Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage

1Millions of individuals are infected yearly with DENV and some of these develop potentially deadly disease states, such as DHF and Dengue Shock Syndrome (DSS), both of which involve increases in vascular permeability, plasma leakage into tissues and hemorrhaging within internal organs. In severe cases, circulatory failure and death can occur. Currently, no targeted treatments exist to stabilize the vasculature during severe DENV complications, in part due to our lack of understanding of the mechanisms of DENV-induced vascular leakage.

2MCs are well-established cellular regulators of vascular integrity, tone and function. They line blood vessels and produce many vasoactive mediators that have redundant functions in inducing vascular permeability. Some of these are pre-stored and can act nearly instantaneously on vascular endothelium, including TNF, proteases (e.g., chymase and tryptase), and heparin. Other de novo synthesized vasoactive factors include leukotrienes, prostaglandins, VEGF and, again, TNF. With their activation, MC-derived factors act in concert to promote the break down of junctions between endothelial cells, plasma leakage and edema within tissues, as well as to reduce clot formation and increase blood flow in the vicinity of MC-activated endothelium. MCs are also versatile detectors of infection that respond strongly to direct activation by DENV as well as indirect, antibody-mediated activation. We also have recently reported that the degranulation response of MCs to DENV does not require active viral replication within MCs, since UV-inactivated virus was sufficient to promote MC degranulation.

3During localized inflammation in vivo, MC-induced vascular permeability promotes the delivery of humoral factors into a tissue and recruitment of immune cells, such as NK cells and T cells to the sites of DENV infection. However, systemic or aberrant activation of MCs is a contributing factor to many pathological conditions associated with leakage of blood vessels, including anaphylaxis, asthma, aneurysm, and others. Severe DENV outcomes in human patients have been associated with high levels of vasoactive factors that MCs produce, high levels of products that enhance MC responses, including IgE, and MC-activation associated symptoms, such as rash and thrombocytopenia. Together, these associations prompted us to examine the role of MC in DENV-induced vasculopathy. We began this work with the hypothesis that MCs modulate the vascular endothelium during DENV infection with pathological consequences.

4First, we sought to visually examine the interactions between MCs and blood vessels in the presence of DENV. The mouse ear tissues presented in Figure 1A–C were prepared for transmission electron microscopy (TEM) 24 hr after sub-cutaneous injection of saline (Figure 1A) or 1 × 105 PFU of a clinical isolate of DENV (Figure 1B–C). This inoculating dose was used since it approximates our understanding of the infective dose instilled into the skin by mosquito injection. Control, saline-injected tissues contained apparently unactivated MCs, located proximal to intact blood vessels, which were densely packed with intra-cellular granules. Several changes to the ultrastructure of DENV-injected tissues were noted, contrasting the quiescence of saline-injected tissues. DENV-injected tissue contained many activated MC located near vessels (Figure 1B). These could be identified by the characteristic lengthened cellular protrusions that remain after degranulation and the presence of residual intracellular granules. Occasionally granules could be identified in the process of being released (circled in Figure 1B). In addition to MC degranulation, evidence of fluid pooling or edema could be observed in infected tissues, also in close proximity to MCs, extracellular MC granules, and vascular endothelium (Figure 1B). These images are representative of our initial observation that MC activation and loss of vascular structure appear intimately associated in DENV-injected tissues and they led us to undertake further studies to examine the role of MCs in DENV-induced vascular leakage.

