Search

Opinion statementDengue viruses (DENV) continue to circulate worldwide, resulting in a significant burden on human health. There are four antigenically distinct serotypes of DENV, an infection of which could result in a potentially life-threatening disease. Current treatment options are limited and rely on supportive care. Although one dengue vaccine is approved for dengue-immune individuals and has modest efficacy, there is still a need for therapeutics and vaccines that can reduce dengue morbidities and lower the infection burden. There have been recent advances in the development of promising drugs for the treatment of dengue. These include direct antivirals that can reduce virus replication as well as host-targeted drugs for reducing inflammation and/or vascular pathologies. There are also new vaccine candidates that are being evaluated for their safety and efficacy in preventing dengue disease. This review highlights nuances in the current standard-of-care treatment of dengue. We also discuss emerging treatment options, therapeutic drugs, and vaccines that are currently being pursued at various stages of preclinical and clinical development.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named “Modulator of cytochrome C oxidase during Inflammation” (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation., Mito-SEPs are small peptides that can modulate oxidative metabolism in mitochondria. Here the authors show that C15ORF48 encodes a mito-SEP, MOCCI, capable of altering mitochondria respiration to suppress inflammation, while C15ORF48 3’ untranslated region also contains a miRNA, miR-147b, that synergizes with MOCCI to modulate host anti-viral responses.

Coronavirus disease-2019 (COVID-19) is caused by the newly emerged virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was recently declared as a pandemic by the World Health Organization. In its severe form, the disease is characterized by acute respiratory distress syndrome, and there are no targeted intervention strategies to treat or prevent it. The immune response is thought to both contribute to the pathogenesis of disease and provide protection during its resolution. Thus, understanding the immune response to SARS-CoV-2 is of the utmost importance for developing and testing vaccines and therapeutics. In this review, we discuss the earliest knowledge and hypotheses of the mechanisms of immune pathology in the lung during acute infection as well at the later stages of disease resolution, recovery, and immune memory formation.

Dengue induces a spectrum of severity in humans from the milder dengue fever to severe disease, or dengue hemorrhagic fever (DHF). Chymase is a candidate biomarker that may aid dengue prognosis. This prospective study aimed to identify whether warning signs of severe dengue, including hypovolemia and fluid accumulation, were associated with elevated chymase. Serum chymase levels were quantified prospectively and longitudinally in hospitalized pediatric dengue patients in Sri Lanka. Warning signs were determined based on daily clinical assessments, laboratory tests and ultrasound findings. Chymase was significantly elevated during the acute phase of disease in DHF or Severe dengue, defined by either the 1997 or 2009 WHO diagnosis guidelines, and persisted longer in the most severe patients. Chymase levels were higher in patients with narrow pulse pressure and clinical warning signs such as severe leakage, fluid accumulation, pleural effusion, gall-bladder wall thickening and rapid haematocrit rise concurrent with thrombocytopenia. No association between chymase and liver enlargement was observed. This study confirms that serum chymase levels are associated with DHF/Severe dengue disease in hospitalized pediatric patients. Chymase levels correlate with warning signs of vascular dysfunction highlighting the possible functional role of chymase in vascular leakage during dengue. Nanyang Technological University Singapore Infectious Diseases Initiative - National Medical Research Council Published version The clinical study was funded by the Lee Kong Chian School of Medicine (A. Wilder-Smith`s start-up grant), monitoring of the study was funded by a global health grant from the Singapore Infectious Diseases Initiative (to Wei-Yee Leong). The laboratory and data analyses were funded by Duke-NUS Start-up funding to Ashley St. John.

Significance Because dengue viruses are spread by mosquitoes during biting, transmission capacity depends on mosquito-biting behavior. For this reason, it is critical to understand how infection in mosquitoes influences biting. To answer this question, we deployed a multidisciplinary approach including high-resolution, multivariate biting behavior monitoring on mice, in vivo transmission assay, and mathematical modeling. We demonstrated that infected mosquitoes are more attracted to mice and bite more often to get the same amount of blood as uninfected mosquitoes. While the effect of increased attraction to host on transmission capacity is trivial, we showed that increased number of bites results in successive transmission. Eventually, we calculated that the infection-induced behavior changes tripled transmission capacity of mosquitoes., Mosquito blood-feeding behavior is a key determinant of the epidemiology of dengue viruses (DENV), the most-prevalent mosquito-borne viruses. However, despite its importance, how DENV infection influences mosquito blood-feeding and, consequently, transmission remains unclear. Here, we developed a high-resolution, video-based assay to observe the blood-feeding behavior of Aedes aegypti mosquitoes on mice. We then applied multivariate analysis on the high-throughput, unbiased data generated from the assay to ordinate behavioral parameters into complex behaviors. We showed that DENV infection increases mosquito attraction to the host and hinders its biting efficiency, the latter resulting in the infected mosquitoes biting more to reach similar blood repletion as uninfected mosquitoes. To examine how increased biting influences DENV transmission to the host, we established an in vivo transmission model with immuno-competent mice and demonstrated that successive short probes result in multiple transmissions. Finally, to determine how DENV-induced alterations of host-seeking and biting behaviors influence dengue epidemiology, we integrated the behavioral data within a mathematical model. We calculated that the number of infected hosts per infected mosquito, as determined by the reproduction rate, tripled when mosquito behavior was influenced by DENV infection. Taken together, this multidisciplinary study details how DENV infection modulates mosquito blood-feeding behavior to increase vector capacity, proportionally aggravating DENV epidemiology. By elucidating the contribution of mosquito behavioral alterations on DENV transmission to the host, these results will inform epidemiological modeling to tailor improved interventions against dengue.

Cocaine is one of the most potent and addictive psychostimulants known and there are no available pharmacotherapies to treat cocaine addiction. Here we describe a novel cocaine vaccine employing the mucosal adjuvant and mast cell-activating oligopeptide, mastoparan-7 (M7), to achieve optimal IgA antibody responses in mucosal secretions and effective induction of humoral immunity using a short immunization protocol. This formulation, using a hapten-carrier system to deliver cocaine as antigen, also reduced cocaine penetration of the blood brain barrier and protected mice from its psychoactive effects by reducing cocaine-induced locomotion. Surprisingly, the magnitude of cocaine-specific antibody titers induced by each adjuvant was not the major determinant of functional protection from cocaine challenge. A side-by-side comparison of the two haptens, cocaine and its analog GNC demonstrated that cocaine haptenation resulted in superior functional protection when used in combination with the novel mucosal adjuvant, M7. These results provide a new potential strategy for combatting cocaine addiction through mucosal vaccination.

Dengue virus (DENV) is the most prevalent vector-borne viral pathogen, infecting millions of patients annually. Thrombocytopenia, a reduction in circulating platelet counts, is the most consistent sign of DENV-induced disease, independent of disease severity. However, the mechanisms leading to DENV-induced thrombocytopenia are unknown. Here, we show that thrombocytopenia is caused by serotonin derived from mast cells (MCs), which are immune cells that are present in the perivascular space and are a major peripheral source of serotonin. We show that during DENV infection, MCs release serotonin, which prompts platelet activation, aggregation, and enhanced phagocytosis, dependent on 5HT2A receptors. MC deficiency in mice or pharmacologic inhibition of MCs reversed thrombocytopenia. Furthermore, reconstitution of MC-deficient mice with wild-type MCs, but not MCs lacking serotonin synthesis resulting from deficiency in the enzyme tryptophan hydroxylase-1, restored the thrombocytopenic phenotype. Exogenous serotonin was also sufficient to overcome the effects of drugs that inhibit platelet activation in vitro and to restore thrombocytopenia in DENV-infected MC-deficient mice. Therapeutic targeting of 5HT2A receptors during DENV infection effectively prevented thrombocytopenia in mice. Similarly, serotonin derived from DENV-activated human MCs led to increased human platelet activation. Thus, MC-derived serotonin is a previously unidentified mechanism of DENV-induced thrombocytopenia and a potential therapeutic target.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.

