Prostacyclin synthase deficiency exacerbates inflammatory reactions in lipopolysaccharide-induced sepsis in vivo

Prostacyclin synthase deficiency exacerbates inflammatory reactions in lipopolysaccharide-induced sepsis in vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prostacyclin synthase deficiency exacerbates inflammatory reactions in lipopolysaccharide-induced sepsis in vivo Tsubasa Ochiai, Toshiya Honsawa, Keishi Yamaguchi, Yuka Sasaki, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3972516/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objectives Sepsis is an inflammatory disorder characterized by life-threatening organ dysfunction resulting from a dysregulated host response to infection. In contrast, prostacyclin (PGI 2 ) is a bioactive lipid produced by PGI synthase (PGIS) and is known to play important roles in inflammatory reactions as well as cardiovascular regulation. However, little is known about the roles of PGIS and PGI 2 in sepsis. Methodology Sepsis was induced by intraperitoneal injection of 5 mg/kg lipopolysaccharide (LPS) in wild type (WT) or PGIS knockout (KO) mice. Selexipag, a selective PGI 2 receptor (IP) agonist, was administered 2 h before LPS injection and again given every 12 h for 3 days. Results Intraperitoneal injection of LPS induced diarrhea, shivering and hypothermia. These symptoms were more severe in PGIS KO mice than in WT mice. The expression of Tnf and Il6 genes was notably increased in PGIS KO mice. In contrast, over 95% of WT mice survived 72 h after the administration of LPS, whereas all of the PGIS KO mice had succumbed by that time. The mortality rate of LPS-administrated PGIS KO mice was improved by selexipag administration. Conclusion Our study suggests that PGIS-derived PGI 2 negatively regulates LPS-induced symptoms via the IP receptor. Selexipag shows promise for the clinical treatment of human sepsis. prostacyclin prostaglandin terminal synthase lipopolysaccharide sepsis selexipag Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Sepsis is defined as life-threatening organ dysfunction due to a dysregulated host response to infection [ 1 ]. Sepsis-related deaths decreased by roughly 52.8% from 1990 to 2017, but in 2017, there were still about 11 million sepsis-related deaths, representing 19.7% of global deaths [ 2 ]. Thus, sepsis is even now one of the leading causes of death in the world. Common signs and symptoms of sepsis include fever, increased heart rate, increased breathing rate and confusion. Immune dysfunction, which in sepsis involves excessive inflammation, activation of the complement system, coagulation and immune suppression, during sepsis contributes to these symptoms. It has been reported that overproduction of proinflammatory mediators in response to infection plays a critical role in sepsis. Pinsly et al. reported that in sepsis patients, substantial amounts of proinflammatory cytokines, including tumor necrosis factor (TNF) α, are released into the bloodstream, further fueling the progression of sepsis. [ 3 ]. Overproduction of prostanoids, which are proinflammatory lipid mediators, has also been detected in the peripheral blood of septic patients [ 4 ]. Prostanoids, including prostaglandin (PG) E 2 , PGF 2α , prostacyclin (PGI 2 ), and thromboxane A 2 (TXA 2 ), are oxygenated metabolites of arachidonic acid produced by sequential catalysis of cyclooxygenase (COX) and specific PG terminal synthases. Widely used nonsteroidal anti-inflammatory drugs (NSAIDs) exert their pharmacologic effects, including anti-inflammatory effects, by reducing prostanoid production via inhibition of COX [ 5 ]. NSAIDs are used to treat various inflammatory diseases, but no reports have conclusively demonstrated that NSAIDs are applicable to clinical use for the treatment of human sepsis. On the contrary, it has been reported that administration of NSAIDs to sepsis patients was associated with increased 28-day mortality [ 6 ]. Several reports using experimental animal models demonstrated that PGF 2a /PGD 2 and PGE 2 have opposing roles in the formation of sepsis symptoms. Maehara et al . reported that inhibition of PGF 2a receptors attenuated LPS-induced systemic inflammation in mice via enhanced IL-10 production [ 7 ]. Ishii et al . showed that gene deletion of CRTH2, a PGD 2 receptor, improved lethality in a murine sepsis model by decreasing proinflammatory cytokine production [ 8 ]. On the other hand, Choudhry et al . reported that PGE 2 has negative roles in sepsis through inhibition of T cells activity by attenuating Ca 2+ signaling [ 9 ]. Unlike NSAIDs, targeting the inhibition of PG terminal synthase, which can selectively suppress specific PG species, may lead to the development of therapeutic strategies for sepsis. PGI 2 is produced by PGI synthase (PGIS), one of PG terminal synthases, that is highly and constitutively expressed in vascular endothelial and smooth muscle cells. COX-2/PGIS-derived PGI 2 has been shown to be crucial for