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The PXR Modulates NLRP3 Inflammasome Activation 45. response to exogenous chemicals at these sites has been well NLRP3 inflammasome activation In this study we report. characterized Koutsounas et al 2013 given their exposure that exposing primed human or mouse macrophages to their. to high concentration of exogenous ligands and xenobiotics respective PXR agonists triggers caspase 1 activation and. In addition the PXR has been shown to regulate tissue interleukin IL 1b secretion through a NLRP3 dependent. inflammation through a reciprocal interaction with nuclear mechanism PXR induced NLRP3 inflammasome activation. factor k light chain enhancer of activated B cells NF kB was abolished by apyrase and selective inhibition of the P2X. Shah et al 2007 Mencarelli et al 2010 2011 Indeed we purinoceptor 7 P2X7 receptor Lastly PXR ligands triggered. and others have reported that PXR agonists inhibit the a rapid and significant release of ATP an effect that is. release of inflammatory mediators from hepatocytes Hu dependent on pannexin 1 and Src kinase activation. et al 2010 Sun et al 2015 and intestinal epithelial cells. Shah et al 2007 Mencarelli et al 2010 2011 by inhibiting. NF kB dependent signaling events and can afford protec Materials and Methods. tion in experimental models of hepatic Wang et al 2010 Reagents. and intestinal inflammation Ma et al 2007 Shah et al. 2007 Dou et al 2012 2013 Terc et al 2014 Garg et al PXR Agonists For experiments in mouse macrophages the. rodent selective PXR agonist pregnenolone 16a carbonitrile PCN. 2016 The PXR can also regulate the expression function of. Sigma Aldrich Canada Oakville Ontario Canada was prepared as. innate immune pattern recognition receptors within the described above For experiments in human macrophages the human. intestinal epithelium an effect that contributes to the proper selective PXR agonists rifaximin and SR12813 Sigma Aldrich. maintenance of intestinal mucosal homeostasis and barrier Canada were dissolved in sterile dimethylsulfoxide and added to. function Venkatesh et al 2014 culture media to attain the final experimental concentrations As a. Beyond the gastrointestinal tract the PXR may contribute vehicle control identical volumes of dimethylsulfoxide were added. to the regulation of inflammation in other cell types Schote and did not exceed a concentration of 1 v v in media The addition of. et al 2007 Zhou et al 2009 Dubrac et al 2010 Casey and exogenous ATP 5 mM Sigma Aldrich Canada was used as a. Blumberg 2012 Beyer et al 2013 In the context of innate positive control in all experiments assessing inflammasome activa. immunity in contrast to its reported anti inflammatory tion as we have done previously Ng et al 2010 Hirota et al 2011. Inhibitors To assess the mechanism s involved in PXR agonist. effects Wang et al 2014a found that stimulation of the. induced NLRP3 inflammasome activation we used the following. PXR in cultured vascular endothelial cells enhanced the. compounds to target reactive oxygen species ROS we employed. expression of a number of innate immune receptors in the broad spectrum antioxidant diphenyleneiodonium chloride. cluding Toll like receptors 2 4 and 9 as well as nucleo DPI 100 mM Jabaut et al 2013 Sigma Aldrich Canada to. tide binding oligomerization domain NOD like receptor chelate intracellular Ca21 and target its effect in NLRP3 inflamma. family members nucleotide binding oligomerization do some activation cells were pretreated with 1 2 Bis 2 aminophenoxy. main leucine rich repeat and CARD domain containing ethane N N N9 N9 tetraacetic acid tetrakis acetoxymethyl ester. 