Differential polarization and activation dynamics of systemic T helper cell subsets after aneurysmal subarachnoid hemorrhage (SAH) and during post-SAH complications

Abstract

Aneurysmal subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. Devastating post-SAH complications, such as cerebral vasospasm (CVS), delayed cerebral ischemia or seizures to mention a few, are mainly responsible for the poor clinical outcome. Inflammation plays an indispensable role during early brain injury (EBI) and delayed brain injury (DBI) phases over which these complications arise. T helper cells are the major cytokine secreting cells of adaptive immunity that can polarize to multiple functionally unique sub-populations. Here, we investigate different CD4+ T cell subsets during EBI and DBI phases after SAH, and their dynamics during post-SAH complications. Peripheral venous blood from 15 SAH patients during EBI and DBI phases, was analyzed by multicolour flowcytometry. Different subsets of CD3+ CD4+ T cells were characterized by differential cell surface expression of CXCR3 and CCR6 into Th1, Th2, Th17, whereas Tregs were defined by CD25^hiCD127^lo. The analysis of activation states was done by the expression of stable activation markers CD38 and HLA-DR. Interestingly, compared to healthy controls, Tregs were significantly increased during both EBI and DBI phases. Different activation states of Tregs showed differential significant increase during EBI and DBI phases compared to controls. HLA-DR- CD38+ Tregs were significantly increased during DBI phase compared to EBI phase in SAH patients developing CVS, seizures and infections. However, HLA-DR- CD38- Tregs were significantly reduced during EBI phase in patients with cerebral ischemia (CI) compared to those without CI. HLA-DR- CD38- Th2 cells were significantly increased during EBI phase compared to controls. A significant reduction in Th17/Tregs and HLA-DR- CD38+ Th17/Tregs ratios was observed during both EBI and DBI phases compared to controls. While HLA-DR- CD38- Th17/Tregs and HLA-DR- CD38- Th1/Th2 ratios were impaired only during EBI phase compared to controls. In conclusion, CD4+ T cell subsets display dynamic and unique activation patterns after SAH and during the course of the manifestation of post-SAH complications, which may be helpful for the development of precision neurovascular care. However, to claim this, confirmatory studies with larger patient cohorts, ideally from different ethnic backgrounds, are required. Moreover, our descriptive study may be the grounds for subsequent lab endeavors to explore the underlying mechanisms of our observations.

Figure 1

Comparison of peripheral blood: (A) lymphocytes (low SSC and high CD45 expressing cells) after SAH during EBI and DBI phases with healthy controls; (B) CD4+ T cells (expressed as %age of CD45 + leukocytes) after SAH during EBI and DBI phases with healthy controls; (C) CD4+ T cells (expressed as %age of lymphocytes) after SAH during EBI and DBI phases with healthy controls; (D) Tregs (CD25^hi CD127^lo, expressed as %age of CD3+ CD4+ T cells) after SAH during EBI and DBI phases with healthy controls; (E) HLA-DR− CD38− Th2 cells (expressed as %age of CD3+ CD4+ T cells) after SAH during EBI and DBI phases with healthy controls; (F) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH during EBI and DBI phases with healthy controls; (G) HLA-DR+ CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH compared to healthy controls during EBI and DBI phases, (H) HLA-DR− CD38− Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH during EBI and DBI phases compared to healthy controls. One way ANOVA followed by Tukey’s multiple comparisons test for normally distributed data. Kruskal Wallis test followed by Dunn’s multiple comparisons test for non-normally distributed data; A p value < 0.05 was considered as a significant difference. *Indicates a p value < 0.05, **indicates a p value < 0.01, ***indicates a p value < 0.001. HC healthy controls (n = 10), EBI early brain injury phase after SAH covering days 1–3 (n = 15), DBI delayed brain injury phase after SAH covering days 7–9 (n = 15).

Figure 2

Comparison of peripheral blood: (A) Tregs (CD25^hi CD127^lo, expressed as %age of CD3+ CD4+ T cells) after SAH in patients with cerebral vasospasm (CVS) and without CVS during EBI and DBI phases; (B) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with CVS and with no CVS during EBI and DBI phases; (C) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with seizures and with no seizures during EBI and DBI phases; (D) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with shunt dependent chronic hydrocephalus and with no shunt dependent chronic hydrocephalus during EBI and DBI phases; (E) HLA-DR− CD38− Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with cerebral ischemia/infarction (CI) and with no CI during EBI and DBI phases; (F) Th1 cells (CXCR3+ CCR6− , expressed as %age of CD3+ CD4+ T cells) after SAH in patients with infections and without infections during EBI and DBI phases; (G) HLA-DR− CD38− Th1 cells (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with infections and without infections during EBI and DBI phases; (H) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with infections and without infections during EBI and DBI phases; (I) HLA-DR− CD38+ Tregs (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with IVH and without IVH during EBI and DBI phases. One way ANOVA followed by Tukey’s/Bonferroni's multiple comparisons test for normally distributed data. Kruskal Wallis test followed by Dunn’s multiple comparisons test for non-normally distributed data; A p value < 0.05 was considered as a significant difference. *Indicates a p value < 0.05, **indicates a p value < 0.01, ***indicates a p value < 0.001. EBI early brain injury phase after SAH covering days 1–3 (n = 15), DBI delayed brain injury phase after SAH covering days 7–9 (n = 15).

Figure 3

Comparison of peripheral blood: (A) Th17/Tregs ratio after SAH during EBI and DBI phases with healthy controls; (B) HLA-DR− CD38+ Th17/Tregs ratio after SAH during EBI and DBI phases with healthy controls; (C) HLA-DR− CD38− Th17/Tregs ratio after SAH during EBI and DBI phases with healthy controls; (D) HLA-DR− CD38− Th1/Th2 cells ratio after SAH during EBI and DBI phases with healthy controls; (E) Th1/Th2 cells ratio after SAH in patients with infections and without infections during EBI and DBI phases; (F) HLA-DR− CD38− Th1/Th2 cells ratio (expressed as %age of CD3+ CD4+ T cells) after SAH in patients with infections and without infections during EBI and DBI phases. One way ANOVA followed by Tukey’s multiple comparisons test for normally distributed data. Kruskal Wallis test followed by Dunn’s multiple comparisons test for non-normally distributed data; A p value < 0.05 was considered as a significant difference. *Indicates a p value < 0.05, **indicates a p value < 0.01, ***indicates a p value < 0.001. HC healthy controls (n = 10), EBI early brain injury phase after SAH covering days 1–3 (n = 15), DBI delayed brain injury phase after SAH covering days 7–9 (n = 15).