Protocol for Evaluating In Vivo the Activation of the P2RX7 Immunomodulator

Background P2RX7 is a purinergic receptor with pleiotropic activities that is activated by high levels of extracellular ATP that are found in inflamed tissues. P2RX7 has immunomodulatory and anti-tumor proprieties and is therefore a therapeutic target for various diseases. Several compounds are developed to either inhibit or enhance its activation. However, studying their effect on P2RX7’s activities is limited to in vitro and ex vivo studies that require the use of unphysiological media that could affect its activation. Up to now, the only way to assess the activity of P2RX7 modulators on the receptor in vivo was in an indirect manner. Results We successfully developed a protocol allowing the detection of P2RX7 activation in vivo in lungs of mice, by taking advantage of its unique macropore formation ability. The protocol is based on intranasal delivery of TO-PRO™-3, a non-permeant DNA intercalating dye, and fluorescence measurement by flow cytometry. We show that ATP enhances TO-PRO™-3 fluorescence mainly in lung immune cells of mice in a P2RX7-dependant manner. Conclusions The described approach has allowed the successful analysis of P2RX7 activity directly in the lungs of WT and transgenic C57BL6 mice. The provided detailed guidelines and recommendations will support the use of this protocol to study the potency of pharmacologic or biologic compounds targeting P2RX7. Supplementary Information The online version contains supplementary material available at 10.1186/s12575-022-00188-6.


Background
The purinergic P2X family of receptors is a family of 7 receptors that are all activated by extracellular ATP (eATP) with various affinities. Unlike the other 6 receptors, P2RX7 requires high levels of eATP (in the range of hundreds of micromolar) to be activated. Such levels of eATP are found in inflamed tissues such as the tumor microenvironment or fibrotic sites [1].
P2RX7 is a receptor with pleiotropic activities. Its activation leads to calcium influx and potassium efflux, a feature it shares with all members of the P2X family and that has been shown to trigger activation of various signaling pathways [2]. However, P2RX7 has its own particularities: its activation also leads to a macropore opening at the plasma membrane that allows the non-selective entry of macromolecules (up to 900 Da) to the cell. Compromising membrane integrity through macropore opening by a prolonged or sustained activation (over 1 hour) of P2RX7 could also lead to cell death [3]. Moreover, activation of P2RX7 leads subsequently to the assembly of the NLRP3 inflammasome and the release of mature IL-1β and IL-18 from the cell [4].
Due to its ability to induce cell proliferation and death but also to modulate the immune response, several Page 2 of 10 Janho dit Hreich et al. Biological Procedures Online (2023) 25:1 approaches are undertaken to either inhibit or enhance its activation, by engineering chemical molecules [5,6] or nanobodies [7] targeting the receptor. Three assays are classically and routinely used to evaluate their ability to modulate P2RX7's activation, based on the 3 hallmarks of P2RX7: • Calcium influx: the goal is to measure intracellular calcium concentrations using cell permeant fluorescent calcium indicators such as Fluo-4 AM or Fura 2. Calcium concentration could be assessed by spectrophotometry, microscopy or flow cytometry. Calcium influx could also be detected by measuring ion fluxes by electrophysiology techniques such as patch-clamp. However, these assays are in vitro or ex vivo-based and rely on using unphysiological medias that could interfere with P2RX7 activation. Indeed, the ionic composition of media is crucial. ATP-induced cell death has been shown to be delayed in low-salt medias [8]. Moreover, macropore formation is impacted by Na + , iodide, thiocyanate and chloride-enriched medias, since increasing concentration of these ions inhibited ethidium uptake by cells [9]. Channel opening is also affected by external anions such as glutamate that potentiates human P2RX7 activation whereas chloride and iodide decreased it [10]. Since P2RX7 activation hangs in the balance of ions concentrations in media, measurement of its activation in vitro and its translation in vivo can be not accurate. For example, under pathological conditions, glutamate levels increase in the extracellular space during nerve injury, that could affect P2RX7 activation [11].
Since compounds targeting P2RX7 are tested in vitro, little is known on their impact on P2RX7 activities in vivo, i.e. in complete physiological conditions. Up to now, in vivo P2RX7 activation state is only seen indirectly through eATP levels or IL-1β/IL-18 production. Therefore, we aimed at setting up a way to measure its activation in vivo and developed a protocol based on intranasal delivery of TO-PRO ™ -3 and fluorescence measurement by flow cytometry.