5Prior to experimentally addressing the role of MC responses to immune pathology and vascular leakage on a systemic level, we undertook studies to validate the use of an immunocompetent animal model that could provide measurable evidence of vascular changes. For DENV, a prominent portion of the literature raises doubt as to whether wild type (WT), immunologically intact, mice can be productively infected by DENV or are valid hosts for undertaking studies to investigate the mechanisms of vascular permeability. In spite of this, studies to address the role of MC-promoted inflammatory responses to DENV require the context of a host that is able to generate normal immune responses, whether productive or pathological. Although WT mice have been shown to be less susceptible to infection than mice lacking the capacity of innate immune activation, we, like others, observe and have previously reported WT mice can sustain replicating DENV infection. Others have also used WT mice to examine DENV-induced vascular hemorrhaging or pathology. Based on these multiple lines of evidence supporting the feasibility of undertaking studies in the WT mouse model, we optimized a system of infecting WT mice to generate systemic DENV infection, by injecting 1 × 106 plaque-forming units (PFU) of DENV, intra-peritoneally (i.p.). This route of infection was chosen in order to bypass the stages of natural, peripheral infection when WT mice can also clear virus quickly. Cohorts of infected WT mice were monitored for evidence of vascular events consistent with DENV symptoms (Figure 2A,B) and for evidence of productive DENV replication (Figure 2C,D) for 5 days after the initiation of systemic infection. Two measures of vascular function that are associated with DENV pathology in human patients are thrombocytopenia and elevated hematocrit values, the latter of which is a key test used to diagnose DENV mild to moderate vascular leakage or hemorrhaging and vascular pathology clinically, although severe hemorrhaging can be accompanied by suddenly lowered hematocrit values. Since hematocrit measures the packed red blood cell volume as a percentage of the total blood volume, it is a measure of plasma loss from the circulation. WT mice experimentally infected with this DENV clinical isolate experience a rise in hematocrit values that peaks 24–48 hr after infection and remains significantly increased over naive mice for 4 days post-infection (Figure 2A), demonstrating that measureable evidence increased vascular permeability can be detected in the WT mouse model over a prolonged time course. In line with additional symptoms of DENV infection in humans, WT mice also experienced a drop in platelets over the first 48 hr of infection before recovering by day 3 (Figure 2B). Thus, with regards to quantitative measures of vascular pathology, WT mice display multiple symptoms consistent with human DENV during infection.

6To further characterize the infection profile of this WT model we also examined the liver and spleen for evidence of viral replication, since these are DENV-target organs in humans. By real time PCR for DENV NS1, we were able to determine that viral replication peaked in the liver at 48 hr and in the spleen at 72 hr (Figure 2C). This evidence of productive DENV infection was supported by a secondary technique of immunofluorescence staining for the replication intermediate, double-stranded RNA (dsRNA). As expected, dsRNA staining could not be observed only 1 hr after infection, prior to when systemic replication could have been initiated on a detectable scale (Figure 2D). However, dsRNA staining could be observed in the spleen beginning at the 24 hr time point and this staining remained apparent throughout the time course, although greatly visually reduced by day 4 post-infection (Figure 2D), consistent with real time PCR quantification of splenic viral copies (Figure 2C). Staining for dsRNA also co-localized with markers for monocytes or macrophages (cd11b+cd11c−) and dendritic cells (cd11b±cd11c+) (Figure 2E), and these are well-established DENV-target cell types. This supports that the DENV clinical isolate, Eden2 is able to replicate in WT mice, with an infection profile consistent with what is known about human DENV infection, in terms of target organs and cell types.

7Having observed that DENV-induced MC activation is linked to microstructural vascular changes (Figure 1) and having validated our model of systemic DENV infection (Figure 2), we undertook studies experimentally to assess the contributions of MCs to DENV-induced vascular pathology. To reveal any contributions of MC to vascular leakage during DENV infection, we compared the responses of immunologically intact WT hosts to mice lacking MCs (Sash). Sash mice are a well-established MC-deficient model, where MCs are almost entirely absent due to a key point mutation in the promoter of the ckit gene that predominately affects its expression within the MC lineage. To determine if DENV-infected WT mice can display systemic levels of MC activation, we measured the MC-specific product, MCPT1, in mouse serum by ELISA in naïve and DENV-infected mice. After intra-peritoneally. injection of DENV as described above, MCPT1 was detectable in the serum of WT mice at 24 hr and continuing beyond 48 hr (Figure 3A). As expected, Sash mice had undetectable serum levels of MCPT1 at baseline, as did uninfected congenic wild-type (WT) controls (Figure 3A). Having verified that our infection model allows systemic detection of a key MC-derived vasoactive product, we next examined readouts of vascular leakage in this system by hematocrit analysis, again, since this is a key measure of both mild and severe vascular leakage with clinical relevance. The results of hematocrit analysis supported a MC-dependency to vascular leakage since MC-sufficient WT animals had elevated hematocrit values with DENV infection, but Sash mice did not (Figure 3B). Importantly, since a few other processes (such as melanocyte migration) are affected in Sash mice, repletion studies were performed in these experiments as a control. To replete MCs in order to establish that MCs are sufficient to restore the WT functional phenotype, bone marrow-derived MCs (BMMCs) were passively infused intra-venously. Since repletion of Sash mice with MCs, alone, was able to restore elevated vascular leakage (Figure 3B), the DENV-induced affect of increased vascular permeability appears to be MC dependent.

