Title: Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway
Abstract: Article2 June 2020Open Access Transparent process Tissue-resident macrophages actively suppress IL-1beta release via a reactive prostanoid/IL-10 pathway Natacha Ipseiz Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Robert J Pickering orcid.org/0000-0003-3332-9868 Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Marcela Rosas Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Victoria J Tyrrell Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Luke C Davies Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Selinda J Orr Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast, UK Search for more papers by this author Magdalena A Czubala Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Dina Fathalla Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK Search for more papers by this author Avril AB Robertson School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Clare E Bryant orcid.org/0000-0002-2924-0038 Immunology Catalyst Programme, GSK, Cambridge, UK Department of Veterinary Medicine, University of Cambridge, Cambridge, UK Search for more papers by this author Valerie O'Donnell Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Philip R Taylor Corresponding Author [email protected] orcid.org/0000-0003-0163-1421 Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK Search for more papers by this author Natacha Ipseiz Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Robert J Pickering orcid.org/0000-0003-3332-9868 Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Marcela Rosas Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Victoria J Tyrrell Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Luke C Davies Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Selinda J Orr Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast, UK Search for more papers by this author Magdalena A Czubala Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Dina Fathalla Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK Search for more papers by this author Avril AB Robertson School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Clare E Bryant orcid.org/0000-0002-2924-0038 Immunology Catalyst Programme, GSK, Cambridge, UK Department of Veterinary Medicine, University of Cambridge, Cambridge, UK Search for more papers by this author Valerie O'Donnell Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK Search for more papers by this author Philip R Taylor Corresponding Author [email protected] orcid.org/0000-0003-0163-1421 Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK Search for more papers by this author Author Information Natacha Ipseiz1,‡, Robert J Pickering1,‡, Marcela Rosas1, Victoria J Tyrrell1, Luke C Davies1, Selinda J Orr1,2, Magdalena A Czubala1, Dina Fathalla1,3, Avril AB Robertson4, Clare E Bryant5,6, Valerie O'Donnell1 and Philip R Taylor *,1,3 1Systems Immunity Research Institute, Heath Park, Cardiff University, Cardiff, UK 2Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast, UK 3UK Dementia Research Institute at Cardiff, Cardiff University, Cardiff, UK 4School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld, Australia 5Immunology Catalyst Programme, GSK, Cambridge, UK 6Department of Veterinary Medicine, University of Cambridge, Cambridge, UK ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +44 02920687328; E-mail: [email protected] EMBO J (2020)39:e103454https://doi.org/10.15252/embj.2019103454 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The alarm cytokine interleukin-1β (IL-1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1β production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1β from its pro-form. However, despite the important role of IL-1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1β processing, providing a previously unrecognized control of IL-1β in tissue-resident macrophages. Synopsis The interleukin-1β (IL-1β) is a potent cytokine, present in the early stages of inflammation and playing a role in autoinflammatory syndromes. Its production requires two independent signals, the first leading to the production of its pro-form (pro-IL-1β) and the second to its maturation and release. Here we identified an additional mechanism, actively suppressing IL-1β production, thereby contributing to the control of inflammation. In response to inflammatory signals (lipopolysaccharide), resident peritoneal macrophages engage ata6-dependant prostacyclin (PGI2) production. PGI2 signals back to the macrophages to induce IL-10 production. IL-10 actively blocks IL-1β processing. In the absence of a secondary signal, the lack of either PGI2 or IL-10 is sufficient to trigger IL-1β maturation and release. Introduction Interleukin-1β (IL-1β) is a pro-inflammatory cytokine, an alarmin which, once released into the extracellular milieu, triggers the inflammatory response. It is commonly accepted that a two-step mechanism is required