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Background: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8 + T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells., Methods: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro , ex vivo and in vivo using RNA and Western blot analyses. CD8 + T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8 + T-cell numbers were determined in patients' peripheral blood using tetramer technology., Results: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro , ex vivo and in vivo . In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8 + T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8 + T-cells compared to healthy controls and asthmatics., Conclusion: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8 + T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections., Competing Interests: Conflict of Interest: H. Ma reports support for the present manuscript from the German Center for Lung Research (DZL). M. Nakayama reports overseas grant from Uehara Memorial Foundation (Japan) and overseas grant from Shiga university of Medical Science, outside the submitted work. A.L. Mora reports support for the present manuscript from NIH (NIH U01 HL1455550-01 and NIH NHLBI R01 HL149825). J.S. Lee reports participation on clinical adjudication committee with Janssen R&D, outside the submitted work. S. Krauss-Etschmann reports support for the present manuscript from the German Center for Lung Research. K. Milger reports consulting fees and lecture honoraria from AstraZeneca, GSK, Janssen, Novartis and Sanofi, outside the submitted work. C.A. Staab-Weijnitz reports support for the present manuscript from Helmholtz Association, German Center for Lung Research (DZL) and Deutsche Forschungsgemeinschaft (DFG) within the Research Training Group GRK2338. K.I. Gaede reports support for the present manuscript from Research Center Borstel – Leibniz Lung Center – BioMaterialBank North, Airway Research Center North, German Center for Lung Research (DZL), PopGen 2.0 Network (P2N). K.I. Gaede also holds a leadership role as member of the Board of Directors of the TMF (www.tmf-ev.de), outside the submitted work. I.E. Kammerl reports support for the present manuscript from ERS (Short Term Fellowship). All other authors have no potential conflicts of interest to declare., (Copyright ©The authors 2023.)

Background: Immune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in chronic obstructive pulmonary disease (COPD), thereby affecting immune cell responses., Methods: We analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMCs) isolated from healthy male young smokers as well as from patients with severe COPD and compared them with matching controls., Results: Proteasome expression was upregulated in COPD patients as assessed by quantitative reverse transcriptase-PCR and mass spectrometry-based proteomic analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modelling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro . Inhibition of the immunoproteasome reduced pro-inflammatory cytokine expression in COPD-derived blood immune cells., Conclusions: Given the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD., Competing Interests: Conflict of interest: I.E. Kammerl has nothing to disclose. Conflict of interest: S. Hardy has nothing to disclose. Conflict of interest: C. Flexeder has nothing to disclose. Conflict of interest: A. Urmann has nothing to disclose. Conflict of interest: J. Peierl has nothing to disclose. Conflict of interest: Y. Wang has nothing to disclose. Conflict of interest: O. Vosyka has nothing to disclose. Conflict of interest: M. Frankenberger has nothing to disclose. Conflict of interest: K. Milger reports personal fees for lectures from AstraZeneca, BerlinChemie, GSK, Novartis and Sanofi, outside the submitted work. Conflict of interest: J. Behr has nothing to disclose. Conflict of interest: A. Koch has nothing to disclose. Conflict of interest: J. Merl-Pham has nothing to disclose. Conflict of interest: S.M. Hauck has nothing to disclose. Conflict of interest: C. Pilette has nothing to disclose. Conflict of interest: H. Schulz has nothing to disclose. Conflict of interest: S. Meiners has nothing to disclose., (Copyright ©The authors 2022.)

Circulating immune cell populations have been shown to contribute to interstitial lung disease (ILD). In this study, we analysed circulating and lung resident monocyte populations, and assessed their phenotype and recruitment from the blood to the lung in ILD. Flow cytometry analysis of blood samples for quantifying circulating monocytes was performed in 105 subjects: 83 with ILD (n=36, n=28 and n=19 for nonspecific interstitial pneumonia, hypersensitivity pneumonitis and connective-tissue disease-associated ILD, respectively), as well as 22 controls. Monocyte localisation and abundance were assessed using immunofluorescence and flow cytometry of lung tissue. Monocyte populations were cultured either alone or with endothelial cells to assess fractalkine-dependent transmigration pattern. We show that circulating classical monocytes (CM) were increased in ILD compared with controls, while nonclassical monocytes (NCM) were decreased. CM abundance correlated inversely with lung function, while NCM abundance correlated positively. Both CCL2 and CX3CL1 concentrations were increased in plasma and lungs of ILD patients. Fractalkine co-localised with ciliated bronchial epithelial cells, thereby creating a chemoattractant gradient towards the lung. Fractalkine enhanced endothelial transmigration of NCM in ILD samples only. Immunofluorescence, as well as flow cytometry, showed an increased presence of NCM in fibrotic niches in ILD lungs. Moreover, NCM in the ILD lungs expressed increased CX3CR1, M2-like and phagocytic markers. Taken together, our data support that in ILD, fractalkine drives the migration of CX3CR1 + NCM to the lungs, thereby perpetuating the local fibrotic process., Competing Interests: Conflict of interest: V. Viteri-Alvarez has nothing to disclose. Conflict of interest: M. Frankenberger has nothing to disclose. Conflict of interest: D. Dietel has nothing to disclose. Conflict of interest: A. Ortega-Gomez has nothing to disclose. Conflict of interest: J.S. Lee reports grants from NIH, personal fees for advisory board work from Genentech, Celgene and Boehringer Ingelheim, outside the submitted work. Conflict of interest: A. Hilgendorff has nothing to disclose. Conflict of interest: J. Behr has nothing to disclose. Conflict of interest: O. Soehnlein has nothing to disclose. Conflict of interest: O. Eickelberg reports grants from Ministry of Research and Education Germany, during the conduct of the study; personal fees for advisory board work from Blade Therapeutics, Pieris and Boehringer Ingelheim, outside the submitted work. Conflict of interest: I.E. Fernandez has nothing to disclose. Conflict of interest: F.R. Greiffo has nothing to disclose., (Copyright ©ERS 2020.)