Regulation of Immunoproteasome Function in the Lung

Impaired immune function contributes to the development of chronic obstructive pulmonary disease (COPD). Disease progression is further exacerbated by pathogen infections due to impaired immune responses. Elimination of infected cells is achieved by cytotoxic CD8+  T cells that are activated by MHC I-mediated presentation of pathogen-derived antigenic peptides. The immunoproteasome, a specialized form of the proteasome, improves generation of antigenic peptides for MHC I presentation thereby facilitating anti-viral immune responses. However, immunoproteasome function in the lung has not been investigated in detail yet. In this study, we comprehensively characterized the function of immunoproteasomes in the human and murine lung. Parenchymal cells of the lung express low constitutive levels of immunoproteasomes, while they are highly and specifically expressed in alveolar macrophages. Immunoproteasome expression is not altered in whole lung tissue of COPD patients. Novel activity-based probes and native gel analysis revealed that immunoproteasome activities are specifically and rapidly induced by IFNγ treatment in respiratory cells in vitro and by virus infection of the lung in mice. Our results suggest that the lung is potentially capable of mounting an immunoproteasome-mediated efficient adaptive immune response to intracellular infections.


Primary lung fibroblast isolation:
Primary mouse or human lung fibroblasts were isolated as described 1 . Mouse fibroblasts were used between passages 2-4, human fibroblasts before passage 6.

Mouse alveolar epithelial cell isolation and culture:
Primary alveolar type II cells (pmATII) were isolated from C57BL/6 mice as described previously 2 .
Protein extracts and Western Blotting: Cells or dismembrated frozen tissue was lysed in ice-cold RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.5), supplemented with protease inhibitor cocktail cOmplete (Roche, Basel, Switzerland). After 20 min incubation on ice, lysates were centrifuged at maximum speed for 20 min at 4°C and supernatants were used for further analysis.
Immunohistochemistry: Human or mouse lung sections (3 µm) were deparaffinized in Xylene and rehydrated. Slides were incubated in solution containing 80% methanol and 1.8% H 2 O 2 for 20 min to quench endogenous peroxidase activity. Heat-induced antigen retrieval was performed in 0.05% citraconic anhydride buffer (pH 7.4). Slides were washed with TBST buffer (20 mM Tris, 135 mM NaCl, 0.02% Tween, pH 7.6), blocked with Rodent Block M (Biocare, Concord, CA, USA) for 30 min, washed and incubated for 60 min with an LMP2 specific antibody (1:600, ab3328, Abcam, Cambridge, UK). After another washing step, slides were incubated with rabbit-polymer coupled to alkaline phosphatase (Biocare) for 30 min and washed again. Vulcan Fast Red (Biocare) was used as substrate and incubated for 12 min. Hematoxylin counterstaining was performed, and slides were dehydrated and mounted in Eukitt ® (Sigma-Aldrich). Slides were evaluated using a MIRAX scanning system (Zeiss, Oberkochen, Germany).
20S proteasome isolation from mouse lungs: Isolation and purification of proteasomes from lung tissue was performed essentially as described by Dahlmann et al. 4 , except for the fact that DEAE-Toyopearl was used for the initial step of anion exchange chromatography and Superose 6 instead of Sepharose for gel chromatography. After chromatography on arginine-Sepharose, the enzyme preparation was concentrated by ultracentrifugation and the precipitate dissolved in TSDG buffer (10 mM Tris/HCl, 25 mM KCl, 1.1 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 1 mM NaN 3 , 10% glycerol, pH 7) containing 2 mM ATP. 20S and 26S proteasomes were then separated by centrifugation in a glycerol gradient (20% -40% dissolved in TSDG buffer). Centrifugation was performed for 24 h at 25,000 rpm in a Beckman SW28 rotor and afterwards the gradient was fractionated into fractions of 0.5 ml.
Determination of proteolytic activity was performed by use of fluorogenic peptide substrates as described by Dahlmann et al. 5 .
For detection of proteasome activity by substrate overlay technique after non-denaturing polyacrylamide gel electrophoresis, the substrate Bz-VGR-MCA was used. This technique as well as non-equilibrium pH gradient and SDS-PAGE were performed as described by Dahlmann et al. 4 .
The tryptic digest was diluted with one equivalent of MALDI matrix consisting of 2,5-dihydroxy-benzoic acid (Sigma-Aldrich) (20 mg/ml in 20% acetonitrile, 0.1% TFA) and 2-hydroxy-5-bethoxybenzoic acid (Fluka) (20 mg/ml in 20% acetonitrile, 0.1% TFA) in a 9:1 ratio (v/v), and spotted onto a steel target plate. Peptide mass fingerprint identification of the sample protein was done by comparing peptide masses of the tryptic digest to the virtually trypsinized Ensembl Mouse protein database (database downloaded from www.ensembl.org).
The database search was performed using the MASCOT Database search engine v1.9 (Matrix Science Ltd.). Search parameter settings were 150 ppm peptide mass tolerance and one allowed missed cleavage. LC-MS/MS analysis was performed on an Ultimate3000 nano HPLC system (Dionex, Sunnyvale, CA) coupled to a LTQ OrbitrapXL mass spectrometer (Thermo Fisher Scientific) by a nano spray ion source. Samples from in-gel digest were acidified using TFA and automatically loaded to the HPLC system as described by Hauck et al. 7 . The acquired spectra (Thermo raw file) were exported to Mascot Deamon and searched against the Ensembl_Mouse protein database. Search parameters included fixed modification Carbamidomethyl (C) and variable modifications Deaminated (NQ) and Oxidation (M).
Peptide tolerance was set to 10 ppm and MS/MS tolerance to 0.6 Da. Only 2, 3 and 4 fold charged peptides were selected for protein identification. Search results were viewed using the Scaffold software (Scaffold 3).