Sensing dengue and chikungunya virus (co-) infections
Aguilar Briseño, Alberto
DOI:
10.33612/diss.172910464
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Publication date:
2021
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Aguilar Briseño, A. (2021). Sensing dengue and chikungunya virus (co-) infections. University of
Groningen. https://doi.org/10.33612/diss.172910464
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CHAPTER 1
Epidemiology of dengue virus and chikungunya virus
Dengue and chikungunya fever are two of the most important mosquito-borne viral diseases worldwide. Their etiological agents are dengue virus (DENV) and chikungunya virus (CHIKV), respectively. Both viruses are transmitted by mosquitoes of the genus Aedes, mainly Aedes
aegypti and Aedes albopictus. Globalization, increased urbanization and global warming are
considered the main factors that have contributed to the upsurge of cases and the geographical expansion of both viruses and mosquitoes (Figure 1)1–3.
Figure 1. Areas at risk of dengue and chikungunya virus infection. The map depicts the countries and regions currently
being at risk of dengue and chikungunya virus infections, as well the area of distribution of their mosquito vectors A.
albopictus and A. aegypti. Adapted from4,5. Figure made in Biorender.com
The oldest record of a dengue-like illness dates back to China (265- 420 A.D). Between 1779 and 1780, outbreaks of a disease comparable to dengue were reported in Africa, Asia and North America6. DENV was originally recognized as the causative agent of dengue in 1943 and since
then the incidence of the disease has dramatically increased7. Since then, four serotypes of
DENV (1-4) have been recognized and these are spread throughout tropical and subtropical regions of the world8. The largest number of cases ever reported worldwide occurred in 2019,
where in the Americas approximately 3.1 million cases were reported with more than 25,000 classifi ed as severe dengue9. To date, approximately 3.9 billion people in over 129 countries are
at risk of DENV infection. The most affected regions are the Americas, the Western Pacifi c and the South-East Asia, the latter representing 70% of the global burden of the disease2,10.
Chikungunya fever was fi rst described as a dengue-like disease during an outbreak in Tanzania (previously the Southern Province of Tanganyika) in 195311. The disease was mainly
characterized by fever, rash and arthralgia, with arthralgia being the main symptom of the disease. Due to the contorted postures of those experiencing severe arthralgia, the disease was
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referred to as chikungunya, the Makonde term that translates to ‘that which bends up’3. For
the next 50 years, small and restricted episodes of transmission occurred only Africa. Later, during 2004-2005, a large outbreak hit Kenya and La Reunion Island, further spreading to India in 2006, where it infected more than 1 million people12. The virus then spread to Europe
where it caused restricted outbreaks in Italy, Croatia, France, Spain and Portugal. The first local episodes of transmission in the Americas were reported in Saint Martin in 2013, which was followed by a rapid spread to South, Central and North America13. To date, CHIKV is endemic
in over 60 countries worldwide, with Asia and the Americas being the most affected regions. The latest CHIKV outbreaks have been reported in Brazil, Pakistan, India, Sudan, Yemen and most recently Chad and Cambodia in 2020.14
Importantly, due to the geographic overlap in distribution of DENV, CHIKV and now Zika virus (ZIKV) and their respective vectors, cases of co-infections with these arboviruses have been reported15–19. The lack of vaccines or antivirals to ameliorate these diseases makes the search for
new tools that aid in development of effective therapies imperative to control arboviral infections.
The viruses
Dengue virus
DENV is an enveloped virus with a single-stranded, positive-sense RNA genome belonging to the Flaviviridae family20. The RNA genome of 11 kb encodes three structural glycoproteins:
envelope (E), precursor membrane (prM, of which the mature form is M), capsid (C); and seven non-structural proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS521. One hundred and
eighty copies of E and M are embedded on the surface of the host-derived lipid membrane of mature virions. Multiple copies of another structural protein, capsid (C), encapsulate the RNA genome to form the nucleocapsid22–24 (Figure 2). The ectodomain of the E protein contains three
structurally defined domains. Domain I functions as the hinge region and is located between domain II and the immunoglobulin-like domain III25. Domain II harbors the fusion loop and
domain III has been postulated to be involved in virus receptor binding. Ninety homodimers of E are arranged in sets of three E head-to-tail dimers that lie in 30 rafts and form a herringbone pattern on the surface of the virus21. E and M are anchored to the viral membrane via their
transmembrane regions. The cytoplasmic tails of the E and M proteins do not interact with the nucleocapsid in the virion26.
