Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis

Abstract

Background: Ebolavirus species Zaire (ZEBOV) causes highly lethal hemorrhagic fever, resulting in the death of 90% of patients within days. Most information on immune responses to ZEBOV comes from in vitro studies and animal models. The paucity of data on human immune responses to this virus is mainly due to the fact that most outbreaks occur in remote areas. Published studies in this setting, based on small numbers of samples and limited panels of immunological markers, have given somewhat different results.

Methodology/principal findings: Here, we studied a unique collection of 56 blood samples from 42 nonsurvivors and 14 survivors, obtained during the five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo. Using Luminex technology, we assayed 50 cytokines in all 56 samples and performed phenotypic analyses by flow cytometry. We found that fatal outcome was associated with hypersecretion of numerous proinflammatory cytokines (IL-1β, IL-1RA, IL-6, IL-8, IL-15 and IL-16), chemokines and growth factors (MIP-1α, MIP-1β, MCP-1, M-CSF, MIF, IP-10, GRO-α and eotaxin). Interestingly, no increase of IFNα2 was detected in patients. Furthermore, nonsurvivors were also characterized by very low levels of circulating cytokines produced by T lymphocytes (IL-2, IL-3, IL-4, IL-5, IL-9, IL-13) and by a significant drop of CD3+CD4+ and CD3+CD8+ peripheral cells as well as a high increase in CD95 expression on T lymphocytes.

Conclusions/significance: This work, the largest study to be conducted to date in humans, showed that fatal outcome is associated with aberrant innate immune responses and with global suppression of adaptive immunity. The innate immune reaction was characterized by a "cytokine storm," with hypersecretion of numerous proinflammatory cytokines, chemokines and growth factors, and by the noteworthy absence of antiviral IFNα2. Immunosuppression was characterized by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of peripheral CD4 and CD8 lymphocytes, probably through Fas/FasL-mediated apoptosis.

Figure 1. Circulating proinflammatory cytokines upregulated in fatal (red plots, D) and non fatal (green plots, S) cases of clinical ZEBOV infection.

Fatal and non fatal cases were each subdivided into two groups according to the interval between symptom onset and blood sampling, as follows: S1 and D1 sampled 1–4 days after symptom onset, S2 and D2 sampled ≥5 days after symptom onset. Given that disease course in all fatal cases lasted 6–7 days, D2 group contains patients sampled in the last 2–3 days before death. Cytokine levels were compared with those found in 30 randomly selected healthy volunteers (blue plots). Results are shown as mean values in each group, and the bars on the plots indicate the standard errors. Asterisks (*) indicate statistically significant differences between patients and healthy controls (p<0.05). Symbols † indicate statistically significant differences between survivors and fatal cases (p<0.05).

Figure 2. Upregulated circulating chemokines in fatal (red plots, D) and non fatal (green plots, S) cases of clinical ZEBOV infection.

Figure 3. Downregulated cytokines mainly secreted by T lymphocytes, in fatal (red plots, D) and non fatal (green plots, S) cases of clinical ZEBOV infection.

Figure 4. Flow cytometry of PBMC (CD3, CD4 and CD8 phenotyping) collected from fatal and non fatal cases of clinical ZEBOV infection, and from convalescent sampled two weeks after recovery.

Results are presented as individual pictures (one individual per group). Mean percentages of gated cells (side and forward scatter) in each group are shown on each picture.

Figure 5. Flow cytometry of PBMC (CD3, CD4, CD8 and CD95 phenotyping) collected from fatal and non fatal cases of clinical ZEBOV infection, and from convalescent sampled two weeks after recovery.

Results are presented as individual pictures (one individual per group). Mean percentages of gated cells (side and forward scatter) in each group are shown on each picture.