Sub-neutralizing concentrations of antibodies in dengue infected patients is a major risk factor for the development of dengue hemorrhagic fever and dengue shock syndrome. Here, we describe a mouse model with a deficiency in mast cells (MCs) in addition to a deficiency in Type-I and II IFN receptors for studying dengue virus (DENV) infection. We used this model to understand the influence of MCs in a maternal antibody-dependent model of severe dengue, where offspring born to DENV-immune mothers are challenged with a heterologous DENV serotype. Mice lacking both MCs and IFN receptors were found susceptible to primary DENV infection and showed morbidity and mortality. When these mice were immunized, pups born to DENV-immune mothers were found to be protected for a longer duration from a heterologous DENV challenge. In the absence of MCs and type-I interferon signaling, IFN-γ was found to protect pups born to naïve mothers but had the opposite effect on pups born to DENV-immune mothers. Our results highlight the complex interactions between MCs and IFN-signaling in influencing the role of maternal antibodies in DENV-induced disease severity.

ELKS1 controls mast cell degranulation by regulating the transcription of Stxbp2 and Syntaxin 4 via Kdm2b stabilization., ELKS1 is a protein with proposed roles in regulated exocytosis in neurons and nuclear factor κB (NF-κB) signaling in cancer cells. However, how these two potential roles come together under physiological settings remain unknown. Since both regulated exocytosis and NF-κB signaling are determinants of mast cell (MC) functions, we generated mice lacking ELKS1 in connective tissue MCs (Elks1f/f Mcpt5-Cre) and found that while ELKS1 is dispensable for NF-κB–mediated cytokine production, it is essential for MC degranulation both in vivo and in vitro. Impaired degranulation was caused by reduced transcription of Syntaxin 4 (STX4) and Syntaxin binding protein 2 (Stxpb2), resulting from a lack of ELKS1-mediated stabilization of lysine-specific demethylase 2B (Kdm2b), which is an essential regulator of STX4 and Stxbp2 transcription. These results suggest a transcriptional role for active-zone proteins like ELKS1 and suggest that they may regulate exocytosis through a novel mechanism involving transcription of key exocytosis proteins.

Currently there are no specific treatments available for acute dengue infection. We considered that rupatadine, a platelet-activating factor receptor inhibitor, might modulate dengue-associated vascular leak. The effects of rupatadine were assessed in vitro, and in a dengue model, which showed that rupatadine significantly reduced endothelial permeability by dengue sera in vitro, and significantly inhibited the increased haematocrit in dengue-infected mice with dose-dependency. We conducted a randomised, placebo-controlled trial in 183 adult patients in Sri Lanka with acute dengue, which showed that rupatadine up to 40 mg daily appeared safe and well-tolerated with similar proportions of adverse events with rupatadine and placebo. Although the primary end-point of a significant reduction in fluid leakage (development of pleural effusions or ascites) was not met, post-hoc analyses revealed small but significant differences in several parameters on individual illness days - higher platelet counts and lower aspartate-aminotransferase levels on day 7 in the rupatadine group compared to the placebo group, and smaller effusions on day 8 in the subgroup of patients with pleural effusions. However, due to the small sample size and range of recruitment time, the potential beneficial effects of rupatadine require further evaluation in large studies focused on recruitment during the early febrile phase.

