Role of Toll-like Receptors in Adjuvant-Augmented Immune Therapies

Effective therapeutic vaccines contain two primary constituents, antigen and adjuvant. Adjuvants consisting of microbial pattern molecules play a central role in vaccination. Successful vaccine requires efficient induction of antibody (Ab), type I interferons (IFN), cytokines/chemokines, cytotoxic T lymphocytes (CTL) and/or NK cells. Toll-like receptors (TLRs) in myeloid dendritic cells (mDC) essentially act as adjuvant receptors and sustain the molecular basis of adjuvant activity. Current consensus is that TLRs and their adapters introduce signals to preferentially induce IFN-α/β, chemokines and proinflammatory cytokines, and mature mDC to augment antigen presentation. Although most of these data were obtained with mice, the results are presumed to be adaptable to humans. Whenever TLR pathway is activated in mDC, NK and/or CTL activation is promoted. For induction of antigen-specific CTL toward phagocytosed material, cross-priming must be induced in mDC, which is also sustained by TLR signaling in mDC. Since the TLR responses vary with different adjuvants, mDC functions are skewed depending on adjuvant-specific direction of mDC maturation. It appears that the directed maturation of mDC largely relies on selection of appropriate sets of TLRs and their adapter signaling pathways. Synthetic chimera molecules consisting of TLR agonists and target antigens are found to be effective in induction of CTL to eliminate target cells in vivo. Here, we review the role of human TLRs and adapters in a variety of host immune responses. We will also describe the relevance of adjuvants in the manipulation of receptors and adapters in vaccine therapy

Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish

Nitro-fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities. We recently demonstrated that nitro-fatty acids activate the Keap1-Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear. A number of chemical compounds with different structures have been reported to activate the Keap1-Nrf2 system, which can be categorized into at least six classes based on their sensing pathways. In this study, we showed that nitro-oleic acid (OA-NO2), one of major nitro-fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators. In transgenic embryos, GFP was induced in the whole body by treatment with OA-NO2, 15d-PGJ2 or diethylmaleate (DEM), but not with hydrogen peroxide (H2O2), when exogenous Nrf2 and Keap1 were co-overexpressed. Induction by OA-NO2 or 15d-PGJ2 but not DEM was observed, even when a C151S mutation was introduced in Keap1. Our results support the contention that OA-NO2 and 15d-PGJ2 share an analogous cysteine code as electrophiles and also have similar anti-inflammatory roles

Tissue-Restricted Expression of Nrf2 and Its Target Genes in Zebrafish with Gene-Specific Variations in the Induction Profiles

The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2

Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction

International audienceDisruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone

The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis

Abstract The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired