Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage.

Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Antibody Profiling of Dengue Severities Using Flavivirus Protein Microarrays.

Dengue is a viral disease transmitted by Aedes aegypti mosquitoes. According to the World Health Organization, about half of the world's population is at risk of dengue. There are four serotypes of the dengue virus. After infection with one serotype, it will be immune to such a serotype. However, subsequent infection with other serotypes will increase the risk of severe outcomes, e.g., dengue hemorrhagic fever, dengue shock syndrome, and even death. Since severe dengue is challenging to predict and lacks molecular markers, we aim to build a multiplexed Flavivirus protein microarray (Flaviarray) that includes all of the common Flaviviruses to profile the humoral immunity and cross-reactivity in the dengue patients with different outcomes. The Flaviarrays we fabricated contained 17 Flavivirus antigens with high reproducibility (R-square = 0.96) and low detection limits (172-214 pg). We collected serums from healthy subjects (n = 36) and dengue patients within 7 days after symptom onset (mild dengue (n = 21), hospitalized nonsevere dengue (n = 29), and severe dengue (n = 36)). After profiling the serum antibodies using Flaviarrays, we found that patients with severe dengue showed higher IgG levels against multiple Flavivirus antigens. With logistic regression, we found groups of markers with high performance in distinguishing dengue patients from healthy controls as well as hospitalized from mild cases (AUC > 0.9). We further reported some single markers that were suitable to separate dengue patients from healthy controls (AUC > 0.9) and hospitalized from mild outcomes (AUC > 0.8). Together, Flaviarray is a valuable tool to profile antibody specificities, uncover novel markers for decision-making, and shed some light on early preventions and treatments.

Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication.

Dengue virus (DENV) infection can induce life-threatening dengue hemorrhagic fever/dengue shock syndrome in infected patients. DENV is a threat to global health due to its growing numbers and incidence of infection in the last 50 years. During infection, DENV expresses ten structural and nonstructural proteins modulating cell responses to benefit viral replication. However, the lack of knowledge regarding the cellular proteins and their functions in enhancing DENV pathogenesis impedes the development of antiviral drugs and therapies against fatal DENV infection. Here, we identified that integrin-linked kinase (ILK) is a novel enhancing factor for DENV infection by suppressing type I interferon (IFN) responses. Mechanistically, ILK binds DENV NS1 and NS3, activates Akt and Erk, and induces NF-κB-driven suppressor of cytokine signaling 3 (SOCS3) expression. Elevated SOCS3 in DENV-infected cells inhibits phosphorylation of STAT1/2 and expression of interferon-stimulated genes (ISGs). Inhibiting ILK, Akt, or Erk activation abrogates SOCS3 expression. In DENV-infected mice, the treatment of an ILK inhibitor significantly reduces viral loads in the brains, disease severity, and mortality rate. Collectively, our results show that ILK is a potential therapeutic target against DENV infection.

Antibodies against the SARS-CoV-2 S1-RBD cross-react with dengue virus and hinder dengue pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally since December 2019. Several studies reported that SARS-CoV-2 infections may produce false-positive reactions in dengue virus (DENV) serology tests and vice versa. However, it remains unclear whether SARS-CoV-2 and DENV cross-reactive antibodies provide cross-protection against each disease or promote disease severity. In this study, we confirmed that antibodies against the SARS-CoV-2 spike protein and its receptor-binding domain (S1-RBD) were significantly increased in dengue patients compared to normal controls. In addition, anti-S1-RBD IgG purified from S1-RBD hyperimmune rabbit sera could cross-react with both DENV envelope protein (E) and nonstructural protein 1 (NS1). The potential epitopes of DENV E and NS1 recognized by these antibodies were identified by a phage-displayed random peptide library. In addition, DENV infection and DENV NS1-induced endothelial hyperpermeability in vitro were inhibited in the presence of anti-S1-RBD IgG. Passive transfer anti-S1-RBD IgG into mice also reduced prolonged bleeding time and decreased NS1 seral level in DENV-infected mice. Lastly, COVID-19 patients' sera showed neutralizing ability against dengue infection in vitro. Thus, our results suggest that the antigenic cross-reactivity between the SARS-CoV-2 S1-RBD and DENV can induce the production of anti-SARS-CoV-2 S1-RBD antibodies that cross-react with DENV which may hinder dengue pathogenesis.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein.