Highlights • At the host-environment interfaces such as skin or mucosae mast cells act as immune sentinels for variety of pathogens including viruses. • Mast cells initiate cellular immune responses at the infection site by recruitment of various subsets of T cells. • Systemic activation of mast cells is associated with vascular pathologies during viral infections. • Targeting of mast cell specific products has a promising therapeutic potential in treating virus induced immune pathologies., Mast cells (MCs) are long-lived immune cells. They are armed with preformed mediators within granules that can be instantaneously released in response to an invading pathogen, including certain viruses. At the skin and mucosae, they initiate innate immune responses and promote the development of adaptive immune responses, through cellular recruitment or antigen presentation. However, systemic MC activation may promote immune pathologies through their vasoactive proteases and biogenic amines. Recently, MC products were identified to contribute to pathologies associated with viral hemorrhagic fever, such vascular leakage and thrombocytopenia. Similar associations of MCs with disease severity have been noted for certain respiratory viral pathogens. Here we discuss the specific MC responses to viruses and their influences on functional immune outcomes during infection.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Dengue is a rapidly spreading mosquito-borne flavivirus infection that is prevalent in tropical and sub-tropical regions. Humans are known to be the main reservoir host maintaining the epidemic cycles of dengue but it is unclear if dengue virus is also maintained in a similar enzootic cycle. The systematic review was conducted in accordance to Cochrane's PRISMA recommendations. A search was done on PubMed, EMBASE, Scopus and Cochrane Library. Key data on animal dengue positivity was extracted and classified according to animal type and diagnostic modes. Of the 3818 articles identified, 56 articles were used in this review. A total of 16,333 animals were tested, 1817 of which were positive for dengue virus by RT-PCR or serology. Dengue positivity was detected in bats (10.1%), non-human primates (27.3%), birds (11%), bovid (4.1%), dogs (1.6%), horses (5.1%), pigs (34.1%), rodents (3.5%), marsupials (13%) and other small animals (7.3%). While majority of dengue positivity via serology suggests potential enzootic transmission, but regular dengue virus spillback cannot be excluded. With the exception of bats, acute infection among animals is limited. Further investigation on animals is critically required to better understand their role as potential reservoir in dengue transmission., Highlights • Besides non-human primates, dengue infection occurred in pigs, marsupials, bats, birds, horses, bovid, rodents and dogs. • There is potential enzootic transmission, but regular dengue virus spillback cannot be excluded. • With the exception of bats, acute dengue infection among animals is still limited in evidence. • Though the role of animals as potential dengue reservoir cannot be concluded, neither can it be excluded yet.

Mast cells (MCs) are immune sentinels, but whether they also function as antigen-presenting cells (APCs) remains elusive. Using mouse models of MC deficiency, we report on MC-dependent recruitment and activation of multiple T cell subsets to the skin and draining lymph nodes (DLNs) during dengue virus (DENV) infection. Newly recruited and locally proliferating γδ T cells were the first T cell subset to respond to MC-driven inflammation, and their production of IFN-γ was MC dependent. MC-γδ T cell conjugates were observed consistently in infected peripheral tissues, suggesting a new role for MCs as nonconventional APCs for γδ T cells. MC-dependent γδ T cell activation and proliferation during DENV infection required T cell receptor (TCR) signaling and the nonconventional antigen presentation molecule endothelial cell protein C receptor (EPCR) on MCs. γδ T cells, not previously implicated in DENV host defense, killed infected targeted DCs and contributed to the clearance of DENV in vivo. We believe immune synapse formation between MCs and γδ T cells is a novel mechanism to induce specific and protective immunity at sites of viral infection.

Infection of fetuses with Zika virus during pregnancy and congenital defects are enhanced by maternal immunity to dengue virus., Zika virus (ZIKV), an emergent flaviviral pathogen, has been linked to microcephaly in neonates. Although the risk is greatest during the first trimester of pregnancy in humans, timing alone cannot explain why maternal ZIKV infection leads to severe microcephaly in some fetuses, but not others. The antigenic similarities between ZIKV and dengue virus (DENV), combined with high levels of DENV immunity among ZIKV target populations in recent outbreaks, suggest that anti-DENV maternal antibodies could promote ZIKV-induced microcephaly. We demonstrated maternal-to-fetal ZIKV transmission, fetal infection, and disproportionate microcephaly in immunocompetent mice. We show that DENV-specific antibodies in ZIKV-infected pregnant mice enhance vertical ZIKV transmission and result in a severe microcephaly-like syndrome, which was dependent on the neonatal Fc receptor, FcRN. This novel immune-mediated mechanism of vertical transmission of viral infection is of special concern because ZIKV epidemic regions are also endemic to DENV.

Dengue is the leading mosquito-borne viral illness infecting humans. Owing to the circulation of multiple serotypes, global expansion of the disease and recent gains in vaccination coverage, pre-existing immunity to dengue virus is abundant in the human population, and secondary dengue infections are common. Here, we contrast the mechanisms initiating and sustaining adaptive immune responses during primary infection with the immune pathways that are pre-existing and reactivated during secondary dengue. We also discuss new developments in our understanding of the contributions of CD4+ T cells, CD8+ T cells and antibodies to immunity and memory recall. Memory recall may lead to protective or pathological outcomes, and understanding of these processes will be key to developing or refining dengue vaccines to be safe and effective. The existence of four different serotypes of dengue virus poses a challenge to vaccine development, as pre-existing immunity can lead to severe disease during infection with a heterologous serotype. This Review analyses the mechanisms of protective and pathological adaptive immune responses in primary and secondary dengue infection.

