The results presented above demonstrate two important and novel findings which have potentially high impact in our understanding of immunological mechanisms of ischemic brain injury. First, a previously unrecognized activity of the PD-1/PD-L co-inhibitory pathway contributes to limit infarct volume and functional neurological deficits, as well as to inhibit activation and recruitment of inflammatory T-cells, granulocytes, macrophages and microglia into the growing infarct. These regulatory activities were significantly decreased in MCAO-treated PD-1 deficient mice, thus implicating unequivocally the protective activity of this regulatory pathway. Second, PD-L1&2 expression was increased on peripheral and CNS B-cells and PD-1 expression was up-regulated on CNS microglia and infiltrating macrophages within the lesioned brain hemisphere 96h after MCAO. Since peripheral B-cells, T-cells and macrophages migrate across the blood-brain barrier to contribute to the ischemic injury, our novel results suggest a previously undescribed regulatory circuit in which PD-L1/2+
, IL-10-secreting B cells (Kozoburo, et al., manuscript submitted) may directly inhibit T-cells and regulate activation and release of neurotoxic factors by PD-1+ MG and macrophages. These putative interactions are illustrated in Supplementary Figure 3
(please see http://stroke.ahajournals.org
). These findings implicate the PD-1/PD-L immunoregulatory pathway as a novel target for protection from CNS damage in experimental stroke.
It is of potential importance that PD-1 has been reported to be constitutively expressed on neurons20
, and it is conceivable that interaction of PD-L+
APC with PD-1 expressed by neurons could be directly neurotoxic. This event would contribute to the MCAO lesion only in WT but not PD-1-KO mice, thus producing larger lesions in WT mice (the opposite of what was observed in our study). In the absence of PD-1, there would be less neuronal death due to PD-L/PD-1 interactions but more neuronal damage from neurotoxic factors released by unregulated microglial cells and macrophages.
Although our results unequivocally implicate PD-1 as a contributor to CNS lesion development during 96h reperfusion after MCAO, a prior report did not observe a change in lesion volumes in PD-1 deficient mice 24h after MCAO21
. This result suggested a lack of involvement of PD-1 as a co-stimulator for adaptive T-cell responses in early stroke, and the 24h observation time in PD-1 deficient mice was apparently too early after occlusion to allow for the production of larger infarct volumes as was observed in our study. Congruent with this interpretation, our study implicates the PD-1 effect at a later time point, involving reduced recruitment and cytokine release by activated T-cells, macrophages and brain microglial cells.
As mentioned above, the cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-based switch motif (ITSM) sequence, and it was later demonstrated that the tyrosine within the ITSM motif is essential for binding the protein tyrosine phosphatases SHP-1 and SHP-222.23
that mediate inhibitory PD-1 function. In T-cells, inhibition of activation requires co-ligation of the TCR23
. In macrophages (and presumably MG), PD-1 can be up-regulated and cell activation inhibited by an interferon-sensitive response element (ISRE)24
, Toll-like receptor (TLR)-2, TLR3 & TLR4 and other agents25
. This is of particular importance in stroke, where MG activation and release of CNS neurotoxic factors is known to occur through ligation of TLR2 and TLR426-28
, possibly by heat-shock protein 60 released from CNS cells undergoing necrotic or apoptotic cell death29
. Thus, ligation of PD-1 expressed on macrophages and MG by PD-L expressed by regulatory B-cells could result in the inhibition of a key neurodestructive process in stroke.
In conclusion, our study provides new insights into the contribution of the PD-1/PD-L co-inhibitory pathway that limits stroke-induced endogenous inflammatory responses.