Interplay between innate immune response and inflammatory mediators

The arbovirus transmission cycle is triggered when a vector ingests microbe-containing fluid during its blood feeding. These viral particles proliferate in the midgut and traverse to the salivary glands. Surprisingly, DENV induces multiple transcription factors that modulate viral replication in the salivary glands, which alter chemosensory attributes. This results in shaping bloodmeal selection by adjusting mosquito host-seeking or probing behaviour, which ultimately leads to increased viral transmission (Ref. Reference Sim, Ramirez and Dimopoulos18). Female mosquitoes utilize a proboscis, which contains 1 × 10⁴–1 × 10⁶ plaque-forming units of the viral particle, that is, to be injected into the epidermis and dermis, thereby inducing an interaction between the pathogen and resident and migratory cells (Ref. Reference Rathore and John19). It is generally accepted that viruses are predominantly injected into the dermis; nevertheless, several investigations have indicated with respect to parasitic infection that pathogens are also found in the epidermis (Ref. Reference Jin, Kebaier and Vanderberg20), which is expected to also occur in arboviral infections owing to analogous saliva composition and probing behaviour (Ref. Reference Rathore and John19). DENV inoculum is rather minuscule; however, it is effective in efficiently invading the host (Ref. Reference Rathore and John19). The cells engaged during transmission have significant implications for the subsequent inflammatory cascade. A more thorough understanding of early responders, along with their ability to enhance replication and propagate, provides further insight into their pathophysiology (Figure 2).

Figure 2. A pictorial representation of the immune response triggered in the skin during dengue infection. The diagram displays the network of immune cell types that encounter DENV in the skin during the early hours after infection. Few viruses are thought to be introduced into the epidermis through natural route infection; however, keratinocytes within the epidermis are one of the primary targets for viral dissemination. DCs, monocytes and macrophages are identified as the infection targets in the dermis. Although MCs are not substantially infected in the skin, they undergo activation by DENV and degranulate. This activation leads to the recruitment of NK cells to the infection site. Skin-homing T cells also migrate to the infected skin sites. Using lymphatics, infected DCs transport DENV to the draining lymph nodes.

Keratinocytes

The skin, an intricate organ, executes diverse defensive roles against exogenous factors, and this protective role is accomplished by its structure, which comprises of three layers: the epidermis, the dermis and the hypodermis. These cells facilitate the pro-inflammatory and antiviral milieu created in the early stages of DENV infection. Consequently, the skin barrier harbours a diverse variety of indigenous and transient immunocompetent cells capable of guiding and triggering an effective immune response aimed at limiting pathogen propagation (Ref. Reference Garcia, Wehbe, Lévêque and Bodet21). Keratinocytes contribute 90% to the integrity and infrastructure of the outer layer of the skin, suggesting the ability of viral particles to multiply in these resident cells constitutes a compelling prospect for host expansion. As previously reported, keratinocytes are more susceptible to DENV infection than dermal fibroblasts (Refs Reference Garcia, Wehbe, Lévêque and Bodet21, Reference Wei, Wei, Liao and Chang22). Pattern recognition receptors (PRRs) expressed in host cells, such as membrane-bound TLRs, nucleotide oligomerization domain (NOD)-like receptors (NLRs), protein kinase R (PKR), retinoic-acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), are the primary interactors in the immune recognition of DENV. Their activation results in eliciting an immune response (Refs Reference Garcia, Wehbe, Lévêque and Bodet21, Reference Wei, Wei, Liao and Chang22, Reference Wichit, Ferraris, Choumet and Missé23, Reference Briant, Desprès, Choumet and Missé24). These PRRs initiate signalling cascades by activating transcription factors such as nuclear factor kappa B (NF-κB), interferon (IFN) regulatory factor 3 (IRF3) and IRF7. Numerous IFN-stimulated genes (ISGs), proinflammatory cytokines and chemokines with varying degrees of antiviral activity are expressed as a consequence of activated IRF3 and IRF7 (Refs Reference Wichit, Ferraris, Choumet and Missé23, Reference Chen, King, Tu, Sanchez, Luster and Shresta25).

Keratinocytes function as an accessory cell in adaptive immune response and are one of the earliest targets, making up for 60% of all infected cells. As keratinocytes do not migrate, they are unable to aid in the dissemination of viral particles, but are equipped to secrete chemotactic factors that draw susceptible cells to the site of infection (Ref. Reference Duangkhae, Erdos, Ryman, Watkins, Falo, Marques and Barratt-Boyes26). Surasombatpattan et al.(Ref Reference Surasombatpattana, Hamel, Patramool, Luplertlop, Thomas, Desprès, Briant, Yssel and Missé27) reported keratinocytes being permissive to DENV-2. The double-stranded viral RNA present intracellularly is sensed by TLR3, thereby exhibiting strong IFN beta (IFN-β) expression. Antimicrobial proteins, comprising human β defensin 2 (hBD2), hBD3, LL37 and RNase 7, known for triggering local innate response and attracting immunocompetent cells, become activated post-infection. Following dengue infection, hBD3 expression immediately spikes, but as this gradually declines, hBD2 expression peaks. Additionally, LL37 is a chemoattractant for mast cells (MCs), neutrophils, monocytes and T-cells, and shows enhanced expression following infection (Refs Reference Surasombatpattana, Hamel, Patramool, Luplertlop, Thomas, Desprès, Briant, Yssel and Missé27, Reference Wang and Li28, Reference Surasombatpattana, Patramool, Luplertlop, Yssel and Missé29). IRF3 expression remained unaltered after DENV inoculation, whereas TLR3 and IRF7 expression increased. Other ISGs, including PKR, 2’-5’-oligoadenylate synthetase 2 (OAS2) and Ribonuclease L, are also activated, leading to heightened antiviral response. Another enhanced protein, Ribonuclease L inhibitors, counterbalance heightened immune response, thereby maintaining an equilibrium (Refs Reference Surasombatpattana, Hamel, Patramool, Luplertlop, Thomas, Desprès, Briant, Yssel and Missé27, Reference Aluri, Cooper and Schuettpelz30). The pro-inflammatory factor interleukin-1beta (IL-1β) secreted by infected keratinocytes mediates the recruitment of macrophages, activates dendritic cells (DCs) and promotes T-cell function. Another function of IL-1β is that it induces C–C motif Chemokine ligand 20 (CCL20), another immunomodulatory factor, to recruit DC at the site of infection. Migration of Langerhans and dermal DCs from skin to lymph nodes requires IL-1β in conjunction with tumour necrosis factor alpha (TNF-α) for its dissemination (Refs Reference Duangkhae, Erdos, Ryman, Watkins, Falo, Marques and Barratt-Boyes26, Reference Wang and Li28). As previously reported, salivary gland extract (SGE) is known to enhance DENV replication by suppressing LL37 and IFN titre, thereby resulting in viral evasion (Refs Reference Surasombatpattana, Patramool, Luplertlop, Yssel and Missé29, Reference Surasombatpattana, Ekchariyawat, Hamel, Patramool, Thongrungkiat, Denizot, Delaunay, Thomas, Luplertlop, Yssel and Missé31). A study of heterologous infection highlighted that secondary dengue infection does not alter keratinocyte activation, indicating no probable role in the severity of infection (Ref. Reference Castanha, Erdos, Watkins, Falo, Marques and Barratt-Boyes32) (Figure 3).

Figure 3. Immune response of keratinocytes. A. DENV is permissible to keratinocytes, which causes viral RNA to be sensed by intracellular TLR3, which then activates TRIF. Activated TRIF binds to TRAF3 and TBK-1, eventually leading to the activation of IRF7, which stimulates the synthesis of IFN-β. B. hBD3 expression heightens in the early stage of infection and gradually declines with an increase in the expression of hBD2. Both the antimicrobial peptides have chemoattractant properties. C. The pathogenic role of keratinocytes, SGE injected during blood feed, results in diminishing the expression of LL37 and IFN, thereby enhancing DENV proliferation. D, E. Schematic representation of the diversified role of IL-1β and LL37 in response to infection.