the regulation of platelet aggregation and vascular tone [ 10 ]. In addition, we previously demonstrated that PGIS has a proinflammatory function in several disease models using PGIS gene knockout (KO) mice [ 11 , 12 ]. On the other hand, other research groups have reported that PGIS-derived PGI 2 has an anti-inflammatory function [ 13 ]. Thus, PGIS has been shown to be an ambivalent regulator of inflammatory reactions, but the role of PGIS in sepsis is not fully understood. To investigate the possibility of PGIS and PGI 2 serving therapeutic targets for sepsis, we here examined the effects of PGIS deficiency on sepsis using a lipopolysaccharide (LPS)-induced mouse sepsis model. Materials and Methods Mice Male 8- to 10-week-old C57BL/6 mice were used. PGIS KO mice were described previously [ 11 ]. Mice were bred in climate-controlled rooms (23°C) under specific pathogen-free conditions on a 12-h light/dark cycle at the Institute of Laboratory Animals of Showa University (Tokyo, Japan). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Showa University. LPS-induced sepsis model Sepsis was induced by a single intraperitoneal (i.p.) injection of LPS (5 mg/kg, Sigma Aldrich (St. Louis, MO, USA); lipopolysaccharides from Escherichia coli 0111: B4) dissolved in saline [ 7 , 14 ]. The control group received the same volume of saline. Rectal temperatures, body weight, diarrhea, and shivering of the mice were monitored before LPS administration and until day 7 after LPS administration using an animal temperature recorder (A & D Company (Tokyo, Japan); KN-91-AD1687). LPS-induced diarrhea was scored using a previously reported protocol as follows: 0, normal; 1, very mild diarrhea; 2, mild diarrhea; 3, diarrhea; 4, watery diarrhea [ 15 ]. Behavioral assessments were scored with slight modifications from the previously described protocol as follows: 0, unchanged; 1, walking with a slight stagger; 2, walking with a stagger; 3, immobility; 4, shivering [ 16 ]. Intraperitoneal fluid was collected with 4 mL of 2% fetal calf serum (FCS)-containing phosphate-buffered saline (PBS). Selexipag, a selective PGI 2 receptor (IP) agonist (1 mg/kg, Selleck Chemicals (Houston, TX, USA)) was dissolved in 1% DMSO containing saline, orally administered (p.o.) 2 h before LPS injection, and given every 12 h for 3 days. Mice in the selexipag control group received the same volume of 1% DMSO containing saline [ 17 ]. Resident peritoneal macrophages (MFs) isolation and culture Resident peritoneal MFs were obtained via peritoneal washing with 4 mL of of 2% FCS-containing PBS. Cells were plated and left to adhere for 3 h at 37°C, in a 5% CO 2 environment using RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine (Thermo Fisher Scientific (Rockford, IL, USA)). Adhered cells were washed twice with PBS and further stimulated with 1 µM A23187 (Abcam) containing 0.1% DMSO for 1 h or 10 ng/mL LPS for 3 h. Quantitative RT-PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). The resultant RNA was spectrophotometrically qualified and quantified at 260 nm and 280 nm using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Thermal cycling was performed using a Step One Plus real-time PCR system (Thermo Fisher Scientific). For each sample, real-time qRT-PCR was then performed using the SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a total volume of 10 µl, at 95°C for 15 sec and at 60°C for 45 min. The target amplicon was detected within the range of 10 to 40 cycles for all primers. PCR efficiency was examined by serially diluting the template cDNA, and the melting curve data were collected to check the PCR specificity. The expression of candidate genes in each group of mice was normalized to the Gapdh RNA level. The results were quantified using the relative standard curve method. Details on the primers details are provided in Table 1. Measurement of PGs by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) Samples were prepared according to a previously described methodology with minor modification [ 11 ]. Briefly, to prepare samples, 750 µL of 0.03% formic acid containing ultrapure water and an internal standard (25 pg of leukotriene (LT) B 4 -d 4 , Cayman (Ann Arbor, MI, USA)) were added to the each 250 µL aliquot of the sample. The prepared samples were then passed through an OASIS cartridge (Waters (Milford, MA, USA)) to obtain the PG fraction. The purified PG fraction was dried with a rotary evaporator. The resulting residues were dissolved in 50 µL mobile phase A (ultrapure water/acetonitrile/formic aced = 63/37/0.02) and injected into the LC-ESI-MS system to quantify PGs with mobile phase A and mobile phase B (acetonitrile/isopropanol = 50/50). To quantify PGs, LC-ESI-MS-based analysis was performed on a Prominence high performance liquid chromatography (HPLC) system (Shimadzu (Kyoto, Japan)) coupled with a QTRAP 5500 hybrid triple-quadrupole linear ion-trap mass spectrometer (AB SCIEX (Framingham, MA, USA)). PGs were subsequently