for IL-1β production in mouse macrophages (MФ). First, pathogen-associated molecular pattern (PAMP) recognition induces transcription and translation of the inactive pro-form of IL-1β (pro-IL-1β). A secondary signal, such as reactive oxygen species (ROS) (Nakahira et al, 2011; Zhou et al, 2011), crystals (Hornung et al, 2008) or potassium efflux (Petrilli et al, 2007), is then needed to induce the classical inflammasome assembly, composed of NOD-like receptor family, pyrin domain containing 3 (Nlrp3) and apoptosis-associated speck-like protein containing a CARD (ASC), also called PYCARD. Once assembled, the NLRP3 inflammasome activates caspase1 which in turn cleaves pro-IL-1β into its mature IL-1β form (Bryant & Fitzgerald, 2009; Dowling & O'Neill, 2012; Latz et al, 2013; Lamkanfi & Dixit, 2014). Despite intensive research, the mechanisms regulating IL-1β maturation and release (Lopez-Castejon & Brough, 2011; Martin-Sanchez et al, 2016) are still under discussion (Cullen et al, 2015; Evavold et al, 2018; Monteleone et al, 2018). Dysregulated IL-1β production has been associated with the development of many inflammatory and autoinflammatory diseases (Lamkanfi & Dixit, 2012, 2014; Yao et al, 2016; Mayer-Barber & Yan, 2017) such as cryopyrin-associated periodic syndromes (CAPS), type 2 diabetes (Jourdan et al, 2013), increased susceptibility to Crohn's disease (Villani et al, 2009) and intestinal inflammation (Shouval et al, 2016), gout (Joosten et al, 2010) and rheumatoid arthritis (Pascual et al, 2005). Macrophages are part of the immune system's first line of defence. Initially simply categorized as phagocytes, evidence of their complexity has accumulated over the years (Ley et al, 2016). Resident peritoneal macrophages (pMФ), a well-studied tissue macrophage population, have essential functions, including modulation of the inflammatory response after pathogen recognition (Dioszeghy et al, 2008; Spight et al, 2008; Leendertse et al, 2009) or injury (Uderhardt et al, 2019), phagocytosis of pathogens (Ghosn et al, 2010) and dying cells (Fond & Ravichandran, 2016), liver repair (Wang & Kubes, 2016; Rehermann, 2017) and maintenance of self-tolerance (Russell & Steinberg, 1983; Mukundan et al, 2009; Munoz et al, 2010; Uderhardt et al, 2012; Ipseiz et al, 2014; Majai et al, 2014; Carlucci et al, 2016). pMФ are part of the first wave of response during peritonitis (Khameneh et al, 2017) and help ensure the survival of the host and the optimal clearance of the infection. Their efficiency is coupled to their optimal cytokine and chemokine secretion which have to be finely tuned, including IL-1β (Topley et al, 1996; Hautem et al, 2017). After the first inflammatory burst following PAMP recognition, macrophages dampen their inflammatory processes by producing anti-inflammatory molecules, such as IL-10 (Bogdan et al, 1991; Berlato et al, 2002; Saraiva & O'Garra, 2010). IL-10 protects against acute inflammation (Howard et al, 1993), and its loss has dramatic effects as observed in IL-10 deficient mice, which develop chronic enterocolitis (Kuhn et al, 1993; Krause et al, 2015). However, the regulatory control of IL-10 production by pMФ (Liao et al, 2016) as well as its mode of action remain unclear. Additionally, after microbial stimulation, peritoneal macrophages rapidly release prostanoids, such as prostaglandin I2 (PGI2), also called prostacyclin (Brock et al, 1999). PGI2 is known to be generated in peritoneal macrophages following inflammatory stimulation, although it is poorly studied and neglected in this context (Yokode et al, 1988; Stewart et al, 1990; Wightman & Dallob, 1990). Despite the paradoxical role of PGI2 in inflammatory diseases (Stitham et al, 2011), synthetic analogues can decrease tumour necrosis factor (TNF) and induce IL-10 in human peripheral mononuclear cells in vitro (Eisenhut et al, 1993; Luttmann et al, 1999) and inhibit function of murine dendritic cells (Zhou et al, 2007), suggesting an active control of inflammation. Here we have studied the inflammatory response of resident pMФ that lack their specialized tissue-programming as a consequence of deletion of the tissue-specific transcription factor Gata6 (Rosas et al, 2014). While wild-type (WT) pMФ need a secondary signal after lipopolysaccharide (LPS) stimulation to produce mature IL-1β, we show that the Gata6-deficient pMФ do not, and they exhibit aberrant production of IL-1β after LPS stimulation. Using Gata6-KOmye pMФ, we identified a Gata6-PGI2-IL-10 axis as a major regulator of IL-1β processing in resident pMФ. This axis actively inhibits IL-1β processing during response to a microbial stimulus in the absence of a second signal and thus ensures proportionate and finely regulated production of IL-1β in response to LPS. Results Gata6-deficient peritoneal macrophages exhibit dysregulated IL-1β release We and others previously identified the transcription factor Gata6 as a major key regulator of tissue-resident