8To provide a second measure of vascular leakage supporting the hematocrit data, we undertook studies to examine the MC-dependency of dye leakage into tissues during DENV infection, using the dye Evans blue. Evans blue leakage studies are commonly used to assess models of viral hemorrhaging, however, we modified the standard technique so as to improve the sensitivity to detect sub-hemorrhagic vascular leakage by flushing excess dye from the vascular system by saline perfusion, prior to quantitation. Following this strategy, mice were injected by tail vein with the tracking dye, Evans blue, 30 min prior to euthanasia at a 24-hr time point after infection. Initial examination of the internal organs of mice did not reveal visible leakage of dye (Figure 3—figure supplement 1), which is consistent with the observations by others that immunocompetent WT mice do not always exhibit gross pathological vascular leakage with DENV infection. However, when mice were perfused with saline, increased in vascular permeability was visible in infected mice due to residual blood and dye remaining in the tissues (Figure 3—figure supplement 1). Therefore, this model allows us to detect the earliest signs of vascular leakage quantitatively and to assess the effects of MC detection of DENV to vascular integrity in vivo.

9Using this Evans blue perfusion model, we determined the extent of vascular leakage induced in 24 hr after intra-peritoneally instillation of 1 × 106 plaque-forming units (PFU) of DENV in WT and Sash mice. Liver tissue was harvested and homogenized to obtain quantitative values regarding the amount of dye leakage into the tissue and also visually examined for evidence of leakage (Figure 3C). After perfusion, WT and Sash-R mice showed visually apparent increases in vascular permeability in the liver that were supported by Evans blue quantitation, however, Sash mice did not (Figure 3C). Additional highly vascularized organs, such as the kidney, also showed visually perceivable evidence of MC-dependent leakage of blood into the tissues that was not apparent in Sash mice after saline perfusion (Figure 3—figure supplement 2). Since the liver of mice contains very few MCs compared to other species such as humans, these findings suggested that systemic MC effects were likely responsible for the observed vascular changes. MC-dependent vascular leakage was also directly correlated to increased MC-derived product MCPT1 (Figure 3A). Together, these findings, combined with the data obtained by hematocrit analysis (Figure 3B) point to a pivotal role for MCs in promoting DENV-induced increases in vascular permeability.

10Due to the contributions of MCs to many pathological inflammatory disorders, there are a number of available drugs that target MCs or products that they produce after activation. Informed by our studies suggesting that MCs augment DENV-induced vascular leakage in animal models (Figure 3), we attempted to block this vascular leakage using a panel of these drugs. We chose two well-established MC-stabilizing compounds that are clinically available, cromolyn and ketotifen, and two treatments that block MC products that are known to promote vascular leakage, an anti-TNF blocking antibody and montelukast, a leukotriene receptor antagonist. None of these drugs act exclusively on MCs or MC-derived products but they are known to modulate the functions of MCs effectively in a clinical context. Drugs were administered once, 30 min after intra-peritoneally instillation of 1 × 106 DENV and vascular leakage was assessed 1 day after infection. Both MC-stabilizing compounds significantly decreased vascular leakage compared to untreated DENV alone, quantitated by Evans blue leakage as well as hematocrit analysis (Figure 4A). Blocking TNF has previously been shown to be effective in limiting vascular leakage at late time points of infection in immunocompromised mice lacking Type I and II Interferon receptors (IFN-α,β,γ-R−/−, or AG129), which is a mouse model of DENV viremia. In our WT mouse model, when TNF-blocking antibodies were administered 1 hr after systemic infection was initiated by intra-peritoneally injection, the average Evans blue dye leakage and hematocrit values were lower; however, this did not reach statistical significance (Figure 4A). Blocking leukotriene function using the drug montelukast also allowed striking, significant reductions in dye leakage compared to untreated mice (Figure 4A), suggesting leukotrienes can also contribute to DENV-induced vascular permeability. Although cromolyn’s inhibitory influence on MCs is well established, to support a direct effect of the MC-stabilization strategy on MCs in this DENV infection model, we again compared serum MCPT1 in untreated and cromolyn-treated DENV-infected mice. As expected, levels of MCPT1 were reduced by cromolyn treatment at 24 hr, and by 48 hr, serum MCPT1 was undetectable in treated mice, yet still elevated in untreated mice (Figure 4B). By 24 hr, there was a trend towards a slight increase in the average number of DENV genome copies in the serum, but this did not reach levels of statistical significance (Figure 4C). Since three drugs from this panel, cromolyn, ketotifen and montelukast, all were successful in restoring both the levels of dye detection in the liver tissue and hematocrit values to levels that were not statistically different from baseline levels (Figure 4A), these data highlight the potential of MCs to serve as therapeutic targets to limit DENV pathology.