1 and nucleotide binding oligomerization domain leucine BAPTA AM 10 mM Ainscough et al 2015 Sigma Aldrich. rich repeat and pyrin domain containing 3 NLRP3 Fur Canada and to assess the role of extracellular ATP and P2X7. thermore endothelial cells treated with PXR agonists receptor activation we used apyrase to break down extracellular. ATP 30 U ml Iyer et al 2009 Sigma Aldrich Canada or treated. displayed features of NLRP3 inflammasome activation. cells with oxidized ATP to selectively inhibit the P2X7 recep. Wang et al 2014a, tor oxidized ATP oATP 100 mM Grahames et al 1999 Sigma. The NLRP3 inflammasome is an innate immune signaling Aldrich Canada To assess the role of pannexin 1 in PXR related. complex that mediates the responses to a variety of pathogen ATP release we used the panx 1 mimetic inhibitory peptide 10Panx. associated molecular patterns PAMPs and endogenous 400 mM Grahames et al 1999 Tocris Cedarlane Burlington. danger associated molecular patterns DAMPs Mangan Ontario Canada along with a scrambled peptide control ScPanx. et al 2018 The NLRP3 inflammasome s activation following 400 mM Grahames et al 1999 Tocris Cedarlane To assess the role. PAMP exposure and the induction of the ensuing innate of Src family kinases SFKs in the induction of ATP release we used. immune response play key roles in host defense against viral 4 Amino 5 4 chlorophenyl 7 t butyl pyrazolo 3 4 d pyrimidine PP2. bacterial and fungal pathogens Mangan et al 2018 10 mM Chang et al 2012 Sigma Aldrich Canada. whereas the recognition of DAMPs and subsequent activation. of the NLRP3 inflammasome play an important role in the Cell Culture. initiation of sterile inflammatory responses following tissue Human Studies The human acute monocytic leukemia cell line. damage Interestingly several studies have reported the THP 1 American Type Culture Collection Cedarlane Burlington. independent contributions of the PXR Wang et al 2014b Ontario Canada was propagated in RPMI 1640 medium supple. and NLRP3 inflammasome activation Imaeda et al 2009 mented with 10 fetal bovine serum and 50 mM 2 ME To differentiate. in the pathogenesis of acute liver injury caused by sterile THP 1 cells into macrophages for inflammasome activation experi. inflammation In this scenario the interplay between direct ments cells were plated at 5 105 cells well in a 24 well plate in. hepatocyte damage DAMP release and activation of resident complete RPMI media and differentiated with phorbol 12 myristate. macrophages is thought to contribute to the sterile inflamma 13 acetate PMA 50 ng ml Invivogen Cedarlane Burlington Ontario. tory response that propagates acute liver damage Hoque Canada for 24 hours as we have done previously Ng et al 2010 Hirota. et al 2011 NLRP32 2 THP 1 cells were generated and propagated as. et al 2012 Although the response of the hepatocyte has been. described previously Lau et al 2018, the focus of much research the role of PXR signaling and Mouse Studies Peritoneal macrophages were isolated from. NLRP3 inflammasome within the macrophage as a driver of Nlrp32 2 and PXR2 2 mice and their littermate wild type counter. inflammation has not been addressed parts male 8 10 weeks of age all bred in house 48 hours after. In the current study we sought to test the hypothesis that receiving an i p injection of 4 thioglycolate injected mice BD Biosci. the PXR plays a critical role in the macrophage by modulating ences San Jose CA as we have done previously Ng et al 2010. 46 Hudson et al, Fig 1 Mouse and human PXR agonists trigger IL 1b release from primed macrophages A The rodent specific PXR agonist PCN 10 and 100 mM for. 6 hours elicits significant IL 1b release from LPS primed peritoneal mouse macrophages as measured by enzyme linked immunosorbent assay. Human specific PXR agonists B SR121813 SR 0 1 1 4 10 mM 6 hours or C rifaximin rifx 1 5 10 100 mM 6 hours stimulated significant IL 1b. release from wild type PMA differentiated THP 1 cells N 3 to 4 P 0 05 for indicated comparison generated by analysis of variance and Tukey s. post hoc test, Hirota et al 2011 Isolated macrophages were plated in complete Viability Assay Promega North America Madison WI as per the. RPMI media at 5 105 cells well of a 24 well plate overnight and manufacturer s instructions and described previously Mortimer. stimulated with 100 ng ml ultra pure lipopolysaccharide LPS et al 2015. Invivogen in serum free Opti MEM for 30 minutes before chal. lenge For experiments with knockout mice littermates were used. as the wild type control group for all experiments to control for. Statistical Analysis, potential microbiota dependent differences in phenotype All stud All data were assessed for distribution using D Agostino Pearson.
