green). Immune cell types include neutrophils (dark blue), monocytes (blue), dendritic cells (light blue), CD4+ T-cells (dark violet), CD8+ T-cells (violet), NK-cells (light violet), B-cells (cyan), and healthy muscle (moss-green). A detailed graph with all probesets labelled is presented in supplemental material (figure S4).
Figure 6
Figure 6. Quantification of cellular infiltration and confirmation of PSMB8/-9 gene activation in myositis muscle samples:
Transcriptome data referenced in table S2 were re-investigated. A) Cell type infiltration was quantified based on the expression of cell specific marker transcripts (figure S5). Signal ratios between muscle biopsy and purified cell type were calculated for each cell type specific transcriptional markers. Taking the median of the ratios in each cell specific marker set and scaling their sum to 100% revealed an estimate of the cellular composition. Increase of monocyte, dendritic cell and T-cell transcription patterns corresponds to the molecular myositis score. (ND = normal donor; NM = necrotizing myopathy). B) The maximum of infiltration related signal intensity was calculated as described in materials and methods. Comparing these expected intensities (x-axis) with real intensities (y-axis) in each sample, PSMB8 and PSMB9 are higher expressed than expected in the majority of IBM and several of the PM muscle. In DM, PSMB8 expression also exceeds the expected signal intensity, although on a lower level. One out of the 8 DM samples (DM_13) showed consistently a pattern similar to the highly inflamed IBM samples. All other conditions including controls remained below the expected intensity. For PSMB10, the increased signal intensity in several samples of IBM seems to correspond to the level of infiltration.
Figure 7
Figure 7. Correlation of immunoproteasome with IFN and IFNR expression in myositis muscle tissue:
Transcriptome data referenced in table S2 were re-investigated. A) Correlation analysis was performed for 133P and 133A datasets independently. Only IFNγ revealed high correlation coefficients with PSMB8, PSMB9 and PSMB10. The corresponding constitutively expressed subunits PSMB5-7 were not or even negatively correlated. B) Comparing the association of IFNγ with PSMB8, -9 and -10 for each sample individually, the increase was much higher for PSMB8/-9 compared to PSMB10 as described before (figure 6).
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German Research Foundation grant DFG FE470/3-1; German Research Foundation grant DFG GRK1631 (MyoGrad); EU IMI grant BeTheCure, contract no 115142-2; European Science Foundation, grant EUMYONET. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.