A major challenge in managing DENV infections is the lack of reliable predictors for disease severity, as well as the absence of early biomarkers that can predict disease progression. In this study, we measured the levels of 41 plasma mediators, which are crucial in both innate and adaptive immune responses and have previously been associated to DENV infection13,14,15. The study included patients with three distinct disease outcomes (AD, DF, and DHF), recruited from the well-defined DENFREE Thailand cohort, and encompassed four key time points throughout the course of SD infection to provide a comprehensive understanding of dengue immunopathology and identify potential biomarker.
Our cross-sectional findings revealed a global dampening of plasma mediators in AD compared to SD patients (DF and DHF). This supports the hypothesis that AD cases regulate cytokine secretion more efficiently, enabling viral clearance without excessive immunopathology. The findings align with our previous study on the same cohort, which showed a lower viral decay rate in AD cases23. Therefore, we suggest that the balanced immune response observed in AD potentially limits immunopathology and symptoms.
Our finding may also support several studies linking cytokine storms, characterized by excessive soluble mediator production, to dengue pathogenesis13,26. Notably, the elevated levels of anti-inflammatory cytokine IL-10 in SD patients have been identified as a hallmark of severe dengue infection14,19,20. Previous studies suggested that elevated plasma IL-10 may be associated with plasmablast differentiation22,27, linking it to antibody-dependent enhancement, a well-known mechanism in dengue pathogenesis. Moreover, the concentrations of IL-10 in severe DHF patients were found to be 25 times higher than in severe COVID-19 patients28, underscoring its potential role in disease severity. Beyond IL-10, prolonged high concentrations of inflammatory cytokines have been linked to severe dengue, in contrast to the rapid decline seen in mild dengue cases29. Moreover, elevated TNF-α and IL-6 levels, in particular, have been implicated in vascular leakage and are thought to play critical roles in dengue pathogenesis30,31,32. Meanwhile, the secretion of anti-inflammatory cytokines such as IL-1RA and IL-10 during the acute phase may help counterbalance inflammatory activities, as evidenced by the positive correlation between IL-10 levels and pro-inflammatory cytokines, including IL-6 and IL-833. In summary, SD is characterized by excessive cytokine production, and this dysregulation likely contributes to a spectrum of outcomes, ranging from mild symptoms to severe disease.
Although we observed a global dampening of cytokines in AD compared to SD patients, a study from the DENFREE Cambodia cohort showed contrasting results, which reported significantly elevated levels of IFN-γ, IL-2, IL-12, and IL-23 in AD cases22. However, the median levels of these cytokines in both AD and SD patients were relatively low (< 5 pg/ml), and the differences between the groups were only 1–5 pg/ml. This raises questions about the biological significance of such small variations in cytokine levels. Additionally, other cytokines, including IL-8, IL-15, TNF-α, IL-6, CCL3, and CCL4, exhibited similar levels in both AD and SD patients in that study22. The discrepancies between our cytokine profiles and those reported in previous research may arise from differences in study cohorts, such as the age range of donors (children vs. adults), the timing of sample collection relative to the course of infection, or the detection limits of the assays used.
In our longitudinal analysis, we observed significant shifts in plasma mediator levels during the febrile, defervescence, and convalescent phases of SD. Our findings are consistent with a study by Chng et al., which measured the levels of 65 cytokines and observed distinct cytokine profiles between the acute and convalescent phases34. Both studies reported elevated levels of IFN-γ, IL-1β, IP-10, and MCP-1 during the acute phase. However, our findings during the convalescent phase differed, with Chng et al. reporting an increase in Eotaxin-2 (CCL24), while we identified elevated levels of several growth factors. Similarly, our findings on anti-inflammatory cytokines, IL-10 and IL-1RA, align with those of a recent study by Vuong et al., which reported increased levels of these cytokines during the acute phase of infection (1–3 days after disease onset), followed by a decline during the convalescent phase (10 days after disease onset)35.
During the acute phase of SD infection, we observed a broad upregulation of cytokines involved in inflammatory responses (TNF-α, IL-1α, IL-6, IL-15, MIP-1α, and MIP-1β), antiviral activity (IFN-γ, IFN-α2), anti-inflammatory control (IL-10, IL-1RA), and chemokines (IL-8, MCP-1, IP-10, and Fractalkine). Previous studies have shown that increasing levels of interferons are associated with the decline of DENV viral load, suggesting their role in viral elimination and heightened cellular antiviral responses during symptomatic infection36,37. Other cytokines, such as IL-8, has been shown to promote neutrophil activation during acute dengue infection, leading to the formation of neutrophil extracellular traps (NETs)25. Elevated levels of IP-10 have been linked to the recruitment of CXCR3-bearing cells, such as activated T cells and NK cells, which amplify the inflammatory response38. Similarly, MIP-1α and MIP-1β, which are predominantly produced by macrophages, exhibit chemotactic and pro-inflammatory activity39, contributing to the inflammatory milieu upon acute dengue infection.