When draining lymph nodes become infected by Yersinia pestis (Y. pestis), a massive influx of phagocytic cells occurs, resulting in distended and necrotic structures known as buboes. The bubonic stage of the Y. pestis life cycle precedes septicemia, which is facilitated by trafficking of infected mononuclear phagocytes through these buboes. However, how Y. pestis convert these immunocytes recruited by host to contain the pathogen into vehicles for bacterial dispersal and the role of immune cell death in this context are unknown. We show that the lymphatic spread requires Yersinia outer protein J (YopJ), which triggers death of infected macrophages by downregulating a suppressor of receptor-interacting protein kinase 1-mediated (RIPK1-mediated) cell death programs. The YopJ-triggered cell death was identified as necroptotic, which released intracellular bacteria, allowing them to infect new neighboring cell targets. Dying macrophages also produced chemotactic sphingosine 1-phosphate, enhancing cell-to-cell contact, further promoting infection. This necroptosis-driven expansion of infected macrophages in buboes maximized the number of bacteria-bearing macrophages reaching secondary lymph nodes, leading to sepsis. In support, necrostatins confined bacteria within macrophages and protected mice from lethal infection. These findings define necrotization of buboes as a mechanism for bacterial spread and a potential target for therapeutic intervention.

Dengue virus (DENV) is the most prevalent vector-borne viral pathogen, infecting millions of patients annually. Thrombocytopenia, a reduction in circulating platelet counts, is the most consistent sign of DENV-induced disease, independent of disease severity. However, the mechanisms leading to DENV-induced thrombocytopenia are unknown. Here, we show that thrombocytopenia is caused by serotonin derived from mast cells (MCs), which are immune cells that are present in the perivascular space and are a major peripheral source of serotonin. We show that during DENV infection, MCs release serotonin, which prompts platelet activation, aggregation, and enhanced phagocytosis, dependent on 5HT

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis., How Japanese encephalitis virus (JEV) penetrates the blood-brain barrier (BBB) remains unclear. Here, using a genetic mouse model and a virulent JEV strain, the authors show that perivascular mast cells (MC) mediate JEV neuroinvasion and identify the MC-protease chymase as a potential therapeutic target.

Zika virus (ZIKV), a recently emerged flaviviral pathogen, has been linked to microcephaly in neonates1. Yet, it is not understood why some fetuses develop severe microcephaly due to maternal ZIKV infection while others do not. The risk for ZIKV-induced microcephaly is greatest during the first trimester of pregnancy in humans2,3, yet this alone cannot account for the varied presentation of microcephaly observed. Given the antigenic similarity between ZIKV and closely related dengue virus (DENV)4, combined with the substantial immunity to DENV in ZIKV target populations in recent outbreaks, we hypothesized that maternal antibodies against DENV could promote ZIKV-induced microcephaly. Here, using immune-competent mice, we show that maternal to fetal transmission of ZIKV occurs, leading to fetal infection and disproportionate microcephaly. DENV-specific antibodies in pregnant female mice enhance vertical transmission of infection and result in a severe microcephaly like-syndrome during ZIKV infection. Furthermore, fetal infection was promoted by the neonatal Fc receptor (FcRN). Our results identify a novel immune-mediated mechanism of vertical transmission of viral infection and raise caution since ZIKV epidemic regions are also endemic to DENV.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage, and shock due to the ability of DENV to infect a variety of immune and nonimmune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs), and infected monocytes, can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling, and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT, and CD8 + T cell activation. The MC-stabilizing drug ketotifen reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen, consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immunocompetent host and supports the further development of targeted immunomodulatory strategies to treat DENV disease. IMPORTANCE Dengue virus (DENV), which causes febrile illness, is transmitted by mosquito vectors throughout tropical and subtropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection, but it is thought that drugs that target the host immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host transcriptional response to infection in multiple DENV target organs using a mouse model of disease. We found that DENV infection induced metabolic dysregulation and inflammatory responses and affected the immune cell content of the spleen and liver. The use of the mast cell stabilization drug ketotifen reversed many of these responses and induced additional changes in the transcriptome and immune cell repertoire that contribute to decreased dengue disease.