General Strategy to Measure P2RX7 Activity In Vivo
Since calcium influx is a common feature of P2X receptors and that NLRP3 assembly is a step downstream of P2RX7 but also not specific to P2RX7, we opted to use macropore opening, which directly represents the activation state of the receptor, as a readout for P2RX7 activity ( Fig. 1). Several dyes are available for measuring macropore opening. We opted to use the TO-PRO ™ -3 dye (Invitrogen) since it is the most sensitive non-permeant DNA intercalating dye and was shown to work best in flow cytometry [12].
When the cell is in steady state (absence of eATP, membrane integrity) TO-PRO ™ -3 does not enter the cell and the cell remains non fluorescent (Fig. 1A). In the presence of eATP, P2RX7 is activated and macropores are open, permitting the entry of TO-PRO ™ -3 into the cell. TO-PRO ™ -3 binds to DNA and renders the cell fluorescent (Fig. 1B). Macropore formation is a reversible mechanism. However, once P2RX7 is activated, the cell remains fluorescent since TO-PRO ™ -3 has already bound to DNA, even if no eATP remains or if P2RX7 is internalized [13,14] or cleaved [15] during a prolonged activation ( Fig. 1C and D), allowing the detection of P2RX7 activation at any point in time.
Channel opening and P2RX7-dependent calcium influx are detected few seconds after ATP stimulation. However, detection of macropore formation is much slower due to the incorporation of fluorescent dyes in DNA. and can take up to 1 hour to detect all activated receptors [12]. We have previously shown in a time course experiment of ATP-induced TO-PRO ™ -3 uptake that a range between 20 and 30 minutes post stimulation is the ideal time point at which the TO-PRO ™ -3 signal is well above background [5]. Moreover, since prolonged activation of P2RX7 (over 1 hour) can also lead to cell death in a macropore-dependent manner and since TO-PRO ™ -3 can also detect dead cells, it is crucial to add a Live/Dead marker. Indeed, excluding dead cells to reduce background noise and macropore-unrelated TO-PRO ™ -3 staining is important for accurate P2RX7-activation measurement. Therefore, to detect P2RX7-related TO-PRO ™ -3 fluorescence and to rule out detection of ATP-induced cell death, we chose to activate P2RX7 for 30 minutes in vivo.

ATP Activates P2RX7 in the Lung and Increases In Vivo TO-PRO ™ -3 Fluorescence
We first analyzed TO-PRO ™ -3 fluorescence in the whole lung. We observed an increase of fluorescence in WT mice receiving ATP compared to PBS ( Fig. 2A and B), indicative of macropore opening and therefore activation of P2RX7. In order to know if this increase is specific of P2RX7, the experiment was also done on p2rx7 −/− mice, where the increase of TO-PRO ™ -3 fluorescence in the ATP group was not observed and was similar to WT-PBS mice (Fig. 2B). No difference was observed in the percentage of TO-PRO ™ -3 positive cells (supp Fig. 1A-C). These results suggest that TO-PRO ™ -3 fluorescence after ATP administration was specific to P2RX7 and indicative of its activation state.
To go further, we analyzed TO-PRO ™ -3 fluorescence in immune and non-immune cells. We show that WT-ATP mice exhibit an enhanced fluorescence compared to WT-PBS mice only in immune cells (Fig. 2C), and that was only observed in WT mice, demonstrating furthermore the requirement of P2RX7 for the TO-PRO ™ -3 signal observed.
We also show that TO-PRO ™ -3 + cells express P2RX7. However, percentage of P2RX7 + cells among TO-PRO ™ -3 + cells was decreased in ATP-mice ( Fig. 2D and E), but not in TO-PRO ™ -3 + CD45 − cells (Fig. 2E). This is not surprising since it has been shown that sustained activation of P2RX7 leads to the internalization of the receptor [14] or its cleavage [15] after its activation, explaining the possibility of TO-PRO ™ -3 + P2RX7 − cells.