ies were approved by the University of Calgary s Health Sciences normality test prior to statistical analysis using GraphPad Prism. Animal Care Committee protocol AC15 0181 All approved activ Multiple comparisons of parametric data were accomplished using an. ities conform to the guidelines and regulation for laboratory animal analysis of variance followed by Tukey s post hoc test For non. use set forth by the Canadian Council for Animal Care parametric data or experiments with small samples sizes N 5 a. Kruskal Wallis test was used followed by a Mann Whitney test with. Assessing Inflammasome Activation a Bonferroni correction for multiple comparisons In all experiments. N denotes individual experiments performed in different cell passages. Western Blots Prior to performing inflammation activation or cells derived from unique animals. experiments isolated macrophages were pulsed with ultra pure LPS. 100 ng ml for 30 minutes Invivogen Cedarlane PMA differentiated. or LPS pulsed mouse peritoneal macrophages were treated with their. respective PXR agonists Following the designated treatment period. culture supernatants were collected cells were washed with ice cold PXR Agonists Trigger Caspase 1 Activation and. phosphate buffered saline and cell lysates were isolated following IL 1b Secretion from Macrophages in a NLRP3. incubating the cells with lysis buffer 150 mM NaCl 20 mM Tris pH Dependent Manner Recent reports suggest that the PXR. 7 5 1 mM EDTA 1 mM EGTA 1 Triton X 100 and protease can regulate NLRP3 inflammasome activity in cultured. inhibitor cocktail phosphatase inhibitor cocktail Complete Minitab. vascular endothelial cells Wang et al 2014a 2017 but this. Complete PhoStop Roche Sigma Aldrich Canada Total protein was. quantified using the Precision Red Advanced Protein Assay Cytoske. has not been assessed in macrophages the prototypical model. leton Cedarlane Burlington Ontario Canada and sample protein for innate immune signaling To test the hypothesis that. concentration was equalized Culture supernatant and cell ly stimulation of the PXR triggers NLRP3 inflammasome acti. sate samples were resolved transferred to polyvinylidene difluoride vation in primed macrophages we first treated LPS primed. membranes 0 2 mm pores Bio Rad Laboratories Mississauga peritoneal macrophages or PMA differentiated THP 1 cells. Ontario Canada and blotted with the following antibodies anti with their respective species specific PXR ligands at concen. caspase 1 sc 622 sc 56036 and sc392736 Santa Cruz Biotechnol trations previously reported to elicit selective responses in. ogy Dallas TX Densitometry was performed using ImageJ and other cell types Garg et al 2016 In primed mouse macro. cleaved caspase 1 was expressed as a percentage of pro caspase 1 phages treatment with the rodent specific PXR agonist PCN. IL 1b Secretion To quantify IL 1b release from PMA. for 6 hours was able to trigger the release of IL 1b Fig 1A. differentiated or LPS pulsed mouse peritoneal macrophages treat. ed with their respective PXR agonists culture supernatants were. In human macrophages stimulation with two structurally. subject to enzyme linked immunosorbent assay human DY201 unique selective human PXR agonists SR12813 or rifaximin. mouse DY401 R D Systems Cedarlane Burlington Ontario for 6 hours also triggered significant IL 1b secretion Fig 1 B. Canada and C, To test whether IL 1b secretion in response to PXR. Assessing ATP Release activation in mouse and human macrophages was triggered. To characterize the mechanism by which PXR agonist triggered by NLRP3 inflammasome activation we next examined the. inflammasome activation in some experiments PMA differentiated ability of PXR agonists to trigger caspase 1 activation and. or LPS pulsed mouse peritoneal macrophages were treated with IL 1b secretion in cells lacking NLRP3 When treated with. their respective PXR agonists culture supernatants were collected PCN 1 10 and 100 mM for 6 hours LPS primed peritoneal. and ATP was quantified using CellTiter Glo Luminescent Cell macrophages isolated from Nlrp32 2 mice failed to trigger. The PXR Modulates NLRP3 Inflammasome Activation 47. Fig 2 Mouse and human PXR agonists evoke caspase 1 activation and IL 1b release in a NLRP3 dependent manner ATP 5 mM positive control and. PCN 1 10 