Dengue viruses (DENV1-4) are mosquito-borne flaviviruses estimated to cause up to ∼400 million infections and ∼100 million dengue cases each year. Factors that contribute to protection from and risk of dengue and severe dengue disease have been studied extensively but are still not fully understood. Results from Phase 3 vaccine efficacy trials have recently become available for one vaccine candidate, now licensed for use in several countries, and more Phase 2 and 3 studies of additional vaccine candidates are ongoing, making these issues all the more urgent and timely. At the "Summit on Dengue Immune Correlates of Protection", held in Annecy, France, on March 8-9, 2016, dengue experts from diverse fields came together to discuss the current understanding of the immune response to and protection from DENV infection and disease, identify key unanswered questions, discuss data on immune correlates and plans for comparison of results across assays/consortia, and propose a research agenda for investigation of dengue immune correlates, all in the context of both natural infection studies and vaccine trials.

Sequential exposures to flaviviruses sharing CD4 epitopes promote protection during subsequent heterologous infections., How previous immunity influences immune memory recall and protection against related flaviviruses is largely unknown, yet encounter with multiple flaviviruses in a lifetime is increasingly likely. Using sequential challenges with dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV), we induced cross-reactive cellular and humoral immunity among flaviviruses from differing serocomplexes. Antibodies against JEV enhanced DENV replication; however, JEV immunity was protective in vivo during secondary DENV1 infection, promoting rapid gains in antibody avidity. Mechanistically, JEV immunity activated dendritic cells and effector memory T cells, which developed a T follicular helper cell phenotype in draining lymph nodes upon secondary DENV1 infection. We identified cross-reactive epitopes that promote recall from a pool of flavivirus serocomplex cross-reactive memory CD4 T cells and confirmed that a similar serocomplex cross-reactive immunity occurs in humans. These results show that sequential immunizations for flaviviruses sharing CD4 epitopes should promote protection during a subsequent heterologous infection.

Patients with acute dengue who present with high serum chymase levels are at greater risk of dengue hemorrhagic fever or dengue shock syndrome. The chymase level is a prognostic biomarker of severe dengue for adult and pediatric patients before they develop clinical warning signs., Background Most patients with dengue experience mild disease, dengue fever (DF), while few develop the life-threatening diseases dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). No laboratory tests predict DHF or DSS. We evaluated whether the serum chymase level can predict DHF or DSS in adult and pediatric patients and the influence of preexisting conditions (PECs) on chymase levels. Methods Serum chymase levels were measured in patients presenting with undifferentiated fever to hospitals in Colombo District, Sri Lanka. The value of serum the chymase concentration and clinical signs and symptoms as predictors of DHF and/or DSS was evaluated by multivariate analysis. We assessed the influence of age, PECs, and day after fever onset on the robustness of the chymase level as a biomarker for DHF and/or DSS. Results An elevated chymase level in acute phase blood samples was highly indicative of later diagnosis of DHF or DSS for pediatric and adult patients with dengue. No recorded PECs prevented an increase in the chymase level during DHF. However, certain PECs (obesity and cardiac or lung-associated diseases) resulted in a concomitant increase in chymase levels among adult patients with DHF. Conclusions These results show that patients with acute dengue who present with high levels of serum chymase consistently are at greater risk of DHF. The chymase level is a robust prognostic biomarker of severe dengue for adult and pediatric patients.

