High-dimensional CyTOF analysis of dengue virus–infected human DCs reveals distinct viral signatures
Rebecca E. Hamlin, Adeeb Rahman, Theodore R. Pak, Kevin Maringer, Ignacio Mena, Dabeiba Bernal-Rubio, Uma Potla, Ana M. Maestre, Anthony C. Fredericks, El-ad D. Amir, Andrew Kasarskis, Irene Ramos, Miriam Merad, Ana Fernandez-Sesma
Rebecca E. Hamlin, Adeeb Rahman, Theodore R. Pak, Kevin Maringer, Ignacio Mena, Dabeiba Bernal-Rubio, Uma Potla, Ana M. Maestre, Anthony C. Fredericks, El-ad D. Amir, Andrew Kasarskis, Irene Ramos, Miriam Merad, Ana Fernandez-Sesma
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Research Article Immunology Virology

High-dimensional CyTOF analysis of dengue virus–infected human DCs reveals distinct viral signatures

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.

Authors

Rebecca E. Hamlin, Adeeb Rahman, Theodore R. Pak, Kevin Maringer, Ignacio Mena, Dabeiba Bernal-Rubio, Uma Potla, Ana M. Maestre, Anthony C. Fredericks, El-ad D. Amir, Andrew Kasarskis, Irene Ramos, Miriam Merad, Ana Fernandez-Sesma

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Figure 1

DENV-2 and DENV-4 demonstrate replication differences in human DCs but not in mosquito cells.

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DENV-2 and DENV-4 demonstrate replication differences in human DCs but n...
(A) Mass cytometry by time-of-flight (CyTOF) analysis of DCs infected at an MOI of 0.5 with dengue virus 2 (DENV-2) and DENV-4. Viral infection was visualized by antibody staining for the DENV envelope (DENV E) and nonstructural 3 (NS3) proteins. DENV-infected cells were gated on live single CD45+ cells. One representative donor of seven is shown. (B) The percentage of DENV-infected DCs was quantified as shown in A by CyTOF analysis for all 7 donors. (C) Mean signal intensity of DENV NS3 protein staining for cells positive for DENV infection for all 7 donors. (D) Replication kinetics of DENV-2 and DENV-4 in DCs, as determined by plaque assay of extracellular infectious particles. Fifteen donors are represented. (E) Replication kinetics of DENV-2 and DENV-4 (MOI of 0.5) in Aag2 cells, as determined by plaque assay of extracellular infectious particles. The graph depicts 3 biological replicates. Mean ± SEM is shown. Black asterisks represent statistical significance by the paired, 2-tailed Wilcoxon signed-rank test, comparing DENV-4 to DENV-2 at each time point. Red asterisks represent statistical significance for either DENV-2 or DENV-4 compared with mock-infected cells at each time point. The Benjamini-Hochberg procedure was performed within each time point to adjust the significance level for multiple comparisons (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).