and 100 mM trigger caspase 1 activation as assessed by detecting the cleaved caspase 1 p20 subunit in the supernatant A and B along. with significant IL 1b release from wild type WT peritoneal macrophages C effects that were absent in macrophages isolated from Nlrp32 2 mice. A C ATP 5 mM positive control SR121813 SR 4 mM and rifaximin Rifx 5 and 10 mM trigger caspase 1 activation as assessed by detecting. the cleaved caspase 1 p10 subunit in the supernatant D and E along with significant IL 1b release F from wild type PMA differentiated THP 1. cells effects that were absent in NLRP3 deficient THP 1 cells D F CL cell lysate SN supernatant All outcomes were measured after a 6 hour. treatment period N 3 6 P 0 05 for indicated comparison generated by analysis of variance and Tukey s post hoc test Western blots are. representative of three separate experiments, caspase 1 activation and IL 1b secretion Fig 2 A C The K1 efflux via activation of the P2X7 receptor In a human. human selective PXR agonists SR12813 4 mM rifaximin colon cancer cell line the expression of the PXR enhanced. 5 and 10 mM for 6 hours also failed to activate caspase 1 and sensitivity to oxidative stress that was associated with. the subsequent secretion of IL 1b in PMA differentiated increased agonist induced ROS production Gong et al. THP 1 cells lacking NLRP3 Fig 2 D F 2006 To assess the role of ROS in the activation of the. Because antagonists of the human and mouse PXR are NLRP3 inflammasome by the PXR in macrophages we used. lacking specificity and exhibit off target effects we used the broad spectrum antioxidant DPI treatment 100 mM. macrophage cells isolated from PXR2 2 mice Fig 3A to Jabaut et al 2013 In PMA differentiated THP 1 cells. validate the role of this receptor in the observed NLRP3 DPI pretreatment had no effect on SR12813 or rifaximin. inflammasome responses In support of our hypothesis LPS induced IL 1b secretion Supplemental Fig 1A suggesting. primed peritoneal macrophages isolated from PXR2 2 mice the ROS production does not mediate the PXR induced. did not exhibit caspase 1 activation nor secrete IL 1b in inflammasome activation within macrophages. response to the selective mouse PXR agonist PCN 10 and Activation of the vitamin D receptor VDR a nuclear. 100 mM Fig 3 B D highlighting the role of the PXR in the receptor closely related to the PXR has been linked with. activation of the inflammasome within macrophages increasing intracellular Ca21 concentrations via an inositol. PXR Mediated NLRP3 Inflammasome Activation Does 1 4 5 trisphosphate dependent process Tien et al 1993 in. Not Involve ROS or Intracellular Ca21 There are a variety epithelial cells as well as activation of the NLRP3 inflammasome. of mechanisms that contribute to NLRP3 inflammasome acti in macrophages Tulk et al 2015 Furthermore Ainscough et al. vation including but not limited to the generation of 2015 reported that intracellular Ca21 and its interaction with. ROS mobilization of intracellular Ca21 and ATP dependent calmodulin were required for nigericin induced IL 1b. 48 Hudson et al, Fig 3 Mouse PXR agonists fail to evoke caspase 1 activation or IL 1b release in peritoneal macrophages isolated from PXR2 2 mice The PXR is. expressed in LPS pulsed peritoneal macrophages A and is required for PCN induced caspase 1 activation as assessed by detecting the cleaved. caspase 1 p20 subunit in the supernatant B and C and subsequent IL 1b release D measured by enzyme linked immunosorbent assay CL cell. lysate SN supernatant All outcomes were measured after a 6 hour treatment period N 3 6 denotes P 0 05 for indicated comparison generated. by analysis of variance and Tukey s post hoc test Western blots are representative of three separate experiments. secretion in macrophages To test whether intracellular apyrase Fig 4A As extracellular ATP is known to activate. Ca 21 plays a role in NLRP3 inflammasome activation the NLRP3 inflammasome by triggering the K1 efflux. mediated by the PXR macrophages were pretreated with through the P2X7 receptor Hung et al 2013 Mortimer. BAPTA AM 10 mM a cell membrane permeable Ca21 et al 2015 we next treated macrophages with oATP. chelating agent As reported previously BAPTA AM sig 100 mM a selective P2X7 receptor antagonist Grahames. nificantly reduced ATP induced