Mast cells

MCs, triggered by mosquito saliva, play an integral role in host defence because of their ability to regulate both immune responses. When infected, they secrete a variety of T_H1-associated cytokines, along with histamine, leukotrienes, prostaglandins and chemokines, which are crucial for trafficking natural killer (NK) cells, monocytes and T cells (Refs Reference Syenina, Saron, Jagaraj, Bibi, Arock, Gubler, Rathore, Abraham and John33, Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34). Many of these compounds have been shown to activate endothelial cells, control the coagulation cascade and/or boost vascular permeability (Ref. Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34). MCs have been reported to be degranulated in response to the viral components of DENV and are involved in DENV-induced vascular leakage. Post-degranulation, dengue viral components can be observed in the extracellular space or cytoplasmic granules. These granules have heparin and heparin-like glycosaminoglycans as their surface receptors, which also have a binding affinity for envelope protein. It implied that DENV can migrate in these extracellular granules from the initial site of infection to the lateral lymph node, another indispensable method for viral transmission (Ref. Reference Troupin, Shirley, Londono-Renteria, Watson, McHale, Hall, Hartstone-Rose, Klimstra, Gomez and Colpitts35).

DENV-induced MC activation causes degranulation and increased secretion of inflammatory mediators such as IL-1β, IL-6, TNF-α, CCL3, CCL4, CCL5, chymase and histamine (Refs Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34, Reference Puerta-Guardo, Biering, Harris, Pavia-Ruz, Vázquez-Prokopec, Ayora-Talavera and Manrique-Saide36, Reference King, Anderson and Marshall37). Li et al. reported that salivary proteins, such as adenosine deaminase and al34K2, of A. albopictus alter the inflammatory response by interacting with MC-secreted proteases, known for inducing vascular leakage (Ref. Reference Li, Ji, Cheng, Åbrink, Shen, Kuang, Shang and Wu38). Similarly, chymase converts endothelin-1, a known factor involved in the pathogenesis of haemorrhagic shock, hypothesizing an associated response of MC and endothelial cells in dengue pathogenesis (Ref. Reference Syenina, Saron, Jagaraj, Bibi, Arock, Gubler, Rathore, Abraham and John33). Multiple studies have documented chymase, a serine protease, to be significantly expressed in severe cases as compared to non-severe cases, suggesting its potential as a prognostic marker of severity (Refs Reference Tissera, Rathore, Leong, Pike, Warkentien, Farouk, Syenina, Eong Ooi, Gubler, Wilder-Smith and John39, Reference Rathore, Senanayake, Athapathu, Gunasena, Karunaratna, Leong, Lim, Mantri, Wilder-Smith and John40, Reference Sahu, Aggarwal, Ekka, Nayer, Bhoi, Kumar and Luthra41, Reference John, Rathore, Raghavan, Ng and Abraham42). A study conducted in Sri Lanka by Tissera et al. concluded chymase to be a more pronounced prognostic biomarker for children and adolescent patients as opposed to adult patients (Ref. Reference Tissera, Rathore, Leong, Pike, Warkentien, Farouk, Syenina, Eong Ooi, Gubler, Wilder-Smith and John39). Additionally, they also reported that 96% of the severe cases were accurately predicted in the early stages of the infection (Ref. Reference Tissera, Rathore, Leong, Pike, Warkentien, Farouk, Syenina, Eong Ooi, Gubler, Wilder-Smith and John39). Histamine, another protease found at high levels in dengue-infected patients, indicates its potential role in vascular leakage, which is known to contribute to severe outcomes (Refs Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34, Reference Puerta-Guardo, Biering, Harris, Pavia-Ruz, Vázquez-Prokopec, Ayora-Talavera and Manrique-Saide36). The increased production of vascular endothelial growth factor (VEGF) following DENV infection aids in viral propagation by utilizing a lymphatic pathway to reach the draining lymph nodes. Furthermore, increased expression of VEGF and neuropilin inevitably leads to vascular leakage (Ref. Reference Troupin, Shirley, Londono-Renteria, Watson, McHale, Hall, Hartstone-Rose, Klimstra, Gomez and Colpitts35). Localized expression is known to elevate cellular recruitment and vasodilation, which leads to DENV clearance, whereas systemic infection induces vascular leakage, culminating in DENV pathogenesis (Ref. Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34).

In response to elevate the pathogenic role of MCs, Syenina et al. reported that MC-stabilizing medications could inhibit DENV-induced enhanced endothelial permeability in vivo and reverse uncontrolled pro-inflammatory expression (Ref. Reference Syenina, Saron, Jagaraj, Bibi, Arock, Gubler, Rathore, Abraham and John33). Similarly, St John et al. hypothesized that utilizing a leukotriene receptor antagonist can be a therapeutic target for treating DENV-induced vasculopathy (Ref. Reference John43). Thorough knowledge of MC-induced severe conditions might provide new perspectives on the development of therapeutics that target MCs for controlled degranulation (Figure 4).

Figure 4. Protective or pathogenic role of mast cell in dengue infection. Viral interaction with receptors expressed on the surface of mast cells causes degranulation. DENV-induced degranulation secretes IL-1β, IL-6, TNF-α, CCL3, CCL4 and CCL5, which act as chemoattractants, recruiting monocytes, NK and T cells, essential for viral clearance. On the contrary, granules released during degranulation express heparin and heparin-like glycosaminoglycan on their surface, which has a binding affinity for envelope protein, resulting in viral dissemination. Other proteins secreted during degranulation, such as leukotrienes, VEGF, neuropilin, histamine and chymase, are responsible for altering endothelial permeability, resulting in vascular leakage.

Dendritic cells

DENV infiltrates DCs via DC-specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) and Mannose Receptor (MR), which are the primary interacting receptors responsible for activation and for initiating the downstream signalling cascade by secreting TNF-α (Ref. Reference Schaeffer, Flacher, Papageorgiou, Decossas, Fauny, Krämer and Mueller44). DCs are a type of specialized antigen-presenting cell comprising of three distinct subpopulations, which are known to play a crucial role in moulding the adaptive immune response. Immature myeloid DCs (mDCs) subsequently transform into an activated/mature state after being stung by a mosquito and secrete an array of cytokines and chemokines; additionally, they also migrate towards sites with a high concentration of T cells. Upon maturation, DCs exhibit diminished expression of DC-SIGN, highlighting their incompetence in getting infected by DENV. Upregulated expression of CD40, CD80, CD83 and CD86 was observed in infected and uninfected bystander DC cells. These upregulated CD markers facilitate interactions between infected and T cells (Refs Reference Libraty, Pichyangkul, Ajariyakhajorn, Endy and Ennis45, Reference Dejnirattisai, Duangchinda, Lin, Vasanawathana, Jones, Jacobs, Malasit, Xu, Screaton and Mongkolsapaya46, Reference Martins, Silveira, Alves, Duarte dos Santos and Bordignon47). Activated DCs secrete enhanced titres of pro-inflammatory cytokines, which stimulate the expression of ISGs and apoptosis of epithelial cells (Ref. Reference Martins, Silveira, Alves, Duarte dos Santos and Bordignon47). IFN gamma (IFN-γ) circulating in the bloodstream could assist in promoting the maturation of immature DC by enhancing IL-12p70 expression, a known variable responsible for T_H1 polarization (Ref. Reference Libraty, Pichyangkul, Ajariyakhajorn, Endy and Ennis45).