11Additional cell types can also produce some of the vasoactive products that MCs produce, including cytokines and eiocosinoid mediators. To determine if DENV elicits products from MCs, directly, that act on vascular endothelial cells, we performed trans-well assays to measure the responses of mouse endothelial cell (EOMA) monolayers to MC-derived products. Supernatants from DENV activated or control mast cells were transferred to monolayers of EOMA cells and the trans-endothelial resistance (TER), a measure of the barrier function of a cell monolayer that decreases as permeability increases, was measured after 24 hr to determine if DENV-activated MCs specifically promote loss of barrier function. We observed a significant drop in the TER reading relative to the baseline for EOMA cells exposed to DENV-activated MC supernatant but not to unactivated MCs or virus alone (Figure 4D). Cromolyn treatment of MCs also prevented this reduced barrier function, as did both blockade of leukotrienes with montelukast or inhibition of chymase with a cocktail of specific inhibitors including chymostatin and soybean trypsin inhibitor (Figure 4D). To confirm the role of MC-derived leukotrienes in promoting endothelial cell permeability in an additional experimental system, we tested whether leukotriene-deficient MCs are able to induce comparable vascular permeability to WT MCs. For this, we purified MCs from the peritoneal and pleural cavities of mice that lack 5-lipoxygenase (5-LO-KO), the enzyme required for leukotriene synthesis for use in trans-well assays. For comparison, MCs were also purified from WT and TNF-KO mice. Purified MCs were stained to verify that the isolated cells demonstrated a granulated MC morphology (Figure 4E, inset). We observed a significant reduction in the TER relative to baseline in EOMA cells exposed to supernatants from DENV-activated WT or TNF-KO MCs that did not occur in EOMA cells exposed to supernatant from DENV activated 5-LO-KO MCs (Figure 4E). 5-LO appears to mediate direct activity of DENV-activated MCs on endothelial cells since DENV-exposed WT MCs have a significantly greater impact on the TER of EOMA cells compared to MCs from 5-LO-KO mice. These data also provide evidence that MCs products have the potential to be direct intermediaries in promoting vascular permeability of endothelial cells that is not induced by virus alone treatment (Figure 4D,E).

12To give our findings context in another established DENV mouse model, we also undertook experiments in IFN-α,β,γ-R−/− mice, since these mice can sustain replicating DENV infection for many days and are a common model used for testing DENV anti-viral drugs. Figure 5A contains images of the mouse mesentery (the membrane that anchors the gut within the peritoneal cavity), stained for blood vessels, MCs and DENV replication, 24 hr after intra-peritoneally injection of 2 × 105 DENV. In control tissues, blood vessels appeared intact and were surrounded by tightly granulated MCs (Figure 5A). However, blood vessels during DENV infection appeared to have lost structural integrity and were surrounded by tiny avidin-staining particles, signifying extensive MC-degranulation within the vicinity of the vessel (Figure 5A). It was apparent that blood vessels in DENV-infected tissues also have decreased staining for the marker of vascular endothelium, CD31 (or PECAM), which functions as an adhesion molecule within endothelial junctions (Figure 5A). Proximal DENV-infected cells were revealed through staining for the DENV non-structural protein 3 (NS3) (Figure 5A), which is produced only during viral replication. Thus, evidence of MC activation and vascular leakage were also observed in these mice.

13In this system, we delayed treatment with cromolyn until 1 day after systemic infection was initiated via intra-peritoneally injection, as performed previously. When blood was collected 3 days after infection and after two daily doses of cromolyn, we again observed that hematocrit values were reduced significantly compared to DENV-infected, untreated mice (Figure 5B). This apparent decrease in vasculopathy with cromolyn treatment even occurred in spite of an approximate doubling in the serum viral titer (Figure 5B). Although cromolyn has additional immunosuppressive functions that are not MC-restricted and these data were obtained in a mouse model having severe defects in anti-viral responses, they appear to emphasize the dual contributions of MCs to immune protection and pathology during DENV infection.