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.

Regulation of innate immune signaling pathways by autophagy in dengue virus infection.

Autophagy is not only an intracellular recycling degradation system that maintains cellular homeostasis but is also a component of innate immunity that contributes to host defense against viral infection. The viral components as well as viral particles trapped in autophagosomes can be delivered to lysosomes for degradation. Abundant evidence indicates that dengue virus (DENV) has evolved the potent ability to hijack or subvert autophagy process for escaping host immunity and promoting viral replication. Moreover, autophagy is often required to deliver viral components to pattern recognition receptors signaling for interferon (IFN)-mediated viral elimination. Hence, this review summarizes DENV-induced autophagy, which exhibits dual effects on proviral activity of promoting replication and antiviral activity to eliminating viral particles.

Overcoming interferon (IFN)-γ resistance ameliorates transforming growth factor (TGF)-ß-mediated lung fibroblast-to-myofibroblast transition and bleomycin-induced pulmonary fibrosis.

Abnormal activation of transforming growth factor (TGF)-ß is a common cause of fibroblast activation and fibrosis. In bleomycin (BLM)-induced lung fibrosis, the marked expression of phospho-Src homology-2 domain-containing phosphatase (SHP) 2, phospho-signal transducer and activator of transcription (STAT) 3, and suppressor of cytokine signaling (SOCS) 3 was highly associated with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts exhibited activation of SHP2, SOCS3, protein inhibitor of activated STAT1, STAT3, interleukin (IL)-6, and IL-10. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decreased expression of phospho-STAT1, IFN-γ-associated genes, and IFN-γ-inducible protein (IP) 10. Microarray analysis showed an induction of fibrotic genes in TGF-ß1-differentiated myofibroblasts, whereas IFN-γ-regulated anti-fibrotic genes were suppressed. Interestingly, BIBF 1120 treatment effectively inhibited both STAT3 and SHP2 phosphorylation in TGF-ß1-differentiated myofibroblasts and BLM fibrotic lung tissues, which was accompanied by suppression of fibroblast-myofibroblast transition. Moreover, the combined treatment of BIBF 1120 plus IFN-γ or SHP2 inhibitor PHPS1 plus IFN-γ markedly reduced TGF-ß1-induced α-smooth muscle actin and further ameliorated BLM lung fibrosis. Accordingly, myofibroblasts were hyporesponsiveness to IFN-γ, while blockade of SHP2 contributed to the anti-fibrotic efficacy of IFN-γ.

Dengue Nonstructural Protein 1 Maintains Autophagy through Retarding Caspase-Mediated Cleavage of Beclin-1.

Dengue virus (DENV) infection is a significant public health threat in tropical and subtropical regions; however, there is no specific antiviral drug. Accumulated studies have revealed that DENV infection induces several cellular responses, including autophagy and apoptosis. The crosstalk between autophagy and apoptosis is associated with the interactions among components of these two pathways, such as apoptotic caspase-mediated cleavage of autophagy-related proteins. Here, we show that DENV-induced autophagy inhibits early cell apoptosis and hence enhances DENV replication. Later, the apoptotic activities are elevated to suppress autophagy through cleavage of Beclin-1, an essential autophagy-related protein. Inhibition of cleavage of Beclin-1 by a pan-caspase inhibitor, Z-VAD, increases both autophagy and viral replication. Regarding the mechanism, we further found that DENV nonstructural protein 1 (NS1) is able to interact with Beclin-1 during DENV infection. The interaction between Beclin-1 and NS1 attenuates Beclin-1 cleavage and facilitates autophagy to prevent cell apoptosis. Our study suggests a novel mechanism whereby NS1 preserves Beclin-1 for maintaining autophagy to antagonize early cell apoptosis; however, elevated caspases trigger apoptosis by degrading Beclin-1 in the late stage of infection. These findings suggest implications for anti-DENV drug design.

