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.

Müller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Müller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Müller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Müller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Müller cell proteomes and secretomes after stimulation with INFγ, TNFα, IL-4, IL-6, IL-10, VEGF, TGFβ1, TGFβ2 and TGFβ3. We used both, primary porcine Müller cells and the human Müller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Müller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Müller cells resulted in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Müller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Müller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation.

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.

BackgroundVirus infections drive COPD exacerbations and progression. Anti-viral immunity centers on the activation of virus-specific CD8+T cells by viral epitopes presented on MHC class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialized intracellular protein degradation machine, which is induced by anti-viral cytokines in infected cells.MethodsWe here analysed the effects of CS 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 CS-exposed Influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of CS on inflammatory antigen presentation in lung cells. IAV-specific CD8+T cell numbers were determined in peripheral patients’ blood using tetramer-technology.ResultsCS impaired the induction of the immunoproteasome by cytokine signaling and viral infection in lung cellsin vitro,ex vivoandin vivo. CS also altered the peptide repertoire of antigens presented on MHC class I under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+T cells was dampened by CS. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+T cells compared to healthy controls and asthmatics.ConclusionOur 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.