peritoneal macrophage (pMФ) specialization (Gautier et al, 2014; Okabe & Medzhitov, 2014; Rosas et al, 2014). To determine its role in the inflammatory function of pMФ, we analysed the response of Gata6-WT and Gata6-KOmye pMФ after toll-like receptor (TLR) ligand stimulation. Surprisingly, we observed that ultra-pure LPS, a specific TLR4 agonist, induced the production of IL-1β by Gata6-KOmye pMФ in the absence of an exogenous secondary signal (Fig 1A). LPS also induced significantly higher production of TNF by Gata6-KOmye pMФ as observed by ELISA (Fig 1B) and flow cytometry (Figs EV1 and 2A). Additionally, the effect of LPS on IL-1β and TNF production was found to be concentration (Fig 1C and D) and time-dependent (Fig 1E and F). Interestingly, inhibiting TNF with etanercept (a fusion protein composed of TNFR2 connected to a human IgG1 Fc tail) slightly reduced IL-1β production from Gata6-KOmye pMФ and blocking the IL-1 signalling pathway using an IL-1 receptor antagonist (rIL-1ra) when stimulating the cells with purified LPS did not dramatically change IL-1β secretion by Gata6-WT or Gata6-KOmye pMФ (Fig 1G). Stimulating the cells with recombinant TNF (recTNF) did not induce IL-1β production from either Gata6-WT or Gata6-KOmye pMФ (Fig 1H). These data suggest that neither IL-1 receptor nor TNF signalling greatly affected IL-1β production. Figure 1. Aberrant cytokine release from LPS-stimulated Gata6-deficient resident peritoneal macrophages A, B. Gata6-WT and Gata6-KOmye peritoneal macrophages (pMФ) were unstimulated (–) or stimulated with TLR2L Pam3CSK4 (500 ng/ml), TLR3L Poly(I:C) (1 μg/ml), TLR4L ultrapure LPS (100 ng/ml), TLR5L flagellin (100 ng/ml), TLR7 and 8L R848 (1 μg/ml) or TLR9L CpG ODN1826 (5 μM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n = 5, two-way ANOVA analysis with Tukey's multiple comparison post-test. C, D. IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMФ stimulated for 24 h with the indicated LPS concentrations. n = 3, two-way ANOVA analysis with Sidak's multiple comparison post-test. E, F. IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMФ stimulated with LPS (100 ng/ml) for the indicated times. n = 3, two-way ANOVA analysis with Sidak's multiple comparison post-test. G. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMФ stimulated for 18 h with 100 ng/ml LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n = 4–5, two-way ANOVA analysis with Tukey's multiple comparison post-test. H. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMФ stimulated for 18 h with 100 ng/ml LPS or 100 ng/ml recombinant TNF (recTNF), n = 4–5, two-way ANOVA analysis with Tukey's multiple comparison post-test. Data information: Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Representative gating strategy of pMФ from Gata6-WT and Gata6-KOmye miceFirst, dead cells are excluded based on FSC and SSC value; then, single cells are gated based on FCS area and FCS linear value. pMФ are then identified as F4/80hiTim4+ (Gata6-WT) or F4/80+Tim4+ (Gata6-KOmye). Download figure Download PowerPoint Figure 2. The aberrant release of IL-1β by Gata6-KO-deficient pMФ follows classical inflammasome activation A. Normalized expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell-sorted Gata6-WT and Gata6-KOmye pMФ. Data are shown as mean ± SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamini–Hochberg procedure. n.d = non-detectable. B. Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMФ. n = 4–7 individual mice per group. C, D. Il1b and Tnf mRNA relative expression (C) and IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates. E. Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng/ml LPS. n = at least three independent experiments. F. Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 3 h. Data shown are pooled from three independent experiments. G. Western blot protein analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 6 h. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates. H, I. IL-1β ELISA (H) and Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or 10 μM MCC950 for 24 h (n = 5). Data shown in (H) are pooled from five independent replicates. J, K. Caspase1 (Casp1) mRNA relative expression (J) and Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are pooled from three independent experiments. L. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of five independent replicates. M. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 μM nigericin. Data shown are pooled of five independent replicates. N. Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 μm. Data information: Data are expressed as mean ± SEM and analysis were performed using two-way ANOVA analysis Tukey's multiple comparison post-test unless otherwise stated. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Activation of the NLRP3 inflammasome by classical stimuli is independent of Gata6 programming To investigate the mechanism behind the aberrant release of IL-1β by Gata6-KOmye pMФ following LPS stimulation, we analysed the components of the classical NLRP3 inflammasome pathway. Gata6-WT and KOmye pMФ showed similar levels of toll-like receptors (Tlr) expression, with an exception for Tlr13 which appeared to be reduced in Gata6-KOmye cells. Tlr4 and Cd14, the receptor and co-receptor, respectively, for LPS were similarly expressed by Gata6-WT and KOmye cells (Fig 2A). Cell surface expression analysis by flow cytometry however showed an increase in TLR2 and TLR4 in KOmye cells (Figs 2B and EV2B). Gata6-WT and KOmye pMФ exhibited similar Il1b mRNA expression (Fig 2C) and comparable production of pro-IL-1β (Figs 2D and EV3A), 3 and 6 h after LPS stimulation, respectively. Interestingly, Gata6-KOmye cells showed a significantly upregulated Tnf expression (Fig 2C), indicating a direct regulation of TNF production on a mRNA level rather than on a protein level. The pro-IL-1β expression was confirmed by flow cytometry analysis (Fig 2E). These results suggest that, despite an increased TLR4 expression in Gata6-KOmye pMФ, both Gata6-WT and KOmye pMФ have a similar response capacity to the primary signal LPS regarding the initiation of IL-1β production and that the aberrant release observed in the Gata6-KOmye cells was likely due to a downstream dysregulation in pro-IL-1β processing. Further investigation revealed that Gata6-KOmye pMФ did not have increased Nlrp3 mRNA expression compared to WT pMФ (Fig 2F), as well as similar protein levels (Figs 2G and EV3B). To determine whether the classical Nlrp3 inflammasome was responsible for the IL-1β release in Gata6-KOmye pMФ, we stimulated Gata6-WT and KOmye pMФ with LPS in the presence of the specific Nlrp3 inhibitor MCC950 (Coll et al, 2015, 2019). We observed an abrogation of IL-1β secretion from Gata6-KOmye cells (Fig 2H and I), confirming the essential role of Nlrp3 in the production of IL-1β by Gata6-KO pMФ. In addition, Gata6-KOmye cells showed upregulated mRNA expression of caspase1 (Casp1) (Fig 2J) but protein expression showed no significant difference (Figs 2K and EV3C). The selective caspase1 inhibitor Ac-YVAD-cmk blocked IL-1β secretion by LPS-stimulated Gata6-KOmye pMФ (Fig 2L). Interestingly, when first primed with LPS for 3 h and then stimulated with a secondary signal (ATP or nigericin) for 30 min, both Gata6-WT and KOmye pMФ released comparable levels of IL-1β (Fig 2M). Confocal immunofluorescence analysis showed that both cell types were able to form ASC specks, a hallmark of the classical NLRP3 inflammasome assembly, when stimulated with LPS and ATP (Fig 2N). Overall, these data indicate a similar NLRP3 inflammasome capacity of both Gata6-WT and KOmye pMФ, suggesting that the aberrant IL-1β release observed in Gata6-KOmye cells after LPS stimulation might be due to alteration of the regulatory mechanisms and not to the inflammasome machinery itself. Click here to expand this figure. Figure EV2. Flow cytometry analysis of intracellular TNF and extracellular TLR2, TLR4 and CD14 expression Representative flow cytometry analysis of TNF expression in Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng/ml LPS and 0.1 % (v/v) GolgiBlock. Data shown are representative of at least three independent experiments. Data are expressed as mean ± SEM. Two-way ANOVA analysis followed by Tukey's multiple comparison post-test was performed. ***P < 0.001. Histogram overlays of flow cytometry analysis of cell surface expression of TLR2, TLR4 and CD14 of Gata6-WT and Gata6-KOmye pMФ. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Western blot quantification of the immature and mature IL-1β and the canonical inflammasome machineryWestern blot quantification of pro-IL-1β and mature IL-1β (A), Nlrp3 (B) and caspase1 (C) of Gata6-WT and Gata6-KOmye pMΦ, unstimulated or stimulated overnight with 100 ng/ml LPS. Results showed are pooled from three independent experiments, normalized to Gata6-WT unstimulated sample and are expressed as mean ± SEM. Two-way ANOVA analysis followed by Tukey's multiple comparison post-test was performed. *P < 0.05. Download figure Download PowerPoint Peritoneal-resident macrophages actively suppress IL-1β production Based on our findings above, we next hypothesized that the Gata6-KOmye pMФ had a defect in an inhibitory pathway, present in the Gata6-WT cells, that basally constrains further processing of pro-IL-1β after LPS stimulation. To investigate this hypothesis, we co-cultured Gata6-WT and KOmye cells and observed a nearly complete inhibition of IL-1β secretion by the Gata6-KOmye pMФ (Fig 3A). This observation suggested that the Gata6-WT cells actively inhibited IL-1β processing by the Gata6-KOmye pMФ. To determine whether this inhibition was due to direct contact between the cells or to a soluble factor secreted by the Gata6-WT pMФ, we performed a Transwell experiment (Fig 3B). In this setting, the Gata6-WT pMФ significantly inhibited IL-1β