Conversely, during the convalescent phase, we observed elevated levels of several growth factors and other mediators that may play a role in recovery following DENV infection. One such mediator, sCD40L, has been shown to enhance platelet activation, leading to the release of platelet granule-derived mediators such as TGF-β, EGF, FGF, PDGF, RANTES, VEGF, IL-8, and PF4 (CXCL4)40. These factors are critical for endothelial cell expansion and extracellular matrix formation, promoting wound healing41,42,43. Given that plasma leakage is a hallmark of dengue pathogenesis, we hypothesize that platelet activation and the secretion of these cytokines may promote endothelial repair during recovery. Interestingly, reduced levels of platelet-derived growth factor have been frequently observed in severe dengue cases44. However, platelet activation has also been reported during the acute phase, where activated platelets exhibit apoptotic characteristics, resulting in thrombocytopenia45, a key signs of bleeding manifestations. These contrasting findings underscore the need for further studies to clarify the role of sCD40L and its association with disease pathogenesis.
Although the cytokine profiles in DF and DHF were generally similar, we observed increased IL-15 levels during the early febrile phase in DHF, which may serve as a promising predictive biomarker for disease severity. IL-15, an inflammatory cytokine with a broad range of biological functions, primarily involves the activation and survival of CD8 T cells and NK cells46. However, few studies have examined the role of IL-15 in the context of DENV infection, with some suggesting a protective role, which contrasts with our findings. One study linked increased IL-15 during the acute phase to the frequency and cytotoxic activity of NK cells47, while another study found higher IL-15 production prior to DENV exposure was associated with subclinical symptoms upon subsequent infection48. Interestingly, IL-15 can also induce the secretion of IL-8 from neutrophils and monocytes49,50, potentially contributing to the elevated IL-8 levels observed during the febrile phase. This dual role of IL-15, both as a predictive biomarker and a contributor to inflammation, warrants further investigation to better understand its impact on disease progression and to support its potential use as a biomarker.
We also observed differing levels of IL-8 and IP-10 between DF and DHF during the critical phase, suggesting that prolonged high levels of these cytokines may be associated with dengue immunopathogenesis and could be potential targets for treating severe disease. Elevated IP-10 levels are associated with higher levels of atypical lymphocytes and T cell apoptosis in severe dengue51,52, and it has been shown to inhibit endothelial cell proliferation and disrupt angiogenesis, which supports its role in causing plasma leakage53,54. Consistently, the level of IP-10 has been shown to increase in patients with significant plasma leakage55. However, a protective role for IP-10 has also been suggested, as the absence of IP-10 and its receptor leads to reduced effector T cell recruitment and increases the mortality rate in dengue-infected mice56. Additionally, one study demonstrated that high IP-10 levels can prevent DENV infection by inhibiting viral binding to heparan sulfate molecules57. Furthermore, McCracken’s study showed that downregulation of IP-10 transcripts during mosquito probing promotes DENV spread and viral replication in vivo, highlighting the role of IP-10 in DENV suppression58.
Despite the limitations of our study, such as the lack of healthy controls for all cytokines and the absence of dengue shock syndrome (DSS) patients, our work addresses important gaps in understanding the soluble immune response during dengue infection. The small sample size, particularly for DF patients during convalescence, limits the generalizability of our findings. Further studies, including in vitro and in vivo experiments, are needed to validate these findings, particularly the role of IL-15 in dengue, and to develop reliable predictive biomarkers. Additionally, future studies incorporating healthy controls to establish baseline cytokine levels would be valuable. This would also help clarify the persistently high levels of certain cytokines observed at the two-month follow-up. Moreover, the predictive value of IL-15, along with other potential biomarkers such as IL-8, IL-10, suPAR, and olfactomedin 4 identified in various studies18,19,20,21, should be validated across multiple independent cohorts to establish their reliability. Future studies utilizing machine learning approaches, along with independent cohort validation involving larger sample sizes and comprehensive clinical parameters, will be essential for designing and developing a robust disease severity prediction model.
In summary, this work highlights the soluble immune signature in asymptomatic dengue (AD) and the cytokine profile throughout the course of symptomatic dengue (SD) infection (Supplementary Fig. 4). We suggest that the limited secretion of multiple cytokines is a key feature of AD, while excessive cytokine secretion is characteristic of symptomatic disease. Prolonged high levels of IL-8 and IP-10 during the critical phase may be associated with hemorrhagic manifestations in DHF patients. Additionally, growth factors and platelet activation may play an essential role in the recovery of endothelial integrity during the convalescent phase. Finally, we propose that IL-15 could serve as a valuable biomarker for predicting disease severity. Ultimately, these findings improve our understanding of the cytokine response in DENV infection and lay the foundation for biomarker development and better treatment strategies.