Vaccination has achieved remarkable successes in the control of childhood viral diseases. To control emerging infections, however, vaccines will need to be delivered to older individuals who, unlike infants, probably have had prior infection or vaccination with related viruses and thus have cross-reactive antibodies against the vaccines. Whether and how these cross-reactive antibodies impact live attenuated vaccination efficacy is unclear. Using an open-label randomized trial design, we show that subjects with a specific range of cross-reactive antibody titres from a prior inactivated Japanese encephalitis vaccination enhanced yellow fever (YF) immunogenicity upon YF vaccination. Enhancing titres of cross-reactive antibodies prolonged YF vaccine viraemia, provoked greater pro-inflammatory responses, and induced adhesion molecules intrinsic to the activating Fc-receptor signalling pathway, namely immune semaphorins, facilitating immune cell interactions and trafficking. Our findings clinically demonstrate antibody-enhanced infection and suggest that vaccine efficacy could be improved by exploiting cross-reactive antibodies. Supplementary information The online version of this article (doi:10.1038/nmicrobiol.2016.164) contains supplementary material, which is available to authorized users., Immunization with inactivated Japanese encephalitis vaccine generates cross-reactive antibodies that enhance immunogenicity on subsequent immunization with live attenuated yellow fever vaccine, demonstrating antibody-dependent enhancement of infection in a clinical trial. Supplementary information The online version of this article (doi:10.1038/nmicrobiol.2016.164) contains supplementary material, which is available to authorized users.

The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines.

A wealth of evidence supports the essential contributions of mast cells (MCs) to immune defense against bacteria and parasites; however, the role of MCs in viral infections has not been defined. We now report that rodent, monkey, and human MCs are able to detect dengue virus (DENV), a lymphotropic, enveloped, single-stranded, positive-sense RNA virus that results in MC activation and degranulation. We observe that the response of MCs to DENV also involves the activation of antiviral intracellular host response pathways, melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene 1 (RIG-I), and the de novo transcription of cytokines, including TNF-α and IFN-α, and chemokines, such as CCL5, CXCL12, and CX3CL1. This multifaceted response of MCs to DENV is consequential to the containment of DENV in vivo because, after s.c. infection, MC-deficient mice show increased viral burden within draining lymph nodes, which are known to be targeted organs during DENV spread, compared with MC-sufficient mice. This containment of DENV is linked to the MC-driven recruitment of natural killer and natural killer T cells into the infected skin. These findings support expanding the defined role of immunosurveillance by MCs to include viral pathogens.

Although mast cells were discovered more than a century ago, their functions beyond their role in allergic responses remained elusive until recently. However, there is a growing appreciation that an important physiological function of these cells is the recognition of pathogens and modulation of appropriate immune responses. Because of their ability to instantly release several pro-inflammatory mediators from intracellular stores and their location at the host–environment interface, mast cells have been shown to be crucial for optimal immune responses during infection. Mast cells seem to exert these effects by altering the inflammatory environment after detection of a pathogen and by mobilizing various immune cells to the site of infection and to draining lymph nodes. Interestingly, the character and timing of these responses can vary depending on the type of pathogen stimulus, location of pathogen recognition and sensitization state of the responding mast cells. Recent studies using mast cell activators as effective vaccine adjuvants show the potential of harnessing these cells to confer protective immunity against microbial pathogens.

A novel immune-modulatory therapy utilizing targeted delivery of cytokines to draining lymph nodes effectively reprograms Th2 allergic responses towards a Th1 and tolerogenic profile, resulting in protection from peanut antigen-induced anaphylaxis.

We report that infection of draining lymph nodes (DLNs) by Salmonella typhimurium results in the specific downregulation of the homeostatic chemokines CCL21 and CXCL13, which are essential for normal DLN organization and function. Our data reveal that the mechanism of this suppression is dependent on S. typhimurium LPS (sLPS). The decrease in CCL21 expression involves interaction between sLPS and CCL21-producing cells within DLNs, triggering a distinct Toll-like receptor 4 (TLR4)-mediated host signaling response. In this response, suppressor of cytokine signaling-3 (Socs3) is upregulated, which negatively regulates mothers against decapentaplegic homolog-3 (Smad3)-initiated production of CCL21. Disruption of lymph node architecture and cellular trafficking enhances S. typhimurium virulence and could represent a mechanism of immune suppression used by pathogens that primarily target lymphoid tissue.

