Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapeutic option mainly for patients with acute leukemias or malignant lymphomas but also for selected non-malignant diseases. Initial transplantation attempts remained ineffective due to the lack of knowledge regarding human leukocyte antigen (HLA) compatibility between donor and recipient and the lack of adequate immunosuppressive drugs. Today, more than 60 years later, immunologic reactions between donor and host still remain one of the major causes of morbidity and mortality after allo-HCT. Here, we discuss the impact of purines as danger-associated molecular patterns (DAMPs) in the context of acute GvHD. We decided to focus on purines and their receptors in GvHD because other danger signals in the context of allo-HCT have been discussed in a previous review (1). Nucleosides and nucleotides bind to the P1 and P2 family of purinergic receptors. Whereas adenosine activates four receptors belonging to the P1 family, UDP (Uridine-5′-diphosphate), uridine-5′-triphosphate (UTP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) activate the large family of P2 receptors with a variable affinity. The P2 receptor family is divided into two subfamilies, the ligand-gated ion channels P2X receptors and the G-protein-coupled P2Y receptors. Purinergic signaling is regulated by the expression of cell surface enzymes known as ectonucleotidases, most prominently CD39 and CD73 that convert ATP/UTP to ADP/UDP and ultimately to the respective nucleosides adenosine and uridine. Purinergic signaling modulates inflammation on multiple levels and contributes to the pathogenesis of a broad variety of diseases besides GvHD. P1 and P2 receptors show a variable distribution among different tissues that ensures a broad spectrum of effects. For instance, the P2Y2 receptor is expressed on immune cells but also on epithelial and endothelial cells and osteoblasts. Expression of the P2X7 receptor is predominant on immune cells, such as antigen-presenting cells, but is also found in the skin and pancreas (2). Expression of the P2Y12 receptor on platelets is a key feature for the use of P2Y12 receptor antagonists in the clinic.

Purinergic signaling has a well-established role in cardiovascular disease on multiple levels. For instance, nucleotides play a role in the formation of atherosclerotic plaques as a result of lipid metabolism dysregulation. In a murine model of atherosclerosis with apolipoprotein E-deficient mice, lack of the P2Y12 receptor was linked to a reduced plaque lesion area, decreased monocyte infiltration, and enhanced fibrous content of the plaque (3). In this same model, deficiency of the P2Y1 receptor significantly decreased the expression of vascular adhesion molecules P-selectin, VCAM-1, and ICAM-1 leading to diminished recruitment of leukocytes to lesion sites (4). Furthermore, endothelial cell cytoskeleton, motility, and adhesion are regulated via activation of the P2Y2 receptor following ATP or UTP binding (5). This is of particular importance due to the fact that the P2Y2 receptor is upregulated in the neointima of injured arteries in rats (6). Nucleotide binding to the P2Y2 receptor results in co-localization of the P2Y2 and VEGFR2 with subsequent upregulation of VCAM-1 that facilitates leukocyte adhesion (7). Endothelial cell migration is enhanced upon binding of ADP to the P2Y1 receptor via activation of the mitogen-activated protein kinase pathways (8). Last but not least, purinergic signaling has long been known to modulate platelet aggregation (9), mostly by ADP binding to the P2Y12 receptor (10). This fact led to the utilization of P2Y12 receptor antagonists, such as clopidogrel for inhibition of platelet aggregation for multiple cardiovascular diseases in patients (Table 1). Taken together, these data indicate that release of nucleotides with subsequent activation of purinergic receptor is a pro-inflammatory stimulus that enhanced leukocyte binding to the endothelium, platelet aggregation, and subsequent atherosclerotic plaque formation. Given the fact that multiple receptors have been implied to play a role, a more general purinergic receptor blockade might be required in order to achieve optimal protection.