Figure 2. DENV structure and genome composition. (A) Cross-sectional representation of a DENV virion depicting the
E dimer (yellow), the capsid protein (C, green), the M protein (red), the RNA genome and the viral membrane. (B) 3D model of a fully mature and immature DENV virion. (C) DENV genome and polyprotein composition. The scissors repre-sent the cleavage sites of furin, NS3 and signal peptidase proteases. Figure adapted from27,28 and made in Biorender.
Dengue virus replication cycle
The fi rst step in DENV virus replication is receptor binding and entry. To date, no specifi c receptor has been identifi ed for DENV infection in human cells29. However, several attachment
factors are known to mediate host cell binding and entry, including the ubiquitously expressed heat-shock proteins 70 and 90 and heparan sulfate30–32. Moreover, the more cell-type specifi c
C-type lectin receptors namely C-type lectin domain family 5 member A (CLEC5A), dendritic cell-specifi c intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN, also known as CD209), L-SING and mannose receptor (CD206) and CD14 have been reported to serve as attachment factors for DENV infection33–37.
The E glycoprotein mediates cell entry via clathrin-mediated endocytosis. Acidifi cation of the endosomal compartments triggers a conformational change that dissociates the E protein dimers, thus exposing the domain II fusion loop. The E protein then inserts into the endosomal membrane, inducing the trimerization of E and subsequently driving fusion of the viral and
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endosomal lipid membranes. Upon fusion, the nucleocapsid is released into the cytoplasm and C becomes degraded by ubiquitin-proteosome-dependent process38–40. Once genome uncoating
occurs, the positive sense RNA genome is translated into a single polyprotein. This polyprotein is further processed co- and post-translationally by cellular and viral proteases into 3 (E, prM and C) structural and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5)21 (Figure 2). The E and prM proteins dimerize and get oriented into the lumen of
the endoplasmic reticulum (ER). The non-structural glycoproteins assemble together to form the replication complex, in which NS5 functions as the RNA-dependent RNA polymerase21,41.
Replication of the genome occurs in ER-derived vesicles. The progeny RNA then exits the vesicles and associates with C proteins to form the nucleocapsid which buds into the ER thereby acquiring an E- and prM-containing lipid bilayer. The presence of the prM protein on newly assembled immature virions protects E from premature fusion from within the acidic compartments of exocytotic pathway. Virion maturation occurs while passing through the trans-Golgi network where the host enzyme furin cleaves prM into M and the pr peptide26,42,43. The
pr peptide remains attached to the mature virus progeny and dissociates from the virus particle after its release to the extracellular milieu42,44,45. Importantly, in case of DENV, the cleavage
of prM into M is generally inefficient and results in the release of a mixture of heterogeneous particles namely mature, partially mature and fully mature virions46. Depending of the infected
cell-type, up to 40% of released DENV virions still contain prM43,47. Generally, fully immature
virions are considered virtually non-infectious47–49.
Chikungunya virus
Chikungunya virus is a member of the family Togaviridae and genus Alphavirus to which Semliki forest virus, rubella virus and Sindbis virus also belong. CHIKV is an enveloped virus with a single-stranded positive-sense RNA genome of 11.8 kb. The RNA genome consists of two separate open reading frames (ORFs), the 5’ORF encodes for the four non-structural proteins (nsP1-4) and the 3’ORF encodes for the 5 structural proteins (C, E1, E2, E3 and 6K)50.
The genome is packaged by 240 copies of the capsid protein (C) to form the nucleocapsid. The nucleocapsid is surrounded by a host-cell derived lipid bilayer in which 240 units of E1 and E2 glycoproteins are anchored. E1 and E2, are arranged into 80 spikes, each spike is a trimer of E2/ E1 heterodimers50,51 (Figure 3). The E1 glycoprotein is a type II membrane protein and has three
domains, DI, DII and DIII. The fusion loop is located at the distal point of DII. The E2 contains three immunoglobulin domains, DA, DB and DC. DB is located at the end of the spike, DA in the center and DC lay proximal to the viral membrane52.
Figure 3. CHIKV structure and genome composition. (A) Cross-sectional representation of a CHIKV virion depicting
the E1-E2 trimer (yellow), the capsid protein (C, green), the RNA genome and the viral membrane. (B) 3D model of a CHIKV virion. (C) CHIKV genome and polyproteins composition. The scissors represent the cleavage sites of furin, nsP2, signal peptidase and capsid proteases. Figure adapted from53,54 and made in Biorender.
