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 immunoproteasomein vitro,ex vivoandin vivousing 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 cellsin vitro,ex vivoandin 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.

Retinal Müller glial cells (RMG) are involved in virtually every retinal disease; however, the role of these glial cells in neuroinflammation is still poorly understood. Since cell surface proteins play a decisive role in immune system signaling pathways, this study aimed at characterizing the changes of the cell surface proteome of RMG after incubation with prototype immune system stimulant lipopolysaccharide (LPS). While mass spectrometric analysis of the human Müller glia cell line MIO-M1 revealed 507 cell surface proteins in total, with 18 proteins significantly more abundant after stimulation (ratio ≥ 2), the surfaceome of primary RMG comprised 1425 proteins, among them 79 proteins with significantly higher abundance in the stimulated state. Pathway analysis revealed notable association with immune system pathways such as “antigen presentation”, “immunoregulatory interactions between a lymphoid and a non-lymphoid cell” and “cell migration”. We could demonstrate a higher abundance of proteins that are usually ascribed to antigen-presenting cells (APCs) and function to interact with T-cells, suggesting that activated RMG might act as atypical APCs in the course of ocular neuroinflammation. Our data provide a detailed description of the unstimulated and stimulated RMG surfaceome and offer fundamental insights regarding the capacity of RMG to actively participate in neuroinflammation in the retina.

There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

Retinal Müller glial cells (RMG) play a crucial role in retinal neuroinflammation, including autoimmune uveitis. Increasing evidence supports their function as active modulators of immune responses and potential atypical antigen-presenting cells (APCs). To further investigate this hypothesis, we conducted a differential proteome analysis of primary equine RMG from healthy controls and horses with equine recurrent uveitis (ERU), a spontaneous model of autoimmune uveitis. This analysis identified 310 proteins with differential abundance. Among these, the Major Histocompatibility Complex (MHC) class II and the enzyme Arginase 1 (ARG1) were significantly enriched in RMG from uveitis-affected horses, whereas Mannose Receptor C-type 2 (MRC2) and its interactor Thrombospondin 1 (THBS1) were more abundant in healthy RMG. The detection of MHC class II in equine RMG, consistent with previous studies, validates the robustness of our approach. Furthermore, the identification of ARG1 and MRC2, together with THBS1, provides new insights into the immunomodulatory and antigen-presenting properties of RMG. Immunohistochemical analyses confirmed the proteomic findings and revealed the spatial distribution of ARG1 and MRC2. ARG1 and MRC2 are thus markers for RMG in the neuroinflammatory or physiological milieu and highlight potential differences in the immune function of RMG, particularly in antigen presentation.

As part of the Pr55^Gag polyprotein, p6 fulfills an essential role in the late steps of the replication cycle. However, almost nothing is known about the functions of the mature HIV-1 p6 protein. Recently, we showed that p6 is a bona fide substrate of the insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease. This phenomenon appears to be specific for HIV-1, since p6 homologs of HIV-2, SIV and EIAV were IDE-insensitive. Furthermore, abrogation of the IDE-mediated degradation of p6 reduces the replication capacity of HIV-1 in an Env-dependent manner. However, it remained unclear to which extent the IDE mediated degradation is phylogenetically conserved among HIV-1. Here, we describe two HIV-1 isolates with IDE resistant p6 proteins. Sequence comparison allowed deducing one single amino acid regulating IDE sensitivity of p6. Exchanging the N-terminal leucine residue of p6 derived from the IDE sensitive isolate HIV-1_NL4-3 with proline enhances its stability, while replacing Pro-1 of p6 from the IDE insensitive isolate SG3 with leucine restores susceptibility towards IDE. Phylogenetic analyses of this natural polymorphism revealed that the N-terminal leucine is characteristic for p6 derived from HIV-1 group M except for subtype A, which predominantly expresses p6 with an N-terminal proline. Consequently, p6 peptides derived from subtype A are not degraded by IDE. Thus, IDE mediated degradation of p6 is specific for HIV-1 group M isolates and not occasionally distributed among HIV-1.

The majority of peptides presented by MHC class I result from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides to MHC class I loading non-canonically. We used an immunopeptidomics workflow combined with prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect the proteasomal processing of immune epitopes presented by A549 cells. The treatment of A549 cells with IFNγ enhanced the proteasomal cleavage probability of MHC class I ligands for both the constitutive proteasome and the immunoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated the HLA allotype-specific proteasomal cleavage probabilities for MHC class I ligands, the peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely, HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. The peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue, suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases.