Cells Stimulated with More Than One Toll-Like Receptor-Ligand in the Presence of a MyD88 Inhibitor Augmented Interferon-ß via MyD88-Independent Signaling Pathway.

Host exposure to pathogens engage multiple pathogen recognition receptors (PRRs) including toll-like receptors (TLRs); recruit intracellular signaling adaptor proteins primarily myeloid differentiation primary response protein 88 (MyD88) for activating downstream signaling cascades, which culminate in the production of type I interferons (IFNs), proinflammatory cytokines, and chemokines; and impede pathogen replication and dissemination. However, recent studies highlight that absence of MyD88 increased antiviral type I IFN induction, and MyD88-/- mice showed a higher survival rate compared with the low survival rate of the MyD88+/+ mice, implicating MyD88 limits antiviral type I IFN response. As a single infectious agent may harbor multiple PRR agonists, which trigger different sets of TLR-initiated immune signaling, we examined whether MyD88 inhibition during stimulation of cells with more than one TLR-ligand would augment type I IFN. We stimulated human U87- and TLR3-transfected HEK293-TLR7 cells with TLR-ligands, such as lipopolysaccharides (LPS) (TLR4-ligand) plus poly I:C (TLR3-ligand) or imiquimod (R837, TLR7-ligand) plus poly I:C, in the presence of compound 4210, a previously reported MyD88 inhibitor, and measured IFN-ß response using an enzyme-linked immunosorbent assay. Our results showed that when U87- or TLR3-transfected HEK293-TLR7 cells were stimulated with TLR-ligands, such as poly I:C plus LPS or poly I:C plus R837, IFN-ß production was significantly increased with MyD88 inhibition in a dose-dependent manner. Collectively, these results indicate that during more than one TLR-ligand-induced immune signaling event, impairment of antiviral type I IFN response was restored by inhibition of MyD88 through MyD88-independent pathway of type I IFN signaling, thus, offer a MyD88-targeted approach for type I IFN induction.

An increase in p62/NBR1 levels in melioidosis patients of Sri Lanka exhibit a characteristic of potential host biomarker.

Introduction. Melioidosis, caused by Burkholderia pseudomallei, in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei, host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim. We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology. We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results. Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions. ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.