Glycosylated NS1 initiates the activation of TLR2/myeloid differentiation primary response gene 88 (MyD88) pathway, which subsequently promotes enhanced expression of inflammatory mediators. Moreover, during primary infection, DC maturation via TLR2/MyD88 activation results in T_H2-based antibody response, whereas this shift in T-helper response leads to severe consequences during secondary dengue infection via ADE-mediated pathogenesis (Refs Reference Dash, Samal, Rout, Behera, Sahu and Das48, Reference George, Kim, Choi, Patil, Hossain, Uyangaa, Hur, Park, Lee, Kim and Eo49, Reference Fernandes-Santos and Azeredo50). TLR2 expression in DC and plasmacytoid DCs (pDCs) is more pronounced in severe cases as opposed to non-severe cases, hypothesizing their involvement in severe outcomes (Ref. Reference Fernandes-Santos and Azeredo50). In contrast, recent studies have reported that TLR9, a receptor responsible for sensing DNA viruses, is activated during dengue infection. Upon dengue infection, the generation of reactive oxygen species (ROS) and inflammasome activation induced the release of mitochondrial DNA (mtDNA) in the cytosol triggering the activation of NF-κB and mitogen-activated protein kinase (MAPK) p38 signalling cascades, which are essential for eliciting antiviral responses. Additionally, TLR9^- exhibited a negative association with the innate immune response and viral load. A study conducted by Lai et al. reported that oxidized mtDNA was more pronounced in activating and upregulating TLR9 expression (Refs Reference Fernandes-Santos and Azeredo50, Reference Lai, Wang, Huang, Wu, Hung, Yang, Ke, Luo, Liu and Ho51). Similarly, Torres et al. reported that both pDCs and mDCs showed elevated expression of TLR9 in DF compared to dengue haemorrhagic fever (DHF), highlighting its role as an antiviral factor by triggering type I IFN response (Ref. Reference Torres, Hernández, Giraldo, Arboleda, Rojas, Smit and Urcuqui-Inchima52).

A study by Sprokholt et al. concluded that post-24 h of inoculation, no host–virus interaction occurred, but viral RNA was necessary for activation. These activations were initiated by the cytoplasmic RIG-I and MDA5. DC-infected cell exhibited heightened expression of IL-1β, IL-6, TNF, IL-12p70, IL-8, C-X-C motif chemokine ligand 9 (CXCL-9), IFN-γ-inducible protein 10 (IP-10), CCL2, CCL3, CCL4 and CCL5. CCL2 and CCL4 aid in recruiting monocytes, while CCL3 potently attracts neutrophils to areas of inflammation. An auto feedback loop occurs in which DENV-infected DCs attract monocytes, upon their activation, results in recruiting additional DCs, thereby heightening the immune response (Refs Reference Martins, Silveira, Alves, Duarte dos Santos and Bordignon47, Reference Sprokholt, Kaptein, van Hamme, Overmars, Gringhuis and Geijtenbeek53). Another research by Palmer et al. observed that DENV-infected DC had enhanced apoptotic gene expression, which also functioned as a stimulant for the bystander DC for maturation. Enhanced expression of IL-10 was additionally discovered, which might indicate the general rationale for suppression of immune response and viral clearance evasion (Ref. Reference Palmer, Sun, Celluzzi, Bisbing, Pang, Sun, Marovich and Burgess54).

pDC, a subpopulation of DC, is identified for secreting localized elevated levels of type I IFNs, recognize DENV in a distinctive mechanism by contact interaction. Following this interaction, viral RNA is sensed by TLR7 and ensures the activation of the signalling cascade, thereby suppressing the DENV infection (Ref. Reference Webster, Werneke, Zafirova, This, Coléon, Décembre, Paidassi, Bouvier, Joubert, Duffy, Walzer, Albert and Dreux55). A study by Gandhini et al. discovered that non-severe patients displayed a heightened frequency of activated circulating pDC indicated by enhanced expression of IFN alpha (IFN-α), and soluble TNF-related apoptosis-inducing ligand (sTRAIL) compared to severe patients, hypothesizing that activated pDC could potentially be utilized as a prognostic marker in the future (Ref. Reference Gandini, Gras, Azeredo, Pinto, Smith, Despres, da Cunha, de Souza, Kubelka and Herbeuval56). Similarly, a study by Upasani et al. concluded two inferences: first, in severe patients, the frequency of circulating pDCs is reduced compared to DF patients; second, a link between IFN-α concentration and the ratio of circulating pDCs. This implies that this subset is solely responsible for type I IFN response and might probably impart protection from severe outcomes (Ref. Reference Upasani, Scagnolari, Frasca, Smith, Bondet, Vanderlinden, Lay, Auerswald, Heng, Laurent, Ly, Duong, Antonelli, Dussart, Duffy and Cantaert57). DENV has evolved multiple strategies to evade host response, one of which is inhibiting type I IFN response. Interestingly, this approach is ineffective for pDC, considering they autonomously activate the signalling cascade as no active viral replication occurs intracellularly (Ref. Reference Schmid, Diamond and Harris58). A study conducted by Bittencourt et al. established that titres of pDCs and mDCs accumulated more in healthy individuals compared to non-severe adult cases, highlighting their modulation during infection (Ref. Reference De Carvalho Bittencourt, Martial, Cabié, Thomas and Césaire59). Another research by Decembre et al. concluded that infected cells secreting immature viral particles enabled IFN-α response via pDCs more potently than cells efficiently producing completely mature virions, disclosing a functional role of immature viral particles in activating innate immunity (Ref. Reference Décembre, Assil, Hillaire, Dejnirattisai, Mongkolsapaya, Screaton, Davidson and Dreux60). High levels of DENV replication in mDCs have been reported, which coincides with DC-SIGN expression. On the contrary, diminished levels of DENV replication were noted in pDCs, but the inflammatory response in pDCs was more pronounced and rapid. The robust TNF-α and IFN-α response aids in regulating viral replication and, additionally, also interacts with other immune cell types, contributing to a pathogenic consequence (Ref. Reference Sun, Fernandez, Marovich, Palmer, Celluzzi, Boonnak, Liang, Subramanian, Porter, Sun and Burgess61). Therefore, it can be concluded that both lineages of DCs serve their role in viral dissemination and inflammatory host response, thereby specifying their part in disease pathogenesis (Figure 5).

Figure 5. Dendritic cell in pathogenesis of dengue infection. A. (a–f) DENV is recognized by DC-SIGN and MR on the surface of myeloid dendritic cells, which is responsible for the internalization of the viral particle. This viral particle is sensed by RIG-1 and MDA5 in the cytoplasm, which induces type I IFN signalling cascade. Upon stimulation, they secrete TNF-α, IFN-α, IL-1β, IL-6, IL-8, CXCL9, IP-10, CCL5, CCL2, CCL4 and CCL3. CCL3 recruits neutrophils, and CCL2 and CCL4 recruit monocytes to the site of inflammation. (h) These monocytes subsequently, in an auto-feedback loop manner, activate dendritic cells, boosting the immune response. (g) IFN-γ in the circulation aids in inducing the expression of IL-12p70, which is essential for T_H1 polarization. (i) As apoptotic gene expression increases, bystander dendritic cell gets activated, culminating in a heightened immune response. (j–m) Upon dengue infection, mtDNA released is sensed by the TLR9 receptor localized in the endosome, which initiates a cascade of events through the activation of NF-κB and MAPKp38 signalling, which is vital for eliciting an antiviral response. B. The mechanism by which plasmacytoid dendritic cells get activated is when a naïve cell interacts with a DENV-infected cell by forming a bridge-like structure composed of actin filaments. Viral particle traverses from the infected to the naïve cell via this framework. TLR7 on naïve cells interacts with viral RNA and induces type I IFN response. Additionally, another Toll-like receptor, TLR2/MyD88, present on the surface of dendritic cells, is crucial for triggering DC maturation and initiating a T_H2-based immune response by activating the NF-κB signalling pathway.

