Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach.

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Molecular characterization, redox regulation, and immune responses of monothiol and dithiol glutaredoxins from disk abalone (Haliotis discus discus).

Glutaredoxins (Grxs) are well-known oxidoreductases involved in a wide range of redox activities in organisms. In this study, two invertebrate Grxs (AbGrx1-like and AbGrx2) from disk abalone were identified and characterized in an effort to gain a deeper understanding into their immune and redox regulatory roles. Both AbGrxs share typical thioredoxin/Grx structures. AbGrx1-like and AbGrx2 were identified as monothiol and diothiol Grxs, respectively. AbGrxs were significantly expressed at the egg and 16-cell stage of early abalone development. Although the expression of both AbGrxs demonstrated similar patterns, the expression of AbGrx1-like was higher than AbGrx2 during development stages. In contrast, AbGrx2 expression was significantly higher than that of AbGrx1-like in adult tissues. Highest AbGrx1-like expression was observed in the hepatopancreas and digestive tract, while highest AbGrx2 expression was found in the gills, followed by the mantle, in healthy adult abalone tissues. The highest expression of AbGrx1-like was observed in the gills at 12 h and 6 h post injection (p.i) of Vibrio parahemolyticus and other stimulants, respectively. The highest expression of AbGrx2 in the gills were observed at 120 h, 6 h, 24 h, and 12 h post injection of V. parahaemolyticus, Listeria monocytogenes, Viral hemorrhagic septicemia virus, and Polyinosinic:polycytidylic acid, respectively. AbGrxs possessed significant 2-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) reduction activity, but AbGrx2 exhibited higher redox activity than AbGrx1-like. Altogether, our results suggest an important role of AbGrx1-like and AbGrx2 in redox homeostasis, as well as in the invertebrate immune defense system. Our findings will aid the development of new disease management strategies for this economically valuable species.

Glutaredoxin 2 from big belly seahorse (Hippocampus abdominalis) and its potential involvement in cellular redox homeostasis and host immune responses.

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H2O2-induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish.

Glutaredoxin 1 from big-belly seahorse (Hippocampus abdominalis): Molecular, transcriptional, and functional evidence in teleost immune responses.

Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 presented the highest homology to the Grx1 ortholog from Hippocampus comes. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 transcripts in all the seahorse tissues tested. Significant modulation (p < 0.05) of HaGrx1 transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The ß-hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1. HaGrx1 utilized 1,4-dithiothreitol, l-cysteine, 2-mercaptoethanol, and reduced l-glutathione as reducing agent with different dehydroascorbate reduction activity levels. Altogether, our results suggested a vital role of HaGrx1 in redox homeostasis as well as the host innate immune defense system.

The bacterial pigment pyocyanin inhibits the NLRP3 inflammasome through intracellular reactive oxygen and nitrogen species.

Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1ß and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 indicated that PCN provokes cytosolic and nuclear but not mitochondrial redox changes. PCN-induced intracellular ROS/RNS inhibited the NLRP3 inflammasome posttranslationally, and hydrogen peroxide or peroxynitrite alone were sufficient to block its activation. We propose that cytosolic ROS/RNS inhibit the NLRP3 inflammasome and that PCN's anti-inflammatory activity may help P. aeruginosa evade immune recognition.

ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation.

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein-deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species-regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.

Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils.

The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Activation of the glutaredoxin-1 gene by nuclear factor κB enhances signaling.

The transcription factor nuclear factor κB (NF-κB) is a critical regulator of inflammation and immunity and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyzes deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice that express a doxycycline-inducible constitutively active version of inhibitory κB kinase-ß (CA-IKKß) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKß also resulted in significant induction of Grx1. A 2-kb region of the Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKß, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression and time-dependent increases in S-glutathionylation of IKKß. Overexpression of Grx1 decreased S-glutathionylation of IKKß, prolonged NF-κB activation, and increased levels of proinflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB and suggests a feed-forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.

Redox atlas of the mouse. Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse.

BACKGROUND: Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available. METHODS: Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry. RESULTS: The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues. CONCLUSION: Our results imply more specific functions and interactions between the proteins of this family than previously assumed. GENERAL SIGNIFICANCE: Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases-the laboratory mouse. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.