14After obtaining data from two separate mouse models implicating MCs in the mechanism of DENV vasculopathy, as well as evidence that drugs that target MCs or their products can restore vascular integrity, we sought to validate this key finding in humans. To determine if MC activation occurred in human DENV patients, we tested serum from patients for the MC-specific product, chymase, by ELISA (Figure 6A). This human homologue of mouse MCPT1 is also known to increase vascular leakage over a prolonged time course. All sera were obtained from a previously described DENV clinical study, where early serum samples were obtained during acute DENV infection (1–3 days after onset of fever), followed by a second sample during the defervescent stage of infection (4–7 days after onset of fever). The sera tested fell into the following groups: (1) control healthy human sera, (2) control sera from individuals with fever that were referred to the study as possible DENV cases but were negative for DENV by laboratory tests employing RT-PCR for DENV RNA, (3) sera from patients that were positive for DF by clinical diagnosis as well as RT-PCR and (4) sera from patients that were DENV positive by clinical diagnosis, molecular tests, and were also diagnosed during the study to have DHF, rather than the less severe DF. Strikingly, we found that those patients that were diagnosed with DF or DHF showed chymase levels in serum obtained during the acute phase of infection were significantly higher than levels in the serum of either healthy controls or individuals with fever that were DENV negative by RT-PCR (Figure 6A). This trend of heightened levels of chymase in the blood of DF and DHF patients persisted to the second time point of blood collection (Figure 6B), illustrating a prolonged course of elevated MC products occurs during infection. During the acute phase of infection, DF patients displayed an increase in serum chymase that was approximately 10 times higher than to healthy individuals or DENV-negative patients while, in DHF patients, chymase levels 30 times higher than healthy controls were detected (Figure 6C). Heightened levels of serum chymase were also maintained in DF and DHF patients relative to healthy controls during the defervescent stage of infection (Figure 6C; Figure 6—figure supplement 1). Since there is an approximate threefold increase in the levels of serum chymase in the blood of DHF patients compared to DF patients, this finding also illustrates a correlation between the levels of chymase and the severity of vasculopathy experienced in DENV-infected humans (Figure 6A–C). Patient serum samples were DENV serotypes 1-, 2-, or 3-positive, and the infecting serotype also appeared to be a significant, but minor influence to levels of chymase detected (Figure 6D). For example, chymase concentrations in DENV2-infected individuals were highest on average in both DF of DHF patients compared to DENV1- and DENV3-infected patients (Figure 6D). Although the serum used here was derived from a study where there was no increased incidence of DHF diagnosis in patients with secondary infection over primary infection, secondary infection is considered to be a risk factor for increased likelihood of DHF. For this reason, we also compared the levels of chymase in these two groups of primary and secondary infection and found that while there were no differences in the chymase levels in patients diagnosed with DF between primary and secondary infection groups (Figure 6E), interestingly, significantly higher chymase levels were detectable in DHF patients with secondary infection compared to DHF patients diagnosed during their primary infection (Figure 6F). We also observed that for DF, there was no discernable correlation between the amount of virus in the serum and serum chymase levels; however, for DHF patients, there appeared to be a strong correlation between high levels of chymase and lower DENV genome copies in the serum (Figure 6G), perhaps, highlighting the potential of MCs to contribute to virus clearance. Human DENV-infection follows a disease course where acute infection is characterized by high viremia and fever, followed by either resolution or hemorrhagic complications that usually occur during the defervescent phase, as viral titers decline. These opposing outcomes are difficult to predict early in infection. Our results suggest that, beyond the potential role of chymase in directly promoting vascular events, it may be a useful biomarker for prediction of severe DENV disease outcomes.

15Our findings highlight the critical role that MCs may play in virus-induced vascular leakage. Since animals that lack MCs have reduced vascular permeability during DENV infection, to baseline levels, it appears that these cells are highly consequential to the initiation of vasculopathy, which is characteristic of severe DENV cases. TNF has previously been identified to promote the severe vascular leakage that occurs in DENV-infected animals and our data that leukotrienes and proteases also promote vascular leakage during infection with DENV suggests that multiple mediators, a substantial portion of them MC-derived, likely act in concert to effectuate the vascular complications that occasionally occur during severe DENV infections (Figure 7). Since DENV is naturally a primarily human-restricted pathogen and information gleaned from each animal model of disease has inherent caveats, we have verified our findings that MCs are activated during DENV by examining human clinical samples. Surprisingly, for those few patients that progress beyond normal DENV-associated malaise to develop hemorrhagic complications, we observe that heightened responses of MCs are associated with DHF and are apparent in an extreme elevation of serum chymase. If corroborated in independent patient populations and prospective human studies, this connection may be of great clinical utility since severe cases are notoriously difficult to predict early in infection at the time points where elevated chymase was first detected in this study. Patient serum was also derived in this case from adults experiencing DENV1, DENV2, or DENV3 infections and further studies will also be required to extend these results to children, a patient group that frequently experiences DHF, and to additional strains of virus. In support of the potential role of MC proteases during DENV infection, another recent study has also observed elevated chymase and tryptase in the serum of human patients. Chymase, itself, can promote vascular leakage over a notably prolonged timecourse and its presence in the serum would signify that additional vasoactive cytokines and mediators would have been released from MCs, potentially, also contributing to vascular permeability.

