Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of Sa˜o Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Author Summary The pathogenesis of severe dengue virus (DENV) infection is still not fully understood. It is hypothesized that it is caused by a cytokine storm as is described in severe sepsis. In the sepsis field, the potent immunostimulator lipopolysaccharide (LPS) is proposed to play an important role in the development of a cytokine storm. In a previous study we have found elevated levels of LPS in children with severe DENV infection. In this study we have investigated if we could confirm that microbial translocation occurs in DENV-infected patients. Moreover, we have determined the levels of thirty cytokines to get more insight in the cytokine storm during DENV infections and we have investigated whether microbial translocation is associated with immune activation. The patients in this cohort were classified according to their clinical presentation. Furthermore, a cluster analysis based on the expression of the determined cytokines was applied to identify patients with similar cytokine profiles. With these two techniques, we identified cytokines that may contribute significantly to the cytokine storm, and we could relate elevated levels of LPS to patients with a pro-inflammatory cytokine profile.

This work was supported by the ‘‘Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazilian Ministry of Science and Technology’’ [CNPq, project number 476088/2009-7 to EGK]. AMdM and EGK’s scholarships were supported by ‘‘CNPq.’’ KIC’s scholarship was supported by the ‘‘Coordenac¸a˜o de Aperfeicoamento de Pessoal de N?´vel Superior, Brazilian Ministry of Education.’’ Moreover, this study was also supported by the ‘‘Virgo consortium,’’ funded by the Dutch government [project number FES0908] and by the ‘‘Netherlands Genomics Initiative (NGI)’’ [project number 050-060-452]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.