analyzed via multiple-reaction monitoring (MRM) in negative-ion mode, as described previously [ 11 ]. Statistics Data are presented as the means ± SEM. Comparisons among all experimental groups were performed using one-way analysis of variance (ANOVA) followed by the Tukey‒Kramer post hoc test (dependent on the results of Levene’s test to determine the equality of variances) or a Kruskal–Wallis test with a Steel‒Dwass multiple comparisons test (if the data were not normally distributed). Survival curves were compared by the Kaplan‒Meier method, and statistical analyses in the survival experiments were performed by a log-rank test. All statistical analyses were performed using JMP Pro version 16 (SAS Institute Inc. (Cary, NC, USA)). The level of significance is indicated in each figure (i.e., ** p < 0.01, * p < 0.05). Results PGIS deficiency exacerbated sepsis-related symptoms caused by LPS Since it has been reported that administration of LPS causes sepsis-related symptoms such as hypothermia [ 18 – 20 ], we first intraperitoneally injected LPS into WT and PGIS KO mice. As shown in Fig. 1 A, in WT mice, rectal temperature began to decrease at 1 h, peaked at 24 h and gradually recovered by day 7 after LPS administration (Fig. 1 A). Furthermore, LPS-induced changes in mouse movement, such as shivering, were also exacerbated along with decreased body temperature (Fig. 1 B). LPS-induced diarrhea was observed at 1 h after LPS injection (Fig. 1 C). These LPS-induced symptoms were more severe in PGIS KO mice than in WT mice (Fig. 1 B, C). PGIS deficiency also exacerbated the reduction in rectal temperature of PGIS KO mice. The peak of hypothermia occurred earlier in PGIS KO mice than in WT mice (Fig. 1 A). In addition, whereas 95% or more of WT survived 72 h after LPS administration, all of the PGIS KO mice died by then (Fig. 1 D). PGI 2 production in peritoneal cavity of nontreated and LPS-administered mice We next analyzed prostanoid production in the peritoneal cavity of nontreated and LPS-administered mice using LC-MS/MS. 6-keto-PGF 1a , a stable metabolite of PGI 2 , was the most detected prostanoids, followed by TXB 2 (a stable metabolite of TXA 2 ), PGE 2 , PGD 2 , and PGF 2a in the peritoneal fluid of nontreated WT mice. At 3 h after LPS administration, PGE 2 production was significantly increased, whereas PGI 2 production tended to be decreased in WT mice. In PGIS KO mice, 6-keto-PGF 1a was not detected, prostanoid production was shunted, and the production of TXB 2 , PGE 2 , PGD 2 , and PGF 2a was increased compared to WT mice (Fig. 2 A). We further investigated the expression of genes involved in prostanoid biosynthesis in the peritoneal cells by qRT-PCR analysis. Expression of the Ptgis (PGIS) gene was downregulated in WT mice following LPS administration. PGIS-expressing resident cells may decrease in response to LPS stimulation. Ptgs1 (COX-1) gene expression was also downregulated, but Ptgs2 (COX-2) gene expression was up-regulated after LPS administration. The upregulation of COX was increased in PGIS KO mice compared to WT mice (Fig. 2 B). We next investigated the prostanoid production from peritoneal resident MFs in vitro . Among the prostanoids detected, 6-keto-PGF 1a was the most prominently observed in MFs of WT mice, and its production was increased upon stimulation by both Ca 2+ -ionophore A23187 and LPS. In contrast, 6-keto-PGF 1a was not detected in culture medium from MFs of PGIS KO mice with or without A23187 or LPS. In response to LPS stimulus, prostanoid production was shunted and the productions of PGE 2 , TXB 2 , PGF 2a , and PGD 2 was increased in PGIS KO MFs compared to WT MFs (Fig. 2 C). PGIS deficiency increased sepsis-related inflammatory responses in the peritoneal cavity We further investigated the effects of PGIS deficiency on LPS-induced inflammation-related gene expression in the peritoneal cells. As shown in Fig. 3 A, the gene expressions levels of proinflammatory cytokines, including Tnf (TNF-α) and Il6 (IL-6), were elevated at 12 h after LPS administration in peritoneal cells from WT mice, and the increases in these gene expressions were upregulated by PGIS deficiency. LPS-induced induction of myeloperoxidase (MPO), which is specifically expressed in neutrophils, and CXCL1, a chemokine related to neutrophil migration, was also enhanced in PGIS KO mice (Fig. 3 B). In PGIS KO mice, LPS-induced neutrophil migration into the peritoneal cavity might be facilitated, leading to the exacerbation of LPS-induced sepsis-related symptoms. IP agonist treatment improved LPS-induced sepsis-related symptoms and lethality in PGIS KO mice We next pretreated WT or PGIS KO mice with selexipag, a selective agonist for the PGI 2 receptor (IP), to reveal the effects of IP stimulation on LPS-induced sepsis-related symptoms. As shown in Fig. 4 , selexipag treatment significantly palliated LPS-induced sepsis-related symptoms in WT mice. Selexipag significantly improved hypothermia at 12 h after