P2RX7 Is Mainly Expressed and Activated in Lung Immune Cells
Since we observed a decrease of P2RX7 expression in TO-PRO-3 + cells ( Fig. 2D and E), we investigated furthermore its expression in whole lungs of mice. We show that the expression of P2RX7 was decreased in all cells ( Fig. 3A) but also in immune cells (Fig. 3B), supporting furthermore the idea of an internalization of P2RX7 after its activation. Moreover, we show that P2RX7 is  3C) and in immune cells (Fig. 3D), meaning that P2RX7 + cells are indeed activated by ATP.
Since TO-PRO ™ -3 fluorescence (Fig. 2C) and P2RX7 expression (Figs. 2E and 3B) were unchanged after ATP delivery in CD45 − cells, we can speculate that P2RX7expressing immune cells are the main cells activated by ATP in the lung. However, we observed a decrease of TO-PRO ™ -3 fluorescence in CD45 − P2RX7 + cells (Fig. 3D).

Discussion
This protocol was set up to assess P2RX7 activation in vivo, by taking advantage of its property of macropore formation. Among P2X receptors, a permeability for large cations was also described for P2RX2 and P2RX4. However, P2RX7 is activated by high concentrations of eATP (0.5 to 1 mM), whereas P2RX2 and P2RX4 are activated by much lower concentrations (3 to 10 μM). Unlike P2RX7, P2RX2 and P2RX4 receptors desensitize after ATP stimulation. Even though P2RX4 is broadly expressed in most tissues of the body, it is preferentially localized in lysosomes where the acidity prevents its activation [16]. Based on these properties, we hypothesized that the macropore activity of P2RX2 and P2RX4 will not interfere with our assay. This is indeed the case since no increase in TO-PRO ™ -3 signal was observed in p2rx7 −/− mice (Fig. 2B).
Our protocol allows the measurement of P2RX7 activation in lungs and is based on TO-PRO ™ -3 uptake by cells and detection by flow cytometry. Flow cytometry was previously shown as a sensitive tool to study P2RX7 activation in vitro [12] and is also efficient for analysis of an in vivo activation of the receptor as shown in this study. Moreover, flow cytometry allows the reduction of background noise by excluding non P2RX7-dependent TO-PRO ™ -3 staining of dead cells. The other advantage of using flow cytometry is coupling TO-PRO ™ -3 with other surface markers to distinguish P2RX7 activation at the single cell level. We only studied its activation in immune versus non-immune cells, however, one can easily increase the number of markers during the surface staining step to precisely identify the subtypes of immune and non-immune cells activated by ATP.
To our knowledge, we show for the first time in a direct manner that P2RX7 is mainly activated in lung immune cells of mice, as per an increase of TO-PRO ™ -3 fluorescence. P2RX7 has a pro-inflammatory role since it releases IL-1β and is at the etiology of inflammatory diseases. Nonetheless, direct activation of the receptor i.e. calcium influx or macropore formation was never assessed in vivo. However, expression of P2RX7 on hematopoietic cells [17,18], ATP levels [19,20] and IL-1β release [21,22] were shown in vivo to be important for airway inflammation in mouse models. Even so, these experiments require the use of transgenic and chimeric mice and do not evaluate the receptor activation status. Our protocol could be used instead for accurate and easier assessment of the activation of P2RX7. We previously showed using human tumor lung adenocarcinoma (LUAD) tissues that P2RX7 is expressed in tumor associated immune cells whereas non-immune cells do not express P2RX7. Further, we showed that the overall activity of P2RX7 is impaired by expression of the C-terminal domain truncated P2RX7B receptor in immune cells [23]. Accordingly, we also show that ATP did not enhance TO-PRO ™ -3 fluorescence in non-immune cells. Lung CD45 − cells comprise endothelial cells, fibroblasts, and alveolar epithelial cells of type I (AEC I) and type II (AEC II). They all express P2RX7 except for AEC II. It has been shown in vitro and ex vivo studies that they express a functional receptor (macropore opening, calcium influx, IL-1β release) [24,25] but we do not seem to detect an activation in vivo with our protocol by measuring TO-PRO ™ -3 fluorescence in CD45 − cells. This could be due to the lower levels of expression of P2RX7 in CD45 − cells than in CD45 + cells (Fig. 3B, supp Fig. 2B) and the heterogeneity of the amplitude of its activation in these cells that is lost when looking at overall CD45 − cells. One possible way to overcome this is to analyze TO-PRO ™ -3 fluorescence in each CD45 − cell type by including specific markers. However, we could not exclude that the overall macropore activity of P2RX7 is impaired by the expression of truncated splice variants as previously described in mouse astrocytes [26]. Surprisingly, we observed that TO-PRO ™ -3 fluorescence was decreased in P2RX7 + CD45 − cells in ATP-mice (Fig. 3D). It should be noted that the intensity of TOPRO ™ -3 fluorescence in P2RX7-expressing CD45 − cells (Fig. 3D) is identical to the background value observed in p2rx7 −/− mice (Fig. 2C) accompanied by unchanged P2RX7 expression (Fig. 3B, supp Fig. 2B). Thus, these observations support the background signal of TO-PRO-3 fluorescence rather than an ATP-related effect.
We have also shown that the expression of P2RX7 was decreased in WT-ATP mice. P2RX7 activation by ATP stimulates membrane internalization [27] that results to the loss of expression of many surface proteins [28,29] including P2RX7 [13,14]. Indeed, prolonged stimulation with high levels of ATP results in decreased surface expression and internalization of the receptor in RAW cells [14], Caski cells and HEK 293 transfected with the human P2RX7 [13], at least 15 to 30 minutes after its activation depending on the cell type. Decreased expression of the receptor was also reported on human and mouse monocytes after sustained stimulation with bzATP [30]. Moreover, it has been shown that sustained activation of P2RX7 induces its cleavage at the plasma membrane by MMP-2, starting 15 minutes after its activation [15]. Altogether, these observations support the decrease of P2RX7 surface expression in lungs of mice due to high levels of ATP administration and duration of P2RX7 activation in this study.
Even though this protocol successfully detects P2RX7 activation in the lungs, it can be adapted for studying its activation in other organs by giving TO-PRO ™ -3 to the mice by intravenous (i.v.) route. YO-PRO-1 (an analog of TO-PRO ™ -3) and propidium iodide (PI) are also used to study macropore formation. They have been given i.v. in mice and rats to study in vivo cell death in lungs [31,32], brain [33,34] and liver [35]. However, TO-PRO ™ -3's time to access and penetrate the organ of interest should be carefully assessed in order to study the activation of P2RX7.
To go further, this protocol could also be helpful to determine the in vivo efficacy of compounds targeting P2RX7, to potentially adapt their administration route or their dosage or even to identify the targeted cell type of the compounds. HEI3090 is a novel positive modulator  [5], it is of interest to study its impact on P2RX7 activation in vivo, especially since HEI3090 is a promising molecule for treatment of lung cancer [5].