Chikungunya virus replication cycle
The fi rst step of CHIKV replication cycle is attachment to the host-cell lipid membrane. Recently, Mxra8 (also known as DICAM or limitrin), which is expressed in myeloid, mesenchymal and epithelial cells, has been reported to be a receptor for several arthritogenic alphaviruses, including CHIKV55. However, several attachment factors, namely prohibitin,
phosphatidylserine-mediated virus entry-enhancing receptors and glycosaminoglycans, have been described to mediated CHIKV entry to the cell56. The E2 glycoprotein facilitates attachment
to the cell surface receptors which promotes entry to the cell mainly by clathrin-mediated endocytosis. Acidifi cation of the endosomal vesicles (pH below 6.2 or 5.9) destabilizes the E1/E2 heterodimer leading to the exposure of the fusion loop and the subsequent fusion of the viral and endosomal lipid membranes57. After fusion occurs, the nucleocapsid is delivered
into the cytoplasm. Disassembly of the capsid and subsequent release of the RNA genome is thought to happen due to its binding to the large ribosomal unit58. The 5’ORF of the viral
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nsP2, nsP3 and nsP4. This polyprotein is further processed in cis by nsP2 generating P123 and nsP459,60 (Figure 3). The newly unstable replication complex will then synthetize
negative-sense RNA intermediates61. When the levels of P123/nsP4 reach a stoichiometric balance, the
polyprotein will be completely processed to its individuals’ non-structural proteins. Meanwhile, the negative-sense RNA intermediates serve as a template for the synthesis of genomic and subgenomic RNAs62. Translation of the subgenomic RNA generates a polyprotein precursor
containing the structural proteins. Auto-processing of the polyprotein precursor will generate the C protein63. Due to the presence of a signal sequence in E3, the rest of the polyprotein will
be directed and translocated to the ER64. Once in the ER, host proteases will cleave the rest
of the polyprotein to generate E1, 6K and the fused form of E3-E265 (Figure 3). Upon further
post-translational modification E2/3 and E1 are transported to the trans-Golgi network where the host protease furin will mediate the cleavage of E2/3 into E2 and soluble E3, leading to the formation of E2/E1 heterodimers, which are transported to the plasma membrane. The viral RNA is packaged by C proteins to form the nucleocapsid. Interactions between the nucleocapsid and E2 proteins drive virion assembly and release at the plasma membrane62.
DENV and CHIKV tropism
DENV and CHIKV viruses are transmitted by the same mosquito’s species, thus both share the same route of entry to the human body. At the site of the mosquito bite, dermal fibroblasts, keratinocytes and skin-resident Langerhans cells are the first targets for DENV and CHIKV virus infection66–70. Inflammatory responses elicited after the mosquito bite induce the recruitment
of mononuclear phagocytic cells, namely monocytes, macrophages and dendritic cells which are the main targets for DENV infection in blood 49,71–78. For CHIKV it is thought that blood
monocytes, which have low susceptibility to infection, contribute to viral dissemination in muscles, joints, eyes and skin79,80. Additionally, DENV antigen has been detected in B cells,
T cells and platelets 81–84. Hepatocytes and endothelial cells have also found to sustain DENV
replication in vitro and in patient material66,85–87.
The innate immune system
Sensing pathogens: cellular effectors
Innate immune cells together with epithelial and endothelial cells constitute the first line of defense against invading pathogens. Innate immunity is largely composed of myeloid cells that can phagocyte and destroy pathogens88. The myeloid cell repertoire includes polymorphonuclear
and mononuclear phagocytic cells. Neutrophils, basophils and eosinophils comprise the polymorphonuclear phagocytes and have key role in controlling infection. Of these cells, neutrophils are of great importance as they are equipped with a variety of weapons to efficiently destroy invading pathogens88,89. The mononuclear phagocytic cells are a heterogeneous
population of cells encompassing monocytes subsets in blood and tissues, dendritic cells (DCs) as well as macrophages 90. Monocytes in blood represent a dynamic cell population composed
of three subsets which differ in phenotype and function91–93. In humans, blood monocytes are
classified by the expression of CD14 and CD1692. Classical monocytes (CM, CD14++CD16-)
account for the 85% of the monocyte pool. The rest of the monocyte population consists of intermediate (IM, CD14++CD16+) and non-classical (NM, CD14+CD16++) monocytes91,94.
CM play a very important role in the initiation of the inflammation by producing a large set of inflammatory mediators such as cytokines, myeloperoxidase and superoxide95. IM, known
as inflammatory monocytes, are rapidly recruited to the sites of inflammation where they contribute with the production of inflammatory cytokines. Additionally, IM can differentiate into inflammatory macrophages96–98. NM, also known as patrolling monocytes, act as protectors of
the endothelium and once activated differentiate into anti-inflammatory macrophages to repair tissue damage99–101. Macrophages are a heterogenous population of cells comprising
tissue-resident macrophages and monocyte-derived macrophages. Their morphology and function vary depending on their microenvironment102. Exposure to cytokines and other stimuli drives what
is known as macrophage polarization. In general, M1, or classically activated macrophages, have an inflammatory profile and mediate pathogen clearance. M2, or alternatively activated macrophages, have an anti-inflammatory profile and promote wound healing and resolution of the inflammation. However, this classification seems insufficient to describe their unique expression profiles102,103. Importantly, macrophages release chemotactic cytokines that recruit
other innate immune cells to the site of infection and can initiate adaptive immunity to invading pathogens via antigen presentation88. Dendritic cells (DCs) are also a heterogenous population
of myeloid cells. They are known to stimulate and induce adaptive immune responses88,104.