LPS administration in WT mice. LPS-induced behavioral change such as shivering was also suppressed by selexipag at 12 h after LPS administration in WT mice. The effects of selexipag on LPS-induced sepsis-related symptoms in PGIS KO mice was more striking than those in WT mice. In PGIS KO, selexipag treatment significantly relieved hypothermia at 12 h and 24 h, followed by a gradual recovery until the endpoint (Fig. 4 B). LPS-induced behavioral changes were attenuated in selexipag-pretreated PGIS KO mice at 3 h, 6 h and 12 h after LPS administration (Fig. 4 A). LPS-induced diarrhea was also suppressed (Fig. 4 C), and the mortality rate of LPS-administered PGIS KO mice has improved by selexipag administration (Fig. 4 E). Discussion In this study, we used genetic and pharmacological approaches to show that PGIS-derived PGI 2 -IP signaling suppresses LPS-induced sepsis-related symptoms. PGIS deficiency exacerbated the sepsis-related symptoms, which the IP agonist conversely alleviated. It has been indicated that PGIS-derived PGI 2 is an ambivalent regulator of inflammatory reactions. We previously reported that PGIS deficiency suppressed hemorrhagic cystitis, contact dermatitis, and acetic acid-induced writhing reaction [ 11 , 12 , 21 ]. On the other hand, IP deficiency augmented acute lung injury and allergic lung inflammation [ 22 – 24 ]. However, the anti-inflammatory functions of PGIS have not been fully elucidated. This is the first report to show the anti-inflammatory roles of PGIS in vivo . Ipseiz et al . demonstrated that the transcription factor Gata6 controlled PGIS expression, and PGIS-derived PGI 2 suppressed the LPS-induced proinflammatory IL-1b production via IL-10 induction in resident peritoneal MFs [ 13 ]. We here found that mouse peritoneal resident MFs produced PGI 2 without stimulation and that there was a large amount of PGI 2 in the peritoneal cavity of nontreated WT mice (Fig. 2 ). Resident MFs produced much more PGI 2 in response to proinflammatory stimuli. While an LPS-induced increase in PGI 2 was not observed throughout the entire cavity, it is possible that the production of PGI 2 might be locally increased to suppress inflammatory reactions in response to LPS. Both constitutive and LPS-induced PGI 2 productions were absent in PGIS KO mice. As a results, PGIS KO mice may exhibit excessive inflammatory responses to LPS and then die. It was noteworthy that PGIS deficiency increased LPS-induced gene expression levels of proinflammatory cytokines such as TNF-a and IL-6, as well as CXCL1 chemokine, in peritoneal cells while also increasing neutrophil infiltration into the peritoneal cavity. Oppositely, in murine hemorrhagic cystitis, PGIS gene deletion suppressed the expressions levels of proinflammatory cytokines and chemokines and neutrophil infiltration. These differences might arise from differences in the types of cells expressing the IP receptor involved in each inflammatory response. Additional studies aimed at identifying the sources and targets of PGI 2 are needed to elucidate the precise mechanism by which PGIS-derived PGI 2 -IP signaling suppresses sepsis-related symptoms. As described above, several reports using experimental animal models demonstrated that each prostanoids has a different role in the formation of sepsis symptoms. Our study indicated that PGIS-derived PGI 2 has an inhibitory effect on sepsis, similar to PGE 2 . Furthermore, it demonstrated that selexipag, an IP agonist, effectively suppresses sepsis-related symptoms. Selexipag, known as a novel IP receptor stimulator, has a milder impact on other PG receptors compared to PGI 2 analogs like beraprost and ilopstost [ 25 , 26 ]. Selexipag is extensively utilized for the treatment of pulmonary hypertension and has been shown to inhibit LPS-induced lung damage [ 17 , 27 ]. Our study indicated that selexipag might be safely used for the treatment of sepsis. Abbreviations COX cyclooxygenase HPLC high performance liquid chromatography IP PGI 2 receptor i.p. intraperitoneal KO knock out LC-ESI-MS liquid chromatography-electrospray ionization-mass spectrometry LPS lipopolysaccharide LT leukotriene MF macrophage MPO myeloperoxidase MRM multiple-reaction monitoring NSAIDs non-steroidal anti-inflammatory drugs PG prostaglandin PGI 2 prostacyclin PGIS PGI 2 synthase qRT-PCR quantitative RT-PCR TNF tumor necrosis factor TX thromboxane WT wild-type Declarations Conflict of Interest The authors state explicitly that there are no conflicts of interest in connection with this article. Author Contribution Tsubasa Ochiai, Hiroshi Kuwata, and Shuntaro Hara designed the experiments for this study. Tsubasa Ochiai, Toshiya Honsawa and Keishi Yamaguchi performed the experiments, analyzed the results, and made the figures. Tsubasa Ochiai and Hiroshi Kuwata adjusted LC-MS/MS. Chieko Yokohama generated and provided the PGIS KO mice. Yuka Sasaki participated in discussions and provided technical support. Tsubasa Ochiai and Shuntaro Hara wrote the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was supported by JSPS KAKENHI Grants-in-Aid for Scientific Research (B) (No. 19H03375 to S.H.), and for Early Career Scientists (No. 22K15283 to T.O.) from the Japan Society for the Promotion of Science, and by Grants-in-Aid for Scientific Research on Innovative Areas (Nos. 23116515 and 25116720 to S.H.) from the Ministry of Education, Sports, Science, Culture, and Technology of Japan. References van der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG. The immunopathology of sepsis and potential therapeutic targets. Nat Rev Immunol. 