Monocytes

Monocytes, one of the most prevalent blood mononuclear phagocytes, are the primary target cells of DENV. Post-interaction with the virus, they can differentiate into tissue macrophages and DCs (Ref. Reference Kwissa, Nakaya, Onlamoon, Wrammert, Villinger, Perng, Yoksan, Pattanapanyasat, Chokephaibulkit, Ahmed and Pulendran62). They display an array of sensory receptors, such as TLRs, RIG-1 and MDA-5, which are known to be involved in pathogen recognition and stimulation of the inflammatory cascade (Ref. Reference Nasirudeen, Wong, Thien, Xu, Lam and Liu63). Monocytes typically fall into three distinct subtypes depending on their phenotype and functionality. The aforementioned categories include the classical, intermediate and non-classical monocytes. The percentage of intermediate monocytes surges after infection during the initial phases and plasmablast levels at later stages, highlighting their contribution to the humoral response by stimulating resting B cells to mature forms (Ref. Reference Kwissa, Nakaya, Onlamoon, Wrammert, Villinger, Perng, Yoksan, Pattanapanyasat, Chokephaibulkit, Ahmed and Pulendran62). Diminishing titres of monocytes is known to enhance systemic viremia. Additionally, they also act as the primary vessels of virus dissemination and inflammatory mediators, which disrupt the integrity of endothelial cells. However, they are equally effective in producing an antiviral response by secreting components that promote systemic viral surveillance. Emphasizing monocytes, like all other innate cells, have a bidirectional involvement with respect to pathogenesis and prophylaxis of the infection.

Wong et al. reported CD16⁺CD14^high cells were the primary secretors of inflammatory cytokines (IL-1β, TNF-α, IL-6, CCL2, CCL3 and CCL4) in response to DENV infection. Prolonged polarization or release of CD16+ monocytes into the circulation, in turn, culminates in severe consequences (Ref. Reference Wong, Chen, Balakrishnan, Toh, Fink and Wong64). Furthermore, some of the cytokines secreted by monocytes following DENV infection, which include TNF-α, IL-1β and IL-8, have been reported to be an influencing factor for endothelial permeability (Ref. Reference Castillo and Urcuqui-Inchima65). Adikari et al. reported that NS1 has lipopolysaccharide (LPS) like activity. As previously established, LPS promotes IL-10 secretion via TLR4-mediated pathway. They proposed that secretion of IL-10, an anti-inflammatory cytokine from monocytes, might occur in a similar manner via the NS1-TLR4 pathway (Ref. Reference Adikari, Gomes, Wickramasinghe, Salimi, Wijesiriwardana, Kamaladasa, Shyamali, Ogg and Malavige66).

Azeredo et al. reported a higher proportion of non-classical monocytes post DENV-2 infection, with a significant level of intercellular adhesion molecule (ICAM-1) -1 expression that serves as an inducement for propagation to the site of infection, along with TLR2 and TLR4 expression, which, on interaction with soluble NS1, as previously declared, activate NF-κB and IRF3 signalling cascade (Ref. Reference Azeredo, Neves-Souza, Alvarenga, Reis, Torrentes-Carvalho, Zagne, Nogueira, Oliveira-Pinto and Kubelka67). Aguilar-Briseño et al. disclosed similar findings, where enhanced levels of TLR2 associate with severe disease progression and decreased TLR2 levels correlate with symptoms of milder severity, thus tackling the TLR2 axis as a potential target that might mitigate the pathogenesis of severe disease by influencing vascular response (Ref. Reference Aguilar-Briseño, Upasani, Ellen, Moser, Pauzuolis, Ruiz-Silva, Heng, Laurent, Choeung, Dussart, Cantaert, Smit and Rodenhuis-Zybert68).

Arias et al. reported increased levels of TNF-α, IL-12, IL-17, soluble suppression of tumorigenicity (sST2), sTRAIL and IL-6 in dengue-infected individuals when compared to healthy controls; when analysed based on severity, IL-6 and sST2 exhibited a pathogenic role, and IL-12 and sTRAIL a protective role (Ref. Reference Arias, Valero, Mosquera, Montiel, Reyes, Larreal and Alvarez-Mon69). Marinho et al. pitched obstructing CR3 signalling inhibited 30% of the viral infection. In addition, they reported diminished expression of CR3, CR4 and CD59 with elevated expression of sC5b-9 in dengue-infected patients, inferring that this modulation is associated with severe consequences (Ref. Reference Marinho, Azeredo, Torrentes-Carvalho, Marins-Dos-Santos, Kubelka, de Souza, Cunha and de-Oliveira-Pinto70). Moreover, Silva et al. reported that phospholipase A₂ (PLA₂) enzymes, prostaglandins and inflammatory mediators, which are released from activated monocytes, are pathogenic factors influencing vascular permeability (Ref. Reference Silva, Gomes, Jeewandara, Ogg and Malavige71).

According to Corrêa et al. extracellular adenosine triphosphate (ATP) via the P2X7R pathway aids in diminishing NS1-infected cells, which ultimately influences the antiviral and inflammatory process. The newly discovered mechanism involving purinergic signalling in the backdrop of DF still requires investigation and could potentially be deployed as a prospective target for therapies (Ref. Reference Corrêa, Lindenberg, Fernandes-Santos, Gandini, Petitinga Paiva, Coutinho-Silva and Kubelka72). Alhoot et al. further advanced the research about the activated surface receptor of monocytes in dengue infection by targeting CD14-associated molecules, known targets of DENV-2 entry. These effectively inhibited the DENV entry and subsequent virus multiplication, assisting as a novel target for inhibiting progression to severe outcomes as well as a promising therapeutic agent for the attenuation of dengue infection (Ref. Reference Alhoot, Wang and Sekaran73).

Another adhesion molecule with dual roles is CD169, frequently referred to as sialic-acid immunoglobulin-like lectins (Siglec-1). In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, CD169 elicited a protective role (Ref. Reference Ullah, Ladinsky, Sharma, Gilani, Rowland, Mack and Uchil74), whereas, upon trans viral attachment, it aided in the dissemination of the human deficiency virus (HIV) (Ref. Reference Izquierdo-Useros, Lorizate, Puertas, Rodriguez-Plata, Zangger, Erikson, Pino, Erkizia, Glass, Clotet, Keppler, Telenti, Kräusslich and Martinez-Picado75). Fenutria et al.’s research revealed increased CD169 titres after dengue and ZIKA infections (Ref. Reference Fenutria, Maringer, Potla, Bernal-Rubio, Evans, Harris, Rahman, Fernandez-Sesma and Ramos76). Similar to previously reported literature, high titres of sSiglec-1 were obtained in patients with DENV infection when compared to healthy controls (Ref. Reference Mishra, Mallik, Paul, Saha and Mukhopadhyay77). Additional investigation into CD169 is essential and could potentially contribute to the development of a more effective prognostic or therapeutic target (Ref. Reference Herzog, Fragkou, Arneth, Mkhlof and Skevaki78) (Figure 6).