HECT E3 Ubiquitin Ligase-Regulated Txnip Degradation Facilitates TLR2-Mediated Inflammation During Group A Streptococcal Infection.

Thioredoxin-interacting protein (Txnip) inhibits the activity of thioredoxin (Trx) to modulate inflammatory responses. The burden of inflammation caused by microbial infection is strongly associated with disease severity; however, the role of Txnip in bacterial infection remains unclear. In Group A Streptococcus (GAS)-infected macrophages, Txnip was degraded independent of glucose consumption and streptococcal cysteine protease expression. Treatment with proteasome inhibitors reversed GAS-induced Txnip degradation. The activation of Toll-like receptor 2 (TLR2) initiated Txnip degradation, while no further Txnip degradation was observed in TLR2-deficient bone marrow-derived macrophages. NADPH oxidase-regulated NF-κB activation and pro-inflammatory activation were induced and accompanied by Txnip degradation during GAS infection. Silencing Txnip prompted TLR2-mediated inducible nitric oxide synthase (iNOS)/NO, TNF-α, and IL-6 production whereas the blockage of Txnip degradation by pharmacologically inhibiting the HECT E3 ubiquitin ligase with heclin and AMP-dependent protein kinase with dorsomorphin effectively reduced such effects. Our findings reveal that TLR2/NADPH oxidase-mediated Txnip proteasomal degradation facilitates pro-inflammatory cytokine production during GAS infection.

Combination of Modified NS1 and NS3 as a Novel Vaccine Strategy against Dengue Virus Infection.

Dengue virus (DENV) causes a range of illness, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. DENV nonstructural protein (NS) 1 has been considered to be a desirable vaccine candidate for its ability to induce Ab and complement-dependent cytolysis of DENV-infected cells as well as to block the pathogenic effects of NS1. However a potential drawback of NS1 as a vaccine is that anti-DENV NS1 Abs can lead to endothelial cell damage and platelet dysfunction by antigenic cross-reactivity. Therefore, we modified the DENV NS1 by replacing the C-terminal cross-reactive epitopes with the corresponding region of Japanese encephalitis virus NS1 to generate a chimeric DJ NS1 protein. Active immunization with DJ NS1 induced a strong Ab response. To enhance cellular immunity, we further combined DJ NS1 with DENV NS3 to immunize mice and showed activation of Ag-specific CD4+ and CD8+ T cells in addition to Ab responses. We further detected NS3-specific CTL activities as well as CD107a expression of effector cells. Importantly, the protective effects attributed by DJ NS1 and NS3 immunization were demonstrated in a DENV-infected mouse model by reduced viral titers, soluble NS1 levels, mouse tail bleeding time, and vascular leakage at skin injection sites. Collectively, the results from this study reveal the humoral and cellular immune responses and the protective effects conferred by DJ NS1 and NS3 immunization in the mouse model of DENV infection and provide a potential strategy for dengue vaccine design.

The monocyte-macrophage-mast cell axis in dengue pathogenesis.

Dengue virus, the causative agent of dengue disease which may have hemorrhagic complications, poses a global health threat. Among the numerous target cells for dengue virus in humans are monocytes, macrophages and mast cells which are important regulators of vascular integrity and which undergo dramatic cellular responses after infection by dengue virus. The strategic locations of these three cell types, inside blood vessels (monocytes) or outside blood vessels (macrophages and mast cells) allow them to respond to dengue virus infection with the production of both intracellular and secretory factors which affect virus replication, vascular permeability and/or leukocyte extravasation. Moreover, the expression of Fc receptors on the surface of monocytes, macrophages and mast cells makes them important target cells for antibody-enhanced dengue virus infection which is a major risk factor for severe dengue disease, involving hemorrhage. Collectively, these features of monocytes, macrophages and mast cells contribute to both beneficial and harmful responses of importance to understanding and controlling dengue infection and disease.

Sodium Hyaluronate/Chitosan Composite Microneedles as a Single-Dose Intradermal Immunization System.

