Schistosomiasis is a chronic intravascular parasitic disease caused by S. mansoni. Endothelial cell injury resulting from worm migration and oviposition leads to the release of DAMPs, which modulate the host immune system through purinergic receptors [11, 24]. Within the metabotropic P2Y1R-like family of purinergic receptors, the P2Y6 receptor (P2Y6R) has been linked to mitogenic and pro-inflammatory effects in myocytes, with its expression typically upregulated in sterile inflammatory conditions and initially characterized as resistant to desensitization [25, 26]. Contrary to these observations, our study reveals a downregulation of endothelial P2Y6R expression and function during schistosomiasis.

Previous investigations unveiled the pivotal role of pyrimidinergic P2Y6R signaling in promoting inflammatory responses within the vasculature. For instance, P2Y6R activation favored leukocyte diapedesis [18] and played an important role in amplifying vascular inflammatory pathways in vivo during systemic LPS challenge [14]. P2Y6R also exacerbated intestinal inflammation in non-infectious disease [27], a condition where endothelial cells may also contribute to pathology [6].

As leukocyte adhesion to endothelial cells is a prerequisite for diapedesis, we investigated in vitro the effect of endothelial P2Y6R activation on monocyte adhesion. In control endothelial cells, 100 µM UDP treatment increased monocyte adhesion, an effect that was completely blocked by the selective P2Y6R antagonist MRS2578 (added 30 min before), thereby confirming that P2Y6Rs are functional in mouse mesenteric endothelial cells. Conversely, endothelial P2Y6R activation did not enhance monocyte adhesion in cells from the infected group.

Previous data from our group demonstrated that endothelial NTPDase2 and −3 expressions, but not NTPDase1, are increased in schistosomiasis compared to control, which promoted extracellular ADP accumulation [8]. Mouse and human NTPDase2 also hydrolyze UTP efficiently to UDP, which in turn is a poor substrate for this enzyme. Moreover, mouse NTPDase3 hydrolyzes UTP with a transient formation of UDP [13]. To evaluate the role of these enzymes, we tested the effect of endothelial NTPDases inhibition with ARL67156. A short exposure (100 µM, 30 min) did not alter UDP response, while prolonged incubation (24 h) restored the P2Y6R effect in response to the agonist in the infected group. This observation is consistent with previous data showing that ARL67156 has stronger inhibitory effects in mouse NTPDases1 and 3 than in NTPDase2 [28]. We further examined whether desensitization induced by endogenous nucleotides could account for the absence of UDP responses. Endothelial cells were treated with apyrase for 24 h to remove extracellular nucleotides, followed by washing and UDP stimulation, but no alteration in leukocyte adhesion was observed. However, molecular analysis provided additional insight, as RT-qPCR and immunocytochemistry analyses revealed reduced endothelial P2Y6R gene transcription and protein expression, respectively, in the infected group compared to the control. Nevertheless, P2Y6R remained partially expressed in endothelial cells from infected animals. Thus, the P2Y6R reduced expression observed in the schistosomiasis model could not fully explain the absence of the UDP-induced effect in the functional assay. Given that P2Y6R is a cysteine-rich GPCR, we therefore explored alternative mechanisms of receptor downregulation.

Among P2Y receptors activated by uridine nucleotide, the P2Y6R has a distinct mechanism of intracellular regulation. Brinson and Harden [29] showed that P2Y6R is more slowly lost from the cell surface in the presence of agonist and fails to rapidly recycle once the agonist is removed. This slow desensitization and internalization dynamics may be explained by the presence of only one potential phosphorylation site by GPCR kinases (GRK) in the entire P2Y6R amino acid sequence, whereas other receptors, such as P2Y4R, contain multiple phosphorylation sites [26].

In case of a GPCR containing several residues of serine and threonine within the third intracellular loop and C terminus, the mechanism of desensitization usually depends on phosphorylation by GRK and β-arrestins coupling. However, instead of serine/threonine residues, P2Y6R contains cysteine residues that possess nucleophilic properties, allowing them to react with oxidants and electrophiles [30]. The cysteine-dependent regulation of P2Y6R is particularly relevant since some selective receptor antagonists, such as MRS2578, contain isothiocyanate electrophile groups [31].

A known form of post-translational GPCR signaling regulation independent of GRK is ubiquitination, in which, for instance, ubiquitin is covalently attached to cysteine residues within the cytoplasmic domains of substrate proteins. Different types of ubiquitination are associated with various functions; however, ubiquitin typically functions as a sorting signal that facilitates the trafficking of mammalian GPCRs from endosomes to lysosomes for degradation [15,16,17].

