Dendritic cells (DCs) reside and migrate into barrier tissues such as the skin and mucosal epithelium that are the sites of pathogen invasion. In the steady state, DCs display high levels of phagocytic activity, take up antigen, and probe for pathogens via pattern-recognition receptors. DCs express Toll-like receptors (TLRs) and C-type lectins as transmembrane proteins as well as intracellular sensors, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA-5), and cyclic GMP-AMP synthase (cGAS) that recognize conserved microbial patterns (1, 2). Upon pathogen recognition, DCs become activated, produce inflammatory cytokines and chemokines, migrate to lymph nodes (LNs), and present antigen to prime naïve T-cells (3).

Subsets of DCs, monocytes, and macrophages (MΦs) reside in different tissues and fulfill distinct functions. Classical DCs (cDCs) display the characteristic DC dendrites, are present in LNs, spleen, and bone marrow as well as skin, lung, liver, and intestine, and have the greatest ability to stimulate naïve T-cells (4). All cDCs can present endogenous antigen from the cytosol via MHC I to CD8⁺ T-cells (5). In addition, most subsets of cDCs present exogenous antigen via MHC II to CD4⁺ T-cells, whereas only specialized subsets can cross-present exogenous antigen via MHC I to CD8⁺ T-cells (6, 7). In addition, plasmacytoid DCs (pDCs) are another DC subset that reside in the spleen, bone marrow, and liver and circulate in the blood. During viral infections, pDCs migrate to infected tissues and secrete up to 1,000-fold higher amounts of interferon (IFN)-α/β than other cell types (8), although their capacity for antigen presentation is still debated (9). Nevertheless, the role of DCs in priming protective immune responses against many human pathogens and their potential contribution to pathogenesis and development of disease need further investigation.

Monocytes circulate in steady-state blood, patrol lymphoid, and non-lymphoid organs, and are recruited to inflamed tissues, where they phagocytize pathogens as well as infected or damaged cells (10). During inflammation, monocytes can differentiate to monocyte-derived DCs (moDCs) (11, 12). In vitro-generated human moDCs are used widely to study DC biology (13). Monocytes are isolated from human peripheral blood, differentiated in the presence of GM-CSF and IL-4 first to immature moDCs and after further stimulation with inflammatory cytokines or pathogen-associated microbial patterns (PAMPs) to mature moDCs (13, 14). The ability of moDCs to prime naïve T-cell responses remains controversial, as this function initially was attributed solely to cDCs (15). Nevertheless, recent studies demonstrated that moDCs can migrate to LNs and prime naïve T-cells during Leishmania major, influenza virus, and bacterial infections (12, 16, 17). In contrast to DCs, MΦs have limited ability to migrate and prime naïve T-cells. MΦs reside within tissues, where they phagocytize, secrete cytokines, present antigen to effector and memory T-cells, and contribute to the healing of injured tissue (18).

Female Aedes aegypti and Aedes albopictus mosquitoes transmit the four dengue virus serotypes (DENV1–4) while feeding on blood vessels in the skin (19). The positive-sense RNA genome of the flavivirus DENV encodes three structural (C, prM/M, E) and seven non-structural (NS) proteins (20). DENV causes the most prevalent arthropod-borne viral disease of humans, with an estimated 390 million infections and 96 million apparent cases per year (21).

The acute febrile illness dengue fever (DF) can progress to a potentially life-threatening vascular leakage syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the latter characterized by hypotension and circulatory failure (22). At present, no vaccine or therapeutic against dengue is approved for use in humans. A major challenge in the development of vaccines and therapies is that although infection with one DENV serotype leads to long-lasting immunity against the same serotype, subsequent infection with a different (heterotypic) serotype is the major risk factor for severe disease (19). To date, the mechanisms by which the host immune response to DENV provides either protection or enhancement in secondary infection remain poorly understood. Antibodies can neutralize infection or conversely trigger “antibody-dependent enhancement” (ADE) (23, 24), whereby cross-reactive anti-DENV antibodies facilitate entry of DENV into Fcγ receptor (FcγR)-bearing cells and thus increase viral load and ultimately disease severity. Some DHF/DSS cases occur during primary (1°) infections, especially in infants 6–9 months of age (25). In this case, it is thought that maternal DENV-specific antibodies transferred via the placenta wane to levels that can enhance a newly acquired DENV infection (26). Thus, the quantity and quality of the antibody response influences the severity of a secondary DENV infection. Similarly, T-cells can provide protection (27–29), but cross-reactive T-cells have been implicated in disease pathogenesis (30–32). Nevertheless, most secondary DENV infections are asymptomatic or mild, suggesting that the immune system can mount protective responses against dengue.