16Although we have identified MCs as critical cellular catalysts of DENV-induced vascular pathology, in our view, this does not diminish the important role of the virulence mechanisms by which DENV achieves systemic or substantial infection. In some studies, but not others, high viral titers have been associated with DENV disease severity. Interestingly, we observe that for DHF patients, a decrease in serum virus genome copies is correlated with increased levels of MC products in the serum. We believe that this observation points to the dual role of MCs in immune protection and pathology. Our findings may provide one mechanistic explanation of how systemic infection may translate into a substantial pro-inflammatory response with negative implications for vascular integrity in terms of its barrier function. Just as many risk factors have been identified for DENV patients that are associated with severe infection and vascular leakage, many factors can theoretically influence the degree of MC activation in vivo in response to a stimulus, including the stimulus concentration, the presence of pre-existing antibodies such as IgE and their concentration, and host-specific differences in the intracellular signaling and sensitization of MC that can result from genetic diversity or existing allergy.

17These studies have also identified several candidate drugs that target MCs or their products that may be effective in limiting or preventing DENV pathology in a clinical setting and each has the added advantage of already being approved for human use in other contexts. Interestingly, all of these drugs would be expected to limit host immune activation without antiviral activity. Although use of the Sash MC-deficient mouse model suggests that MCs are major contributors of the vasoactive factors that promote vascular permeability during DENV infection, we have used drugs to block MCs and MC products that are not entirely specific to inhibiting MC function. For example, cromolyn can reduce the activation levels of additional cell types. Although leukotrienes were targeted because they are produced by MCs in copious amounts and we show that MC-derived leukotrienes can directly induce permeability of endothelial cell monolayers, they are also produced by cell types in addition to MCs. Therefore, the benefits of MC targeting may be in part due to the blockade of inflammatory products from other cellular sources. Our observation that MC stabilization also promotes modestly increased viremia, at least when used in an immunocompromised mouse model where virus replication cannot be effectively controlled, also appears to support the contrasting role of MCs in the effective containment of DENV infection, occurring in this case independent of interferon responses. Although we do see that viral replication occurs in our WT mouse model over a time course of several days, it is interesting to point out that the mechanism of MC degranulation in response to DENV is not dependent upon intracellular replication of virus. Experimental infection of MCs with DENV is possible in vitro and could potentially occur in vivo where it would likely enhance the cytokine responses to infection; however, MCs appear to directly detect the structure of DENV virus particles during degranulation. When this occurs as an early detection event during cutaneous infection, detection of DENV can be protective for the host, for example, by promoting the recruitment of NK cells and NKT cells into a site of infection to aid in viral clearance. Widespread MC activation may become pathological when viral titers are high on a systemic level and, as in additional pathological contexts, MC responses in the context of systemic infection could reach a point where they become detrimental to the host. In addition to the evidence that direct sensing of DENV structure occurs by MCs, other indirect mechanisms exist for MCs to degranulate in response to pathogens in vivo which could provide additional activating stimuli for MCs during infection. For example, MCs are able to degranulate in response to components of the complement pathway and complement activation has also been associated with DENV severity and plasma leakage in human patients.

18Many infections are capable of generating events such as cytokine storm in infected hosts; yet, the pathogen-specific manifestations of DENV (including the substantial degree to which vascular leakage can occur) highlights that unique mechanisms underlie the interaction between the virus and host during this infection. MCs appear to have highly specific responses to unique viruses and very few viruses have been identified that are capable of causing direct degranulation of MCs. Interestingly, respiratory viruses are primarily thought to not induce direct degranulation of MCs (without antibody sensitization or augmentation of MC responses due to host factors). This is consistent with our data from non-DENV febrile human patients, most of which had been diagnosed as having respiratory viral infections, where systemic MC activation was not detected in terms of chymase levels. Another virus that was recently recognized to induce MC-degranulation is vaccinia virus, which is not a hemorrhagic virus and is also primarily a cutaneous and not blood borne virus. These observations point to a DENV-specificity of the MC response that may be dependent on the ability of virulent DENV to reach systemic titers and elevate MC products in the blood. This may have further implications in the context of related and unrelated viral hemorrhagic fevers and suggests that further investigations are warranted into the role of MCs in hemorrhaging induced by additional viral pathogens.