IL 1b secretion Brough et al 1999 As with apyrase cotreatment oATP signi. et al 2003 but had no effect on the activation of the ficantly attenuated IL 1b secretion in response to PXR. inflammasome by PXR agonists SR12813 and rifaximin agonists Fig 4B Importantly neither apyrase nor oATP. Supplemental Fig 1B suggesting mobilization of intra had any effect on cell viability over the course of our. cellular Ca21 is not playing a role in PXR mediated NLRP3 experiments Supplemental Fig 2 Taken together these. inflammasome activation data suggest that the PXR mediated activation of the NLRP3. Activation of the PXR Triggers ATP Release Which inflammasome involves the release of ATP and its subse. Mediates NLRP3 Inflammasome Activation Reports quent activation of the P2X7 receptor. have characterized the activation of the NLRP3 inflamma PXR Stimulation Triggers the Rapid Release of. some through the autocrine paracrine signaling of ATP re ATP from Macrophages via Pannexin 1 To further. leased into the extracellular environment via pannexin 1 strengthen the link between the PXR ATP release and. Hung et al 2013 Mortimer et al 2015 Although there is NLRP3 inflammasome activation we directly quantified. no reported role for PXR inducing the release of ATP bile ATP release from mouse peritoneal macrophages wild. acids agents known to activate a variety of nuclear receptors type versus PXR 2 2 and PMA differentiated THP 1 cells. including the PXR have been shown to induce ATP release in at different time points In macrophages isolated from. liver cells Nathanson et al 2001 Furthermore activation of wild type mice the rodent specific PXR agonist PCN. the farnesoid X receptor FXR a nuclear receptor closely triggered rapid and significant extracellular ATP release. related to the PXR by bile acids induces rapid release of ATP that could be detected within 15 seconds and was sustained. in pancreatic cell lines Kowal et al 2015 Taken together we for up to 60 seconds before tapering off after 300 seconds. next sought to test the hypothesis that PXR mediated NLRP3 5 minutes ATP release following PCN treatment was. inflammasome activation could be driven by the release of completely absent in cells isolated from PXR2 2 mice. ATP and subsequent activation of P2X7 First to identify a Fig 5A Similar ATP release responses were observed. role for ATP in our system we cotreated PMA differentiated in THP 1 cells treated with either SR12813 or rifaximin. THP 1 cells with apyrase 30 U ml to break down extracel Fig 5B Collectively these data indicate that the activa. lular ATP Iyer et al 2009 As in our previous experi tion of the PXR triggers a rapid release of ATP that fits the. ments SR12813 and rifaximin triggered IL 1b secretion but kinetics reported by other NLRP3 inflammasome activa. this response was abolished in macrophages cotreated with tors Mortimer et al 2015. The PXR Modulates NLRP3 Inflammasome Activation 49. PXR driven ATP release and NLRP3 inflammasome activa. tion we pretreated PMA differentiated THP 1 cells with the. Src kinase inhibitor PP2 10 mM Chang et al 2012 and. exposed them to PXR agonists Selective inhibition of SFKs. abolished the rapid ATP release triggered by SR12813. and rifaximin Fig 6B Taken together these data suggest. that PXR agonists trigger ATP efflux through a SFK and. pannexin 1 dependent process,Discussion, In the current study we found that stimulation of the PXR. in primed macrophages triggered the activation of the NLRP3. inflammasome resulting in caspase 1 activation and IL 1b. secretion Mechanistically PXR activation induced NLRP3. inflammasome signaling was reliant on pannexin 1 dependent. ATP release and subsequent stimulation of the P2X7 re. ceptor a well characterized driver of inflammasome activa. tion Mangan et al 2018 In this process PXR dependent. ATP release occurs as early as 15 seconds following receptor. stimulation and involves SFK signaling suggesting a cyto. solic function for the PXR in macrophages Altogether our. data support a novel role for the PXR in triggering a. host defense response in macrophages linking xenobiotic. sensing and innate immunity in a system that may protect. against xenobiotics and other chemical contaminants of. foreign origin, The PXR is a member of the nuclear receptor superfamily.