Figure 6. Role of monocytes in dengue infection. (a–i) DENV is sensed by intracellular RIG-1 and MDA5, which activate TBK-1. This interaction leads to the phosphorylation of IRF3. Activated IRF3 translocates to the nucleus and stimulates type I IFN synthesis. This response leads to the activation of IFNAR and subsequently STAT1, which induces the transcription of Siglec-1 and TRIM27 mRNA. Post-translational modification leads to siglec-1 being expressed on the surface of the cell and matured Trim27 degrades TBK-1, leading to impaired IRF3 signalling, hypothesizing a modulation of the immune response. (j–o) Another pathway by which monocytes aid in viral clearance is by activating the purinergic signalling. ROS generated in the cell induces ATP to be released from the cell; this extracellular ATP binds with P2X7R, which further activates NLRP3 inflammasome. This activation leads to the secretion of IL-1β and IL-18 into the circulation. (p–s) Soluble form of NS1 present in the circulation interacts with TLR2 and TLR4 and results in the activation of IRF3. Phosphorylated IRF3 translocates to the nucleus and triggers the synthesis of immune response genes such as TNF-α, IL-8, IL-1β, IL-12 and IL-17. (t) Blocking CR3 signalling reduced 30% of viral infection, a probable future therapeutic target.

Macrophages

One of the first target cells exposed to the DENV during blood feeding is the macrophage. Macrophages have been grouped into two fundamental classifications: pro-inflammatory M1 and anti-inflammatory M2. Macrophage polarization has been deemed to play a crucial role in illness manifestation and dissemination. Polarization into M1 phenotype is predicted to enhance inflammation and give rise to potent antiviral immune responses, whereas M2 macrophages are involved in tissue remodelling and immunosuppression (Ref. Reference Lee, Tseng, Chen, Kuo, Wang, Lin, Huang, Wang, Lin and Hung79). Lee et al. discovered in their study that diminished M2 activation could expose individuals to bleeding risk and thrombocytopenia, potentially leading to severe dengue outcome, therefore hypothesizing M2 as a biomarker for distinguishing severe paediatric cases (Ref. Reference Lee, Tseng, Chen, Kuo, Wang, Lin, Huang, Wang, Lin and Hung79). IFN-γ, secreted by lymphoid cells, is a principal mediator for the interaction between macrophages and lymphocytes. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is one of the necessary factors for the alteration of dormant macrophages into M1 macrophages, which have an increased ability to present antigens and engulf pathogens and can recruit lymphocytes, NK cells and neutrophils at the site of infection (Refs Reference Liu, Zou, Chai and Yao80–Reference Hwang, Hur, Koo, Oh, Kim, Jung, Baek, An, Park and Hong82).

The DENV NS1 in the bloodstream interacts with macrophages by engaging with TLR4, therefore inducing their stimulation and the release of mediators. Moreover, platelets that have been activated by NS1 via TLR4 form aggregates and are subjected to being engulfed by macrophages (Ref. Reference Chao, Wu, Lai, Tsai, Perng, Lin and Yeh83). Furthermore, given the background of viral evasion, specific salivary components are involved in reducing the invasion of macrophages at the site of the bite. This reduction may be attributable to lower amounts of pro-IL-1β and CXCL2 at the bite site (Ref. Reference Sekaran, Liew, Yam and Raju81). Similarly, Barros et al. reported that expression of SGE, a salivary component of A. aegypti minimized inducible nitric oxide synthase (iNOS) and proinflammatory expression, while enhancing IL-10 production. This interaction disrupted the polarization of M1 macrophages, without influencing the M2 polarization (Ref. Reference Barros, Lara, Fonseca, Moretti, Filgueiras, Martins, Capurro, Steiner and Sá-Nunes84).

Fink et al. described macrophages as one of the innate cells involved in limiting systemic viral transmission (Ref. Reference Fink, Ng, Nkenfou, Vasudevan, van Rooijen and Schul85). Previous studies by Züst et al. on a macrophage-type I IFN knockout model rendered mice susceptible to lethal DENV infection, highlighting their crucial role in triggering an efficient immune response (Ref. Reference Züst, Toh, Valdés, Cerny, Heinrich, Hermida, Marcos, Guillén, Kalinke, Shi and Fink86).

Activated M1 macrophages secrete increased levels of pro-inflammatory mediators, such as TNF-α, IFN-α, IL-1β, IL-6, IL-8, IL-12, CCL3, CCL5 and reduced levels of IL-10 as a result, triggering a T_H1 response (Refs Reference Wan, Wu-Hsieh, Lin, Chen, Huang and Anderson34, Reference Liu, Zou, Chai and Yao80, Reference Wu, Chen, Yang, Lin, Lin, Chen, Tsai, Li and Hsieh87). A separate investigation reported that M1 is more susceptible to DENV infection and yields an elevated level of IL-18 and IL-1β via C-type lectin domain family 5 member A (CLEC5A) stimulating the NLRP3 inflammasome pathway, thereby contributing to disease severity through heightened vascular permeability (Ref. Reference Wu, Chen, Yang, Lin, Lin, Chen, Tsai, Li and Hsieh87). Independent of CLEC5A, another receptor crucial for viral uptake and attachment is MR on macrophages, which interacts with the envelope protein of DENV through its carbohydrate-recognition domain (Ref. Reference Miller, de Wet, Martinez-Pomares, Radcliffe, Dwek, Rudd and Gordon88). Studies done by Chen et al. on the dynamics of inflammatory mediators secreted by activated macrophages uncovered that following DENV infection, IL-8 production was most prominent, followed by that of CCL3 and CCL5. The levels of IFN-α, IL-12 and TNF-α were intermediary, whereas minimal levels of IL-1β were secreted (Ref. Reference Chen and Wang89). In addition, upon dengue infection, proinflammatory cytokines, such as IL-6, TNF-α and IFN-γ, induce IL-10 secretion, which thereby results in triggering of macrophages expressing CD163 (Ref. Reference Ab-Rahman, Rahim, AbuBakar and Wong90). Moreover, CD163, a known marker of macrophage activation, is altered in multiple infectious diseases. A study conducted in the south-eastern part of India reported elevated but not statistically significant levels of soluble CD163 (sCD163) in severe dengue when compared to non-severe cases. Nonetheless, it showed a statistically significant difference when compared between primary and secondary dengue infection, with the latter being more pronounced, indicating its potential as a tool for monitoring disease progression (Ref. Reference Pillai, Ramachandrappa, S., K., Dhodapkar, Kah and Rajendiran91). In a similar context, another study conducted by Ab-Rahman et al. reported significantly elevated expression of sCD163 in severe cases when compared to non-severe cases (Ref. Reference Ab-Rahman, Rahim, AbuBakar and Wong90). This alteration might be due to the reduced no. of cases in the severe category. Similar to the previous report, Vuong et al., in 2022, highlighted that sCD163 was elevated but not statistically significant in the severe leakage category when compared with the no and/or moderate leakage category (Ref. Reference Vuong, Cheung, Periaswamy, Vi, Duyen, Leong, Binte Hamis, Gregorova, Ooi, Sessions, Rivino and Yacoub92). More extensive studies with a larger sample size in the severe category are essential to denote sCD163 as a marker of disease prognosis (Figure 7).

Figure 7. Macrophage role in dengue infection. A. (a–d) Circulating NS1 interacts with TLR4 expressed on the surface of macrophages, which in turn results in the activation of IRF7. Activated macrophage culminates in secreting TNF-α, IFN-α, IL-1β, IL-6, IL-8, IL-12, CCL3 and CCL5. (e–g) Another pathway by which macrophage gets stimulated is by interacting with CLEC5A with viral components. This interaction results in activation of the NLRP3 inflammasome, which causes IL-1β and IL-18 activation, which is essential for the induction of T_H17 cells. (h) Mannose receptor interacts with envelope protein, aiding in viral internalization, which further modulates the immune response. (i) Upon infection, inflammatory cytokines trigger macrophages to express CD163, which, upon proteolytic cleavage, results in the formation of sCD163, a known disease severity marker. B. SGE secreted during blood feeding promotes downregulation of iNOS and pro-inflammatory cytokine expression and upregulation of IL-10; this signalling suppresses M1 macrophage polarization, which assists in the viral evasion mechanism.