19Within the context of treatment for DENV vasculopathy, the dual role of MCs in protection and pathology also emphasizes the importance of carefully testing any therapeutic regimen targeting MCs in human patients and perhaps considering a combination therapy approach that also would limit viral replication when such a strategy is available. Since those individuals with the highest levels of MC products during this study also experienced the most clinically severe disease, defined by vascular leakage or hemorrhaging, our data also highlight the possibility of using MC products as biomarkers to identify individuals at highest risk for hemorrhagic complications. Investigating the mechanism of DENV-induced degranulation may also reveal additional targets for blocking MC activation by DENV. For additional pathogens, such as E. coli, unique receptors on MCs have been identified to promote MC activation. Identification of receptors and signaling components involved in the direct activation of MCs by DENV is likely to impact our understanding of both DENV protection and pathology.

20MC-deficient mice (Wsh/Wsh; “Sash”), Alox5-deficient (5-LO-KO), TNF-KO, and control mice (+/+; wild type, on a C57BL/6 background), were originally purchased from Jackson Laboratories. Additional C57BL/6 mice were purchased from the National Cancer Institute. Sash mice were repleted with in vitro matured bone marrow–derived MCs from congenic controls to generate reconstituted Sash (Sash-R) mice as previously described. Sv/129 mice deficient in type I and II IFN receptors (strain AG129, denoted as IFN-α,β,γ-R−/−) were originally purchased from B&K Universal and provided as a gift by Dr Subhash Vasudevan. All experiments were performed according to protocols approved by the DUKE-NUS or Duke University Division of Laboratory Animal Resources and their respective University Institutional Animal Care and Use Committees.

21For infections, DENV serotype 2, strain Eden2, was used. This clinical isolate was derived from the same study (Early Dengue Infection and Outcomes Study, Eden; ) as the patient sera in Figure 6. The culture conditions we used to maintain this strain and its interactions with MCs were previously described. Mouse infections were performed by intra-peritoneally injection of 1 × 106 PFU (WT and Sash mice) or 2 × 105 PFU (IFN-α,β,γ-R−/− mice). Cromolyn (3 mg/mouse/day) and ketotifen (0.6 mg/mouse/day) (both from Sigma) were injected intra-peritoneally in PBS as vehicle either 1 hr (WT and Sash mice) or 24 hr (IFN-α,β,γ-R−/− mice) after infection. Montelukast (brand Singulair; Merck) was administered (0.4 mg/mouse) by oral gavage 1 hr after infection by crushing tablets using a mortar and pestle and resuspending in PBS.

22Vascular leakage was assessed at the reported time points after infection using an Evans blue leakage assay. Evans blue was prepared as a 1% solution in PBS, syringe filtered and 100 μl of this solution was injected by tail vein, 30 min prior to euthanasia. Immediately following death, animals were perfused with 15 ml of PBS (approximately fivefold the mouse blood volume) per animal by opening the chest cavity, creating an incision in the right atrium of the heart and injecting the full PBS volume in the left ventricle of the heart. Blood was, thus, flushed fully from the mouse circulatory system. Tissue biopsies were then obtained, their mass recorded, and homogenized in PBS. Cellular debris was cleared by centrifugation, and the OD-600 was measured to quantify the amount of dye within tissues, based on a standard curve. These values were normalized to the tissue mass. For hematocrit analysis, heparinized blood that had been collected from individual mice via the maxillary vein was run on an automatic hematology analyzer to obtain values. ELISAs for MCPT1 were performed using a kit from R&D Systems, according to manufacturer’s instructions.

23Spleen sections were frozen in OCT compound (Tissue-Tek), and frozen sectioned (10 μm thick) using a cryostat (Leica). Sections were acetone-fixed at 4°C, then blocked with PBS containing 1% BSA prior to staining with primary antibodies against anti-CD11b, anti-CD11c (eBiosciences) and dsRNA (clone J2; English and Scientific Consulting Bt.). The following secondary antibodies were then used: anti-mouse-AlexaFluor555, streptavidin-AlexaFluor488, and anti-rat-Cy5 (Invitrogen). Slides were then mounted and imaged by confocal microscopy. For whole mounting, mouse mesentery was dried onto slides, fixed with formaldehyde, and permeabilized/blocked using 0.1% saponin (Sigma) in PBS containing 1% BSA. Primary staining was then performed in permeabilization/blocking buffer at 4°C, overnight, using a rat antibody against CD31 (eBioscience), a rabbit antibody against DENV NS3 (GeneTex), and the MC-specific probe, Avidin conjugated to FITC (Sigma). After extensive washing with PBS, tissues were incubated overnight with secondary antibodies: AlexaFluor 660-conjugated anti-rat (Molecular Probes), and Cy3-conjugated anti-rabbit (Jackson Immunoresearch). Images were obtained under epifluorescence.

