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REVIEW ARTICLEAutoimmunity in dengue pathogenesisShu-Wen Wan a,b, Chiou-Feng Lin a,b,c,d, Trai-Ming Yeh b,c,e,Ching-Chuan Liu b,f, Hsiao-Sheng Liu a,b,c, Shuying Wang a,b,c, Pin Ling a,b,c,Robert Anderson a,b,g,h,i, Huan-Yao Lei a,b,c,j, Yee-Shin Lin a,b,c,*aDepartment of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, TaiwanbCenter of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, TaiwancInstitute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, TaiwandInstitute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, TaiwaneDepartment of Medical Laboratory Science and Biotechnology, National Cheng Kung University Medical College, Tainan,TaiwanfDepartment of Pediatrics, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, TaiwangDepartment of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, CanadahDepartment of Pediatrics, Dalhousie University, Halifax, Nova Scotia, CanadaiCanadian Center for Vaccinology, Dalhousie University, Halifax, Nova Scotia, CanadaReceived 20 October 2012; accepted 9 November 2012KEYWORDSautoimmunity;dengue;immunopathogenesisDengue is one of the most important vector-borne viral diseases. With climate change and theconvenience of travel, dengue is spreading beyond its usual tropical and subtropical bound-aries. Infection with dengue virus (DENV) causes diseases ranging widely in severity, fromself-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shocksyndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical mani-festations associated with severe DENV infection, yet the mechanisms remain unclear. Besidesthe direct effects of the virus, immunopathogenesis is also involved in the development ofdengue disease. Antibody-dependent enhancement increases the efﬁciency of virus infectionand may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cellsand overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules leadto their abnormal activation or dysfunction. Molecular mimicry between DENV proteins andhost proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no* Corresponding author. Department of Microbiology and Immunology, National Cheng Kung University Medical College, 1 University Road,Tainan 701, Taiwan.E-mail address: email@example.com (Y.-S. Lin).jDr Huan-Yao Lei passed away during the preparation of this manuscript. This review article is dedicated to Dr Lei.0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.http://dx.doi.org/10.1016/j.jfma.2012.11.006Available online at www.sciencedirect.comjournal homepage: www.jfma-online.comJournal of the Formosan Medical Association (2013) 112, 3e11
licensed dengue vaccine is yet available, several vaccine candidates are under development.For the development of a safe and effective dengue vaccine, the immunopathogenic compli-cations of dengue disease need to be considered.Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.IntroductionDengue virus (DENV) belongs to the genus Flavivirus of thefamily Flaviviriade. Based on neutralization assay data,four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) canbe distinguished. DENV is transmitted to humans mainly byAedes aegypti and Aedes albopictus.1About 50 milliondengue infection cases, with around 500,000 cases per yearof severe dengue, have mainly been reported in the Asia-Paciﬁc region, the Americas, and Africa. All four DENVserotypes are now circulating in these areas.2The trans-mission efﬁciency and disease expression between theserotypes are still uncertain, but DENV-2 and DENV-3 mightcontribute the most to disease severity and mortality.3There have been several major outbreaks of dengue inTaiwan, particularly in 1981, 1987e1988, 2001e2002, and2007. Dengue outbreaks involve various combinations ofdengue serotypes, with certain serotypes predominating,such as DENV-2 in the year 2002.4,5Recent reports haveclariﬁed the usual pattern in Taiwan outbreaks: starting byimport from abroad in early summer, spreading out locally,and ending in the winter. Dengue is primarily an adultdisease in Taiwan. Most cases of dengue fever (DF) havebeen reported in individuals in the 50e54-year age rangeand most cases of dengue hemorrhagic fever (DHF) in the60e64-year range.4However, dengue usually occurs inchildren in hyperendemic Southeast Asia. Secondaryinfection of DENV-2 was prevalent in the year 