In the context of airway inflammation, purinergic signaling also plays a significant role in the activation of immune cells (Table 1). For instance, increased ATP levels following allergen challenge recruit airway-specific myeloid cells and induce Th2 cell polarization and eosinophilic airway inflammation, which are major features of allergic asthma in humans. Neutralization of ATP signaling abrogated airway inflammation in response to allergens (11). Further studies emphasized the role of P2X7 receptor expression on dendritic cells (DCs) in this context (12). However, purinergic receptor expression is not limited only to the immune cell compartment. The P2Y6 receptor was found on airway epithelial cells with abundant expression upon allergen challenge and its inhibition by synthetic antagonists or the genetic deletion reduced IL-6 and IL-8 secretion by epithelial cells and improved disease outcome in a murine model (13). More recent studies suggest that purinergic signaling might be regulated by microRNAs. There is evidence that the immunomodulatory miR-155 is necessary for intact purinergic signaling. Lack of miR-155 resulted in impaired DC chemotaxis toward pro-inflammatory stimuli with subsequently reduced airway inflammation in mice (14).

Purine nucleotide-mediated signaling has been implied in autoimmune diseases, including multiple sclerosis (MS), psoriasis, and nephritis among others (Table 1).

P2X7 receptor activation by ATP binding triggers oligodendrocyte excitotoxicity and increases MS plaque formation in an experimental autoimmune encephalomyelitis model. More importantly, P2X7 receptor upregulation is observed in healthy tissue of MS patients, suggesting that inhibition of purinergic signaling might be a novel therapeutic target (15). In line with these data, a signal nucleotide polymorphism in the human p2x7r gene that leads to a gain of function amino acid exchange, occurs more frequently in MS patients than in healthy controls (16). P2X7 receptor upregulation was also observed in lesional and non-lesional skin of psoriasis patients. In lupus-associated nephritis, P2X7 receptor antagonists reduced nephritis severity, pro-inflammatory serum cytokines, and NLRP3 inflammasome activation underlying once more the broad therapeutic potential of these pathways (17).

ATP is a molecule with a high intracellular concentration that is released upon cell stress. In the absence of tissue damage, the intracellular ATP concentration ranges from 3 to 10 mM, while extracellular ATP levels are as low as 10 nM. This balance is regulated by ectonucleotidases, such as CD39 and CD73, which dephosphorylate ATP to ADP, AMP, and ultimately generate adenosine (2, 18, 19). The P2X7 receptor is a cation channel activated by high concentrations of ATP (20). P2X7 plays a central role for IL-1β secretion via activation of the NACHT, LRR, and PYD domains-containing protein 3 (Nlrp3) inflammasome (21, 22). We observed that release of ATP from damaged cells after allo-HCT amplified acute GvHD (23) via enhanced maturation of APCs and reduced Treg numbers. Besides activation via P2X7, the Nlrp3 inflammasome can be activated by uric acid (24) and Syk signaling (25). We found that uric acid enhanced GvHD in the early phase after allo-HCT (26). Inhibition of Syk reduced GvHD-related mortality in the mouse model without impairing anti-MCV or anti-leukemia responses (27). After tissue damage due to chemotherapy of irradiation, neutrophil granulocytes (neutrophils) and inflammatory monocytes reach a site of inflammation particularly early and participate in the first line of defense. Different studies have shown the chemotactic role of ATP for neutrophil chemotactic recruitment (28, 29). It was shown that purinergic signaling causes strong activation of human neutrophils (30) and activated neutrophils can release ATP through pannexin-1 hemichannels by an active process, which means that the process consumes energy (30). Neutrophils release reactive oxygen species (ROS) upon activation with bactericidal activity and the potential to cause local tissue damage (31, 32) that was shown to enhance GvHD (33) (Table 1). The mechanism as to how DAMPs, PAMPs, and neutrophils may contribute to GvHD is depicted in Figure 1. Besides neutrophils (33–35), other myeloid cell populations in particular DCs (36), macrophages (37), and certain monocyte subsets (38, 39) were found to enhance or reduce GvHD. Different purinergic receptors were found to be expressed by these myeloid cells (2) and their activation modifies the immune response elicited by the respective myeloid cell type. Myeloid-derived suppressor cells (MDSC) that lack a function Nlrp3 inflammasome are more protective against GvHD compared to WT MDSC (40), indicating that a functional Nlrp3 inflammasome modifies the inflammatory phenotype of this myeloid cell type. Besides MDSCs, DCs were shown to be influenced by different signals from purinergic receptors. To present the antigen that was taken up at the site of inflammation by a DC, costimulation is required. ATP is involved in this process as it enhances the maturation of human monocyte-derived DCs with increased levels of costimulatory molecules (41, 42). Recently, the central role of donor-derived colonic CD103⁺ DCs in Ag presentation to donor T cells that then induce GvHD was reported (43). These reports from different groups support the concept that P2X7 activation in myeloid cells enhances their inflammatory phenotype that then promotes T cell priming and inflammation that ultimately lead to GvHD.