Aedes mosquitoes that take a blood meal from a human with acute dengue viremia become infected and, after DENV spreads to the salivary glands, transmit the virus when feeding on a new individual. Mosquito saliva contains components that counteract the host hemostatic response and modulate immunity (33, 34). The addition of saliva from Ae. aegypti mosquitoes was found to decrease DENV infection of moDCs in vitro (35). In contrast, mosquito saliva or transmission via infected mosquitoes prolonged DENV serum viremia and fever in “humanized” mice as compared to inoculation with DENV alone (36). Furthermore, saliva that was inoculated by non-infected mosquitoes prior to needle inoculation of DENV blocked the upregulation of genes involved in innate pathogen recognition and increased serum viremia in mice deficient in IRF3 and IRF7 (37). Although certain in vivo studies suggest that mosquito saliva can facilitate DENV infection by generating an environment that favors early virus replication, the impact of saliva on skin DCs requires further study.

Identifying the targets of DENV infection is crucial for understanding virus spread and disease pathogenesis. Human autopsies revealed staining for DENV structural proteins and negative-sense viral RNA, indicative of virus replication, in MΦs in LNs, spleen, lung, and liver and monocytes in clotted blood from patients with lethal dengue disease (38, 39). Staining of the non-structural protein NS3 confirmed DENV replication in phagocytes (including monocytes, MΦs, and DCs) in LNs and spleen, as well as in MΦs in the lung in other autopsy studies (40, 41). In earlier stages of the disease, most DENV-infected cells in the peripheral blood of acute dengue patients were identified as CD14⁺ CD11c⁺ activated monocytes, with higher proportions of monocytes and DENV-infected total cells in the blood in DHF compared to DF patients (42).

Although in humans, DENV efficiently suppresses the IFN response, replicates, and causes disease, DENV fails to antagonize mouse IFN responses, and thus wild-type (WT) mice generally do not sustain DENV replication or develop disease (43). In comparison, mice deficient in IFN-α/β receptor (Ifnar^−/−) and also -γ receptor (AG129) are susceptible to DENV infection and display a tropism similar to humans (40, 44–46). DENV replicated in murine MΦs that were isolated from the peritoneum (47), as well as in MΦs in LNs and spleens of AG129 mice (48, 49). Treatment with clodronate liposomes that deplete monocytes and MΦs decreased viral load in AG129 mice on day 2 but increased viral load on day 4 post-inoculation with DENV2 (48). Monocytes and MΦs thus play an important role as targets for early DENV replication as well as in subsequent control of DENV infection.

Human moDCs generated in vitro support DENV infection (50), with immature moDCs being more susceptible to DENV infection than mature moDCs, monocytes (51), or MΦs (52). Analogously, CD11c^high cells in the spleen of AG129 mice (47) that likely comprised both moDCs and cDCs supported DENV replication in vivo. Recent studies in Ifnar^−/− mice have shown that early after infection, monocytes are recruited to the dermis and differentiate to moDCs, where they become primary targets for DENV replication (53). Although DENV can infect monocytes and MΦs directly, these studies emphasize the greater permissiveness of DCs to DENV infection in the absence of enhancing antibodies, such as during 1° infection conditions.

