Prolonged Activation of Invariant Natural Killer T Cells and TH2-Skewed Immunity in Stroke Patients

Abstract

Background: Infection is highly prevalent and contribute significantly to mortality of stroke patients. In addition to the well described robust systemic lymphocytopenia and skewed T helper 2 (T_H2)-immunity after stroke, emerging experimental evidence demonstrate that the development of infection poststroke is attributed by the activation of invariant natural killer T (iNKT) cells. In this prospective study, we examined the levels of a broad spectrum of inflammatory mediators, the activation status of iNKT cell in the blood of patients with various degree of stroke severity, and investigate whether these parameters differ in patients who later develop poststroke infections.

Methods and results: We obtained blood from stroke patients and matching controls to perform flow cytometry and multiplex measurement of inflammatory mediators. Our data suggest a pronounced activation of iNKT cells in stroke patients as compared with matched Healthy and Hospital control patients. The magnitude of iNKT activation is positively correlated with the severity of stroke, supporting the hypothesis that iNKT cells may contribute in the modulation of the host immune response after stroke-associated brain injury. In addition, stroke severity is closely correlated with decreased T_H1/T_H2 ratio, increased production of interleukin (IL)-10, with infected stroke patients showing exacerbated production of IL-10.

Conclusion: Stroke triggers a robust and sustained shift in systemic immunity in patients, including specific lymphopenia, robust activation of iNKT cells, systemic production of IL-10, and a prolonged T_H2-skewed immunity, all are potential contributors to severe immune suppression seen in patients after stroke. Future studies with large sample size will provide potential causality relationship insights.

Keywords: IL-10; iNKT cells; immune suppression; infection; stroke.

Figure 1

The number of circulating invariant natural killer T (iNKT) cells unchanged after stroke. The number of circulating white blood cells [WBC; (A)], lymphocytes (B), CD3⁺ T cells (C), CD4⁺ T cells (D), CD8⁺ T cells (E), and iNKT cells (F) were quantified in Healthy (n = 10) and Hospital (n = 9) controls as well as stroke patients at Admission (n = 36) and 1 day (n = 36), 2 days (n = 36), 7 days (or at discharge, DC; n = 14), and 90 days (n = 27) after stroke onset. Error bars, SEM. *p < 0.05, **p < 0.01 vs Healthy controls unpaired two-tailed Student’s t-test. N.S. denotes no statistical significance.

Figure 2

Rapid invariant natural killer T (iNKT) cell activation after stroke. (A) The expression of CD69 in iNKT cells was quantified in Healthy (n = 10) and Hospital (n = 9) controls as well as stroke patients at Admission (n = 36) and 1 day (n = 36), 2 days (n = 36), 7 days (or at discharge, DC; n = 14), and 90 days (n = 27) after stroke onset. Error bars, SEM. *p < 0.05 vs Healthy controls, unpaired two-tailed Student’s t-test. The activation of iNKT cells of stroke patients with various National Institute of Health Stroke Scale (NIHSS) at Admission was also measured (B). Error bars, SEM. **p < 0.01 one-way ANOVA with Bonferroni multiple comparisons post hoc test.

Figure 3

Stroke-induced systemic shift toward T_H2. The plasma cytokine T_H1/T_H2 ratio of stroke patients at Admission (n = 36) and 1 day (n = 36), 2 days (n = 36), 7 days (or at discharge, DC; n = 14), and 90 days (n = 27) after stroke onset was presented in the box and whisker plot (A). The plasma cytokine T_H1/T_H2 ratio of stroke patients with various National Institute of Health Stroke Scale at admission was also calculated (B). Dotted line denotes Hospital controls. Error bars, SEM. **p < 0.01, one-way ANOVA with Bonferroni multiple comparisons post hoc test.

Figure 4

Stroke-induced production of interleukin (IL)-10. The plasma levels of IL-10 in Healthy (n = 10) and Hospital (n = 9) controls as well as stroke patients at Admission (n = 36) and 1 day (n = 36), 2 days (n = 36), 7 days (or at discharge, DC; n = 14), and 90 days (n = 27) after stroke onset were measured (A). Error bars, SEM. **p < 0.01, *p < 0.05 vs Hospital controls, unpaired two-tailed Student’s t-test. Correlation coefficient was calculated between plasma IL-10 levels and invariant natural killer T (iNKT) cell activation (B) and National Institute of Health Stroke Scale (NIHSS) (C) at Admission. Pearson’s correlation coefficient (r) with 95% confidence interval expressed in brackets. Plasma level of IL-10 at Admission was measured in stroke patients with different NIHSS (D). Error bars, SEM. *p < 0.05, one-way ANOVA with Kruskal–Wallis test. Plasma level of IL-10 at Admission was measured in stroke patients who later developed poststroke infection (E). Error bars, SEM. *p < 0.05 vs no infection, unpaired two-tailed Mann–Whitney U test.