P2Y2R Signaling Is Involved in the Onset of Glomerulonephritis.

Endogenously released adenosine-5'-triphosphate (ATP) is a key regulator of physiological function and inflammatory responses in the kidney. Genetic or pharmacological inhibition of purinergic receptors has been linked to attenuation of inflammatory disorders and hence constitutes promising new avenues for halting and reverting inflammatory renal diseases. However, the involvement of purinergic receptors in glomerulonephritis (GN) has only been incompletely mapped. Here, we demonstrate that induction of GN in an experimental antibody-mediated GN model results in a significant increase of urinary ATP-levels and an upregulation of P2Y2R expression in resident kidney cells as well as infiltrating leukocytes pointing toward a possible role of the ATP/P2Y2R-axis in glomerular disease initiation. In agreement, decreasing extracellular ATP-levels or inhibition of P2R during induction of antibody-mediated GN leads to a reduction in all cardinal features of GN such as proteinuria, glomerulosclerosis, and renal failure. The specific involvement of P2Y2R could be further substantiated by demonstrating the protective effect of the lack of P2Y2R in antibody-mediated GN. To systematically differentiate between the function of P2Y2R on resident renal cells versus infiltrating leukocytes, we performed bone marrow-chimera experiments revealing that P2Y2R on hematopoietic cells is the main driver of the ATP/P2Y2R-mediated disease progression in antibody-mediated GN. Thus, these data unravel an important pro-inflammatory role for P2Y2R in the pathogenesis of GN.

Extracellular ATP is a danger signal activating P2X7 receptor in a LPS mediated inflammation (ARDS/ALI).

Acute respiratory distress syndrome (ARDS) is a life-threating lung condition resulting from a direct and indirect injury to the lungs [1, 2]. Pathophysiologically it is characterized by an acute alveolar damage, an increased permeability of the microvascular-barrier, leading to protein-rich pulmonary edema and subsequent impairment of arterial oxygenation and respiratory failure [1]. This study examined the role of extracellular ATP in recruiting inflammatory cells to the lung after induction of acute lung injury with lipopolysaccharide (LPS). However, the precise mechanism is poorly understood. Our objective was to investigate the functional role of the P2X7 receptor in the pathogenesis of acute respiratory distress syndrome (ARDS/ acute lung injury (ALI)) in vitro and in vivo. We show that intratracheally applied LPS causes an acute accumulation of ATP in the BALF (bronchoalveolar lavage) and lungs of mice. Prophylactic and therapeutic inhibition of P2X7R signalling by a specific antagonist and knock-out experiments was able to ameliorate the inflammatory response demonstrated by reduced ATP-levels, number of neutrophils and concentration of pro-inflammatory cytokine levels in the BALF. Experiments with chimeric mice showed that P2X7R expression on immune cells was responsible for the observed effect. Consistently, the inflammatory response is diminished only by a cell-type specific knockdown of P2X7 receptor on non-stationary immune cells. Since the results of BALF from patients with acute ARDS or pneumonia simulated the in vivo data after LPS exposure, the P2X7 receptor may be a new therapeutic target for treatment in acute respiratory distress syndrome (ARDS/ALI).

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5'-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.

P2rx4 deficiency in mice alleviates allergen-induced airway inflammation.

Compelling evidences point out a crucial role for extracellular nucleotides such as adenosine triphosphate (ATP) during inflammatory conditions. Once released into the extracellular space, ATP modulates migration, maturation and function of various inflammatory cells via activating of purinergic receptors of the P2Y- and P2X- family. P2RX4 is an ATP-guided ion channel expressed on structural cells such as alveolar epithelial and smooth muscle cells as well as inflammatory cells including macrophages, dendritic cells (DCs) and T cells. P2RX4 has been shown to interact with P2RX7 and promote NLRP3 inflammasome activation. Although P2RX7 has already been implicated in allergic asthma, the role of P2RX4 in airway inflammation has not been elucidated yet. Therefore, we used a selective pharmacological antagonist and genetic ablation to investigate the role of P2RX4 in an ovalbumin (OVA) driven model of allergen-induced airway inflammation (AAI). Both, P2RX4 antagonist 5-BDBD treatment and P2rx4 deficiency resulted in an alleviated broncho alveolar lavage fluid eosinophilia, peribronchial inflammation, Th2 cytokine production and bronchial hyperresponsiveness. Furthermore, P2rx4-deficient bone marrow derived DCs (BMDCs) showed a reduced IL-1ß production in response to ATP accompanied by a decreased P2rx7 expression and attenuated Th2 priming capacity compared to wild type (WT) BMDCs in vitro. Moreover, mice adoptively transferred with P2rx4-deficient BMDCs exhibit a diminished AAI in vivo. In conclusion our data suggests that P2RX4-signaling contributes to AAI pathogenesis by regulating DC mediated Th2 cell priming via modulating IL-1ß secretion and selective P2RX4-antagonists might be a new therapeutic option for allergic asthma.

NTPDase1/CD39 and aberrant purinergic signalling in the pathogenesis of COPD.