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

SummaryMast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that, during footpad infection with E. coli in MC-deficient mice, as compared to their MC-sufficient counterparts, the serum antibody response is significantly diminished and less protective following passive immunization in a urinary tract infection (UTI) model in wild-type mice. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) during a prolonged time course. This pattern of trafficking was facilitated by MC-generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection.

Mast cells (MCs), which are granulated tissue-resident cells of hematopoietic lineage, constitute a major sensory arm of the innate immune system. In this review we discuss the evidence supporting the dual role of MCs, both as sentinels for invading pathogens and as regulatory cells throughout the course of acute inflammation, from its initiation to resolution. This versatility is dependent on the ability of MCs to detect pathogens and danger signals and release a unique panel of mediators to promote pathogen-specific clearance mechanisms, such as through cellular recruitment or vascular permeability. It is increasingly understood that MCs also contribute to the regulated contraction of immune activation that occurs within tissues as inflammation resolves. This overarching regulatory control over innate immune processes has made MCs successful targets to purposefully enhance or, alternatively, suppress MC responses in multiple therapeutic contexts.

Dengue Virus (DENV), a flavivirus spread by mosquito vectors, can cause vascular leakage and hemorrhaging. However, the processes that underlie increased vascular permeability and pathological plasma leakage during viral hemorrhagic fevers are largely unknown. Mast cells (MCs) are activated in vivo during DENV infection, and we show that this elevates systemic levels of their vasoactive products, including chymase, and promotes vascular leakage. Treatment of infected animals with MC-stabilizing drugs or a leukotriene receptor antagonist restores vascular integrity during experimental DENV infection. Validation of these findings using human clinical samples revealed a direct correlation between MC activation and DENV disease severity. In humans, the MC-specific product, chymase, is a predictive biomarker distinguishing dengue fever (DF) and dengue hemorrhagic fever (DHF). Additionally, our findings reveal MCs as potential therapeutic targets to prevent DENV-induced vasculopathy, suggesting MC-stabilizing drugs should be evaluated for their effectiveness in improving disease outcomes during viral hemorrhagic fevers. DOI: http://dx.doi.org/10.7554/eLife.00481.001, eLife digest Dengue fever is an infectious tropical disease that is transmitted by mosquitoes carrying dengue virus. Almost half of the world’s population lives in dengue-plagued regions and it is estimated that between 50 and 100 million people are infected annually. However, there is no effective vaccine and researchers have only a limited understanding of the mechanisms behind the disease. While most infected individuals experience fever, headache, muscle and joint pain, and a skin rash, a small percentage go on to develop a life-threatening condition known as dengue hemorrhagic fever (DHF). This is marked by internal bleeding, and by the leakage of water and salts from blood vessels (vascular leakage). At present, it is difficult to tell which patients will develop this complication, making it hard to tailor treatment appropriately. Here, St John et al. reveal that the vascular leakage that occurs in DHF is triggered by mast cells, which line blood vessels and regulate their permeability through the release of molecules such as histamines and leukotrienes. They also found that mice deficient in mast cells did not show dengue-induced vascular leakage, and that wild-type animals treated with drugs that block the actions of proteins produced by these cells, showed less vascular leakage than controls. Moreover, levels of an enzyme called chymase, another mast cell product, are higher in human patients with DHF than in those with dengue fever. Since chymase release occurs early in infection, tests for the presence of this enzyme could be used to predict which patients are likely to develop DHF. The work of St John et al. suggests new lines of inquiry into the mechanisms that lead some individuals infected with dengue fever to develop DHF, and indicates that drugs that target mast cells could offer an effective treatment. DOI: http://dx.doi.org/10.7554/eLife.00481.002

Mast cells (MCs) have been implicated in orchestrating the host's early innate immune and adaptive immune responses in several models of acute bacterial infections. Most of this activity results in early clearance of the bacteria and timely resolution of infection. However, during chronic infections because of the prolonged nature of MC-bacterial interactions, the role of the MC in determining the fate of infection is markedly more complex. Depending on the nature of the pathogen, severity of infection, and its association with a preexisting inflammatory disease, MCs may promote rather than contain chronic infections and exacerbate their pathological sequellae.