which includes such members as the FXR VDR glucocorticoid. Fig 4 PXR associated NLRP3 inflammasome activation is sensitive to receptor and retinoid X receptor Germain et al 2006 The. ATP degradation and inhibition of the P2X7 receptor A Apyrase APY. 30 U ml pretreatment to degrade extracellular ATP significantly PXR which is highly expressed in the liver and in intestinal. reduces SR12813 SR 4 mM and rifaximin Rifx 10 mM induced IL 1b epithelial cells is best characterized for its ability to regulate. release from PMA differentiated THP 1 cells B oATP 100 mM a the expression of enzymes involved in drug metabolism. selective P2X7 receptor antagonist significantly reduces SR12813 SR. 4 mM and rifaximin Rifx 10 mM induced IL 1b release from PMA. detoxification and excretion Koutsounas et al 2013 The. differentiated THP 1 cells All outcomes were measured after a 6 hour PXR is also expressed in a variety of immune cells including. treatment period N 6 P 0 05 for indicated comparison generated by T cells macrophages and dendritic cells Schote et al 2007. analysis of variance and Tukey s post hoc test and its signaling has been reported to modulate their function. through mechanisms that are less understood Zhou et al. The transmembrane channel pannexin 1 has been impli 2009 Dubrac et al 2010 Casey and Blumberg 2012 Beyer. cated in ATP release and NLRP3 inflammasome activation et al 2013 More recently the PXR s direct and indirect. in macrophages Hung et al 2013 Mortimer et al 2015 regulation of innate immune signaling has been reported in a. and thus we sought to determine whether this mechanism number of systems In a seminal report Venkatesh et al. was at play following PXR stimulation in our studies First 2014 found that the PXR functions as a negative regulatory. to assess the role of pannexin 1 in PXR driven ATP release of Toll like receptor gene expression in the intestinal epithe. PMA differentiated THP 1 cells were exposed to pannexin lium thereby indirectly modulating innate immune signaling. 1 blocking peptide 10Panx 400 mM Grahames et al in the intestinal mucosa These data build on a body of. 1999 or a scrambled peptide control ScPanx 400 mM prior literature that suggests that PXR can negatively regulate. to stimulation with SR12813 or rifaximin PXR activation NF kB dependent inflammatory signaling in a variety of cell. triggered rapid and significant ATP release in the scram types Xie and Tian 2006 Zhou et al 2006 Moreau et al. bled peptide treated cells Fig 6A a response that was 2008. significantly attenuated by pannexin 1 channel blockade In contrast to the notion that the PXR exhibits solely anti. with 10Panx Fig 6A without affecting cell viability inflammatory effects Wang et al 2014a reported that the. Supplemental Fig 2 PXR could enhance NLRP3 inflammasome activity in vascu. Although nongenomic roles for other NRs have been lar endothelial cells The authors found that PXR stimula. described little is known about how the PXR regulates tion upregulated the expression of NLRP3 and pro IL 1b. intracellular signaling processes within the cytosol Others and that prolonged stimulation triggered caspase 1 activa. have reported the involvement of SFKs in the cytosolic effects tion and IL 1b processing indicative of NLRP3 inflamma. of NRs Vertino et al 2005 Buitrago and Boland 2010 Peng some activation Although the kinetics of activation reported. et al 2012 Buitrago et al 2013 Interestingly receptor in this study do not conform to the traditional view of the. mediated gating of pannexin 1 has been shown to involve NLRP3 inflammasome as an expeditious effector our data. SFK dependent phosphorylation events Lohman et al suggest that PXR activation can trigger immediate ATP. 2015 Weilinger et al 2016 To assess the role of SFKs in efflux from macrophages a prerequisite for inflammasome. 50 Hudson et al,Fig 5 PXR agonists trigger rapid and significant. ATP release from mouse and human macrophages,A PCN a mouse PXR agonist triggers rapid and. significant ATP release from LPS pulsed peritoneal. macrophages isolated from wild type mice an effect. that is absent in cells isolated from PXR2 2 mice,N 3 P 0 05 for wild type WT PCN treated. macrophages compared with naive and PXR2 2 cells, generated by analysis of variance and Tukey s post. hoc test B Human PXR agonists rifaximin Rifx,and SR12813 SR trigger rapid and significant ATP.