Purinergic receptor activation via extracellular ATP is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Nucleoside triphosphate diphosphohydrolase-1/CD39 hydrolyses extracellular ATP and modulates P2 receptor signalling.We aimed to investigate the expression and function of CD39 in the pathogenesis of cigarette smoke-induced lung inflammation in patients and preclinical mouse models. CD39 expression and soluble ATPase activity were quantified in sputum and bronchoalveolar lavage fluid (BALF) cells in nonsmokers, smokers and COPD patients or mice with cigarette smoke-induced lung inflammation. In mice, pulmonary ATP and cytokine concentrations, inflammation and emphysema were analysed in the presence or absence of CD39.Following acute cigarette smoke exposure CD39 was upregulated in BALF cells in smokers with further increases in COPD patients. Acute cigarette smoke exposure induced CD39 upregulation in murine lungs and BALF cells, and ATP degradation was accelerated in airway fluids. CD39 inhibition and deficiency led to augmented lung inflammation; treatment with ATPase during cigarette smoke exposure prevented emphysema.Pulmonary CD39 expression and activity are increased in COPD. CD39 deficiency leads to enhanced emphysema in mice, while external administration of a functional CD39 analogue partially rescues the phenotype. The compensatory upregulation of pulmonary CD39 might serve as a protective mechanism in cigarette smoke-induced lung damage.

Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

BACKGROUND: Bronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs. METHODS: Primary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants. RESULTS: RV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium. CONCLUSIONS: RV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

Ceramide-1-phosphate inhibits cigarette smoke-induced airway inflammation.

Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.

CD39 is a negative regulator of P2X7-mediated inflammatory cell death in mast cells.

BACKGROUND: Mast cells (MCs) are major contributors to an inflammatory milieu. One of the most potent drivers of inflammation is the cytokine IL-1ß, which is produced in the cytoplasm in response to danger signals like LPS. Several controlling mechanisms have been reported which limit the release of IL-1ß. Central to this regulation is the NLRP3 inflammasome, activation of which requires a second danger signal with the capacity to subvert the homeostasis of lysosomes and mitochondria. High concentrations of extracellular ATP have the capability to perturb the plasma membrane by activation of P2X7 channels and serve as such a danger signal. In this study we investigate the role of P2X7 channels and the ecto-5'-nucleotidase CD39 in ATP-triggered release of IL-1ß from LPS-treated mast cells. RESULTS: We report that in MCs CD39 sets an activation threshold for the P2X7-dependent inflammatory cell death and concomitant IL-1ß release. Knock-out of CD39 or stimulation with non-hydrolysable ATP led to a lower activation threshold for P2X7-dependent responses. We found that stimulation of LPS-primed MCs with high doses of ATP readily induced inflammatory cell death. Yet, cell death-dependent release of IL-1ß yielded only minute amounts of IL-1ß. Intriguingly, stimulation with low ATP concentrations augmented the production of IL-1ß in LPS-primed MCs in a P2X7-independent but caspase-1-dependent manner. CONCLUSION: Our study demonstrates that the fine-tuned interplay between ATP and different surface molecules recognizing or modifying ATP can control inflammatory and cell death decisions.

P2Y6 deficiency limits vascular inflammation and atherosclerosis in mice.

OBJECTIVE: Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y6 (P2Y6) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y6 in atherogenesis. APPROACH AND RESULTS: Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y6-deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y6 than respective controls. Finally, P2Y6 (-/-)/low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6 (+/+)/low-density lipoprotein receptor-deficient mice. Bone marrow transplantation identified a crucial role of P2Y6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y6-deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y6-deficient macrophages took up less modified low-density lipoprotein cholesterol. CONCLUSIONS: We show for the first time that P2Y6 deficiency limits atherosclerosis and plaque inflammation in mice.

High levels of SOX5 decrease proliferative capacity of human B cells, but permit plasmablast differentiation.

Currently very little is known about the differential expression and function of the transcription factor SOX5 during B cell maturation. We identified two new splice variants of SOX5 in human B cells, encoding the known L-SOX5B isoform and a new shorter isoform L-SOX5F. The SOX5 transcripts are highly expressed during late stages of B-cell differentiation, including atypical memory B cells, activated CD21low B cells and germinal center B cells of tonsils. In tonsillar sections SOX5 expression was predominantly polarized to centrocytes within the light zone. After in vitro stimulation, SOX5 expression was down-regulated during proliferation while high expression levels were permissible for plasmablast differentiation. Overexpression of L-SOX5F in human primary B lymphocytes resulted in reduced proliferation, less survival of CD138neg B cells, but comparable numbers of CD138+CD38hi plasmablasts compared to control cells. Thus, our findings describe for the first time a functional role of SOX5 during late B cell development reducing the proliferative capacity and thus potentially affecting the differentiation of B cells during the germinal center response.

Dependence of immunoglobulin class switch recombination in B cells on vesicular release of ATP and CD73 ectonucleotidase activity.

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Purinergic P2Y2 receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury.

BACKGROUND & AIMS: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS: Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.