Surface expression of viral attachment factors determines the susceptibility to DENV infection. DC-SIGN (dendritic-cell-specific ICAM3-grabbing non-integrin, CD209) is a C-type lectin expressed on the surface of DCs and MΦs that recognizes mannose-type sugars on the surface of bacterial, fungal, and viral pathogens. Signals via DC-SIGN induce the phagocytosis of pathogens and contribute to host defense (54). However, DC-SIGN also interacts with carbohydrates on DENV glycoproteins and mediates the attachment of DENV to moDCs (55–57). Human immature moDCs express high levels of DC-SIGN and are highly susceptible to DENV infection (50, 58, 59). Of note, DC-SIGN mediates virus attachment to the cell surface, but not endocytosis into moDCs (60). Activation of immature moDCs via inflammatory cytokines results in downregulation of DC-SIGN, explaining in part why mature moDCs are less susceptible to DENV infection (51, 58). Further, human cDCs freshly isolated from blood do not express DC-SIGN and, accordingly, become highly susceptible to DENV infection only after culture with GM-CSF and IL-4 that induces DC-SIGN expression (61). Similarly, treatment of monocytes with IL-4 or IL-13 increased DC-SIGN expression and DENV infection (62), but it remains unclear whether these monocytes had differentiated to moDCs or MΦs. Consistent with these findings, higher levels of DC-SIGN expression on cDCs of different blood donors correlated with higher DENV infection (61). Furthermore, a polymorphic variant of the DC-SIGN promoter with a decreased transcriptional activity correlated with protection against DF in humans (63). In summary, changes in DC-SIGN expression on different myeloid cell subsets through differentiation correlate with DENV infection (Figure 1).

Although Aedes mosquitoes transmit DENV when probing for blood vessels in the skin, most studies have focused on DENV infection in tissues after the virus has spread via the blood. Few studies have examined DENV infection and the immune response in the skin. DENV infects epidermal Langerhans cells (LCs) in healthy human skin explants in vitro (58, 64). Infection of LCs was confirmed in AG129 mice after intradermal inoculation of DENV2 (48). However, the dermis of intradermally inoculated Ifnar^−/− mice contains 100-fold more DENV-infected cells than the epidermis (53). Recent studies indicate that dermal cDCs, and to a lesser extent MΦs, are the initial targets of DENV replication after intradermal inoculation of Ifnar^−/− mice (53) or infection of skin explants from healthy human donors (65). Subsequently, de novo-recruited monocytes differentiate into moDCs, which become primary targets for DENV replication in the dermis (53).

Regarding the source of infectious virus, DENV produced in mosquito cells interacts with DC-SIGN and infected human immature moDCs in vitro (66). In contrast, DENV that was produced in human moDCs did not bind to DC-SIGN or infect moDCs but instead was infectious for cells expressing the homolog L-SIGN, such as monocytes and endothelial cells (66). The difference in binding to DC-SIGN or L-SIGN was likely due to different N-linked glycosylation patterns present on DENV particles produced in mosquito or mammalian cells. This may explain how DCs in the skin are the initial targets for DENV infection immediately after transmission. Characterization of the initial targets and immune response to DENV in the skin may foster new strategies to block DENV replication and abort pathogenesis.

Dengue virus-specific adaptive immune responses, in particular subneutralizing concentrations of antibodies, can enhance DENV entry and infection. In cell culture, subneutralizing amounts of DENV-immune human serum or monoclonal antibodies enhance infection of monocytes (67, 68) and mature moDCs, but not immature moDCs (51). Human splenic MΦs showed low levels of DENV infection at baseline in vitro, but at least 10-fold greater infection in the presence of enhancing concentrations of diluted DENV-immune human (69). In vivo, CD14⁺ monocytes in the blood of acute dengue patients contained significantly higher levels of DENV genomic RNA in severe DHF compared to DF cases, and in secondary compared to 1° infections (70). Thus, ADE mediates efficient DENV infection of monocytes, mature moDCs, and MΦs.