NK cells

NK cells are identified as innate, transient effectors having a requisite role in regulating the innate and adaptive immune responses following DENV infection. Both subgroups of NK cells, namely CD56^bright and CD56^dim, exhibit distinctive roles in triggering an immune response, and the juxtaposition of different activation and inhibitory receptors possesses significance in NK-cell-mediated immune response. These immune cells secrete cytokines and cytotoxic proteins, including granzyme B and perforin. These secreted mediators then trigger an array of signalling events that aid in recruiting additional lymphocytes to combat against the infection (Ref. Reference Shabrish, Karnik, Gupta, Bhate and Madkaikar93). DCs and macrophages are one of the primary targets of the DENV, which play an indispensable part in initiating the immune response. When these immune cells are infected with the DENV, they stimulate NK cells through two distinct mechanisms. The first mechanism involves the infected cells releasing immunomodulators, such as IL-12, IL-15, IL-18, TNF-α and type I IFNs. The second approach involves the interaction between the activatory or inhibitory receptors on NK cells and ligands present on the dengue-infected cells (Refs Reference Lim, Yawata, Selva, Li, Tsai, Yeong, Liong, Ooi, Chong, Ng, Leo, Yawata and Wong94, Reference Maucourant, Petitdemange, Yssel and Vieillard95).

Virus-infected cells commonly trigger or enhance the expression of ligands at their surface, allowing for the interaction between NK cell cytotoxic receptors, including NKp30, NKp44 and NKp46, and activatory or inhibitory receptors comprising activating killer cell immunoglobulin-like receptors (KIRs) and others, which interact with DENV peptides exhibited by human leukocyte antigen (HLA) molecules (Ref. Reference Mathew96). NK cells can discern cells infected with DENV by interacting with their activating receptors. However, the elevated levels of class I molecules of HLA may enable them to bypass the NK-cell-mediated immune response (Ref. Reference Beltrán and López-Vergès97). Previous studies highlight that KIR genes are in control of NK cell response; these findings indicate that the expression of certain KIRs on NK cells could systematically alter and influence the immune response (Ref. Reference Mathew96). Interaction between KIR3DL1 and HLA-B57, a conserved NS1 peptide, resulted in inhibition of NK cell activation; activated NK cells’ titre increases with reduction in viremia (Ref. Reference Townsley, O’Connor, Cosgrove, Woda, Co, Thomas, Kalayanarooj, Yoon, Nisalak, Srikiatkhachorn, Green, Stephens, Gostick, Price, Carrington, Alter, McVicar, Rothman and Mathew98). Similarly, interaction between HLA-C0102, a conserved NS3 peptide with KIR2DS2, and the envelope protein with NKp44 results in NK cell activation. On the contrary, KIR2DL1, another inhibitory receptor, interacts with DENV peptides presented by HLA-C molecules, and hinders NK cell activation (Ref. Reference Mathew96). Combining these research findings indicates that an intricate equilibrium is projected to exist between the activation and inhibition of NK cells, and they are likely to play a role in influencing NK cell responses, which culminates in having a functional status during the infection (Ref. Reference Hershkovitz, Rosental, Rosenberg, Navarro-Sanchez, Jivov, Zilka, Gershoni-Yahalom, Brient-Litzler, Bedouelle, Ho, Campbell, Rager-Zisman, Despres and Porgador99). IL-18, often referred to as IFN-γ-inducing factor, is a cytokine capable of enhancing the production of IFN-γ by NK cells. Additionally, it leads to maturation and boosting the cytotoxic response. Occasionally, only the presence of IL-18 is adequate to induce IFN-γ production; however, in vivo conditions, IL-12 and IL-18 played a crucial role in ensuring the functionality of NK cells. It has been demonstrated that the IL-18 level spiked in cases of dengue infection, but revealed no association with NS1 or the infection type (Ref. Reference Nanda, Ho, Satria, Jhan, Wang and Lin100).

Siglec-7 (CD328) is an inhibitory receptor expressed on NK cells and is known for modulating cytotoxic signals. Diminished receptor expression and elevated circulating levels of siglec-7 in both HIV and Hepatitis C virus infections have been observed, highlighting it as an early indicator for NK dysfunctionality (Refs Reference Brunetta, Fogli, Varchetta, Bozzo, Hudspeth, Marcenaro, Moretta and Mavilio101, Reference Varchetta, Mele, Lombardi, Oliviero, Mantovani, Tinelli, Spreafico, Prati, Ludovisi, Ferraioli, Filice, Aghemo, Lampertico, Facchetti, Bernuzzi, Invernizzi and Mondelli102). Siglec-9 (CD329), another inhibitory receptor on NK cells, is known to play a role in immunosurveillance. Enhanced levels of Siglec9+/NK+ cells were inversely related to viremia in HIV-infected individuals (Ref. Reference Adeniji, Kuri-Cervantes, Yu, Xu, Ho, Chew, Shikuma, Tomescu, George, Roan, Ndhlovu, Liu, Muthumani, Weiner, Betts, Xiao and Abdel-Mohsen103). In another study, Zhao et al. reported that reduced siglec-9 titres were associated with enhanced HBV proliferation; moreover, inhibiting siglec-9 restored the NK cell functionality (Ref. Reference Zhao, Jiang, Xu, Yang, Gao, Li, Gao, Ma and Liang104). As the status of siglec-7 and -9 is being disclosed in other viral illnesses, it remains undetermined in one of the most widespread viral infections, that is, dengue. Combining current improvements on siglec-7/9 indicates their probable potential as future therapeutic targets in viral infections (Figure 8).

Figure 8. Interaction of dengue viral component with NK cell receptor and their role in pathophysiology. (a, b) Dengue NS1 presented by HLA-B57 suppresses the KIR3DL1 receptor on NK cell, an alternate receptor, KIR2DL1 on NK cells interacts with HLA-C, both the interaction further inhibits NK cell function, resulting in reduced cytotoxicity. (c–f) On the contrary, NKp44 interacts with the envelope protein, and KIR2DS2 binds with HLA-C0102, presenting NS3 leads to activation of NK cells, triggering enhanced IFN-γ expression, which leads to efficient viral clearance. (g) Siglec-7/9 interacts with sialic acid expressed on the target cell or pathogen itself; this interaction leads to phosphorylation of the ITIM region present at the cytosolic side. The phosphorylated ITIM recruits SHP-1 and SHP-2, which inhibit NK cell cytotoxicity. Its role in the modulation of dengue pathophysiology still remains to be explored. (h) An alternative pathway for NK cell activation is getting stimulation by IL-12, IL-15, IL-18, TNF-α and type I IFNs released by DENV-infected cells.

Neutrophil

Neutrophils, a type of innate immune cell, are an essential component of the body’s defence mechanism. Their heterogeneity, prolonged response time and inadequacy in regulation contribute equally to beneficial and harmful effects. They have been extensively studied for their ability to undergo NETosis, a process by which they capture and render viruses inactive, hence restricting the viral dissemination. Additionally, they also have the ability to phagocytose viral components and secrete proteolytic enzymes, leading to enhanced inflammation, contributing to adverse consequences (Ref. Reference Chan, Morovati, Karimi, Alizadeh, Vanderkamp, Kakish, Bridle and Karimi105). Multiple observations implied that neutrophils are not simply spectators during DENV infection; moreover, increased levels of pro-inflammatory mediators secreted by innate immune cells, such as IL-8 and TNF-α, could stimulate neutrophils. Additionally CCL3 is potent for chemoattracting neutrophils (Refs Reference Sprokholt, Kaptein, van Hamme, Overmars, Gringhuis and Geijtenbeek53, Reference Opasawatchai, Amornsupawat, Jiravejchakul, Chan-In, Spoerk, Manopwisedjaroen, Singhasivanon, Yingtaweesak, Suraamornkul, Mongkolsapaya, Sakuntabhai, Matangkasombut and Loison106, Reference Patra, Mallik, Saha and Mukhopadhyay107). In the study carried out by Rawat et al. they established that DENV-2 triggers CD16^bright/CD62L^dim subtype of neutrophil, thereby unleashing mediators that serve a paradoxical role during dengue pathogenesis (Ref. Reference Rawat, Kumar, Duggal and Banerjee108). Similarly, proinflammatory mediators secreted, such as IL-8, IP-10 and CCL2, post-activation have been postulated to influence endothelial cell permeability (Ref. Reference Chua, Morales, Chia, Yeo and Teo109).