2002, butprimary infection of DENV-1 or DENV-3 in 2004e2007. Inaddition, adults or the elderly have a greater risk ofdeveloping the severe dengue disease.4DENV is a lipid-enveloped, single-positive-RNA virus,with a genome of about 10.7 kb. RNA of the virus is trans-lated to three structural proteins, namely capsid protein(C), precursor membrane protein (prM), and envelopeprotein (E). Besides the structural proteins, there are sevennonstructural proteins (NS), which are involved in variousfunctions affecting viral replication and disease pathogen-esis.6,7The replication cycle of DENV begins when thevirions attach to the surface of host cells and subsequentlyenter the cells by receptor-mediated endocytosis. Acidiﬁ-cation of the endosomal vesicle triggers conformationalchanges in the virion, which results in the fusion of the viraland cell membranes. After the fusion has occurred, thenucleocapsid is released into the cytoplasm. The positive-sense RNA is translated into a single polyprotein that isprocessed cotranslationally and post-translationally by viraland host proteases. Genome replication occurs on intra-cellular membranes. Virus assembly occurs on the surfaceof the endoplasmic reticulum (ER) when the structuralproteins and the newly synthesized RNA bud into the lumenof ER. The virion is maturated in the Golgi compartmentand exits by the secretory pathway. Two processes areinvolved in virus maturation. First, the prM protein iscleaved by host furin and forms the M protein in the trans-Golgi network. Second, the E protein undergoes a majorconformational rearrangement during the maturation ofvirus particles during exocytosis.7,8Infection with DENV causes diseases ranging from mildDF to severe DHF and dengue shock syndrome (DSS). DHF/DSS usually occurs in patients who are secondarily infectedwith heterotypic DENV, but it also occurs in case of primaryinfection.9DF presents with an onset of fever accompaniedby severe headache, retro-orbital pain, myalgia, arthralgia,abdominal pain, rash, and minor hemorrhage in the form ofpetechiae, epistaxis, or gingival bleeding. Leukopenia isa common ﬁnding in laboratory tests, whereas thrombocy-topenia may occasionally be observed in DF patients.10Inaddition to all the symptoms of DF, DHF is characterized bysevere hemorrhage (positive tourniquet test or spontaneousbleeding), thrombocytopenia (platelet counts <100,000/mm3), plasma leakage (increased hemoconcentration orﬂuid effusion in chest or abdominal cavities), and hepato-megaly (elevation of serum transaminases). The WorldHealth Organization (WHO) classiﬁes DHF into four grades(IdIV). DHF grades I and II represent relatively mild caseswithout shock, whereas grades III and IV cases are moresevere and may lead to disseminated intravascular coagu-lation.11e13There has been a systematic literature reviewsummarizing the difﬁculties in applying the criteria for DHFin the clinical situation. For example, the positive tourni-quet test indicative of hemorrhagic manifestation does notsigniﬁcantly distinguish between DHF and DF. In addition,the incidences of major manifestations (hemorrhage,thrombocytopenia, and plasma leakage) observed in DHFpatients span a large range.13Accordingly, the WHO clas-siﬁcation system is currently being reconsidered to be moresuitable for clinical practice. The new guidelines includedengue without warning signs, dengue with warning signs,and severe dengue. From recent studies, 13.7% of denguecases could not be classiﬁed using the DF/DHF/DSS classi-ﬁcation, whereas only 1.6% could not be classiﬁed using therevised classiﬁcation.14Hence, assessments of the newclassiﬁcation are still continuing, and the potential imple-mentation of the revised classiﬁcation has been proposed.The pathogenic mechanisms in DHF/DSS are complicatedand not fully resolved. Several mechanisms are involved inthe pathogenesis of DHF/DSS progression, including viralpathogenesis and immunopathogenesis. Viral pathogenesisreﬂects the pathology directly caused by the virus, and issubject to serotypic or genotypic differences. In contrast,immunopathogenesis encompasses other factors involvingthe host immune response, which may be involved in thepathogenesis.15For example, during secondary infection,the critical phase of disease occurs when the viralburden declines. This has led to the suggestion that4 S.-W. Wan et al.