2017;17:407–20; https://doi.org/10.1038/nri.2017.36 Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the global burden of disease study. 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The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta. 2000;1483:285 – 93; https://doi.org/10.1016/s1388-1981(99)00164-x Sitbon O, Channick R, Chin KM, et al. Selexipag for the treatment of pulmonary arterial hypertension. N Engl J Med. 2015;373(26):2522–33; https://doi.org/10.1056/NEJMoa1503184 Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Apr, 2024 Reviews received at journal 23 Mar, 2024 Reviewers agreed at journal 19 Mar, 2024 Reviewers invited by journal 18 Mar, 2024 Editor assigned by journal 26 Feb, 2024 Submission checks completed at journal 26 Feb, 2024 First submitted to journal 20 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3972516","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274969675,"identity":"e0ddd52b-282d-42b8-acb9-7ad1c9eeceaf","order_by":0,"name":"Tsubasa Ochiai","email":"","orcid":"","institution":"Showa University","correspondingAuthor":false,"prefix":"","firstName":"Tsubasa","middleName":"","lastName":"Ochiai","suffix":""},{"id":274969676,"identity":"618530ef-8622-4625-8256-48c0ba954958","order_by":1,"name":"Toshiya Honsawa","email":"","orcid":"","institution":"Showa University","correspondingAuthor":false,"prefix":"","firstName":"Toshiya","middleName":"","lastName":"Honsawa","suffix":""},{"id":274969677,"identity":"faade414-1c55-42c3-9d03-6faa5526e9f1","order_by":2,"name":"Keishi Yamaguchi","email":"","orcid":"","institution":"Showa University","correspondingAuthor":false,"prefix":"","firstName":"Keishi","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":274969678,"identity":"1e3c4325-a7dc-448a-892e-dfa87de51d0f","order_by":3,"name":"Yuka Sasaki","email":"","orcid":"","institution":"Showa University","correspondingAuthor":false,"prefix":"","firstName":"Yuka","middleName":"","lastName":"Sasaki","suffix":""},{"id":274969679,"identity":"bec31cf9-64f5-488b-88e9-e6a01724d64a","order_by":4,"name":"Chieko Yokoyama","email":"","orcid":"","institution":"Kanagawa Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chieko","middleName":"","lastName":"Yokoyama","suffix":""},{"id":274969680,"identity":"d750b468-0bb1-4bb5-9452-490c848e185d","order_by":5,"name":"Hiroshi Kuwata","email":"","orcid":"","institution":"Showa University","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Kuwata","suffix":""},{"id":274969681,"identity":"04f48631-1036-4bb8-94f3-fd91dddf0b66","order_by":6,"name":"Shuntaro Hara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBACAzBZwZAAphkbEDLM+LWcIVkLYxsWLTiBOfvZhx9/zruTxz/7dALjzx02if0NPAYMP2oY2M1xaLHsSTeW5t32rFjiXO4GZt4zaYkzDvAYMPYcY2C2xGGlwYE0BmnGbYcTG87wbmBmbDuc23D/jQEDbwMDs8EBHFrOP2P++XPO4cT5QC2MP4Fa5oNs+YtPy400NgnehsOJG4BaGHiBWjYAtTDjteXGMzZrnmPPEjcCtRzmbUur33iAreCwzDEJ3H45n8Z880fNncR5Z3g3PvzZZmMsd4B548M3NTbJuEIMCg4gkRCGRLIBMVpQgB0BLaNgFIyCUTByAABU+WCDyu6jOwAAAABJRU5ErkJggg==","orcid":"","institution":"Showa University","correspondingAuthor":true,"prefix":"","firstName":"Shuntaro","middleName":"","lastName":"Hara","suffix":""}],"badges":[],"createdAt":"2024-02-20 10:34:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3972516/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3972516/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51730769,"identity":"8196a56b-d888-4653-a23a-5ee604a5a62a","added_by":"auto","created_at":"2024-02-28 04:38:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGIS deficiency exacerbated LPS-induced sepsis-related symptoms \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Rectal temperature of mice up to day 7 after LPS administration. Mice were divided into four groups: a control group of WT and PGIS KO mice, and LPS-administered groups of WT and PGIS KO mice. \u003cstrong\u003e(B)\u003c/strong\u003e Evaluation of behavioral changes caused by LPS administration in WT and PGIS KO mice. \u003cstrong\u003e(C) \u003c/strong\u003eDiarrhea assessment 1 h after LPS administration in WT and PGIS KO mice. \u003cstrong\u003e(D)\u003c/strong\u003e Survival rate of WT and PGIS KO mice up to 7 days after LPS administration. Data are shown as the means±SEM. N.D., not detectable. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the groups indicated by lines. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the LPS-administered WT mice. Data were analyzed by the Kruskal–Wallis test with a Steel–Dwass multiple comparisons test. In the survival experiments, survival curves were compared by the Kaplan–Meier method and statistical analyses were performed by a log-rank test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/18957c07a4f0d5d8c4121715.png"},{"id":51730768,"identity":"868131af-4eec-424a-9586-4b817e3c74dc","added_by":"auto","created_at":"2024-02-28 04:38:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProstanoid production in the peritoneal cavity and peritoneal resident MFs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) quantification of 6-keto-PGF\u003csub\u003e1a\u003c/sub\u003e (a stable metabolite of PGI\u003csub\u003e2\u003c/sub\u003e), TXB\u003csub\u003e2\u003c/sub\u003e (a stable metabolite of TXA\u003csub\u003e2\u003c/sub\u003e), PGE\u003csub\u003e2\u003c/sub\u003e, PGD\u003csub\u003e2\u003c/sub\u003e, and PGF\u003csub\u003e2a\u003c/sub\u003e in the peritoneal fluid of vehicle- or LPS-administered WT and PGIS KO mice.\u003cstrong\u003e (B)\u003c/strong\u003e \u003cem\u003ePtgis, Ptgs1\u003c/em\u003e, and \u003cem\u003ePtgs2\u003c/em\u003e mRNA expression levels in the peritoneal cells from WT and PGIS KO mice were analyzed by quantitative qRT-PCR. \u003cstrong\u003e(C) \u003c/strong\u003eAnalysis of prostanoids production from peritoneal resident MFs of WT and PGIS KO mice by LC-ESI-MS. Data are shown as the means±SEM. N.D., not detectable. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the groups indicated by lines. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 compared to the vehicle-administered WT mice. Data were analyzed by one-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/c1097f91a424c1d0eeb21901.png"},{"id":51731014,"identity":"0123de4a-13bd-4c9d-bbff-6935a728d9a1","added_by":"auto","created_at":"2024-02-28 04:46:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of LPS-induced inflammation-related genes was upregulated in PGIS-deficient mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e mRNA expression levels of proinflammatory cytokines (\u003cem\u003eTnf\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e) in peritoneal cells from saline- or LPS-administered WT mice and PGIS KO mice were analyzed using quantitative qRT-PCR. (\u003cstrong\u003eB\u003c/strong\u003e) mRNA expression levels of\u003cem\u003eMpo\u003c/em\u003e and \u003cem\u003eCxcl1\u003c/em\u003e in the peritoneal cells from saline- or LPS-administered WT mice and PGIS KO mice were analyzed by qRT-PCR. Data are shown as the means±SEM. N.D., not detectable. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the groups indicated by lines. Data were analyzed by the Kruskal–Wallis test with a Steel–Dwass multiple comparisons test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/ca20e52813c9df319c77c002.png"},{"id":51730772,"identity":"a8ca48d6-86bd-4583-a43a-c0513a248b10","added_by":"auto","created_at":"2024-02-28 04:38:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with selexipag, an IP agonist, improved LPS-induced sepsis-related symptoms.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eEvaluation of LPS-induced behavioral change in mice treated with or without selexipag at 3 h, 6 h, and 12 h after LPS administration.\u003cstrong\u003e (B)\u003c/strong\u003e Rectal temperatures of mice treated with selexipag up to day 7 after LPS administration. Mice were divided into four groups: LPS-administered groups of WT and PGIS KO mice and LPS+selexipag-administered groups of WT and PGIS KO mice.\u0026nbsp; \u003cstrong\u003e(C)\u003c/strong\u003e Diarrhea assessment 6 h after LPS stimulation in WT and PGIS KO mice treated with or without selexipag. \u003cstrong\u003e(D)\u003c/strong\u003e Survival rate of WT mice treated with or without selexipag up to 7 days after LPS administration.\u003cstrong\u003e (E)\u003c/strong\u003e Survival rate of PGIS KO mice treated with or without selexipag up to 7 days after LPS administration. Data are shown as the means±SEM. N.D., not detectable. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the groups indicated by lines in Fig. A and C or compared to the PGIS KO mice with selexipag in Fig. B. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the WT mice without selexipag in Fig B. Data were analyzed by the Kruskal–Wallis test with a Steel–Dwass multiple comparisons test. In the survival experiments, survival curves were compared by the Kaplan–Meier method and statistical analyses were performed by a log-rank test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/29ba336b0b9b923b9d525b16.png"},{"id":51731247,"identity":"5d8e361d-c51c-4e9a-b796-b25413376ebf","added_by":"auto","created_at":"2024-02-28 04:54:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":963551,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/6bfbf32d-aa6c-4beb-ac83-e9c4fffb39a9.pdf"},{"id":51730771,"identity":"8dc2238e-868d-4719-890f-46d4096511f7","added_by":"auto","created_at":"2024-02-28 04:38:49","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10127,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3972516/v1/131b899257e372d96f2f98be.