Research undertaken in Singapore, which enrolled 1,921 patients, concluded that only 11.8% of patients had neutropenia; a homogenous conclusion was reported by Agarwal et al. (Refs Reference Thein, Lye, Leo, Wong, Hao and Wilder-Smith110, Reference Agrawal and Sharma111). As previously observed, DENV infection facilitates degranulation and potential release of elastase, a well-researched variable accountable for endothelial barrier damage. They demonstrated that neutrophil elastase titres were higher in DHF patients when compared to DF, indicating their involvement in the inflammatory processes and their probable association with severity (Ref. Reference Kunder, Lakshmaiah and Moideen Kutty112). Opasawatchi et al. reported that priming by the pro-inflammatory milieu present during acute DENV infection activates neutrophils for ROS generation, which is an essential factor for ROS-dependent neutrophil extracellular trap (NET) formation. Additionally, they revealed that neutrophils, upon activation, are sensitive to delobulation, a preliminary factor of NET formation. They concluded that variables responsible for NET formation or disintegration play a crucial role in the outcomes of the infection (Ref. Reference Opasawatchai, Amornsupawat, Jiravejchakul, Chan-In, Spoerk, Manopwisedjaroen, Singhasivanon, Yingtaweesak, Suraamornkul, Mongkolsapaya, Sakuntabhai, Matangkasombut and Loison106). Similarly, Moreno-Altamirano et al. revealed that DENV-2 suppressed phorbol 12-myristate 13-acetate-induced NET formation by 80% (Ref. Reference Moreno-Altamirano, Rodríguez-Espinosa, Rojas-Espinosa, Pliego-Rivero and Sánchez-García113). To broaden current understanding of NETs, Garishah et al. intended to identify the association between NETs and distinctive features of dengue, notably thrombocytopenia and endothelial dysfunction. Platelet-derived microparticles generated by platelets in response to DENV interaction stimulated the development of NETs via crosstalk with CLEC5A and TLR2 present on neutrophils. They uncovered that dengue infection correlates with NS1-dependent NOX-independent NET formation, which is associated with platelet activation, endothelial disruption and enhanced vascular permeability (Refs Reference Garishah, Rother, Riswari, Alisjahbana, Overheul, van Rij, van der Ven, van der Vlag and de Mast114, Reference Chen, Li, Sun, Hu, Liu, Herrmann, Zhao and Muñoz115). Similarly, Sung et al. reported that blocking CLEC5A or TLR2 reduced NET formation, thereby imparting protection against vascular leakage (Ref. Reference Sung, Huang and Hsieh116). Lien et al. uncovered that envelop protein domain III unilaterally triggered neutrophil NETosis response in vitro and in vivo, and was additionally associated with cytokine storm, a known consequence of severe dengue (Ref. Reference Lien, Sun, Hung, Wu and Chang117). Comprehending the precise mechanism responsible for NETosis and its protective or pathogenic impact on disease outcomes might provide perspective on feasible therapeutic approaches for DENV infections (Refs Reference Opasawatchai, Amornsupawat, Jiravejchakul, Chan-In, Spoerk, Manopwisedjaroen, Singhasivanon, Yingtaweesak, Suraamornkul, Mongkolsapaya, Sakuntabhai, Matangkasombut and Loison106, Reference Rawat, Kumar, Duggal and Banerjee108) (Figure 9).

Figure 9. Overview of neutrophil in dengue pathogenesis. (a–j) DENV stimulates CLEC2 expressed on platelets to secrete exosomes and microvesicles. These platelet-derived microparticles, such as exosomes and microvesicles, interact with CLEC5A and TLR2 present on neutrophils, respectively. These receptors then induce MAPK signalling, which in turn activates PAD4. This culminates in, unfolding of chromatin and damaging the nuclear membrane, thereby enhancing DENV-induced NET formation. (k-l) Inflammatory mediators released by DENV-infected cells activate neutrophils, which results in the secretion of IL-8, IP-10 and MCP-1(CCL2). Upon activation, the neutrophil degranulates and elastase is released. Inflammatory mediators in conjunction with elastase enhance endothelial cell permeability. (m) Envelope protein domain III can also independently interact with CLEC5A to stimulate NET formation.

The interplay between the innate and adaptive immune response is one of the vital aspects of the immune response to pathogens. These innate lymphoid cells have numerous functions, including triggering adaptive immune response, which is essential for pathogen clearance, limiting severe consequences and developing memory cells, which might prove disadvantageous for subsequent infection.

During the initial stages of infection, the DENV infects innate cell, which, upon activation, secretes a distinct spectrum of inflammatory mediators for host defence. In addition, they amplify the infection by disseminating the infected cells to lymph nodes for further downstream immune cell activation. Moreover, infected cells, upon activation, also trigger the adaptive immune response (Ref. Reference Libraty, Pichyangkul, Ajariyakhajorn, Endy and Ennis45). A subset of DCs, that is, pDCs, secrete IL-6 and type I IFN, which promotes differentiation and activation of B cells (Ref. Reference Jego, Palucka, Blanck, Chalouni, Pascual and Banchereau118). Similarly, another subset of DCs, that is, mDCs, secrete IL-12 in the circulation, which are known stimulators of T-cell differentiation and cell-mediated immune response (Ref. Reference Libraty, Pichyangkul, Ajariyakhajorn, Endy and Ennis45). In conjunction with DCs, monocytes, NK cells, type I IFN and circulating cytokines are the main orchestrators of initiating the immune response, which aids in viral clearance.

The host response to invading pathogens is orchestrated by a complex network of innate cells and plasma proteins that are mainly represented by, but not limited to, the aforementioned axis.

Moreover, a concise summary of the differential expression of inflammatory mediators with respect to primary and secondary dengue infection, as well as the immune response to specific flavivirus infection, is delineated in the tables 1 and 2 listed below.

Table 1. Modification in plasma protein profile during primary and secondary dengue infection

Table 2. Comparative analysis of PRRs and plasma protein secretion to flavivirus infections

Current status of vaccine and therapeutic research

Several studies have demonstrated that dengue severity is regulated by numerous host factors, of which uncontrolled stimulation of inflammatory mediators is the key element. While there are vaccine candidates, no approved vaccine or therapeutics are available for treatment to date. Currently, four dengue vaccine types are in their phase trials, including the inactivated vaccine, subunit vaccine and DNA vaccines, and the remaining live attenuated vaccine, CYD-TDV, commonly referred to as Dengvaxia®, the only licensed vaccine, and others such as TV003/TV005 and DENVax, are currently in phase III. They stimulate immunological responses by targeting the viral components (Refs Reference Deng, Yang, Wei, Chen, Wang and Peng143-Reference Wilder-Smith144). Dengvaxia® has two significant hurdles: it is restricted to being given to seropositive individuals over the age of 9 years, as it manifests increased likelihood of severe outcomes in seronegative individuals, and its weak efficacy towards the DENV-2 serotype (Refs Reference Dayan, Langevin, Forrat, Zambrano, Noriega, Frago, Bouckenooghe, Machabert, Savarino and DiazGranados145–Reference Malisheni, Khaiboullina, Rizvanov, Takah, Murewanhema and Bates147). Since there are gaps in the understanding of immune response and pathophysiology of severe conditions, thereby impedes the development of DENV vaccines. Careful investigation of DENV-induced immune responses might be able to assist in the refinement of already developed dengue vaccines and propose alternative and/or heterologous strategies.