immunopathogenic mechanisms, such as the adaptiveimmune response, inﬂammatory mediators, and autoim-munity, are important in the pathogenesis of denguedisease. Such mechanisms play signiﬁcant roles in majormanifestations of DHF, including hemorrhage, thrombocy-topenia, plasma leakage, and hepatomegaly (summarizedin Fig. 1).15e17Viral pathogenesisVirus variationVirus variation indicates the capacity of a virus to producedisease in a host. In the case of dengue, genetic differencesamong DENV isolates contribute to the severity of denguedisease. There are four antigenically distinct serotypes ofDENV, each of which can cause an outbreak of denguedisease.2However, DENV-2 and DENV-3 may contribute themost to disease severity and mortality.3Viral genetic18e20and structural21differences have been shown to inﬂuencehuman disease severity. Recently, viral genetic differenceswere demonstrated to be a contributing factor to virulencein a mouse model.22However, it remains to be determinedwhether these serotypic or genotypic differences observedin vitro or in mouse models, respectively, contribute tovirulence differences in humans.Cell and tissue tropismCell and tissue tropism of DENV likely have a major impacton the outcome of DENV infection. Langerhans cells(dermal dendritic cells) are generally proposed to be theinitial target for DENV infection at the site of the mosquitobite,23followed by the systemic infection of macrophages/monocytes24and viral entry into the blood. From autopsiesof fatal cases, DENV has been found in the skin, liver,spleen, lymph node, kidney, bone marrow, lung, thymus,and brain.11Besides the primary targets (dendritic cells andmacrophages) of DENV, other potential target cellsincluding hepatocytes, endothelial cells, and neuronal cellshave been detected in mouse models. DENV can not onlyreplicate in these cells but also contribute to their damageand/or dysfunction. For example, mice inoculated withDENV by intraperitoneal,25intradermal,26,27or intra-cerebal28routes have been shown to display liverpathology, hemorrhagic or neurological symptoms. Eleva-tion of serum transaminases, hemorrhage, and fatalencephalitis have been observed in these mouse models,and provide mechanistic insights for the manifestations ofdengue disease.25e28The range of these cell or tissue typesinfected with DENV suggest that the receptors of DENV arediverse or broadly distributed. The afﬁnity of DENV withthose receptors might inﬂuence virus infectivity as well asvirulence. A single amino acid mutation on E protein of theﬂavivirus (Murray Valley encephalitis virus) have beendemonstrated to cause altered cell tropism, includingdifferences in entry kinetics, attachment to mammaliancells, and virulence in mice.29In summary, the factors thatdetermine the numbers and fates of infected cells atspeciﬁc sites likely contribute to the pathology of denguedisease.ImmunopathogenesisAntibody-dependent enhancementAntibody-dependent enhancement (ADE) is a well-knownhypothesis of dengue disease pathogenesis. Epidemiolog-ical evidence suggests that the presence of pre-existingsubneutralizing antibodies (Abs) is a major factor fordeveloping DHF/DSS in both infants and adults.30EnhancingAbs increase the efﬁciency of virus attachment andFigure 1 A hypothetical model of dengue pathogenesis. Viral and immunological factors contribute to clinical manifestations,including severe hemorrhage, thrombocytopenia, plasma leakage, and hepatomegaly. DENV Z dengue virus.Autoimmunity in dengue 5
