Intensive cytokine induction in pandemic H1N1 influenza virus infection accompanied by robust production of IL-10 and IL-6

Abstract

Background: The innate immune system is the first line of defense against viruses by inducing expression of cytokines and chemokines. Many pandemic influenza H1N1 virus [P(H1N1)] infected severe cases occur in young adults under 18 years old who were rarely seriously affected by seasonal influenza. Results regarding host cytokine profiles of P(H1N1) are ambivalent. In the present study we investigated host cytokine profiles in P(H1N1) patients and identified cytokines related to disease severity.

Methods and principal findings: We retrieved 77, 59, 26 and 26 sera samples from P(H1N1) and non-flu influenza like illness (non-ILIs) cases with mild symptoms (mild patients), P(H1N1) vaccinees and healthy individuals, respectively. Nine and 16 sera were from hospitalized P(H1N1) and non-ILIs patients with severe symptoms (severe patients). Cytokines of IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-γ and TNF-α were assayed by cytokine bead array, IL-17 and IL-23 measured with ELISA. Mild P(H1N1) patients produced significantly elevated IL-2, IL-12, IFN-γ, IL-6, TNF-α, IL-5, IL-10, IL-17 and IL-23 versus to healthy controls. While an overwhelming IL-6 and IL-10 production were observed in severe P(H1N1) patients. Higher IL-10 secretion in P(H1N1) vaccinees confirmed our observation that highly increased level of sera IL-6 and IL-10 in P(H1N1) patients may lead to disease progression.

Conclusion and significance: A comprehensive innate immune response was activated at the early stage of P(H1N1) infection with a combine Th1/Th2/Th3 cytokines production. As disease progression, a systemic production of IL-6 and IL-10 were observed in severe P(H1N1) patients. Further analysis found a strong correlation between IL-6 and IL-10 production in the severe P(H1N1) patients. IL-6 may be served as a mediator to induce IL-10 production. Highly elevated level of sera IL-6 and IL-10 in P(H1N1) patients may lead to disease progression, but the underlying mechanism awaits further detailed investigations.

Figure 1. Sera cytokine levels of P(H1N1) patients, non-ILIs and healthy controls.

Geomean production of IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, IL-6 and IL-8 expression in the sera samples from pandemic H1N1 influenza [P(H1N1)] (white bars, n = 77), non-flu influenza like illness (non-ILIs) (Light grey bars, n = 59) and healthy controls (Dark grey bars, n = 26) were graphed as figure 1A and 1B. Bars represent geomeans ±95% Confidence Interval (CI). * Indicates significant differences (p≤0.05) between P(H1N1) patients and non-ILIs patients, or between P(H1N1) patients and healthy controls.

Figure 2. Sera cytokine levels of severe P(H1N1) and non-ILIs patients of 2011.

Geomean production of IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, IL-6 and IL-8 expression in sera samples from severe P(H1N1) patients (White bars, n = 9) and severe non-ILIs (Grey bars, n = 16) were graphed as figure 2A and 2B. Bars represent geomeans ±95% CI. * Indicates significant differences (p≤0.05) between 2 study groups.

Figure 3. Sera cytokine levels of P(H1N1) vaccinees and healthy controls.

Geomean production of IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, IL-6 and IL-8 levels in sera samples from P(H1N1) vaccinees (Grey bars, n = 26) and healthy controls (White bars, n = 26) were graphed as figure 4A and 4B. Bars represent geomeans ±95% CI.* Indicates significant differences (p≤0.05) between 2 study groups.

Figure 4. Sera cytokine levels between people got mild and severe infection.

Geomean production of IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, IL-6 and IL-8 in sera samples from severe (Grey bars, n = 9) and mild P(H1N1) patients (White bars, n = 77) were graphed as figure 4A and 4B. Geomean production of IFN-γ, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, IL-6 and IL-8 in sera samples from severe (Grey bars, n = 16) and mild non-ILIs (White bars, n = 59) were graphed as figure 4C and 4D. Bars represent geomeans ±95% CI. * Indicates significant differences (p≤0.05) between 2 study groups.

Figure 5. The associations between IL-6 level and body temperature, and between IL-6 and IL-10.

Associations of the of body temperature (graph 6A) and production of IL-10 (graph 6B)) together with sera IL-6 levels in mild P(H1N1) patients from year 2009 were analyzed using Pearson correlation. Correlations of the of body temperature (graph 6C) and production of IL-10 (graph 6D)) together with sera IL-6 levels in severe P(H1N1) patients from year 2011 were analyzed using Pearson correlation. The “r” (correlation coefficient) and p values are indicated on respective graphs obtained from the correlation analysis.

Figure 6. The schematic diagram summarizing the association observed in this study.

The schematic diagram was designed to describe the innate immune response of individuals who have been immunized with the pandemic H1N1 influenza vaccine, infected with P(H1N1) virus, or infected with other non-influenza respiratory agents. The sera cytokines and chemokine secretion status serve as indicators of the innate immune response. ↑Indicates markedly up-regulated cytokine levels; ↑↑Indicates robust up-regulated cytokines levels.