Moreover, DCs contribute to inflammation by producing pro-inflammatory cytokines and interferons upon infection. The DC repertoire in blood was initially comprised by conventional DC1 and DC2, and plasmacytoid DCs (pDCs)104. However, with the current emergence of
single-cell technologies, such as single-cell RNA sequencing, this repertoire has now expanded to seven subsets105,106. Additionally, each tissue also contains its own collection of
tissue-resident dendritic cells104.
Sensing pathogens: pattern recognition receptors
Cells of the innate immune system perform immune surveillance by utilizing membrane-bound and cytoplasmic pattern recognition receptors (PRRs). During infection, several PRRs work together to sense specifically pathogen-associated molecular patterns (PAMP’s) and host-derived danger signals that are released by infected cells termed tissue-host-derived danger-associated molecular patterns (DAMP’s). PRRs are germline encoded, constitutively expressed and show different pattern of expression among cell types. Engagement of these PRRs in turn activates specific signaling cascades leading to the production of cytokines and chemokines 107,108. PRRs
can be classified in several families based on domain homology encompassing the membrane-bound Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and the intracellularly located retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding domain,
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leucine-rich repeat (LRR)-containing receptors (NLRs or NOD-like receptors), AIM2-like receptors (ALRs) and cGAS (Figure 4)107,109,110. From all these families of PRRs, TLRs and RLRs
have been widely study for their role in sensing RNA viruses111. ALRs and cGAS are cytoplasmic
DNA sensors, thus commonly sensing DNA viruses107. RLRs comprise three cytosolic proteins,
retinoic acid-inducible gene-I (RIG-I), melanoma differentiation gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). These receptors sense specific features of RNA genomes like the presence of 5’triphosphate RNA, single or double-stranded RNA and characteristic regions of genomes like the poly-uridine region of the hepatitis C virus107. Following the
sensing of the viral genomes, RLRs translocate to the mitochondria or the peroxisomes where the adaptor molecule mitochondrial antiviral protein signaling protein (MAVS) is present. Depending on MAVS localization, specific signaling pathways will be triggered leading to the activation of transcription factors nuclear factor kappa B (NF-κB) and interferon regulatory factors 3 and 7 (IRF3 and IRF7, respectively). Ergo, inducing the production of Type-1 IFNs and pro-inflammatory cytokines107,109,110. Binding of IFNs to their specific receptors will activate the
Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) pathway and subsequent production of interferon stimulated genes (ISGs)107.
TLRs were originally identified as homologues of the Toll receptor in the fruit fly Drosophila
melanogaster112. In mammals, these receptors function as sentinels of the innate immune system.
Ten functional TLRs are present in humans and 13 in mice. They are type I transmembrane proteins that contain three domains, an extracellular domain with long-leucine-rich repeat regions that mediates protein-protein or protein-PAMP sensing, a transmembrane domain and a cytoplasmic toll/interleukin-1 receptors (TIR) domain113. Plasma membrane-bound TLR1,
TLR2, TLR4, TLR5, TLR6 and TLR10 sense lipids, proteins, lipoproteins and glycoproteins while TLR3, TLR7, TLR8, TLR9, TLR11 and TLR13 are localized inside the endosomal compartments where they sense nucleic acids110,113. Upon PAMP recognition, sorting adaptors
molecules toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) and TIR domain-containing adaptor protein inducing IFNβ (TRIF)-related adaptor molecule (TRAM) will recruit and assemble supramolecular organizing centers (SMOCs), the myddosome and the triffosome, respectively. The myddosome consists of the myeloid differentiation primary response protein 88 (MyD88) and several copies of the IL-1 receptor-associated kinases (IRAK) serine-threonine kinases. The triffosome, on the other hand, consists of TRIFF which is believed to recruit TNF receptors-associated factor 3 (TRAF3) to form this complex110,113,114. Activation
of all TLRs will lead to the production of cytokines and chemokines through NF-κB and activator protein 1 (AP-1) activation via the myddosome. Production of interferons, however, is induced by TLRs localized in the endosomal compartments and its mediated by the interferon regulatory factors (IRF) family of transcription factors via the triffosome and myddosome, with the triffosome complex most associated with these responses113,114. The production of cytokines
and interferons will trigger a variety of inflammatory responses including the recruitment of immune cells and the induction of adaptive immunity114.