A tabular representation of different types of vaccines, their formulation and efficacy is mentioned in Table 3.

Table 3. Different types of DENV vaccine and their formulation, phase, dosage and efficacy

Furthermore, researchers have dedicated decades to the development of antiviral agents for dengue therapy, but have achieved limited progress. Antiviral therapy aims to minimize the viral load, thereby lowering the disease burden (Ref. Reference Lee, Wu and Poh158). Currently, no animal models are capable of mimicking human dengue symptoms or clinical manifestations (Ref. Reference Lee, Wu and Poh158). Non-human primates are seldom utilized for evaluating the efficacy of antivirals because of their high manufacturing costs and breeding challenges. Murine models, such as AG129, BALB/c and C57BL/6 mice, are frequently used to assess in vivo therapeutic effectiveness. Antiviral drugs against DENV have been developed using methodologies that target host attachment factors, DENV viral components and post-infection stages (Ref. Reference Lee, Wu and Poh158).

Tomatidine, an antiviral compound, does not have a direct virucidal effect but is reported to inhibit all four serotypes by hampering the viral production stage (Ref. Reference Diosa-Toro, Troost, van de Pol, Heberle, Urcuqui-Inchima, Thedieck and Smit159). Some of the common therapeutics targeted by the above-mentioned methodologies are listed in Table 4

Table 4. List of compounds involved in the antiviral effect in dengue infection

.

Despite the enormous number of molecules that show antiviral activity in vitro, only a handful have been subsequently researched and evaluated in clinical trials; nonetheless, none of them have sufficient efficacy to be utilized as antiviral medications (Ref. Reference Lee, Wu and Poh158).

Chen et al. hypothesized that as CLEC5A is involved in DENV-induced inflammatory signalling cascade, blocking CLEC5A attenuated the inflammation and exhibited a potential to act as a therapeutic target by blocking severe outcomes (Ref. Reference Chen, Lin, Huang, Wu, Cheng, Lei, Lee, Chiou, Wong and Hsieh180). C–C chemokine receptor 5 (CCR5), a chemoattractant involved in recruiting mononuclear leukocytes at the site of infection, is required for DENV-2 replication and progression. In respect to that, Marques et al. demonstrated blocking of CCR5 resulted in poor DENV-2 replication in macrophages, indicating an alternative approach to therapeutics by targeting chemokine receptors (Ref. Reference Marques, Guabiraba, Del Sarto, Rocha, Queiroz, Cisalpino, Marques, Pacca, Fagundes, Menezes, Nogueira, Souza and Teixeira181). These findings emphasize the need for ongoing studies to uncover novel targets and compounds that may assist in avoiding or diminishing DENV infection.

Conclusion and future perspective

The increasing prevalence of dengue cases due to urbanization and climate change poses a significant burden; despite that, there is no vaccine or antivirals. Having a comprehensive understanding of the host’s innate immune response to infection is crucial for identifying key therapeutic targets.

CLEC5A, TLRs, RIG-I, MDA5 and DC-SIGN play a crucial role in activating a wide range of innate cells and regulating the downstream signalling cascade. Moreover, uncontrolled activation leads to viral dissemination and proliferation, prompting a future target for therapeutics. Another distinct plausible target for therapeutics might be exploring the type I IFN axis, which, upon activation, induces a protective mechanism. A precise equilibrium between protective and pathogenic responses must be maintained to attain favourable outcomes. Various research on therapeutics targeting host-viral interacting molecules has been undertaken, but unfortunately, none of them have been established as DENV-specific antivirals. One of the possibilities would be that the host response is a complex interplay of multiple innate cells, so targeting a single aspect of the inflammatory response might not provide sufficient protection. Utilizing combination therapies might be a probable alternative for maximizing efficacy and minimizing adverse effects. Further research is necessary to elucidate the mechanism underlying the innate immune response in identifying novel targets and markers for disease severity and monitoring.

In a similar line of reasoning, Siglecs can be a novel and obvious target for theragnostic, as in prior investigations, Siglec-6-specific chimeric antigenic receptor-T cell conferred anti-leukemia reactivity (Ref. Reference Jetani, Navarro-Bailón, Maucher, Frenz, Verbruggen, Yeguas, Vidriales, González, Rial Saborido, Kraus, Mestermann, Thomas, Bonig, Luu, Monjezi, Mougiakakos, Sauer, Einsele and Hudecek182), Siglec-15 has been reported as a prognostic marker for giant cell tumour of bone. Additionally, anti-Siglec-15 was utilized as a prophylactic therapy in juvenile osteoporosis (Refs Reference Fan, Zhang, Xie, Liu, Zhang and Wang183, Reference Sato, Takahata, Ota, Fukuda, Hasegawa, Yamamoto, Amizuka, Tsuda, Okada, Hiruma, Fujita and Iwasaki184). sSiglec-1 and sSiglec-5 are known type-I IFN signatures for systemic lupus erythematosus and prognostic markers for colorectal cancer, respectively (Refs Reference Oliveira, Karrar, Rainbow, Pinder, Clarke, Rubio García, Al-Assar, Burling, Morris, Stratton, Vyse, Wicker, Todd and Ferreira185, Reference Montalbán-Hernández, Cantero-Cid, Lozano-Rodríguez, Pascual-Iglesias, Avendaño-Ortiz, Casalvilla-Dueñas, Bonel Pérez, Guevara, Marcano, Barragán, Valentín, Del Fresno, Aguirre and López Collazo186). In the recent threat of SARS-CoV-2, the need for an antiviral guided the development of a Siglec-9 agonist, which inhibited neutrophilic hyperinflammation (Ref. Reference Delaveris, Wilk, Riley, Stark, Yang, Rogers, Ranganath, Nadeau, Blish and Bertozzi187). As previously reported, targeting the IFN axis might be a promising approach. On the same notion, Faunza et al. hypothesized that targeting the viral component responsible for disrupting IFN response can be a potential target for suppressing infection (Ref. Reference Fanunza, Frau, Corona and Tramontano188). Moreover, incorporation of recent findings into the existing understanding of dengue pathogenesis could be beneficial in researching for more advanced therapeutic targets and medications.

Another aspect of advancing dengue vaccine studies is through stimulation of cytokine-induced memory-like (CIML) NK cells. CIML is a less differentiated subset of NK cells, which is known to have an enhanced ability to secrete IFN-γ. Wagstaffe et al. reported two significant points: one being the addition of adjuvants targeting the CIML population, manifested enhanced effectiveness, and the other being the difference in immunogenicity post vaccination might probably be due to the difference in CIML percentage, indicating CIML might potentially be a promising candidate for future translational dengue vaccine studies (Ref. Reference Wagstaffe, Mooney, Riley and Goodier189). In-depth research is necessary for understanding the mechanism and potential benefits of CIML-NK cells in dengue vaccine research, and to determine whether incorporating CIML-NK cell-stimulating agents in dengue vaccine formulation brings advancement to existing vaccines (Figure 10).

Figure 10. Hypothetical model of CIML NK cell in dengue vaccine research. A. Activation of APCs by vaccine could induce the release of cytokines which, in turn, might lead to the upregulation of CD107a and CD25, indicating increased NK cell cytotoxicity as well as differentiation into CIML-NK cell, respectively. B. Addition of adjuvants (AS03 / AS01?) in vaccine formulation might promote more production of CIML-NK cells, which in turn may stimulate more T and B cells along with IgG2a class switching. C. This formulation, on secondary exposure, might yield more enhanced protection and effectiveness.