Regulation of immunoproteasome function in the lung

The lung is constantly exposed to acute environmental agents such as noxious gases, aerosols, and pathogens1. Efficient clearance and defense mechanisms are thus indispensable to protect the lung from injury and maintain lung function. Failure of these defense mechanisms results in sustained inflammation and activation of the immune system, contributing to chronic pulmonary diseases with impaired lung structure and function2. This is particularly evident for chronic obstructive pulmonary disease (COPD): lungs of COPD patients show increased levels of inflammatory cytokines such as tumor necrosis factor α (TNFα) and interferon-γ (IFNγ) as well as increased numbers of both innate and adaptive immune cells2,3. In addition, bacterial or viral infections in COPD patients often result in acute exacerbations and accelerate disease progression, suggesting that, amongst others, the adaptive immune system is unable to efficiently detect and eliminate infected lung cells to terminate pathogen amplification. Intracellular antigens are detected by pathogen-specific activated CD8+  T cells that patrol the lungs for pathogen-derived peptides presented in complex with major histocompatibility complex (MHC) I on the cell surface of infected cells. The ubiquitin-proteasome system is the major peptide provider for MHC I antigen presentation. It degrades more than 90 % of all cellular proteins - including old and damaged ones - into small peptides4,5,6. The proteasome consists of a barrel-shaped 20S proteolytic core particle which is activated by different proteasome regulators to form for instance the 26S, which degrades poly-ubiquitinated proteins in an ATP-dependent manner, and hybrid proteasomes7. The 20S core is composed of four heptameric rings comprising α- and β-subunits with α7β7β7α7 structure. In standard proteasomes, three of the seven β-subunits - namely β1, β2, and β5 - exhibit proteolytic activity. A replacement of these β-subunits by their immunosubunit counterparts, i.e. low molecular mass protein (LMP) 2, multicatalytic endopeptidase complex-like 1 (MECL-1), and LMP7, also termed β1i, β2i, and β5i, respectively, results in formation of so-called immunoproteasomes. Immunoproteasomes are constitutively present in lymphoid cells but their synthesis can be induced rapidly also in non-immune cells by IFNγ, or TNFα, e.g. upon viral or bacterial infection8. The newly assembled immunoproteasomes have altered cleavage kinetics compared to their 20S standard counterparts and generate antigenic peptides that are preferentially presented by MHC I molecules9. As such, rapid and specific induction of immunoproteasomes is required for efficient elimination of infected cells via the adaptive immune system. Increasing evidence suggests impairment of proteasome function by smoke exposure and in COPD10,11,12,13, however, until now it is not known whether immunoproteasome function is affected as well. Moreover, cell-specific expression of immunoproteasomes in the lung has not been analyzed so far and it is unclear to which degree immunoproteasome activity can be induced upon virus infection in vivo. In this study, we comprehensively characterized immunoproteasome function, i.e. activity, in the lung by dissecting IFNγ-mediated regulation of specific catalytic activities of the immunoproteasome in different respiratory cell types in vitro and upon MHV-68 infection of the lung in vivo.