Figure 4. Pattern recognition receptors and innate immune signaling. General overview of the different families of
PRRs utilized by innate immune cells to perform immune surveillance. These families encompass surface bound and endosomal expressed TLRs, RIG-I-like receptors, NLRs, ALRs and the cGAS/STING pathway. Activation of these PRRs trigger signaling pathways leading to nuclear translocation of transcription factors IRFs, NF-κB and AP-1, and the subse-quent production of infl ammatory and antiviral cytokines. Figure made in Biorender.com
Endothelial cells
Blood vessels encompassing veins, venules, arteries, arterioles and capillaries are known as the vasculature. Their function is to transport the blood throughout the body115. The vascular
endothelium is a semi-permeable layer of endothelial cells (EC) that line the macro- and microvasculature. Its function is to provide a barrier between the vessel’s lumen and the underlying tissue116. EC are sealed to each other by tight junctions, adherens junctions and
VE-cadherin117. In addition, the glycocalyx barrier located over the EC regulates vascular
permeability, protects the EC against invading pathogens, infl ammatory mediators and controls the interactions between EC and immune cells in blood118. During homeostasis (Figure 5),
Angiopoietin 1 released by pericytes binds to Tie2 receptor ensuring barrier maintenance and endothelium integrity119. Activation of the endothelium can occur either due to direct sensing of
PAMPs and DAMPs by PRR expressed on EC or indirectly due to pre-established infl ammatory responses. Upon activation, EC will respond by producing angiopoietin 2, infl ammatory and chemoattractant cytokines (e.g. IL-6, IL-8 and MCP-1) and induce the enhanced surface expression of adhesion molecules (E-selectin, P-selectin, vascular cell adhesion protein 1 or VCAM-1, and intercellular adhesion molecule 1 or ICAM-1)120–122. This activation will
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and subsequently induce endothelium disruption and vascular permeability122–125. Endothelium
activation and dysfunction are two independent processes, with the latter resulting in the EC not being able to restore the homeostatic healthy state. Exacerbated infl ammation and increased leukocyte traffi cking are some of the best-known factors that can contribute to the endothelium dysfunction. Importantly, the endothelial responses to infl ammation as well as its regulatory functions are organ specifi c126. Hence, understanding these responses and functions are crucial
for selecting the right specifi c treatments to alleviate endothelium-associated pathogenesis.
Figure 5. Endothelium during homeostasis. Endothelial cells form a semi-permeable layer that line the micro and macro
vasculature known as the endothelium. Keeping the integrity of the endothelium is crucial for the proper functioning and maintenance of organs. Several molecules work in consonance to maintain the endothelium in homeostasis. For instance, pericytes release angiopoetin-1 that binds the Tie2 receptor and promoting homeostasis. Tight and adherens junctions seal ECs making the endothelium a semi-permeable layer. The glycocalyx barrier protects EC from infl ammatory mediators and invading pathogens. Furthermore, ECs are equipped with PRRs that detect pathogens and/or their components, which will lead to the activation of the endothelium and the production of adhesion molecules and infl ammatory and antiviral cytokines. Figure made in Biorender.
Dengue disease pathogenesis and innate immune responses
Dengue viruses are responsible for an estimated number of 390 million of infections in which the disease is clinically apparent in 96 million cases8. Clinical manifestations range from an
acute self-limited dengue with or without warning sings (previously termed dengue fever) to a life-threatening severe dengue (previously known as dengue hemorrhagic fever and dengue shock syndrome)127,128. The clinical course of dengue is depicted in Figure 6. While most DENV
infections are asymptomatic (75%), few result in a mild febrile disease (25%) characterized by fever, muscular pain, headache, eye pain and rash. Moreover, an estimated 1-5% of patients further develop thrombocytopenia, vascular permeability and plasma leakage, hallmarks of severe disease. The dengue mortality rate is low (<1%), with most of the patients recovering after 7 days post-illness. To date, there are no specifi c treatments available to ameliorate severe disease.
0 Febrile Phase 1 2 3 4 5 6 Critical Phase
Time (days)
• Viremia • Host immune responses • Shock • Bleeding • Organ impairment 10 7 8 9 Recovery Phase • Resolution plasma leakage • No viremia • Antibody responsesFigure 6. Clinical course of dengue. The clinical course of DENV infection consist of three phases. The febrile phase
is characterized by high viral titers which coincides with the clinical manifestations of the disease. An exacerbated and uncontrolled immune response to the infection can lead to the development of vascular permeability and plasma leakage, clinical manifestations that hallmark severe dengue (critical phase). The critical phase is followed by the recovery phase in which plasma leakage is resolved and the virus is cleared. Adapted from129.