FcγR expression determines the susceptibility of cells to ADE during DENV infection via uptake of virus-antibody complexes (Figure 1). Most myeloid cells express FcγRs, which bind the Fc region of antibodies and thus are an important link between cellular effector functions and antigen recognition via the antibody. Different types of FcγRs recognize distinct isotypes of IgG with varying affinity and can transmit activating or inhibitory signals to the cells (71). Attachment of DENV-antibody complexes to ectopically expressed FcγRI (CD64) (72) and FcγRIIA (CD32) (73) in fibroblast cell lines mediated ADE, independently of FcγR-signaling. DENV2 infection in primary human monocytes was increased 50-fold in the presence of enhancing DENV-immune human serum and depended on binding of antibody-virus complexes to FcγRI or FcγRIIA (68). Consistent with these data, blocking of FcγRIIA, but not of FcγRIIB, abrogated ADE of mature moDCs (51). Although mature and immature moDCs express similar levels of FcγRIIA, only mature moDCs sustain antibody-enhanced DENV infection because immature moDCs express high levels of DC-SIGN and thus do not require FcγR for DENV attachment or entry (51). Inflammation may lead to activation and differentiation of monocytes and moDCs, as well as altered expression of FcγRs and DC-SIGN, which modulates ADE of DENV infection in a cell-type specific manner.

Different classes of FcγRs transmit activating or inhibitory signals and play different roles during ADE. Although activating FcγRIIA and inhibitory FcγRIIB similarly bind DENV-antibody complexes, only FcγRIIA mediates enhanced DENV infection (74). In contrast to activating FcγRs, the inhibitory FcγRIIB may help prevent ADE. DENV-antibody aggregates cross-linked the inhibitory low-affinity FcγRIIB, which inhibited the phagocytosis and infection that would have occurred through activating FcγRs (75). This study showed evidence that the size of antibody-DENV aggregates may contribute to the neutralizing versus enhancing capacity of DENV-immune sera. More detailed studies on the expression of activating and inhibitory FcγRs are needed to understand the neutralization or enhancement of DENV infection of diverse cell subsets in different tissues. Along with greater infection in vitro, ADE may contribute to severe dengue disease in humans. Secondary DENV infection with a heterologous serotype is associated with an increased risk of DHF/DSS (76–79). Consistent with a possible role for ADE in vivo, polymorphisms in FcγR genes affect binding affinities for IgG subclasses and may influence the susceptibility to severe disease. Homozygotes for the arginine variant at position 131 (R/R131) of the FcRIIA gene, who have less capacity to opsonize IgG₂ antibodies, showed reduced risk of developing DHF (80, 81). In contrast, the histidine variant H/H131 of FcRIIA was associated with an increased risk of developing DHF (81). Binding affinity of FcγRs to DENV-antibody complexes and the ratio of activating and inhibitory receptors likely determine DENV infection and disease outcome in the setting of pre-existing anti-DENV antibody.

Animal models also have been used to study ADE in vivo. AG129 mice develop mild disease after intravenous or intradermal inoculation with DENV2 in the absence of pre-existing antibody and lethal disease after passive transfer of subneutralizing levels of DENV-immune mouse or human serum prior to infection with otherwise sublethal doses of DENV (53, 82–84). The vascular leakage syndrome that develops in DENV-infected AG129 mice during ADE is similar to that observed after high-dose lethal DENV infection and recapitulates many features of severe dengue disease in humans (40, 44). Monoclonal antibodies directed against DENV protein E or prM can mediate ADE in vitro and in vivo (82, 83, 85). Addition of antibodies that block FcγR binding, F(ab)’₂ fragments that lack the Fc domain, or recombinant monoclonal antibodies that lack the ability to bind FcγR all prevented ADE (82, 83). DENV-specific monoclonal antibodies with modified Fc domains that do not mediate ADE show therapeutic potential in vivo (85–87).

Mouse models have helped to define possible cellular targets for antibody-enhanced DENV infection. While the same cell types become DENV infected in the presence or absence of enhancing antibodies, DENV infection increases during ADE (82). In addition, MHC II⁺ cells in the intestinal lamina propria and sinusoidal endothelial cells in the liver of AG129 mice were infected by DENV mostly in the presence of enhancing concentrations of antibody (83). More detailed studies using human cells in vitro, clinical samples, and animal models are needed to clarify how cellular activation and differentiation modulate FcγR expression and impacts DENV infection and pathogenesis.