release from PMA differentiated THP 1 cells N 3,P 0 05 for indicated comparison generated by. analysis of variance and Tukey s post hoc test, activation in response to a variety of stimuli Mangan et al responses is not without precedent For instance activation of. 2018 Indeed the notion that a nuclear receptor can activate the FXR by bile acids induces rapid release of ATP in pancreatic. intracellular signaling events that culminate in rapid cellular cell lines Kowal et al 2015 Tulk et al 2015 also reported. Fig 6 Blockade of pannexin 1 and inhibition of SFKs. attenuates human PXR agonist induced ATP and IL 1b. release from PMA differentiated THP 1 cells A Selective. inhibition of pannexin 1 with a pannexin 1 channel. blocking peptide 10Panx 400 mM or scrambled control. peptide ScPanx abolishes rifaximin Rifx and SR12813. SR induced ATP release from PMA differentiated THP 1. cells B Inhibition of SFKs with PP2 10 mM significantly. reduces rifaximin Rifx and SR12813 SR induced ATP,release from PMA differentiated THP 1 cells N 4 P. 0 05 for indicated comparison generated by analysis of. variance and Tukey s post hoc test, The PXR Modulates NLRP3 Inflammasome Activation 51. Fig 7 Proposed model for the events that link PXR activation to NLRP3 inflammasome activation and IL 1b release Ligation of the PXR and. heterodimerization with the retinoid X receptor triggers the release of intracellular ATP through pannexin 1 channels Extracellular ATP then binds to. the P2X7 receptor an event that prompts the assembly of the NLRP3 inflammasome and subsequent activation of caspase 1 Caspase 1 then cleaves. pro IL 1b into its active secreted IL 1b, that stimulation of the VDR in primed macrophages of NLRP3 and pro IL 1b expression Indeed Luo et al 2017.
triggered rapid NLRP3 inflammasome activation and reported that pretreating macrophages with baicalein an. IL 1b release Our data suggest PXR stimulation involves agent reported to activate the PXR Dou et al 2012. the rapid gating of pannexin 1 through a SFK dependent attenuated LPS induced NLRP3 and pro IL 1b expression. mechanism resulting in ATP efflux and subsequent P2X7 through its inhibition of NF kB signaling In this context. receptor activation to trigger NLRP3 inflammasome baicalein s inhibition of signal 1 attenuated subsequent ATP. activation induced IL 1b secretion Luo et al 2017 It is important to. Although our findings support a role for the PXR in highlight that in our studies we assessed the impact of PXR. initiating cell signaling that activates the NLRP3 inflamma activation in primed macrophages that have already received. some to induce IL 1b processing and release others have signal 1 Thus in the context of a primed cell that exhibits. reported contrasting observations Sun et al 2015 reported abundant expression of NLRP3 and pro IL 1b PXR stimula. that pretreating hepatocytes with PXR agonists attenuated tion acts as signal 2 gating pannexin 1 to allow the efflux of. LPS induced IL 1b release Furthermore a recent report by ATP which culminates in caspase 1 activation and IL 1b. Wang et al 2017 described a model wherein statins inhibit release Fig 7. inflammasome activity in vascular endothelial cells through Although the functional impact of the PXR s modulation of. the PXR dependent inhibition of NF kB driven NLRP3 gene NLRP3 inflammasome activation has yet to be elucidated. transcription Some clarity could be provided to these dispar xenobiotic and endobiotic sensing mechanisms are thought to. ities by interpreting them in context of the temporal nature of add an additional level of defense in the gastrointestinal tract. inflammasome activation NLRP3 inflammasome output i e of multicellular organisms Dussault and Forman 2002 For. IL 1b processing and release requires two signals The first example Caenorhabditis elegans upregulates xenobiotic re. stimulus often termed signal 1 often involves Toll like sponse genes and initiates avoidance behaviors in the pres. receptor driven NF kB activation to prime the cells inducing ence of pathogens and or specific pathogenic factors in a. the expression of the inflammasome components including process believed to enhance survival in the context of infection. NLRP3 and pro IL 1b Mangan et al 2018 In order for a or exposure to environmental contaminants Melo and Ruv. functional NLRP3 inflammasome to elaborate a second signal kun 2012 Jones et al 2013 Mechanistically these re. also termed signal 2 is required which usually takes the sponses are mediated by a family of nuclear receptors that. form of a DAMP or PAMP and results in NLRP3 oligomeri exhibit functional similarities to the mammalian PXR Jones. zation capsase 1 activation resulting in IL 1b processing et al 2013 As a ligand activated receptor the PXR s flexible. and release Mangan et al 2018 The wealth of data binding domain allows a variety of receptor ligand interac. implicating the PXR as a negative regulator of NF kB tions to occur Kliewer et al 2002 Chang and Waxman 2006. signaling suggests its inhibitory effect on IL 1b release may Staudinger et al 2006 Gupta et al 2008 Chang 2009. be due to its inhibition of signal 1 i e inhibiting the induction Shukla et al 2011 Thus the interplay between xenobiotics. 