Factors like comorbidities, bacterial co-infections and/or translocations and the presence of cross-reactive infectious-enhancing antibodies (raised during previous heterologous infections) have been linked to severe disease development130–135. Myeloid cells like monocytes, macrophages and
dendritic cells produce Type-1 IFNs and pro-inflammatory cytokines during DENV infection that, on the one hand, can be protective for some patients or, on the other hand, can promote severe disease development. Among the three monocyte subsets in blood, increased frequencies of intermediate or inflammatory monocytes have been associated with onset of severe dengue 72,136,137.
Macrophages can contribute to the overall immune responses driving severe disease by producing inflammatory mediators upon DENV infection 138,139. DCs, have been shown to contribute to the
innate immune responses in course of DENV infection by inducing the production of Type-1 IFNs 140–142. Interestingly, the presence of heterologous antibodies from previous DENV infections
that bind to FCγR-expressing cells can enhance DENV entry and infection, a phenomenon called antibody-dependent enhancement (ADE). In vitro DENV infections in the presence of human serum have demonstrated that monocytes, macrophages and mature dendritic cells can support ADE49,77,139,143. Knowing the important role that monocytes, macrophages and DCs play
during DENV infection and pathogenesis, future research should focus in addressing the overall contribution of these cells to severe dengue development.
As previously mentioned, severe dengue is hallmarked by vascular permeability and plasma leakage, indicating a major role of the endothelium in severe disease development. Exacerbated inflammation due to cytokine storm is thought to be the principal contributor to these endothelial disturbances and the immunopathogenesis of severe dengue. Indeed, proinflammatory cytokines IL-1β and TNFα, which are known to induce endothelium activation, have been found to be elevated in patients with dengue129,144,145. During severe dengue, several systems in
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charge of maintaining endothelium integrity are compromised. For instance, EC release high levels of angiopoietin 2 which in turn bind to Tie2 impeding its binding with angiopoietin 1, thus disrupting the EC integrity146,147. The coagulation system also gets affected as DENV
inhibits the conversion of prothrombin intro thrombin leading to the development of internal bleeding148. Additionally, shedding of the glycocalyx during severe dengue has been shown to
promote plasma leakage149.
PRR-expressing cells can indirectly contribute to endothelial activation and dysfunction by sensing specific features of DENV during the course of the infection, subsequently leading to the production of pro-inflammatory mediators. For instance, cytoplasmic RIG-I and MDA5, and endosomal-expressed TLR3 and TLR7, are known to sense DENV RNA genome leading to the production of Type-1 IFNs and proinflammatory cytokines. Interestingly, the production of Type-1 IFNs generally has a protective antiviral role impacting DENV replication. In contrast, signaling cascades triggered by these endosomal and cytoplasmatic sensors can also lead to the production of TNFα, IL-6 and IL-1β, which are cytokines associated with severe dengue. Moreover, DENV and presumably other flaviviruses can induce mitochondrial damage-associated mitochondrial DNA release leading of the activation of cGAS/STING and TLR9 pathways and the subsequent production of Type-I IFNs150–152. Importantly, DENV
non-structural proteins (NSs) target key molecules of these signaling pathways leading to the production of Type-1 IFNs as a mechanism of immune evasion. For instance, NS2B and NS2B3 target the cGAS/STING pathway to prevent mitochondrial (mt)DNA sensing during DENV infection150,151,153. NS4 blocks the interaction between adaptor molecule MAVS and
RIG-I. Furthermore, NS4B, NS2A and NS2B3 impede the phosphorylation and activation of IKKԑ and TBK1. With regard to the IFNα/β receptor signaling, NS4 and NS5 prevent the phosphorylation of STAT1 and NS5 also targets STAT2 for degradation via proteosome further inhibiting the production of ISGs. DENV virus immune evasion of TLRs pathways hasn’t been described yet. However, DENV NS1 is known to induce TLR activation and contributes to severe dengue development. TLR4 expressed on the surface of monocytes can sense DENV NS1, which is released from DENV infected cells, leading to the production of cytokines and further activation of the endothelium154,155. In addition, in vitro and ex vivo models have shown
that NS1 can induce vascular permeability and plasma leakage by directly interacting with glycocalyx components156–159. DENV can also be sensed by CLRs, indeed, it has been reported
that the C-type lectin domain 5 member A (CLEC5A) upon sensing of DENV particles can induce potent immune responses that contribute to severe disease in an in vivo model of ADE33.
