Here, we show that eATP (extracellular ATP) is present in substantial amounts early after cerebral ischemia and that the cells responding to the eATP challenge are microglia rather than infiltrating macrophages. Blocking P2X7 on microglia diminishes the tissue damage caused by the ischemia. Intracerebroventricular injection is necessary to bypass the BBB and render microglia accessible for P2X7-specific nanobodies.
Mounting evidence indicates that stroke triggers a strong inflammatory response. The injured tissue releases a myriad of molecules that can activate the surrounding or infiltrating immune cells. Potent activators of local immune responses are danger-associated molecular patterns (DAMPs). These include for example eATP, NAD, HSP, and HMGB1. Some of these endogenous danger signals can induce activation of the inflammasome and the secretion of proinflammatory cytokines by innate immune cells [4, 21]. Using transgenic mice that express luciferase on the outer layer of the cell membrane, we could show that similar to traumatic brain injury [22], eATP is released very early during ischemic tissue damage. In addition, the signal is sustained over 24 hours clearly indicating an ongoing release of eATP in the ischemic tissue. Therefore, it is likely that eATP and its cognate receptors play an important role for the initiation of the inflammatory reaction following stroke. eATP activates purinergic receptors. While the microglial P2Y12 receptor is important for the microglia neuron interaction, the pro-inflammatory response by microglial is likely triggered by P2X7, which is highly expressed by microglia (Fig. 2A)[23–25]. Three polypeptide subunits, each with two transmembrane domains, form an ion-permeable channel upon eATP activation. Opening of the channel induces Na+ and Ca2+ influx and K+ efflux, resulting in plasma membrane depolarization and initiation of Ca2+ signaling cascades. The K+ efflux through the P2X7 receptor is upstream of the NLRP3-mediated inflammasome complex, which cleaves pro-caspase 1 and leads to a subsequent cleavage of pro-IL-1β and pro-IL-18 into their biologically active forms [5]. The amount of accessible intracellular pro-IL-1β and pro-IL-18, however, depends on a first signal transmitted by receptors such as the toll like receptors (TLR), particularly TLR4, or TNF-receptors and subsequent NFκB.
After ischemic stroke, expression of P2X7 is increased on microglia [26] and can induce cell death in ischemic microglia [27]. We and others have shown that experimental stroke in P2X7-/- mice results in smaller infarcts and that blockade of P2X7 with brilliant blue G (BBG) reduces cerebral ischemic damage [8, 28, 29]. In addition, the inhibition of pannexin 1 decreases the amount of damage after cerebral ischemia, but there is no additional benefit if P2X7 is also blocked [30].These data are still controversial [31]. Yanagisawa and colleagues saw an exacerbation of ischemic brain damage when P2X7 was blocked. Similar findings were also reported by Kang et al. [32], who observed an effect on CNTF production but no effect on lesion size.
One explanation for these discrepancies is the use of BBG. Small molecule inhibitors are often only semi-specific and toxic. Particularly, BBG is not specific for P2X7 and is known to have dose dependent off target effects. On the other hand, nanobodies, recombinant single domain antibodies (sdAbs) derived from camelid heavy chain antibodies, are a promising new technology platform. The first nanobody-based reagents developed by Ablynx-Sanofi have entered clinical trials and achieved FDA approval (targeting TNF-α, von Willebrand Factor, RANKligand, and IL-6 receptor [33]. Nanobodies, named for their small size (3 nm, 12 kDa), offer several key advantages compared to small molecule inhibitor. These include low toxicity, no off target effects and in the case of P2X7R a more potent inhibition [13]. Even compared to conventional antibodies, nanobodies tend to be advantageous. The advantages of nbs over conventional abs include a higher propensity for binding to functional epitopes on proteins, high stability, better solubility, lower immunogenicity, rapid and targetable in vivo biodistribution. In addition, the possibility of assembling nanobody multimers, and the low costs and easiness of production makes them ideal candidates for treatment [34]. Fusion of a nanobody (monomer or multimer) to the Fc domain of a conventional antibody yields a heavy chain antibody with reconstituted Fc-mediated effector functions, including binding to Fc receptors, extended half-life, and complement activation. This allows a much broader tailoring of nanobodies than of conventional antibodies to the particularities of different pathophysiologies [35].
The BBB is a major obstacle for the treatment of brain disease with biologicals. Under healthy conditions, it is only permeable for lipophilic molecules of up to 400 kDa of size [36]. In addition, the delivery of conventional antibodies to the brain is further hampered by the Fc-receptor mediated efflux to the blood [37]. Therefore, nanobodies lacking an Fc-part represent a promising alternative to reach targets behind the BBB. Yet, under non-pathological conditions monovalent nbs do not reach sufficient concentrations for in vivo brain imaging [38] or therapeutic purposes [39]. In stroke, a biphasic BBB breakdown is caused by activated matrix metalloproteinase -2 (MMP-2), MMP-3 and MMP-9 [40, 41]. The breakdown of the BBB is initially reversible but becomes permanent with the mounting release of MMP-3 and MMP-9 [42]. These findings suggest that antibodies or nanobodies would have an easier access to the brain. However, as we can show here, only a minor portion of the intravenously injected nanobodies reached the brain. While macrophages from the blood stream were quickly covered with nanobodies, when they reached the brain, microglia did not carry any nanobodies and their function was unimpaired (Fig. 4). These observations are similar to observations in antibody crossing of the BBB where usually a direct shuttle system such as the transferrin receptor is needed in order to enter the brain [43].
In stroke, microglia are the first immune cell to respond while macrophages enter the brain at later stages [15]. Therefore, it is not surprising that there was no difference in ischemic lesion size after iv nanobody injection. In contrast, after an intra-ventricular injection of P2X7 nanobody, we could reach an up to 95% of the microglia. This level of P2X7R blockage was sufficient to inhibit microglial IL-1β secretion and improved outcome. Our study shows that an inhibition of signaling by eATP is only effective if it is done early and reaches the microglia population. In humans, microglial P2X7R blockage could be accomplished by lumbar injection into the cerebral spinal fluid. This way of direct injection in the CNS is already used as a therapeutic option for other neurological diseases like neuronal ceroid lipofuscinosis [44].