We previously described that CD8 + CD161 + cells are elevated in the acute phase of aSAH in patients that developed DCI.[7] In the present study, we observed that monocytes, NK cells and T cells expressing CD8 + CD161 + in aSAH samples. Among these cells types, we identified a subpopulation of monocytes that expressed high levels of CXCL10. Microglial marker analysis suggested a peripheral origin for this subpopulation. Furthermore, we observed that CXCL10 was significantly elevated in aSAH compared to naSAH at the mRNA level.
Aneurysmal rupture leads to the release of various signals that increase the permeability of the blood brain barrier (BBB) by inducing endothelial injury.[23] A damaged BBB favors migration of activated neutrophils, T cells and monocytes into the CNS.[24] Hemoglobin deposition in the subarachnoid space promotes inflammation, increased BBB dysfunction and activation of peripheral inflammatory cells and microglia.[25] scRNAseq of CD8 + CD161 + cells in the CSF of patients with aSAH led to the profiling of three different cells populations: monocytes, NK and T cells. Monocytes are especially relevant to the immune response in aSAH as multiple studies have shown increased monocytes counts in the CSF of patients with aSAH.[2] Roa et al identified that blockage of monocyte infiltration in the CNS has prevented vasospasm in animal models.[7] The clinical significance of CD8 + CD161 + monocytes in aSAH CSF samples is unclear, but it is highly suggestive of monocyte activation.
Monocytes can be activated through direct cellular interaction or by circulating cytokines.[26] An altered BBB may allow peripheral monocyte infiltration. CXCL10 production by infiltrating monocytes can recruit more monocytes and amplify the immune response. We observed that mRNA levels of CXCL10 were higher in aSAH compared to naSAH CSF. CXCL10 is strong chemoattract for T cells, NK cells and monocytes leading to recruitment and migration.[27, 28] It has been postulated that increased leukocyte counts lead to late complications of aSAH.[29] [30] In our study, CXCL10 was mainly expressed in the CD8 + CD161 + samples. The main cell type expressing CXCL10 were monocytes. Although CXCL10 can be expressed by various cell types,[31] previous studies have demonstrated secretion of CXCL10 mainly by human monocytes.[27, 28] In our samples, monocytes expressed markers that suggested a peripheral origin (Fig. 3). CD44 was widely expressed, this marker is known to be expressed in the peripheral leukocytes but not by the microglia.[22] Moreover, intermediate monocytes (CD14 + CD16+, Fig. 2), the main subtype present in our CD8 + CD161 + samples, actively secrete cytokines that favor inflammation.[32] Increased levels of intermediate monocytes have also been associated with a worse neuropsychiatric outcomes during inflammatory states.[33] This finding supports the potential role of monocytes in generating signals for leukocyte recruitment in aSAH.
CXCL10 may also facilitate cellular injury through direct neurotoxicity. CXCL10 signaling through CXCR3 can induce apoptosis in fetal neurons through intracellular calcium dysregulation.[34] Moreover, CXCL10 blockage results in improved neurologic function and halting of disease progression in mice with induced multiple sclerosis.[30] However, further research is needed considering most studies are animal based and were not done in aSAH.
CXCL10 may also mediate the cellular environment in the unruptured aneurysm. Prior to rupture, CXCL10 is elevated in the unruptured aneurysmal sac.[3] Moreover, CXCL10 was identified in a group of genes that predicted the presence of unruptured aneurysms.[35] CD8 + CD161 + cells are present in the aneurysmal wall of unruptured aneurysms.[7] It is unclear if CD14 + monocytes expressing CD8 + CD161 + may play a role in aneurysm formation. Further molecular profiling of CXCL10 in aSAH could help in the identification of potential therapeutic targets.
LIMITATIONS
The present study has several limitations that should be acknowledged. The scRNAseq analysis in this study was conducted on a small sample size. Although this sample size is typical for scRNAseq studies, it may limit the generalizability of the findings. Additionaly, the CSF samples were collected at different time points during the posthemorrhage phase. The study's focus was not on examining the chronological changes in the immune response over time but rather providing a broad characterization of the role of CD8 + CD161 + cells. Future studies should investigate how cellular subpopulations mature and evolve over time. The study primarily focused on CD8 + CD161 + cells in aSAH, given previous research suggesting their activation. However, multiple cell lineages are involved in the immune response following aSAH, and the characterization of all these cell lineages was beyond the scope of this pilot study. Lastly, the analysis was limited to ten genes that were previously described in the literature as potential players in the immune response following aSAH. It is important to explore other genes that may be activated or involved in aSAH to gain a comprehensive understanding of the immunological response.