Enhancing the immune response to vaccines and minimizing the need for repeated inoculations remain a challenge in clinical vaccination. This study developed a composite microneedle (MN), composed of a sodium hyaluronate (HA) tip and a chitosan base, for biphasic antigen release and evaluated the potential of using this MN formulation as an intradermal delivery system for single-dose vaccination. Upon skin insertion, the dissolvable HA tip dissolved within the skin for rapid release of the encapsulated antigens, thus priming the immune system, while the biodegradable chitosan base remained in the dermis for prolonged antigen release for 4 weeks, thus further boosting the stimulated immunity. Our results showed that a single immunization with the HA/chitosan MN containing ovalbumin (OVA) (100 µg × 1) stimulated both T helper type 1 (Th1) and Th2 immune responses in rats and induced considerably higher and more durable antibody responses than a traditional two-dose (100 µg OVA × 2) or double-dose (200 µg OVA × 1) subcutaneous vaccination. Thus, the proposed MN exerts strong adjuvanticity to greatly augment the antigen's immunogenicity. Moreover, given its unique rapid and sustained release properties, the HA/chitosan MN formulation has the potential to replace the conventional prime-boost regimen to serve as an effective single-dose vaccine formulation.

Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection.

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease.

Resveratrol treatment reveals a novel role for HMGB1 in regulation of the type 1 interferon response in dengue virus infection.

Dengue is one of the most significant mosquito-borne virus diseases worldwide, particularly in tropical and subtropical regions. This study sought to examine the antiviral activity of resveratrol (RESV), a phytoalexin secreted naturally by plants, against dengue virus (DENV) infection. Our data showed that RESV inhibits the translocation of high mobility group box 1 (HMGB1), a DNA binding protein that normally resides in the nucleus, into the cytoplasm and extracellular milieu. HMGB1 migrates out of the nucleus during DENV infection. This migration is inhibited by RESV treatment and is mediated by induction of Sirt1 which leads to the retention of HMGB1 in the nucleus and consequently helps in the increased production of interferon-stimulated genes (ISGs). Nuclear HMGB1 was found to bind to the promoter region of the ISG and positively regulated the expression of ISG. The enhanced transcription of ISGs by nuclear HMGB1 thus contributes to the antiviral activity of RESV against DENV. To the best of our knowledge, this is the first report to demonstrate that RESV antagonizes DENV replication and that nuclear HMGB1 plays a role in regulating ISG production.

Antibodies against nonstructural protein 1 protect mice from dengue virus-induced mast cell activation.

Dengue virus (DENV) infection causes dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). DHF/DSS patients have been reported to have increased levels of urinary histamine, chymase, and tryptase, which are major granule-associated mediators from mast cells. Previous studies also showed that DENV-infected human mast cells induce production of proinflammatory cytokines and chemokines, suggesting a role played by mast cells in vascular perturbation as well as leukocyte recruitment. In this study, we show that DENV but not UV-inactivated DENV enhanced degranulation of mast cells and production of chemokines (MCP-1, RANTES, and IP-10) in a mouse model. We have previously shown that antibodies (Abs) against a modified DENV nonstructural protein 1 (NS1), designated DJ NS1, provide protection in mice against DENV challenge. In the present study, we investigate the effects of DJ NS1 Abs on mast cell-associated activities. We showed that administration of anti-DJ NS1 Abs into mice resulted in a reduction of mast cell degranulation and macrophage infiltration at local skin DENV infection sites. The production of DENV-induced chemokines (MCP-1, RANTES, and IP-10) and the percentages of tryptase-positive activated mast cells were also reduced by treatment with anti-DJ NS1 Abs. These results indicate that Abs against NS1 protein provide multiple therapeutic benefits, some of which involve modulating DENV-induced mast cell activation.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.10.

Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages.