internalization through Fcg receptor (FcgR)-dependent30orFcgR-independent mechanisms.31Enhancing Abs alsocontribute to the binding of DENV to platelets.32Recently, a new hypothesis (termed intrinsic ADE)postulates that FcgR-mediated DENV internalizationsuppresses the type I interferon (IFN)-mediated antiviralresponses by inhibiting antiviral genes and enhancinginterleukin-10 (IL-10) production, which suppresses theIFN-g signaling pathway and promotes T-helper-2responses.33e35T-helper-1 responses are required for virusclearance; however, T-helper-2 responses have limitedantiviral effect and enhance the production of Abs. Thismay lead to high levels of both viral loads and Abs in denguepatients. Besides the ampliﬁcation of viral output, ADEenhances cytokine and chemokine production,36e39cellapoptosis,40and tumor necrosis factor-a (TNF-a)-mediatedendothelial cell activation.36,41Cellular immune responseAlthough memory T cells, which cross-react with heterolo-gous viruses, can provide partial protective immunity, theymay cause immunopathology.42According to the “originalantigenic sin” model, low-afﬁnity memory T cells generatedduring primary DENV infection expand selectively duringthe secondary infection of another virus serotype, prior tothe activation of naı¨ve T cells of higher avidity for thesecond DENV serotype. The cross-reactive T cells producehigh concentrations of inﬂammatory cytokines and maycontribute to the pathogenesis of plasma leakage in denguedisease.11,43e45DENV-speciﬁc human CD4þcytotoxic T cellshave been demonstrated to lyse bystander target cellsin vitro.46This mechanism may provide an explanation forlymphocyte activation and hepatocyte damage in a DENV-infected mouse model.47A recent study demonstratedthat regulatory T-cell frequencies and regulatory T-cell/effector T-cell ratios are increased in acute dengueinfection.48Soluble factorsSeveral studies have indicated that the concentrations ofcytokines, chemokines, or other mediators might besigniﬁcantly increased during DENV infection. Higher levelsof IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-18, monocyte che-moattractant protein-1 (MCP-1), macrophage migrationinhibitory factor (MIF), transforming growth factor-b, TNF-a, and IFN-g have been found in the plasma of severedengue patients.43,49e58These mediators play central rolesin regulating the immune response to dengue. In particular,TNF-a produced by dengue-infected monocytes36as well asby mast cells41triggers the activation of vascular endo-thelial cells. Also, some studies demonstrated that TNF-a contributes to endothelial permeability and hemorrhageduring DENV infection in animal models.26,59In addition toTNF-a,60several studies demonstrated that IL-8,61MCP-1,57MIF,62and metalloproteinase 963,64promoted increasedendothelial permeability in vitro. Furthermore, IL-6 and IL-8 have been found to be associated with the activation ofcoagulation and ﬁbrinolysis.65e67IL-8 levels have been re-ported to be increased in most dengue patients andcorrelated with degranulation of neutrophils.68In addition,levels of IL-10 have been shown to correlate with the loss ofplatelets and failure of platelet function.69High levels of C3a and C5a have been detected in thesera from severely affected dengue patients.70,71C3a andC5a, the products of C3 and C5 cleavage, are anaphylo-toxins, which promote chemotoxis of immune cells andcontribute to inﬂammatory responses. Soluble NS1 andanti-DENV Abs have also been reported to activatecomplement, by binding on the surface of infected endo-thelial cells.70,72High plasma levels of NS1 and terminalcomplement complex have been detected in DENV-infectedpatients, and these were correlated with vascular leakageas well as disease severity.70High levels of regulatoryfactors D and H have also been reported in DHF patientscompared to those in DF patients. The imbalance of factorsD and H caused alternative complement pathway deregu-lation and might correlate with disease severity.72AutoimmunityAutoimmunity and molecular mimicry have been demon-strated in various viral infections, such as Coxsackievirusand EpsteineBarr virus, and have been implicated in humanautoimmune diseases.73Autoantibodies represent anotherimportant factor involved in dengue disease pathogenesis.Several studies showed that the generation of autoanti-bodies against platelets,74e76endothelial cells,77,78andcoagulatory molecules77e81was associated with denguedisease. Molecular