Search Results (11)

Hepatitis C virus (HCV) is a major public health concern, and the development of an effective HCV vaccine plays an important role in the effort to prevent new infections. Supramolecular co-assembly and co-presentation of the HCV envelope E1E2 heterodimer complex and core protein presents an attractive vaccine design strategy for achieving effective humoral and cellular immunity. With this objective, the two antigens were non-covalently assembled with an immunostimulant (TLR 7/8 agonist) into virus-mimicking polymer nanocomplexes (VMPNs) using a biodegradable synthetic polyphosphazene delivery vehicle. The resulting assemblies were characterized using dynamic light scattering and asymmetric flow field-flow fractionation methods and directly visualized in their vitrified state by cryogenic electron microscopy. The in vivo superiority of VMPNs over the individual components and an Alum-formulated vaccine manifests in higher neutralizing antibody titers, the promotion of a balanced IgG response, and the induction of a cellular immunity—CD4+ T cell responses to core proteins. The aqueous-based spontaneous co-assembly of antigens and immunopotentiating molecules enabled by a synthetic biodegradable carrier offers a simple and effective pathway to the development of polymer-based supramolecular nanovaccine systems. Full article

Introduction: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Methods: Gene expression of TLR1–10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Results: Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. Conclusions: The expression of TLR1–10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC. Full article

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV–1–derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV–1–derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases. Full article

The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides. Full article

While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases. Full article

Hybrid resequencing of the antibacterial innate immune genes coding for toll-like receptors, namely TLR1, TLR2, TLR4, TLR5, and TLR6, using HiSeq and PacBio technologies of pooled population samples of Czech Simmental (Czech Red) cattle allowed us to determine haplotypes formed by the polymorphisms present. Directly determined haplotypes within the range of the large proximal amplicon in TLR2 formed two clusters in the network tree graph. The distribution of the statistically reconstructed haplotypes based on individual genotyping of the present SNPs was consistent. Similarly, the statistically reconstructed haplotypes in TLR5 and TLR6 formed two clusters. The trend of bimodal distribution was also observed in TLR4, while the limited diversity of TLR1 did not allow for any conclusion. The observed bimodal distribution is consistent with earlier reports for cattle populations worldwide. The stability of this phenomenon cannot be ascribed to historical origin but rather to a long-term effect of balancing selection. The equilibrium might be based on two different essential functions performed by the TLR genes or their products. The formation of two kinds of heterodimers by the TLR2 product, namely, TLR2/TLR1 and TLR2/TLR6 with different ligand specificities, is considered to be a particular case. On the other hand, the better expression of the bimodal groups in the 5′-proximal SNPs supports the localization of the selection targets in the upstream regulatory regions or the functional interactions in the proximal part of the transcripts. Full article

The African Swine Fever Virus (ASFV) is an economically important, large DNA virus which causes a highly contagious and frequently fatal disease in domestic pigs. Due to the acute nature of the infection and the complexity of the protective porcine anti-ASFV response, there is no accepted vaccine in use. As resistance to ASFV is known to correlate with a robust IFN response, the virus is predicted to have evolved strategies to inhibit innate immunity by modulating the IFN response. The deletion of virus host evasion gene(s) inhibiting IFN is a logical solution to develop an attenuated virus vaccine. One such candidate, the ASFV ORF I329L gene, is highly conserved in pathogenic and non-pathogenic virus isolates and in this study we confirm and extend the conclusion that it has evolved for the inhibition of innate immunity initiated through Toll-like receptors (TLRs). Specifically, the ASFV I329L extracellular (ECD) and intracellular (ICD) domains inhibit TLR signalling by two entirely different mechanisms. Bioinformatics modelling suggests that the ECD inhibits several TLR signalling pathways through a short sequence homologous to the conserved TLR dimerization domain, here termed the putative dimerization domain (PDD). Remarkably, both full length and PDD constructs of I329L were demonstrated to inhibit activation, not only of TLR3, but also TLR4, TLR5, TLR8 and TLR9. Additionally, the demonstration of a weak association of I329L with TLR3 is consistent with the formation of a non-signalling I329L-TLR3 heterodimer, perhaps mediated through the PDD of I329L. Finally, the ICD associates with TRIF, thereby impacting on both TLR3 and TLR4 signalling. Thus, I329L offers potential as a general inhibitor of TLR responses and is a rational candidate for construction and testing of an I329L deletion mutant vaccine. Full article

Calprotectin, a heterodimer of S100A8 and S100A9 EF-hand calcium-binding proteins, is an integral part of the innate immune response. Calprotectin (CP) serves as a ligand for several pattern recognition cell surface receptors including the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and cluster of differentiation 33 (CD33). The receptors initiate kinase signaling cascades that activate inflammation through the NF-kB pathway. Receptor activation by CP leads to upregulation of both receptor and ligand, a positive feedback loop associated with specific chronic inflammatory syndromes. Hence, CP and its two constituent homodimers have been viewed as potential targets to suppress certain chronic inflammation pathologies. A variety of inhibitors of CP and other S100 proteins have been investigated for more than 30 years, but no candidates have advanced significantly into clinical trials. Here, current knowledge of the interactions of CP with its receptors is reviewed along with recent progress towards the development of CP-directed chemotherapeutics. Full article

Opioids, a kind of peptide hormone involved in the development of hypertension, cause systemic and cerebral inflammation, and affects regions of the brain that are important for blood pressure (BP) control. A cause-and-effect relationship exists between hypertension and inflammation; however, the role of blood pressure in cerebral inflammation is not clear. Evidence showed that AT1R and μOR heterodimers’ formation in the NTS might lead to the progression of hypertension. In this study, we investigated the formation of the μOR/AT1R heterodimer, determined its correlation with μORs level in the NTS, and explored the role of TLR4-dependent inflammation in the development of hypertension. Results showed that Ang II increased superoxide and Iba-1 (microgliosis marker: ionized calcium-binding adaptor molecule (1) levels in the NTS of spontaneously hypertensive rats (SHRs). The AT1R II inhibitor, losartan, significantly decreased BP and abolished superoxide, Iba-1, TLR4 expression induced by Ang II. Furthermore, losartan significantly increased nNsOS^S1416 phosphorylation. Administration of a μOR agonist or antagonist in the NTS of WKY and SHRs increased endogenous μ-opioids, triggered the formation of μOR/AT1R heterodimers and the TLR4-dependent inflammatory pathway, and attenuated the effect of depressor nitric oxide (NO). These results imply an important link between neurotoxicity and superoxides wherein abnormal increases in NTS endogenous μ-opioids promote the interaction between Ang II and μOR, the binding of Ang II to AT1R, and the activation of microglia. In addition, the interaction between Ang II and μOR enhanced the formation of the AT1R and μOR heterodimers, and inactivated nNOS-derived NO, leading to the development of progressive hypertension. Full article

The disruption of the immune tolerance induces autoimmunity such as systemic lupus erythematosus and vasculitis. A chromatin-binding non-histone protein, high mobility group box 1 (HMGB1), is released from the nucleus to the extracellular milieu in particular environments such as autoimmunity, sepsis and hypoxia. Extracellular HMGB1 engages pattern recognition receptors, including Toll-like receptors (TLRs) and the receptor for advanced glycation endproducts (RAGE). While the HMGB1-RAGE axis drives inflammation in various diseases, recent studies also focus on the anti-inflammatory effects of HMGB1 and RAGE. This review discusses current perspectives on HMGB1 and RAGE’s roles in controlling inflammation and immune tolerance. We also suggest how RAGE heterodimers responding microenvironments functions in immune responses. Full article

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB. Full article