Proteasomes are multi-subunit protein complexes that belong to the ubiquitin-proteasoma system (UPS) and are responsible for maintaining cellular  proteostasis. The GPCR downregulation induced by polyubiquitinated protein relies on receptor translocation into the interior of the proteasome complex, where active protease subunits cleave it into peptide fragments [32,33,34]. This mechanism can be blocked by proteasome inhibitors, which have been investigated as therapeutic strategies for cancer, neurodegenerative disorders, inflammatory, and infectious diseases [35, 36].

Previously, we reported an increased redox-sensitive signaling by endothelial cells during schistosomiasis leading to IL-1β release [7]. The proteasome system is activated during an increased oxidative stress mediated by reactive oxygen species (ROS), which are molecules derived from O2. The free radicals signaling peroxidizes lipids, generating adducts with proteins, causing cell damage [21, 22, 37]. Evidence also suggests that immunoproteasome induction serves as an adaptive response to facilitate the clearance of damaged proteins [23]. The immunoproteasome is a specialized form of proteasome activation induced by oxidative stress and/or cytokines such as IL-1β. It plays a central role in inflammatory responses, operating dynamically and coordinately to regulate both oxidative stress and immunity, while helping cells cope with oxidative damage, including modifications of cysteine residues [38].

In this work, we observed increased ROS, mainly superoxide anion (O2−), and lipid peroxidation in endothelial cells from the infected group. Previously, we reported that the positive cooperation between P2X7R and P2Y2R resulted in an increased redox-sensitive release of IL-1β by mesenteric endothelial cells during schistosomiasis, which was prevented by antioxidant treatment [7]. Therefore, proteasomal degradation, triggered by redox-dependent modifications of cysteine residues in P2Y6R [30], could be a key mechanism for receptor downregulation during schistosomiasis-associated inflammation and oxidative stress. Indeed, treatment with the proteasome inhibitor MG132 restored P2Y6R levels and receptor function in endothelial cells from the infected group. Even though MG132, a peptidyl aldehyde inhibitor, may also inhibit calpains at higher concentrations, the concentration used in the present work inhibits proteasome activity by approximately 90% [39]. We acknowledge that the present work did not establish a direct causal relationship between infection-induced oxidative stress and ubiquitination or proteasomal targeting of P2Y6R, and that future investigations, such as the evaluation of receptor ubiquitination under infection and antioxidant treatment, or the identification of endothelial members of UPS,  will be important to elucidate in detail how the disease and ROS impact this system.

Using transfected HEK293 cells expressing P2Y6R Nishiyama et al. [30] proposed that the mechanism of redox-dependent internalization and degradation of P2Y6R depends on the covalent modification of the conserved cysteine residue (Cys220) within the third intracellular loop [40]. In vivo, the induction of colitis in the global P2Y6R knockout mice resulted in a markedly attenuated disease progression [30], suggesting that upregulated P2Y6R signaling is relevant for intestinal inflammatory conditions. In another in vivo study, the deletion of NTPDase8 triggered the opposite effect, leading to increased intestinal inflammation driven by enhanced UDP–P2Y6R signaling [27]. Therefore, we could hypothesize that P2Y6R activation is part of the mechanism of acute inflammation, while its downregulation by a heterologous regulation limits an excessive inflammatory response, and in our model, it probably reflects an adaptive response to chronic infection. Moreover, previous studies have also linked the absence of P2Y₆R signaling to a reduction in atherogenic vascular lesions [18], and it has been suggested that schistosomiasis might confer an athero-protective effect in ApoE−/− mice aorta [41]. Therefore, the interplay between schistosomiasis-induced mesenteric endothelial cell oxidative stress and P2Y6R regulation highlights a complex alteration in endothelial cell function during chronic infection.

Another purinergic receptor, the adenosine A2B receptor, but not P2Y2R, also has Cys220 residue on the third intracellular loop and is subject of downregulation induced by electrophilic compounds. Interestingly, P2Y6R Cys220 is conserved among mammalian species including humans and mice. Moreover, the introduction of Cys220 in P2Y2R confers a redox-sensitive downregulation of this receptor subtype [30], suggesting that it is not exclusive to P2Y6R, but may be a conserved mechanism of P2Y6R regulation.

To the best of our knowledge, this study is the first work that links schistosomiasis to alterations in the host endothelial cell proteasome. Our findings unveiled that chronic inflammation reshapes P2Y6R–mediated pathways, which may underlie broader alterations in host physiology beyond the scope of the present study.