mimicry between platelets, endothelialcells, and coagulatory molecules with NS1, prM, and Eproteins may explain the cross-reactivity of anti-NS1, anti-prM, and anti-E Abs, respectively, to host proteins. Theconsequences of these cross-reactive Abs are plateletdysfunction, endothelial cell apoptosis, coagulation defect,and macrophage activation.73,82A schematic model ofimportant dengue manifestations induced by cross-reactiveautoantibodies is illustrated in Fig. 2.Our studies showed that the levels of antiplatelet andantiendothelial cell autoantibodies are higher in the sera ofDHF/DSS patients than in that of DF patients. Immuno-globulin M (IgM) present in the sera of DHF patients playeda more dominant role than IgG in the cross-reactivity withplatelets and endothelial cells. Absorption experimentsrevealed that anti-DENV NS1 Abs in patients’ sera areresponsible for the cross-reactivity, resulting in plateletdysfunction and endothelial cell apoptosis.74,78,83Theseﬁndings suggest that DENV-induced antoantibodies might beassociated with thrombocytopenia and plasma leakage.Anti-DENV NS1 Abs, which were generated from mice,cross-reacted with endothelial cells and triggered apoptosisby nitric oxide production.84In addition, anti-DENV NS1 Absinduced endothelial cells to express IL-6, IL-8, MCP-1, andintercellular adhesion molecule-1. The activation of endo-thelial cells by anti-DENV NS1 Abs demonstrated theinvolvement of anti-DENV NS1 Abs in the vasculopathy ofDENV infection.85Furthermore, mice actively immunizedwith NS1 proteins or passively administrated with anti-DENVNS1 Abs showed a hepatitis-like pathologic effect. Theseresults revealed that anti-DENV NS1 Abs might play a role inliver damage, which is an important manifestation of6 S.-W. Wan et al.
dengue disease.86From proteomic analysis, the potentialcandidate proteins on endothelial cells, recognized by anti-DENV NS1 Abs, include ATP synthase b-chain, vimentin,heat shock protein 60, and protein disulﬁde isomerase. TheC-terminal amino acid (a.a.) 311e352 region of DENV NS1shows certain degrees of homology with the candidateproteins.87Protein disulﬁde isomerase was recognized byanti-DENV NS1 Abs both on endothelial cells and on plate-lets.87,88We also found that the C-terminal region of NS1was responsible for cross-reactivity with platelets. Thedeletion of C-terminal region (a.a. 277e352) of NS1 abol-ished anti-NS1-mediated platelet aggregation and bleedingtendency.89These results suggest a mechanism of molec-ular mimicry in which Abs against DENV NS1 cross-reactwith endothelial cells and platelets.Previous studies in our laboratory identiﬁed importantcross-reactive epitopes on the C terminus (a.a. 271e352) ofDENV NS1 proteins.87e90Recent studies also indicated thata.a. 116e119 of DENV NS1 shared sequence similarity withhuman LYRIC protein (lysine-rich CEACAM1 co-isolated) a.a.334e337.82Furthermore, despite the absence of an argi-nineeglycineeaspartic acid (RGD) motif in the DENV NS1protein sequence, RGD structural mimicry exists within theNS1 protein. Since RGD is an important motif for matrix-integrin-mediated cell adhesion, anti-NS1 Abs could blockRGD-mediated cell adhesion.91These ﬁndings suggest theexistence of still other cross-reactive epitopes, whichshould be investigated in the future.Besides thrombocytopenia and plasma leakage,abnormal coagulopathy can also be observed in severedengue patients. Hemostatic parameters altered in DHF/DSS include prolonged thrombin time and activated partialthromboplastic time, decreased levels of ﬁbrinogen, andincreased levels of ﬁbrinogen degradation products.92Several studies suggested that autoantibodies may partici-pate in abnormal hemostasis during DENV infection. Absagainst NS1 and E proteins have been shown to cross-reactwith human blood coagulation factors, ﬁbrinogen, andplasminogen.77,79,80By sequence alignment, DENV proteins,including core, E, prM, and NS1, have shown differentlevels of sequence similarity with