production from the Gata6-KOmye pMФ, however to a lesser extent than in the direct co-culture experiments. These data may suggest that the Gata6-WT pMФ might be secreting a soluble molecule, albeit with a short half-life based on the reduced effect observed in the Transwell setting, inhibiting the pro-IL-1β processing pathway. Transcriptomic analysis of Gata6-WT and Gata6-KOmye pMФ (Rosas et al, 2014) showed alterations in many such potential candidate molecules (GEO: GSE47049); however, one of the greatest differentially expressed genes was prostaglandin I2 synthase (Ptgis) which converts prostaglandin H2 (PGH2) into prostacyclin (PGI2). Naïve Gata6-KOmye pMФ expressed significantly reduced amount of Ptgis mRNA (Fig 3C) and protein (Fig 3D) and significantly upregulated thromboxane A synthase 1 (Tbxas1) (Fig 3C), a direct competitor to Ptgis for the conversion of PGH2 into thromboxane A2 (TXA2). The expression of the two other enzymes implicated in the processing pathway of arachidonic acid (AA), cyclooxygenase 1 (Ptgs1) converting AA into prostaglandin G2 (PGG2) followed by PGH2 and prostaglandin E synthase 2 (Ptges2) processing PGH2 into prostaglandin E2 (PGE2) was less dramatically changed between Gata6-WT and Gata6-KOmye pMФ (Fig 3C). PGI2 has a very short half-life (< 2 min in vivo) and is rapidly hydrolysed to form 6-keto-prostaglandin F1α (6-keto-PGF1α), a metabolite that is readily detectable by mass spectrometry (Kunze & Vogt, 1971; Hamberg & Samuelsson, 1973; Jogee et al, 1983; Lewis & Dollery, 1983; Stitham et al, 2011). Therefore, to assess the impact of the Ptgis deficiency in Gata6-KOmye cells on prostanoid production, we performed mass spectrometric analysis of the oxylipins in supernatants from Gata6-WT and KOmye pMФ cultured with or without 100 ng/ml LPS for 3 h. As expected, a significant reduction of 6-keto-PGF1α coupled with increased thromboxane B2 (TXB2) was observed from Gata6-KOmye pMФ after LPS stimulation (Fig 3E). Notably, prostaglandin E2 (PGE2) was also significantly decreased in Gata6-KOmye pMФ after LPS stimulation although the expression of the enzyme regulating its production (Ptges2) was unchanged (Fig 3C). These results confirmed an imbalanced prostanoid response in Gata6-KOmye cells upon LPS stimulation (Fig 3F). It is important to note that both TXB2 and PGE2 were produced at much lower levels when compared to 6-keto-PGF1α, in WT LPS-stimulated cells (approximately 10% of the levels). This suggests that PGI2 may normally be the dominant effector on downstream signalling in pMФ (Norris et al, 2011). Altogether, these data suggest that Gata6-WT pMФ are actively inhibiting the processing of IL-1β upon LPS stimulation and that a candidate for this effect may be PGI2 produced by the Gata6-dependent Ptgis enzyme. Figure 3. Prostanoid production is imbalanced in Gata6-KOmye pMФ IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMФ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng/ml LPS. Data shown are pooled from three independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed. IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMФ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng/ml LPS. Data shown are pooled from three independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed. Microarray analysis of Ptgis, Tbxas1, Ptges2 and Ptgs1 expression from Gata6-WT and Gata6-KOmye pMФ isolated from naïve mice. Data are shown as mean ± SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamini–Hochberg procedure. Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice. Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMФ unstimulated (–) or stimulated for 3 h with 100 ng/ml LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed. Representation of the variation of the prostanoid synthesis pathway in Gata6-KOmye pMФ created using Cytoscape software. Circle shape represent lipids, diamond shape enzymes, yellow downregulation and purple upregulation of the expression/production in Gata6-KOmye cells. The size of the circles represents relative levels observed in Gata6-KOmye cells. Data information: Data is shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Prostacyclin inhibits IL-1β production via IL-10 induction in a Gata6-dependant pathway PGI2 and LPS have both been previously shown to induce IL-10 production, including in MФ (Fiorentino et al, 1991; Luttmann et al, 1999; Zhou et al, 2007). It is also known that pMФ are predisposed to the production of IL-10 after stimulation with microbial products (Liao et al, 2016). Here, we observed that Gata6-KOmye pMФ produced significantly less IL-10, compared to Gata6-WT cells, after LPS stimulation (Fig 4A). To determine if this could be a consequence of reduced PGI2 levels, the impact of beraprost, cicaprost and iloprost (PGI2 analogues with various IP receptor affinities and specificities; Clapp & Gurung, 2015) on IL-10 generation was assessed (Fig 4B). When combined with LPS,