Activation of the P2RX7/IL-18 pathway in immune cells attenuates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease associated with progressive and irreversible deterioration of respiratory functions that lacks curative therapies. Despite IPF being associated with a dysregulated immune response, current antifibrotics aim only at limiting fibroproliferation. Transcriptomic analyses show that the P2RX7/IL18/IFNG axis is downregulated in IPF patients and that P2RX7 has immunoregulatory functions. Using our positive modulator of P2RX7, we show that activation of the P2RX7/IL-18 axis in immune cells limits lung fibrosis progression in a mouse model by favoring an antifibrotic immune environment, with notably an enhanced IL-18-dependent IFN-γ production by lung T cells leading to a decreased production of IL-17 and TGFß. Overall, we show the ability of the immune system to limit lung fibrosis progression by targeting the immunomodulator P2RX7. Hence, treatment with a small activator of P2RX7 may represent a promising strategy to help patients with lung fibrosis.

The Role of IL-18 in P2RX7-Mediated Antitumor Immunity.

Cancer is the leading cause of death worldwide despite the variety of treatments that are currently used. This is due to an innate or acquired resistance to therapy that encourages the discovery of novel therapeutic strategies to overcome the resistance. This review will focus on the role of the purinergic receptor P2RX7 in the control of tumor growth, through its ability to modulate antitumor immunity by releasing IL-18. In particular, we describe how the ATP-induced receptor activities (cationic exchange, large pore opening and NLRP3 inflammasome activation) modulate immune cell functions. Furthermore, we recapitulate our current knowledge of the production of IL-18 downstream of P2RX7 activation and how IL-18 controls the fate of tumor growth. Finally, the potential of targeting the P2RX7/IL-18 pathway in combination with classical immunotherapies to fight cancer is discussed.

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.

The Purinergic Landscape of Non-Small Cell Lung Cancer.

Lung cancer is the most common cancer worldwide. Despite recent therapeutic advances, including targeted therapies and immune checkpoint inhibitors, the disease progresses in almost all advanced lung cancers and in up to 50% of early-stage cancers. The purpose of this review is to discuss whether purinergic checkpoints (CD39, CD73, P2RX7, and ADORs), which shape the immune response in the tumor microenvironment, may represent novel therapeutic targets to combat progression of non-small cell lung cancer by enhancing the antitumor immune response.

To inhibit or to boost the ATP/P2RX7 pathway to fight cancer-that is the question.

Despite new biological insights and recent therapeutic advances, many tumors remain at baseline during treatments. Therefore, there is an urgent need to find new therapeutic strategies to improve the care of patients with solid tumors. P2RX7 receptor (P2XR7), an ATP-gated ion channel characterized by its ability to form large pore within the cell membrane, is described by most of the investigators as a "chef d'orchestre" of the antitumor immune response. The purpose of this review is to detail the recent information concerning different cellular mechanisms linking P2RX7 to hallmarks of cancer and to discuss different progresses in elucidating how activation of the ATP/P2RX7/NLRP3/IL-18 pathway is a very promising approach to fight cancer progression by increasing antitumor immune responses.

A Novel Screen for Expression Regulators of the Telomeric Protein TRF2 Identified Small Molecules That Impair TRF2 Dependent Immunosuppression and Tumor Growth.

Telomeric repeat-binding factor 2 (TRF2) is a subunit of the shelterin protein complex, which binds to and protects telomeres from unwanted DNA damage response (DDR) activation. TRF2 expression plays a pivotal role in aging and cancer, being downregulated during cellular senescence and overexpressed during oncogenesis. Cancers overexpressing TRF2 often exhibit a poor prognosis. In cancer cells, TRF2 plays multiple functions, including telomere protection and non-cell autonomous roles, promoting neo-angiogenesis and immunosuppression. We present here an original screening strategy, which enables identification of small molecules that decrease or increase TRF2 expression. By screening a small library of Food and Drug Agency (FDA)-approved drugs, we identified two molecules (AR-A014418 and alexidine·2HCl) that impaired tumor growth, neo-angiogenesis and immunosuppression by downregulating TRF2 expression in a mouse xenograft model. These results support the chemotherapeutic strategy of downregulating TRF2 expression to treat aggressive human tumors and validate this cell-based assay capable of screening for potential anti-cancer and anti-aging molecules by modulating TRF2 expression levels.

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

Alternative splicing of P2RX7 pre-messenger RNA in health and diseases: Myth or reality?

Alternative splicing (AS) tremendously increases the use of genetic information by generating protein isoforms that differ in protein-protein interactions, catalytic activity and/or subcellular localization. This review is not dedicated to AS in general, but rather we focus our attention on AS of P2RX7 pre-mRNA. Whereas P2RX7 mRNA is expressed by virtually all eukaryotic mammalian cells, the expression of this channel receptor is restrained to certain cells. When expressed at the cell membrane, P2RX7 controls downstream events including release of inflammatory molecules, phagocytosis, cell proliferation and death and metabolic events. Therefore, P2RX7 is an important actor of health and diseases. In this review, we summarize the general mechanisms leading to AS. Further, we recapitulate our current knowledge concerning the functional regions in P2RX7, identified at the genetic or exonic levels, and how AS may affect the expression of these regions. Finally, the potential of P2RX7 splice variants to control the fate of cancer cells is discussed.