Programmed death-1 pathway limits central nervous system inflammation and neurologic deficits in murine experimental stroke

Abstract

Background and purpose: Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion strongly implicates a mixture of both pathogenic and regulatory immune cell subsets that affect stroke outcome. Our goal was to evaluate the contribution of the well-described coinhibitory pathway, programmed death (PD)-1, to the development of middle cerebral artery occlusion.

Methods: Infarct volumes, functional outcomes, and effects on infiltrating immune cell populations were compared in wild-type C57BL/6 versus PD-1-deficient mice after 60 minutes middle cerebral artery occlusion and 96 hours reperfusion.

Results: The results clearly demonstrate a previously unrecognized activity of the PD-1 pathway to limit infarct volume, recruitment of inflammatory cells from the periphery, activation of macrophages and central nervous system microglia, and functional neurological deficits. These regulatory functions were associated with increased percentages of circulating PD-ligand-1 and PD-ligand-2 expressing CD19(+) B-cells in blood, the spleen, and central nervous system with the capacity to inhibit activation of inflammatory T-cells and central nervous system macrophages and microglial cells through upregulated PD-1.

Conclusions: Our novel observations are the first to implicate PD-1 signaling as a major protective pathway for limiting central nervous system inflammation in middle cerebral artery occlusion. This inhibitory circuit would likely be pivotal in reducing stroke-associated Toll-like receptor-2- and Toll like receptor-4-mediated release of neurotoxic factors by activated central nervous system microglia.

Figure 1. Increased expression of PD-L on peripheral B-cells and PD-1 on CNS microglia and macrophages after MCAO

PD-L1 (A) and PD-L2 (B) levels were strongly up-regulated on CD19⁺ B-cells from blood and spleen, and PD-1 expression was strongly up-regulated on CD11b^highCD45^low microglia (C) and Gr1^-CD11b^highCD45^high macrophages (D) from the right ipsilateral brain hemisphere after 60min MCAO treatment and 96h reperfusion in WT mice. Results represent at least two independent experiments with three mice per group.

Figure 2. Deficiency of PD-1 exacerbates ischemic infarct volume and worsens behavioral recovery after MCAO

(A) Infarct volumes in Cortex, Striatum and Hemisphere, corrected for the presence of edema, were significantly increased (**, P<0.01; ***, P<0.0001) in PD-1-KO (n=9) versus WT (n=9) mice after 60min MCAO and 96h reperfusion. Values represent Mean ± SEM. (B) Representative TTC-stained cerebral sections of the MCAO modeled to analyze infarct volume. (C) Neurological dysfunction scores (Mean ± SEM) after reperfusion were significantly worsened (*, P<0.05; **, P<0.01) in PD-1 KO (n=25) versus WT (n=26) mice.

Figure 3. PD-1 reduces the infiltration of inflammatory cells into the ischemic brain after MCAO

Absolute numbers of CD3⁺ T cells (A), Gr1⁺ neutrophils (B), CD11b⁺CD45^low microglia (C), and CD11b⁺CD45^high macrophages (D) were significantly increased (**, P<0.001; ***, P<0.0001) in ipsilateral right (R) but not contralateral left (L) brain hemispheres of PD-1-KO versus WT mice after 60min MCAO and 96h reperfusion but not Sham-treatment. Values represent Mean ± SEM from five mice per group.

Figure 4. PD-1 inhibits cytokine production by T-cells in blood, spleen and ischemic brain hemisphere after MCAO

Percentages and absolute numbers of CD3⁺ T-cells expressing TNF-α (A&C) and IFN-γ (B&D) were significantly increased (*, P<0.05, ***, P<0.0001) in blood and spleen (A&B) and ipsilateral right (R) but not contralateral left (L) brain hemispheres (C&D) after 60min MCAO (M) treatment and 96h reperfusion but not Sham (S) treatment of PD-1-KO versus wild-type (WT) mice. Values represent mean ± SEM from five mice per group.

Figure 5. PD-1 inhibits the activation of microglia and infiltrating macrophages in ischemic brain after MCAO

FACS analysis of counted MHCII⁺ microglia (A), TNF-α⁺ microglia (B), MHCII⁺ macrophages (C) and TNF-α⁺ macrophages (D) in the contralateral left (L) and ipsilateral right (R) hemispheres of Sham and MCAO treated wild-type (WT) and PD-1 knockout (KO) mice. CD11b^highCD45^low and Gr1^-CD11b^highCD45^high populations identified microglia and macrophages, respectively. Significant differences between sample means (± SEM) from five mice per group are indicated: **, P < 0.01, ***, P < 0.0001.