The proteasome inhibitor PI31 competes with PA28 for binding to 20S proteasomes

AbstractPI31 is a previously described inhibitor of 20S proteasomes. Using recombinant PI31 we have analyzed its effect on proteasomal hydrolyzing activity of short fluorogenic substrates and of a synthetic 40-mer polypeptide. In addition, we investigated its influence on the activation of 20S proteasome by the proteasome activator PA28. PI31 inhibits polypeptide degradation already at concentrations which only partially inhibit fluorogenic substrate turnover and immunosubunits do not influence the PI31 binding affinity. Furthermore our data demonstrate that PI31 is a potent competitor of PA28-mediated activation

Hsp70 and nf-kb mediated control of innate inflammatory responses in a canine macrophage cell line

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.http://www.mdpi.com/journal/ijmspm2021Veterinary Tropical Disease

Dissecting antigen processing and presentation routes in dermal vaccination strategies

The skin is an attractive site for vaccination due to its accessibility and presence of immune cells surveilling this barrier. However, knowledge of antigen processing and presentation upon dermal vaccination is sparse. In this study we determined antigen processing routes that lead to CD8(+) T cell activation following dermal DNA tattoo immunization, exploiting a model antigen that contains an immunoproteasome-dependent epitope. In agreement with earlier reports, we found that DNA tattoo immunization of wild type (WT) mice triggered vigorous responses to the immunoproteasome-dependent model epitope, whereas gene-deficient mice lacking the immunoproteasome subunits β5i/LMP7 and β2i/MECL1 failed to respond. Unexpectedly, dermal immunization both of irradiated bone marrow (BM) reconstituted mice in which the BM transplant was of WT origin, and of WT mice transplanted with immunoproteasome subunit-deficient BM induced a CD8(+) T cell response to the immunoproteasome-dependent epitope, implying that both BM and host-derived cells contributed to processing of delivered model antigen. Depletion of radiation-resistant Langerhans cells (LC) from chimeric mice did not diminish tattoo-immunization induced CD8(+) T cell responses in most mice, illustrating that LC were not responsible for antigen processing and CD8(+) T cell priming in tattoo-immunized hosts. We conclude that both BM and non-BM-derived cells contribute to processing and cross-presentation of antigens delivered by dermal DNA tattoo immunization

Pre-existing virus-specific CD8+ T-cells provide protection against pneumovirus-induced disease in mice

Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8 + T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8 + T-cells expand relatively late. Induction of CD8 + T-cell memory against a single CD8 + T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8 + T-cells, covering the entire PVM-specific CD8 + T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8 + T-cells offer significant protection to PVM-induced disease. Thus, CD8 + T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine

Immunogenicity Testing of Lipidoids In Vitro and In Silico: Modulating Lipidoid-Mediated TLR4 Activation by Nanoparticle Design.

Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate murine antigen-presenting cells in vitro. This agonistic effect was further confirmed in silico using a prediction model based on crystal structures. Also, lipidoids formulated as lipoplexes or as stable nucleic acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation design

Basophil-derived Amphiregulin is essential for UVB irradiation-induced immune suppression

UVB irradiation (290–320nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell–derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell–mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin.A stimulation grant from the University of Utrecht and a grant from the Dutch Technology Foundation (STW-NWO).http://www.nature.com/jid/hb201

An unexpected major role for proteasome-catalyzed peptide splicing in generation of T cell epitopes: Is there relevance for vaccine development?

Efficient and safe induction of CD8 + T cell responses is a desired characteristic of vaccines against intracellular pathogens. To achieve this, a new generation of safe vaccines is being developed accommodating single, dominant antigens of pathogens of interest. In particular, the selection of such antigens is challenging, since due to HLA polymorphism the ligand specificities and immunodominance hierarchies of pathogen-specific CD8 + T cell responses differ throughout the human population. A recently discovered mechanism of proteasome-mediated CD8 + T cell epitope generation, i.e., by proteasome-catalyzed peptide splicing (PCPS), expands the pool of peptides and antigens, presented by MHC class I HLA molecules. On the cell surface, one-third of the presented self-peptides are generated by PCPS, which coincides with one-fourth in terms of abundance. Spliced epitopes are targeted by CD8 + T cell responses during infection and, like non-spliced epitopes, can be identified within antigen sequences using a novel in silico strategy. The existence of spliced epitopes, by enlarging the pool of peptides available for presentation by different HLA variants, opens new opportunities for immunotherapies and vaccine design