The activation of P2Y2 was shown to promote tissue damage in airway inflammation (44, 45) and acute liver injury (46). However, P2Y2 was also shown to have protective effects in a model of lung infection induced by pneumonia virus of mice (47). P2Y2 can be activated by different nucleotides, the P2Y2 ligand ATP was found in different inflammatory diseases, including inflammatory bowel disease (48), glomerulonephritis (49), asthma (11), and diabetes (48). We recently reported that P2y2 deficiency of the recipient caused lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD (50). Selective deficiency of P2Y2 in inflammatory monocytes lead to reduced GvHD severity (50) and P2y2^−/− inflammatory monocytes had defective ERK activation and ROS production. Besides these results in the mouse model, histochemical analysis of patient samples revealed that the frequency of P2Y2⁺ cells in inflamed GvHD lesions correlated with histopathological GvHD severity.

Transplanation of the donor immune system into the allo-HCT recipient provides the graft-versus-tumor (GvT) effect that ensures long-term control of the underlying malignancy. Due to the fact that most treatments reduce the activation of the immune system, preserving the GvT effect is a major issue in post-transplant care. Interestingly, reduction of GvHD by application of the broad-spectrum P2R inhibitor PPADS did not interfere with the GvT effect, likely due to the fact that CD8⁺ T cell function is independent of purinergic signaling (23). On the other hand, A2A-AR expression on T cells allows adenosine to reduce allo-reactivity in a murine allo-HCT model, so that blockade of adenosine production allows for a more potent GvT effect at the cost of aggravated GvHD (51). These data suggest that purinergic receptor expression on T cells is crucial for the modulation of the GvT effect and offers a few therapeutic perspectives. On the one hand, in cases where a functional GvT effect is required, such as high-risk malignancies, a P2R inhibition might be a successful approach that reduces GvHD but leaves the GvT effect untouched. On the contrary, when performing allo-HCT in a patient with a benign hematopoietic disease without a necessity for a GvT activity, adenosine signaling might be enhanced as a therapy strategy for GvHD.

Purinergic signaling belongs as a DAMP to the intrinsic mechanisms for inflammation regulation without pathogen exposure. Differential receptor expression is observed on various cell and tissue types, indicating distinct roles of purines depending on the particular disease context. In general, nucleotides as ADP, ATP, UDP, and UTP serve as “alarmins” and activate neutrophil granulocytes, macrophages, DCs, and platelets. On the other hand, adenosine produced by regulatory T cells or mesenchymal stem cells counteracts the effects of the nucleotides by binding to P1 receptors. The findings from multiple groups in different models of pathogenic inflammation indicate a central function of different purinergic receptors, such as P2X7 and P2Y2, in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD. Current efforts are concentrating on the development of bioavailable and efficient compounds for the conduct of clinical trials.

The review article was designed and written by RZ together with the first author PA. Both performed literature review and critical discussion of the published literature.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.