different coagulatory-associated molecules such as factor X, factor XI, and plas-minogen.73Although the effects of these autoantibodies oncoagulatory factors are still unclear, some reports demon-strated that DENV-induced autoantibodies might interferewith human ﬁbrinolysis.93,94In our previous studies, the titers of DENV-inducedautoantibodies reached peak levels in the acute phase,declined during the convalescent stage, and lasted forseveral months.74,78This time course is different fromchronic virus infection-associated autoimmune disease.73A recent case report showed a dengue patient withnumerous autoimmune features,95and another reportshowed a dengue patient in whom dengue evolved intosystemic lupus erythematous and lupus nephritis aftera month.96A follow-up study reported that dengue-infected individuals have long-term persistence of clin-ical symptoms with complement factors, rheumatoidfactor, C-reactive protein, antinuclear Abs, and immunecomplexes.97From these studies, it appears that DENVinfection may trigger abnormal immune responsescausing autoimmune reactions. Therefore, autoimmuneFigure 2 A schematic model of autoantibody-mediated immunopathogenesis in DENV infection. Molecular mimicry betweenplatelets, endothelial cells, and coagulatory molecules with NS1, prM, E, and C proteins underlies the cross-reactivity of anti-NS1,anti-prM, anti-E, and anti-C Abs, respectively, to host proteins. Abs Z antibodies; C Z capsid protein; DENV Z dengue virus;E Z envelope protein; NS Z nonstructural protein; prM Z precursor membrane protein.Autoimmunity in dengue 7
complications should be considered when developinga safe dengue vaccine.Dengue vaccine strategyAlthough no licensed dengue vaccine is yet available,several vaccine candidates are under development.Although live viral vaccines have advanced to clinical trials,they encountered new difﬁculties, such as viral interfer-ence among the four serotypes in tetravalent formulations.For safety concerns, nonviral vaccines have also beendeveloped, particularly subunit vaccines mostly focused onthe E protein or its derivatives. However, the challenge ofeliciting balanced levels of neutralizing Abs to each of thefour viral serotypes remains a major concern.12,98NS1 is not a virion-associated protein, and anti-NS1 Absdo not enhance DENV infection. Anti-DENV NS1 Abs ﬁxcomplement and trigger complement-mediated lysis ofDENV-infected cells.99Previous studies showed that activeimmunization with NS1 proteins and passive immunizationwith anti-NS1 Abs could provide protection to mice againstDENV challenge.99,100However, anti-NS1 Abs still showsome pathogenic effects both in vitro and in vivo.15,73Further mapping and/or genetic manipulation of the rele-vant pathogenic epitopes will be important for the devel-opment of a safe dengue NS1 vaccine.ConclusionsDengue is one of the most important vector-borne viraldiseases in the world. The complexity of dengue immuno-pathogenesis increases the difﬁculties associated with thedevelopment of a dengue vaccine. A successful denguevaccine must be effective against all four serotypes, avoidpotential ADE-associated pathogenic effects, as well as befree of potential autoimmune complications.AcknowledgmentsThis work was supported by grants NSC100-2321-B006-004and NSC100-2325-B006-007 from the National ScienceCouncil, Taiwan; NHRI-100A1-PDCO-0209115 from theNationalHealth Research Institutes,Taiwan; andDOH101-TD-B-111-002 from the Multidisciplinary Center of Excellence forClinical Trial and Research, Department of Health, Taiwan.References1. Monath TP. Dengue: the risk to developed and developingcountries. Proc Natl Acad Sci U S A 1994;91:2395e400.2. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J,Gubler DJ, et al. Dengue: a continuing global threat. Nat RevMicrobiol 2010;8:S7e16.3. Guzman A, Isturiz RE. Update on the global spread of dengue.Int J Antimicrob Agents 2010;36(Suppl. 1):S40e2.4. 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