The P2Y₂ receptor as a sensor of nucleotides and cell recruitment during inflammatory processes of the liver

Article summary

Sepsis is a debilitating condition that is characterised by an excessive host immune response to bacterial infections. A common outcome in this scenario is liver injury. Recently, in a novel study, Arunachalan et al. [1]. demonstrated, using two distinct models of liver injury, lipopolysaccharide/galactosamine (LPS/GalN) and cecal ligation and puncture (CLP), how stimulation of the P2Y₂, receptor, a G protein-coupled receptor, can have a negative effect on the liver. The liver is a critical organ for maintaining metabolic homeostasis and host response functions, as it is involved in amino acid processing, protein synthesis and detoxification. Liver injury releases ATP/UTP into the extracellular environment, activating the purinergic signaling cascade, including activation of the P2Y₂ receptor, which is expressed in hepatocytes and non-parenchymal cells.

The authors first used the LPS/GalN to induce acute liver injury and found that knocking out the P2Y₂ receptor attenuated necrotic lesions, loss of hepatic parenchyma, and infiltration of inflammatory cells (e.g., leukocytes and macrophages). Furthermore, activation of JNK signaling and release of inflammatory mediators are important hallmarks of liver injury and phospho-JNK, TNF-α, IL-1β, MIP2, MCP-1, ICAM, and iNOS levels were attenuated in P2Y₂^−/− mice. These animals also had fewer apoptotic cells and improved survival.

They also performed in vitro experiments using RAW 264.7 macrophages and AML-12 hepatocytes to test the impact of pretreatment with the P2Y₂ receptor antagonist, AR-C118925, on the release of cytokines and chemokines in response to LPS, a toll-like receptor 4 (TLR4) agonist. They showed that P2Y₂ receptor blockade reduced MCP-1, IL-6, and IL-10 levels. Additionally, macrophage-conditioned media pretreated with AR-C118925 inhibited the expression of iNOS, cleaved-PARP-1, and cleaved caspase-3 in AML-12 hepatocytes. Another model of liver injury, CLP, demonstrated the same responses as the endotoxin model, which consists of attenuated hepatic lesions, infiltrated inflammatory cells and the release of markers of inflammation in P2Y₂^−/− mice. Likewise, they highlighted that in CLP, the lack of the P2Y₂ receptor protected against the dysregulation of amino acid homeostasis and bacterial translocation. These results emphasize the importance of the P2Y₂ receptor in the inflammatory response mediated by the purinergic signaling cascade, which is necessary for the activation of innate immune cells, including macrophages, avoiding the early induction of “cytokine storm” and its deleterious consequences in liver injury, systemic arginine depletion, bacteremia, morbidity, and mortality.

Commentary

The liver is a vital organ that can be exposed to antigens and microbial products [2]. Immune and non-immune cells survey the liver to detect these pathogens and antigens, as well as danger signals crucial for inducing inflammatory responses [3]. In addition, the liver contributes to the host’s defense against pathogens; however, its dysfunction is related to the severity of sepsis [4]. Sepsis is an inflammatory condition in which pro-inflammatory mediators, such as cytokines and chemokines, are released to induce the recruitment of effector cells (e.g. macrophages, monocytes, and neutrophils) to dampen and resolve the inflammatory process. Nonetheless, this response is accompanied by tissue injury and the liver is one of the most affected organs in this context [5, 6].

Hepatocytes per se release nanomolar levels of ATP into the extracellular space to preserve its function and homeostasis. Under pathophysiological conditions, injured hepatocytes release more ATP, an important danger signal in the pathophysiology of liver diseases. Its increase in the extracellular milieu acts as a first signal, thus activating components of purinergic signaling, such as P2 receptors [7, 8]. It has been reported that the expression of P2Y₂ receptors in the bone marrow compartment is essential for neutrophil infiltration in the liver and subsequent liver injury [9]. Neutrophil chemotaxis induces autocrine feedback dependent on the P2Y₂ receptor, Pannexin-1 (PANX1) channel, CD39 (NTPDase1) and the A_2A receptor. Furthermore, this mechanism is part of a sensory system that includes mTORC signaling and mitochondrial activation [10, 11]. Muller et al. found that the ATP-P2Y₂ receptor axis induced dendritic cell and eosinophil migration to the lungs in a model of allergic inflammation [12]. However, in the liver, Arunachalan et al. demonstrated a possible role of the P2Y₂ receptor in the chemotaxis of macrophages, as the main immune cells recruited after LPS/GalN injury [1]. They also reported that liver-resident macrophages release MCP1 and MIP2, which are implicated in leukocyte infiltration into the hepatic parenchyma. RAW264.7 macrophages treated with LPS upregulated P2Y₂ receptor mRNA and increased ATP release, indicating the involvement of these cells and this receptor in inflammatory processes [13]. These outstanding data might corroborate the role of P2Y₂ receptor acting as a sensor of nucleotides released by apoptotic cells during inflammatory conditions [14].

