Dengue virus NS1 protein activates immune cells via TLR4 but not TLR2 or TLR6

The secreted hexameric form of the dengue virus (DENV) non-structural protein 1 (NS1) has recently been shown to elicit inflammatory cytokine release and disrupt endothelial cell monolayer integrity. This suggests that circulating NS1 contributes to the vascular leak that plays a major role in the pathology of dengue haemorrhagic fever and shock. Pathways activated by NS1 are thus of great interest as potential therapeutic targets. Recent works have separately implicated both toll-like receptor 4 (TLR4) and the TLR2/6 heterodimer in immune cell activation by NS1. Here we have used mouse gene knockout macrophages and antibodies blocking TLR function in human peripheral blood mononuclear cells to show that recombinant NS1, expressed and purified from eukaryotic cells, induces cytokine production via TLR4 but not TLR2/6. Furthermore, the commercial Escherichia coli-derived recombinant NS1 preparation used in other work to implicate TLR2/6 in the response is not correctly folded and appears to be contaminated by several microbial TLR ligands. Thus TLR4 remains a therapeutic target for DENV infections, with TLR4 antagonists holding promise for the treatment of dengue disease

Systems serology detects functionally distinct coronavirus antibody features in children and elderly

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics

Subversion of innate defenses by the interplay between DENV and pre-existing enhancing antibodies: TLRs signaling collapse

Background: The phenomenon of antibody dependent enhancement as a major determinant that exacerbates disease severity in DENV infections is well accepted. While the detailed mechanism of antibody enhanced disease severity is unclear, evidence suggests that it is associated with both increased DENV infectivity and suppression of the type I IFN and pro-inflammatory cytokine responses. Therefore, it is imperative for us to understand the intracellular mechanisms altered during ADE infection to decipher the mechanism of severe pathogenesis. Methodology/Principal Findings:In this present work, qRT-PCR, immunoblotting and gene array analysis were conducted to determine whether DENV-antibody complex infection exerts a suppressive effect on the expression and/or function of the pathogen recognition patterns, focusing on the TLR-signaling pathway. We show here that FccRI and FccRIIa synergistically facilitated entry of DENV-antibody complexes into monocytic THP-1 cells. Ligation between DENV-antibody complexes and FcR not only down regulated TLRs gene expression but also up regulated SARM, TANK, and negative regulators of the NF-kB pathway, resulting in suppression of innate responses but increased viral production. These results were confirmed by blocking with anti-FcγRI or anti-FcγRIIa antibodies which reduced viral production, up-regulated IFN-β synthesis, and increased gene expression in the TLR-dependent signaling pathway. The negative impact of DENV-ADE infection on the TLR-dependent pathway was strongly supported by gene array screening which revealed that both MyD88-dependent and -independent signaling molecules were down regulated during DENV-ADE infection. Importantly, the same phenomenon was seen in PBMC of secondary DHF/DSS patients but not in PBMC of DF patients. Conclusions/Significance: Our present work demonstrates the mechanism by which DENV uses pre-existing immune mediators to defeat the principal activating pathway of innate defense resulting in suppression of an array of innate immune responses. Interestingly, this phenomenon specifically occurred during the severe form of DENV infection but not in the mild form of disease

The many faces of the flavivirus NS1 protein offer a multitude of options for inhibitor design

The flavivirus non-structural protein, NS1, is an unusual viral gene product. Despite the recent unveiling of its atomic structure (Akey et al., 2014), and a growing list of host molecules with which it has been found associated, the primary function of NS1 remains elusive. It assumes many diverse roles including direct participation in the flaviviral replication complex and virion maturation. In its secreted form it is a hexameric lipoparticle that is involved in systemic immune and endothelial cell modulation. In this review we highlight recent advances in elucidating the molecular mechanisms underpinning NS1 function and present the current state of play and some future prospects for NS1 targeted antiviral strategies. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research

Mechanisms of immune evasion induced by a complex of dengue virus and preexisting enhancing antibodies

We have found that dengue virus (DENV) not only uses preexisting enhancing antibodies to promote its entry into Fc receptor-bearing cells but also exploits enhancing antibodies for intracellular immune evasion through 2 mechanisms. In the first mechanism, entry of DENV-antibody complexes into human monocytic cells activates negative regulators, dihydroxyacetone kinase and autophagy-related 5-autophagy-related 12, which then disrupt the retinoic acide incucible gene I and melanoma differentiation associated gene 5 signaling cascade and disable type 1 interferon production, leading to suppression of interferon-mediated antiviral responses. In the second mechanism, the immune evasion was found to be mediated by the suppressive cytokine interleukin 10 (IL-10). High levels of IL-10 activated expression of suppressor of cytokine signaling 3 gene, which subsequently inactivated the Janus kinase-signal transducer and activator of transcription pathway. Inhibition of IL-10 production by small interfering RNA down-regulated suppressor of cytokine signaling 3 gene expression, restored inducible nitric oxide synthase gene expression, and suppressed DENV replication. Importantly, we were able to demonstrate that these 2 loops of suppression occurred in patients with severe secondary dengue infection (denguehemorrhagic fever) but not in patients with mild secondary dengue infection (dengue fever)

Osteoclastogenesis induced by CHIKV-infected fibroblast-like synoviocytes: a possible interplay between synoviocytes and monocytes/macrophages in CHIKV-induced arthralgia/arthritis

Fibroblast-like synoviocytes are known to migrate from joint to joint and are proposed to be one of the key players in the inflammatory cascade amplification in rheumatoid arthritis patients. In the recent CHIKV epidemic, patients developed arthritis-like syndrome and the synoviocyte is one of the suspected players in CHIKV-induced polyarthritis. Thus, to learn more on this syndrome, the responses of fibroblast-like synoviocytes to chikungunya virus (CHIKV) infection, and the interaction between CHIKV-infected synoviocytes and phagocytes, were investigated. Primary human fibroblast-like synoviocyte (HFLS) cultures were infected with clinical isolates of CHIKV at an MOI of 0.001pfu/cell. Data indicated that HFLS are permissive to CHIKV replication, generating peak titers of 10-10pfu/ml. Interestingly, CHIKV-infected HFLS cultures secreted mainly the mediators that are responsible for phagocytes recruitment and differentiation (RANKL, IL-6, IL-8 and MCP-1) but not arthritogenic mediators (TNF-α, IL-1β, MMP-1, MMP-2 or MMP-13). The interaction between CHIKV-infected synoviocytes and phagocytes was studied using UV-irradiated, CHIKV-infected HFLS supernatant. Data revealed that supernatants from CHIKV-infected HFLS cultures not only induced migration of primary human monocytes, but also drove monocytes/macrophages into osteoclast-like cells. These differentiated osteoclast-like cells produced high levels of TNF-α and IL-6, principal mediators of arthritis. This data suggests a potential interplay between infected HFLS and recruiting phagocytes which may responsible for the arthralgia/arthritis in CHIKV-infected patients