24Human serum samples were derived from a bank of samples from adult febrile or healthy control individuals in Singapore. Samples used for this study were derived from patients that ranged in age from 18 to 77 years, with a mean of 40 years; 42% of samples were obtained from females, 58% from males. Dengue positive samples were determined based on physician diagnosis as well as molecular tests including RT-PCR viral RNA. The DF and DHF patient sera used in this study were previously determined to be positive for serotypes 1, 2, or 3 by RT-PCR. Of the samples included in this study, approximately 47% of the DHF cases and 50% of the DF cases were secondary infections, which had been determined previously by testing for dengue-specific IgM and IgG. Non-DENV positive patients were febrile patients that were referred to the original study as suspected DF patients, but were negative for confirmatory DENV markers by molecular tests. Data is derived from sera obtained at the time of enrollment on days 1–3 after the start of fever and from a paired sample that was obtained days 4–7 after the start of fever. The patient’s physician made DHF versus DF diagnosis during the original study, based on WHO 1997 guidelines. These diagnoses have also been given the designation of ‘severe’ (DHF) and ‘mild’ (DF) within the Eden study. All patients supplied written informed consent. The methods of the original study from which these samples were obtained, which was approved by the National Healthcare Group IRB, are included as references and. ELISA kits for Human chymase were obtained from Antibodies-Online and performed according to manufacturer’s instructions. Relative chymase levels in the serum displayed in Figure 6C were calculated by normalizing the concentration in each sample to the average concentration of chymase in healthy control serum donors. To quantify viral genome copies, viral RNA was isolated from 100 μl of serum using the QIAamp Viral RNA Mini Kit (Qiagen), followed by amplification and detection using the Qiagen OneStep RT-PCR Kit, both used according to manufacturers instructions. We used serotype-specific primers for DENV1-3 designed by the CDC that were described previously. A subset of samples was used for PCR detection of virus and this selection was based solely on having sufficient remaining serum to perform the assay.

25Mouse vascular endothelial cell lines, EOMA and SVEC4-10EHR1, were obtained from ATCC through the Duke University Tissue Culture Repository. Cells were grown on transwells with 3-μm pores (BD Biosciences) for 4 days for a monolayer to form. To isolate MCs, the peritoneal and pleural cavities were flushed twice with PBS containing 0.5% BSA and 2 mM EDTA. Cells were then washed and resuspended in anti-c-kit primary antibody (eBioscience) at a concentration of 5 μg/ml and incubated for 1 hr at 4°C. After washing, the MACS method was used to purify MCs by incubating labeled cells with anti-rat-IgG magnetic beads prior to isolation using LS columns (both from Miltenyi), according to manufacturer’s instructions. Some purified cells were cytospun onto slides and stained to visualize MC morphology. Isolated cells were resuspended in culture medium and either incubated alone, or treated with DENV at an MOI of 5 for 1 hr at 37°C in a tissue culture incubator. Media containing DENV alone was incubated concurrently for use as a control. To isolate MC supernatants, after incubation, cells were centrifuged to pellet them and a volume equivalent to supernatant from 5 × 105 cells per well) was applied gently to the trans-well insert for a final volume of 400 μl. The TER of the endothelial monolayers was measured at baseline, prior to treatment and at 12 or 24 hr after exposure.

26For trans-well assays using BMMCs, an MOI of 1 was used and drugs were used to inhibit the actions of MCs or their products on the endothelial cells. For cromolyn treatment, cromolyn (10 μM; Sigma) was incubated with BMMCs during DENV activation. Immediately prior to exposing cells to MC ± DENV supernatants, inhibitors for leukotrienes (montelukast, 10 μM final concentration; Sigma) or chymase (Soybean Trypsin Inhibitor, 100 μM final concentration and Chymostatin, 30 μM final concentration; both from Sigma) were added to trans-wells. Supernatants were then added to each insert.

27Images were prepared for publication using ImageJ. Prism 5 and Excel were used to determine statistical significance. For direct comparisons of infected and control samples, Student’s un-paired t-test was used. For comparisons of multiple groups, ANOVA was performed with Bonferroni’s post-test to determine statistical significance. Two-way ANOVAs were used in the case of assessing data for multiple groups at multiple time points. Data were considered significant at p≤0.05.