52 Hudson et al, and innate immune signaling through the PXR may be broad pregnane X receptor but not constitutive androstane receptor mediated CYP3A4. and multidrug resistance transporter 1 induction studies with anti human im. reaching Furthermore the impact of endogenous PXR ligands munodeficiency virus protease inhibitors Drug Metab Dispos 36 1172 1180. of microbial origin and the regulation of intestinal mucosal Hirota SA Ng J Lueng A Khajah M Parhar K Li Y Lam V Potentier MS Ng K. Bawa M et al 2011 NLRP3 inflammasome plays a key role in the regulation of. inflammasome signaling require further attention Ulti intestinal homeostasis Inflamm Bowel Dis 17 1359 1372. mately our findings suggest the existence of a complex Hoque R Sohail MA Salhanick S Malik AF Ghani A Robson SC and Mehal WZ. 2012 P2X7 receptor mediated purinergic signaling promotes liver injury in. interplay between xenobiotic sensing mechanisms and innate acetaminophen hepatotoxicity in mice Am J Physiol Gastrointest Liver Physiol. immunity that may function as a conserved mechanism to 302 G1171 G1179. protect the host from exposure to chemical agents of foreign Hu G Xu C and Staudinger JL 2010 Pregnane X receptor is SUMOylated to repress. the inflammatory response J Pharmacol Exp Ther 335 342 350. origin Additional work will be required to determine the Hung SC Choi CH Said Sadier N Johnson L Atanasova KR Sellami H Yilmaz. functional role for this interplay in the context of health and and Ojcius DM 2013 P2X4 assembles with P2X7 and pannexin 1 in gingival. epithelial cells and modulates ATP induced reactive oxygen species production and. disease inflammasome activation PLoS One 8 e70210, Imaeda AB Watanabe A Sohail MA Mahmood S Mohamadnejad M Sutterwala FS. Acknowledgments Flavell RA and Mehal WZ 2009 Acetaminophen induced hepatotoxicity in mice is. dependent on Tlr9 and the Nalp3 inflammasome J Clin Invest 119 305 314. We thank J Staudinger University of Kansas Lawrence KS for Iyer SS Pulskens WP Sadler JJ Butter LM Teske GJ Ulland TK Eisenbarth SC. providing Pxr2 2 Nr1i22 2 mice Florquin S Flavell RA Leemans JC et al 2009 Necrotic cells trigger a sterile. inflammatory response through the Nlrp3 inflammasome Proc Natl Acad Sci USA. 106 20388 20393, Jabaut J Ather JL Taracanova A Poynter ME and Ckless K 2013 Mitochondria. Authorship Contributions targeted drugs enhance Nlrp3 inflammasome dependent IL 1b secretion in asso. Participated in research design Hirota Mani Chang Hudson ciation with alterations in cellular redox and energy status Free Radic Biol Med. 60 233 245, Conducted experiments Hudson Flannigan Venu Alston Jones LM Rayson SJ Flemming AJ and Urwin PE 2013 Adaptive and specialised. Sandall transcriptional responses to xenobiotic stress in Caenorhabditis elegans are regu. Contributed new reagents or analytic tools Sandall MacDonald lated by nuclear hormone receptors PLoS One 8 e69956. Kliewer SA Goodwin B and Willson TM 2002 The nuclear pregnane X receptor a. Muruve key regulator of xenobiotic metabolism Endocr Rev 23 687 702. Performed data analysis Hudson Hirota Flannigan Koutsounas I Theocharis S Patsouris E and Giaginis C 2013 Pregnane X receptor. Wrote or contributed to the writing of the manuscript Hirota PXR at the crossroads of human metabolism and disease Curr Drug Metab 14. Hudson Flannigan Chang Mani MacDonald Kowal JM Haanes KA Christensen NM and Novak I 2015 Bile acid effects are. mediated by ATP release and purinergic signalling in exocrine pancreatic cells. References Cell Commun Signal 13 28, Ainscough JS Gerberick GF Kimber I and Dearman RJ 2015 Interleukin 1b Lau A Chung H Komada T Platnich JM Sandall CF Choudhury SR Chun J.
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Wang K Damjanov I and Wan YJ 2010 The protective role of pregnane X receptor 3330 Hospital Drive NW Health Sciences Room 1845 Calgary Alberta. in lipopolysaccharide D galactosamine induced acute liver injury Lab Invest 90 Canada T2N4N1 E mail shirota ucalgary ca.

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