Moreover, it has been recently described that CLEC5A in partnership with TLR2 can sense extracellular vesicles released by platelets in vivo, further promoting the release of cytokines and neutrophil extracellular traps (NETs) by neutrophils. The release of these inflammatory effectors by neutrophils results in vascular permeability160. Furthermore, breakdown of ECs
tight junctions leading to vascular leakage has been linked to the release of tryptase by mast cells upon sensing DENV161. Taken together, rapid antiviral responses coupled with controlled
inflammatory responses are crucial for the containment of DENV infection. The precise molecular mechanisms involved in the development of severe dengue are not fully understood
yet. However, our understanding of the role of PRRs in the sensing of DENV particles by innate immune cells as well ECs has increased over the years. Further research should focus on using agonists or antagonists of these receptors and their associated downstream signaling pathways in relevant settings to develop new therapies aiming to control severe dengue development.
Chikungunya disease pathogenesis and innate immune
responses
After the mosquito bite, CHIKV rapidly spreads to several tissues throughout the bloodstream. CHIKV titers in serum samples from infected patients have been reported to reach up to 109 CHIKV particles /mL162,163. The high viremia coincides with the occurrence of clinical
symptoms such as fever, rash, headache, myalgia and arthralgia which lasts up to 5-7 days164.
However, contrary to dengue, which is transmitted by the same mosquito’s species, 50 to 97% of the CHIKV-infected individuals will develop the aforementioned clinical manifestations163.
Interestingly, even though CHIKF is a mild self-limiting disease, severe cases requiring hospitalization have been reported165,166. During the outbreak in La Réunion island in 2006,
complications such as myocarditis, encephalitis and multiorgan failure following CHIKV infection in newborns, the elderly and diabetic patients were associated with disease mortality. In addition, atypical clinical manifestations such as Guillain-Barré syndrome have been reported in CHIKV-infected patients during La Réunion and French Polynesia outbreaks167,168.
Importantly, considering the fact that the clinical symptoms of CHIKF highly resemble those of dengue and Zika, the correct diagnosis of the disease remains challenging. To date, there is no vaccine and no specific therapeutic agent available to prevent or ameliorate CHIKF.
Nearly 40% of patients with acute CHIKF will develop chronic disease hallmarked by polyarthralgia, which can last up to five years after acute infection164 (Figure 7). Presence
of viral antigen and potent inflammation in the joints are thought to be the cause of chronic disease. Indeed, the presence of viral antigen in macrophages, monocytes and lymphocytes has been observed in synovial fluids and tissues of a one-year CHIKV chronically infected patient. Additionally, several inflammatory mediators such as MCP1, IP-10, MIG, IL-6, TNFα and IL-1β have been detected in plasma samples in acute CHIKV patients and non-human primates169. From those, MCP1, IL-6 and IL-8 have been detected in synovial fluids
of chronic CHIKV patients and their presence is indicative of chronic disease development (Figure 7)163. Importantly, the use of in vitro and in vivo mouse models and non-human primate
models to study CHIKV pathogenesis have increased the current understanding of the CHIKV pathogenesis and associated pathologies169. Those models have underscored the importance of
monocytes and macrophages in CHIKV chronic disease. Namely, both cell types were found to be recruited to inflammation sites via MCP1. There, monocytes and macrophages not only contribute to CHIKV pathogenesis by producing inflammatory mediators, but also serve as a reservoir for viral replication. In NHP, CHIKV RNA has been detected in joints and lymphoid organs after clearance in blood (up to 9 days post infection). Additionally, after acute infection
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the virus is mainly detected in macrophages. The use of CCR2 defi cient mice, which function as a receptor for MCP1, has shown altered immune responses and pathogenesis following CHIKV infection as evidenced by the development of severe and prolonged arthritis. Furthermore, the use of Bindarit, a drug that specifi cally downregulates gene expression of MPC1170, has been
shown to prevent monocyte infi ltration and CHIKV-mediated arthritis in mice171. Moreover,
infl ammatory monocytes and macrophages infi ltrating into bone tissue can differentiate and/or interact with osteoblasts causing bone erosion, thus contributing with the severity of the CHIKV-mediated arthritis172. In CHIKV patients, this process has been associated with presence of high
levels of receptor activator of nuclear factor kappa-B ligand (RANKL), a protein that controls bone regeneration and remodeling171,173. Another cell type found present in high numbers in
infl amed joints of mice are natural killer cells (NKs). The importance of NKs in patients is evidenced by their strong activation during early CHIKV infection, which has been correlated with balanced adaptive immune responses to CHIKV antigens169,174,175.