Conclusions
We described a protocol assessing P2RX7 activation in vivo, by taking advantage of its unique property of macropore formation. Given the immunomodulatory functions of P2RX7 and the increasing number of molecules aiming to modulate its activity, this protocol represents an important advance in the field of purinergic signaling.

Methods
The described methods include the description of the work, along with general guidelines that can be used for implementing the strategy with various compounds described to modulate P2RX7 activity.

Animals
Eight weeks old WT C57BL/6 J Olahsd mice are from Envigo, (Gannat, France), and 8 weeks old p2rx7 −/− (B6.129P2-P2rx7tm1Gab/J) backcrossed more than 10 times with WT C57BL/6 J Olahsd and are inbreeded in our animal facility. Mice were housed under standardized light-dark cycles in a temperature-controlled airconditioned environment under specific pathogen-free conditions at IRCAN, Nice, France, with free access to food and water. All experiments were approved by the committee for Research and Ethics of the local authorities (CIEPAL #772, protocol number MESRI APAFIS #33150-2021091413316813) and followed the European directive 2010/63/UE, in agreement with the ARRIVE guidelines. Experiments were performed in accord with animal protection representative at IRCAN.
Protocol description is summarized in Fig. 4. Measuring P2RX7 activity in vivo requires 5 sequential and mandatory steps as illustrated below.