Thrombocytopenia is an important clinical manifestation of dengue disease. The hypotheses concerning the pathogenesis of thrombocytopenia include decreased production and increased destruction or consumption of platelets. We previously suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DENV) nonstructural protein 1 (NS1) cross-react with platelets. Furthermore, several lines of evidence show activation of endothelial cells (ECs) and macrophages are related to dengue disease severity. Previous studies also suggested that Ab-opsonised platelets facilitate the engulfment of platelets by macrophages. Here we show that TNF-α-activated ECs upregulate adhesion molecule expression to enhance the binding of platelets and macrophages and lead to anti-DENV NS1 Ab-mediated platelet phagocytosis. We further demonstrate that the interaction between macrophages and TNF-α-activated ECs requires binding of FcγR with the Fc region of platelet-bound anti-DENV NS1 Abs. Importantly, the binding of anti-DENV NS1 Abs to platelets did not interfere with platelet adhesion to ECs. The adhesion molecules ICAM-1 and ß3 integrin expressed on ECs as well as the FcγR expressed on macrophages were critical in anti-DENV NS1 Ab-mediated platelet phagocytosis on activated ECs. Moreover, anti-DENV NS1 Abs dramatically enhanced platelet engulfment by macrophages in a murine model of DENV infection. Our study provides evidence for a novel role for anti-DENV NS1 Abs in the pathogenesis of thrombocytopenia in dengue disease by enhancing platelet phagocytosis by macrophages.

Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

Mast cell-macrophage dynamics in modulation of dengue virus infection in skin.

Dengue virus (DENV) infection causes dengue fever, dengue haemorrhagic fever, or dengue shock syndrome. Mast cells have been speculated to play a role in DENV disease although their precise roles are unclear. In this study, we used mast cell-deficient Kit(W-sh/W-sh) mice to investigate the involvement of mast cells after intradermal DENV infection. An approximately two- to three-fold higher level of DENV NS3 antigen was detected at the skin inoculation site in DENV-infected Kit(W-sh/W-sh) mice than in DENV-infected wild-type (WT) mice (using a dose of 1 × 10(9) plaque-forming units/mouse). Moreover, as an indicator of heightened pathogenesis, a more prolonged bleeding time was observed in DENV-infected Kit(W-sh/W-sh) mice than in WT mice. Monocytes/macrophages are considered to be important targets for DENV infection, so we investigated the susceptibility and chemokine response of DENV-infected peritoneal macrophages from Kit(W-sh/W-sh) and WT mice both ex vivo and in vivo. There was a tendency for higher DENV infection and higher secretion of CCL2 (MCP-1) from peritoneal macrophages isolated from Kit(W-sh/W-sh) mice than those from WT mice. In vivo studies using intradermal inoculation of DENV showed about twofold higher levels of infiltrating macrophages and CCL2 (MCP-1) at the inoculation site in both mock control and DENV-inoculated Kit(W-sh/W-sh) mice than in corresponding WT mice. In summary, compared with WT mice, Kit(W-sh/W-sh) mice show enhanced DENV infection and macrophage infiltration at the skin inoculation site as well as increased DENV-associated bleeding time. The results indicate an intriguing interplay between mast cells and tissue macrophages to restrict DENV replication in the skin.

Correlation between serum levels of anti-endothelial cell autoantigen and anti-dengue virus nonstructural protein 1 antibodies in dengue patients.

We have previously shown that anti-dengue virus nonstructural protein 1 (anti-DENV NS1) antibodies cross-react with endothelial cells, and several autoantigens have been identified. This study shows that the antibody levels against these self-proteins are higher in sera from patients with dengue hemorrhagic fever (DHF) than those in control sera. Anti-protein disulfide isomerase (PDI) and anti-heat shock protein 60 (anti-HSP60) IgM levels correlated with both anti-endothelial cells and anti-DENV NS1 IgM titers. A cross-reactive epitope on the NS1 amino acid residues 311-330 (P311-330) had been predicted. We further found that there were higher IgM and IgG levels against P311-330 in DHF patients' sera than those in the control sera. In addition, correlations were observed between anti-PDI with anti-P311-330 IgM and IgG levels, respectively. Therefore, our results indicate that DENV NS1 P311-330 is a major epitope for cross-reactive antibodies to PDI on the endothelial cell surface, which may play an important role in DENV infection-induced autoimmunity.