CHIKV infection 2-4 days Acute Phase Months-years • IFN response • MCP1, TNFα, IL-1β, IL-6 Chronic Phase • Monocyte/Macrophage infiltration to joints • Arthritis • Bone loss Viremia 3-5 days Symptoms • IFN response • MCP1, TNFα, IL-1β, IL-6 Viremia Symptoms
Figure 7. Clinical course of Chikungunya. After the mosquito bite there is an incubation period of 2-4 days followed by
a rapid increase in CHIKV titers in blood. This coincides with development of clinical manifestations in the acute phase which last 3-days. During this phase, immune cells like monocyte and macrophages produce huge amounts of infl ammatory mediators. Activated monocytes and macrophages then infi ltrate to joint and bone tissue causing infl ammation and promoting chronic disease-associated symptoms, such as polyarthralgia and bone loss, that can persist from several months up to several years. Adapted from169.
The host immune response is crucial for the containment of CHIKV infection and mainly driven by the production of antiviral Type-1 IFNs. PRRs such as RIG-I-, MDA5- and TLR3 are most likely to sense CHIKV infection based on their function. Indeed, CHIKV genome- and replication intermediates-sensing by these PRRs have been shown to be involved in triggering the aforementioned antiviral responses in in vitro/in vivo models176–178. Importantly, experiments
in mice have provided evidence that an MyD88-dependent sensor such as TLR2, TLR4 or TLR7 may also control viral dissemination and spread177. However, the involvement of any of those
PRRs during CHIKV infection in patients or human in vitro systems hasn’t been described yet. Additionally, it was recently reported that mtDNA is released into the cytoplasm during CHIKV
infection, further inducing Type-1 interferon responses via the cGAS/STING pathway179.
Interestingly, CHIKV counteracts the cGAS/STING pathway by inducing the degradation of cGAS through an ATG7 autophagy-dependent mechanism and via direct interaction of nsp1 to STING resulting in signaling inhibition179. Furthermore, CHIKV nsp2 inhibits the JAK/STAT
signaling pathway by impeding the translocation of STAT1 into the nucleus, preventing the transcription of ISGs180.
Scope of the thesis
The research presented in this thesis focuses on the innate immune responses elicited by dengue and chikungunya virus infections. Although the immunopathologies of dengue and chikungunya virus infections have been described, the mechanisms underlying the exacerbated inflammation observed during severe dengue and CHIKV-mediated arthritis are not completely understood. Interestingly, monocytes in blood represent primary targets for DENV and CHIKV infections, and due to their associated role as innate immune sentinels, also contribute to disease pathogenesis by producing inflammatory and antiviral cytokines. Here, by utilizing human primary cell in vitro systems, we aim to decipher the contribution of monocytes in the immunopathology of dengue and CHIKV infections.
Due to the number of dengue and chikungunya cases augmenting over the last decades, the incidence of co-infections with both viruses has dramatically increased. In chapter 2, by using primary human PBMCs, we aimed to describe the viral kinetics and innate immune responses of DENV and CHIKV co-infections in vitro. By co-infecting with UV-inactivated particles, we delineate the contribution of viral replication in such responses. Moreover, multiplex analysis of cytokines allowed us to determine the innate immune signature of DENV and CHIKV mono-/co-infections. As PBMCs comprise several types of cells such as lymphocytes and monocytes, we investigated in the subsequent chapters which of these cells contribute to the immunopathogenesis of DENV and CHIKV infections.
Ex vivo analysis of PBMCs has previously shown than CHIKV infection increases the frequency
of intermediate inflammatory monocytes. In chapter 3, we used a previously established in vitro model of CHIKV infection to rewire PBMC responses and delineate the role of intermediate monocytes in virus replication and innate responses.
As sentinels of the innate immune system, monocytes use toll-like receptors to sense pathogens and induce innate immune responses. In chapter 4, by using human primary PBMCs and endothelial cells in vitro models, we identified the role of TLR2 as a sensor and regulator of the innate immune responses following DENV infection. Furthermore, by analyzing the TLR2 expression and monocyte subset frequencies in a DENV-infected pediatric cohort, we identified a prognostic value of TLR2 expression in disease pathogenesis.
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Since virtually non-infectious immature DENV particles are released in large numbers by DENV infected cells, we aimed to investigate the contribution of these particles in the innate immune responses during dengue virus infection. In chapter 5, we indicate that the maturation status of the DENV is likely to influence the kinetics and extent of inflammatory responses during DENV infection. Additionally, we scrutinized the role of TLR2 in these responses using primary human PBMCs and endothelial cell in vitro systems.
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