Distinctive effects of T cell subsets in neuronal injury induced by cocultured splenocytes in vitro and by in vivo stroke in mice

Abstract

Background and purpose: T cells and their subsets modulate ischemic brain injury. We studied the effects of the absence of T cell subsets on brain infarction after in vivo stroke and then used an in vitro coculture system of splenocytes and neurons to further identify the roles of T cell subsets in neuronal death.

Methods: Stroke was induced by middle cerebral artery suture occlusion in mice and infarct sizes were measured 2 days poststroke. Splenocytes were cocultured with neurons, and neuronal survival was measured 3 days later.

Results: A deficiency of both T and B cells (severe combined immunodeficiency) and the paucity of CD4 or CD8 T cells equally resulted in smaller infarct sizes as measured 2 days poststroke. Although a functional deficiency of regulatory T cells had no effect, impaired Th1 immunity reduced infarction and impaired Th2 immunity aggravated brain injury, which may be due to an inhibited and enhanced inflammatory response in mice deficient in Th1 and Th2 immunity, respectively. In the in vitro coculture system, wild-type splenocytes resulted in dose-dependent neuronal death. The neurotoxicity of splenocytes from these immunodeficient mice was consistent with their effects on stroke in vivo, except for the mice with the paucity of CD4 or CD8 T cells, which did not alter the ratio of neuronal death.

Conclusions: T cell subsets play critical roles in brain injury induced by stroke. The detrimental versus beneficial effects of Th1 cells and Th2 cells both in vivo and in vitro reveal differential therapeutic target strategies for stroke treatment.

Figures

Fig. 1

The effects of immune deficiency on infarct size and neurological scores post-stroke. Data were divided into part I and part II to focus on comparing T cell subsets and CD4 T cell subsets, respectively. Part I includes the deficiency or paucity of T and B cells, CD4 or CD8 cells. Part II includes the paucity of CD4 T cells or their functional deficiency (Th1, Th2 or Treg). The deficiency for MHCII and Tap-1 that markedly reduce the number of CD4 T cells and CD8 T cells, respectively are indicated on the figure. A. Infarct sizes. Infarct sizes were measured 2 days post stroke. The top panels are representative ischemic brains with TTC staining. The bottom bar graphs represent average infarct sizes. Although Th1 deficiency resulted in smaller infarct volumes than the paucity of CD4 T cells, no significant difference was observed. B. Neurological scores. Neurological scores were measured 2 days after stroke. Bar graphs corresponding to infarct sizes compare the effects of T cell and CD4 T cell subset deficiencies on neurological scores. * WT, wild type. *, **, *** vs WT, P<0.05, 0.01, 0.001, respectively.

Fig. 2

Th1 deficiency inhibited while Th2 deficiency promoted inflammation after stroke. A. The top picture shows a representative coronal brain section with cresyl/violet staining on which the square represents the area where pictures of immunostaining were taken and cells were counted. The bottom two panels are representative immunostaining of the macrophage activity marker, CD68, and neutrophil activity marker, MPO, at 48 hrs after stroke in WT-, Th1- and Th2-deficient mice. B. Statistical results of CD68- and MPO-positive cell numbers. The immune positive cells were counted and the numbers for WT-, Th1- and Th2-deficient mice are portrayed in the bar graphs. *, ** vs WT, P

Fig. 3

Establishment of in vitro co-culturing system of splenocytes with pure neurons. A. Splenocytes dose-dependently caused neuronal death in pure neuronal culture. Splenocytes were added to pure neuronal culture at various ratios (1:0.4 to 1:10) of neurons to splenocytes. Higher density of splenocytes resulted in more neuronal death. Neuronal survival was measured by the CCK-8 kit 3 days after co-culturing, and transformed into a ratio of neuronal death. B. Splenocytes did not cause neuron death in mixed culture of neurons and astrocytes with the ratio of 1:5 (mixed neurons and astrocytes:splenocytes). Note that survival rate was not transferred into a ratio of neuronal death in this part of the figure. C. The microscopy study suggests that splenocytes were washed away from the co-culture system before neuronal survival was measured. To confirm that splenocytes were removed before measuring neuronal survival, EGFP positive splenocytes were added to the pure neuronal culture. Pictures were taken 3 days later before or after washing. The phase contrast image shows that neurons (arrows) and lymphocytes (arrow heads) co-existed in the culture. The EGFP fluorescent image further shows the presence of EGFP-positive lymphocytes in the culture before washing. However, after washing with media, almost all EGFP splenocytes were washed away, and only MAP-2 positive neurons remained. The picture taken from the culture with vehicle treatment without lymphocytes shows higher densities of MAP-2 positive neurons than the culture treated with lymphocytes. Scale bar, 20µm.

Fig. 4

The effects of splenocytes on neuronal death in the co-culture system. Similarly to Fig.1, the data were divided into Part I and Part II. Part I. Comparison of the paucity of T cell subsets on neuronal death. Splenocytes with total lymphocyte deficiency (T and B cells in SCID mice) and the paucity of CD4 T cells or CD8 T cells in co-culture resulted in less neuronal death. Part II: The effects of the deficiency of individual functional CD4 T cell subsets on neuronal death. The deficient genes and corresponding phenotypes are also labeled. The experimental numbers on the bar graphs represent the total well numbers for each cell type, which were repeated 3 times on different days. WT, wild type. **, *** vs WT, P<0.01, 0.001, respectively.