The P2Y₂ receptor also influences glycogen metabolism, bile secretion, and hepatocyte proliferation [7, 15]. Interestingly, purinergic signaling has been described in sepsis as an important way to manage this condition [16]. Previously, the role of P2Y₂ receptors in sepsis was only reported following lung tissue damage, where neutrophil recruitment depended on this receptor [17]. Regarding liver injury, Arunachalan et al. demonstrated a novel function for P2Y₂ receptors in other main organs affected by sepsis. Furthermore, liver injury induced by sepsis can also be limited by the inhibition of the P2 × 7 receptor and activation of the A_2A receptor [18]. In addition, P2 × 7 receptor genetic deletion attenuated the infiltration of inflammatory cells in the liver during sepsis [19], as well as in the absence of P2Y₂ receptors. This may explain the complex mechanism of purinergic signaling in the context of liver diseases.

In a previous study, RAW264.7 macrophages stimulated with EtOH (Ethanol) before treatment with ATP, UTP or suramin, had increased release of the inflammatory cytokines IL-6, IL-1β, and TNF-α when compared to macrophages only exposed to EtOH alone, indicating that extracellular ATP and P2Y₂ receptor activation are important in alcohol-induced inflammation [20]. Arunachalan et al., in a different inflammatory model, also demonstrated increased cytokines (IL-6, IL-10, TNF-α, and GM-CSF) and chemokine (MCP-1) in RAW 264.7 macrophages stimulated by LPS. In contrast, treatment with a P2Y₂ antagonist attenuated MCP-1, IL-6, and IL-10 release, which suggests that P2Y₂ receptor is necessary for LPS-mediated induction of cytokine release [1]. It is known that LPS reprograms the metabolism and pro-inflammatory polarization of macrophages through TLR4 signaling [21]. Thus, measurement of the concentration of ATP in macrophages exposed to LPS and the relative expression of TLR4 would help to better understand the inflammatory response mediated via the P2Y₂ receptor.

AML-12 hepatocytes have been used to better understand the mechanisms of inflammation in liver damage. AML-12 cells exposed to EtOH had increased in IL-1β and TNF-α levels. In addition, P2Y₂ receptor mRNA levels and protein expression were increased, which was not found in cells pre-treated with suramin, a P2Y₂ receptor antagonist [22]. Also, to explore whether P2Y₂ receptors affect the apoptosis mechanism of AML-12 cells, P2Y₂ receptor siRNA produced a decrease in the expression of cleaved-caspase 3. Likewise, AML-12 cells pretreated with the P2Y₂ antagonist, AR-C118925, and activated by conditioned media, had showed less iNOS production and the cleavage of caspase 3 and PARP-1, which are involved in apoptosis in hepatocytes [1]. Both studies demonstrate an important role of the P2Y₂ receptor in the cell death pathway through apoptosis in AML-12 cells and help to improve comprehension of the mechanisms involved in inflammation and the response of hepatocytes to liver injury. In vivo studies have also demonstrated that P2Y₂ receptor inhibition protects against liver injury due to EtOH, reducing oxidative stress and lipid accumulation [23, 24].

This study led by Arunachalan et al. reported that the P2Y₂ receptor is an important sensor of nucleotide release and cell recruitment. They demonstrated that the P2Y₂ receptor has a deleterious effect on inflammatory processes, such as sepsis and endotoxemia. In addition, the absence of this receptor resulted in less liver injury, due to attenuated cell recruitment, mortality, bacterial load, and inflammatory markers. These findings highlight the function of the P2Y₂ receptor as a mediator of hepatic inflammation. However, further studies are necessary to clarify the mechanisms by which the P2Y₂ receptor releases ATP and recruits inflammatory cells to local sites of injury. In addition, therapeutic intervention antagonizing this receptor positively improves outcomes in sepsis and liver injury, with a beneficial response to these conditions.

Author contributions

VSR, FCR and RCS conceived the idea. VSR and FCR wrote the main manuscript text. All authors reviewed the manuscript.

Funding

This study was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil – CNPq (306839/2019-9). Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (E-26/010.002422/2019; E-26/201.086/2022).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors have no conflicts of interest to declare that are relevant to the content of this article.