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prostacyclin synthase deficiency exacerbates inflammatory reactions in lipopolysaccharide-induced sepsis in vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is defined as life-threatening organ dysfunction due to a dysregulated host response to infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Sepsis-related deaths decreased by roughly 52.8% from 1990 to 2017, but in 2017, there were still about 11\u0026nbsp;million sepsis-related deaths, representing 19.7% of global deaths [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thus, sepsis is even now one of the leading causes of death in the world. Common signs and symptoms of sepsis include fever, increased heart rate, increased breathing rate and confusion. Immune dysfunction, which in sepsis involves excessive inflammation, activation of the complement system, coagulation and immune suppression, during sepsis contributes to these symptoms. It has been reported that overproduction of proinflammatory mediators in response to infection plays a critical role in sepsis. Pinsly \u003cem\u003eet al.\u003c/em\u003e reported that in sepsis patients, substantial amounts of proinflammatory cytokines, including tumor necrosis factor (TNF) α, are released into the bloodstream, further fueling the progression of sepsis. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverproduction of prostanoids, which are proinflammatory lipid mediators, has also been detected in the peripheral blood of septic patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Prostanoids, including prostaglandin (PG) E\u003csub\u003e2\u003c/sub\u003e, PGF\u003csub\u003e2α\u003c/sub\u003e, prostacyclin (PGI\u003csub\u003e2\u003c/sub\u003e), and thromboxane A\u003csub\u003e2\u003c/sub\u003e (TXA\u003csub\u003e2\u003c/sub\u003e), are oxygenated metabolites of arachidonic acid produced by sequential catalysis of cyclooxygenase (COX) and specific PG terminal synthases. Widely used nonsteroidal anti-inflammatory drugs (NSAIDs) exert their pharmacologic effects, including anti-inflammatory effects, by reducing prostanoid production via inhibition of COX [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. NSAIDs are used to treat various inflammatory diseases, but no reports have conclusively demonstrated that NSAIDs are applicable to clinical use for the treatment of human sepsis. On the contrary, it has been reported that administration of NSAIDs to sepsis patients was associated with increased 28-day mortality [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Several reports using experimental animal models demonstrated that PGF\u003csub\u003e2a\u003c/sub\u003e/PGD\u003csub\u003e2\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e have opposing roles in the formation of sepsis symptoms. Maehara \u003cem\u003eet al\u003c/em\u003e. reported that inhibition of PGF\u003csub\u003e2a\u003c/sub\u003e receptors attenuated LPS-induced systemic inflammation in mice via enhanced IL-10 production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ishii \u003cem\u003eet al\u003c/em\u003e. showed that gene deletion of CRTH2, a PGD\u003csub\u003e2\u003c/sub\u003e receptor, improved lethality in a murine sepsis model by decreasing proinflammatory cytokine production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On the other hand, Choudhry \u003cem\u003eet al\u003c/em\u003e. reported that PGE\u003csub\u003e2\u003c/sub\u003e has negative roles in sepsis through inhibition of T cells activity by attenuating Ca\u003csup\u003e2+\u003c/sup\u003e signaling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Unlike NSAIDs, targeting the inhibition of PG terminal synthase, which can selectively suppress specific PG species, may lead to the development of therapeutic strategies for sepsis.\u003c/p\u003e \u003cp\u003ePGI\u003csub\u003e2\u003c/sub\u003e is produced by PGI synthase (PGIS), one of PG terminal synthases, that is highly and constitutively expressed in vascular endothelial and smooth muscle cells. COX-2/PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e has been shown to be crucial for the regulation of platelet aggregation and vascular tone [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, we previously demonstrated that PGIS has a proinflammatory function in several disease models using PGIS gene knockout (KO) mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. On the other hand, other research groups have reported that PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e has an anti-inflammatory function [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, PGIS has been shown to be an ambivalent regulator of inflammatory reactions, but the role of PGIS in sepsis is not fully understood. To investigate the possibility of PGIS and PGI\u003csub\u003e2\u003c/sub\u003e serving therapeutic targets for sepsis, we here examined the effects of PGIS deficiency on sepsis using a lipopolysaccharide (LPS)-induced mouse sepsis model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMale 8- to 10-week-old C57BL/6 mice were used. PGIS KO mice were described previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mice were bred in climate-controlled rooms (23\u0026deg;C) under specific pathogen-free conditions on a 12-h light/dark cycle at the Institute of Laboratory Animals of Showa University (Tokyo, Japan). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Showa University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLPS-induced sepsis model\u003c/h2\u003e \u003cp\u003eSepsis was induced by a single intraperitoneal (i.p.) injection of LPS (5 mg/kg, Sigma Aldrich (St. Louis, MO, USA); lipopolysaccharides from \u003cem\u003eEscherichia coli\u003c/em\u003e 0111: B4) dissolved in saline [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The control group received the same volume of saline. Rectal temperatures, body weight, diarrhea, and shivering of the mice were monitored before LPS administration and until day 7 after LPS administration using an animal temperature recorder (A \u0026amp; D Company (Tokyo, Japan); KN-91-AD1687). LPS-induced diarrhea was scored using a previously reported protocol as follows: 0, normal; 1, very mild diarrhea; 2, mild diarrhea; 3, diarrhea; 4, watery diarrhea [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Behavioral assessments were scored with slight modifications from the previously described protocol as follows: 0, unchanged; 1, walking with a slight stagger; 2, walking with a stagger; 3, immobility; 4, shivering [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Intraperitoneal fluid was collected with 4 mL of 2% fetal calf serum (FCS)-containing phosphate-buffered saline (PBS). Selexipag, a selective PGI\u003csub\u003e2\u003c/sub\u003e receptor (IP) agonist (1 mg/kg, Selleck Chemicals (Houston, TX, USA)) was dissolved in 1% DMSO containing saline, orally administered (p.o.) 2 h before LPS injection, and given every 12 h for 3 days. Mice in the selexipag control group received the same volume of 1% DMSO containing saline [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eResident peritoneal macrophages (MFs) isolation and culture\u003c/h2\u003e \u003cp\u003eResident peritoneal MFs were obtained via peritoneal washing with 4 mL of of 2% FCS-containing PBS. Cells were plated and left to adhere for 3 h at 37\u0026deg;C, in a 5% CO\u003csub\u003e2\u003c/sub\u003e environment using RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin and 2 mM L-glutamine (Thermo Fisher Scientific (Rockford, IL, USA)). Adhered cells were washed twice with PBS and further stimulated with 1 \u0026micro;M A23187 (Abcam) containing 0.1% DMSO for 1 h or 10 ng/mL LPS for 3 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). The resultant RNA was spectrophotometrically qualified and quantified at 260 nm and 280 nm using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Thermal cycling was performed using a Step One Plus real-time PCR system (Thermo Fisher Scientific). For each sample, real-time qRT-PCR was then performed using the SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a total volume of 10 \u0026micro;l, at 95\u0026deg;C for 15 sec and at 60\u0026deg;C for 45 min. The target amplicon was detected within the range of 10 to 40 cycles for all primers. PCR efficiency was examined by serially diluting the template cDNA, and the melting curve data were collected to check the PCR specificity. The expression of candidate genes in each group of mice was normalized to the \u003cem\u003eGapdh\u003c/em\u003e RNA level. The results were quantified using the relative standard curve method. Details on the primers details are provided in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of PGs by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)\u003c/h2\u003e \u003cp\u003eSamples were prepared according to a previously described methodology with minor modification [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, to prepare samples, 750 \u0026micro;L of 0.03% formic acid containing ultrapure water and an internal standard (25 pg of leukotriene (LT) B\u003csub\u003e4\u003c/sub\u003e-d\u003csub\u003e4\u003c/sub\u003e, Cayman (Ann Arbor, MI, USA)) were added to the each 250 \u0026micro;L aliquot of the sample. The prepared samples were then passed through an OASIS cartridge (Waters (Milford, MA, USA)) to obtain the PG fraction. The purified PG fraction was dried with a rotary evaporator. The resulting residues were dissolved in 50 \u0026micro;L mobile phase A (ultrapure water/acetonitrile/formic aced\u0026thinsp;=\u0026thinsp;63/37/0.02) and injected into the LC-ESI-MS system to quantify PGs with mobile phase A and mobile phase B (acetonitrile/isopropanol\u0026thinsp;=\u0026thinsp;50/50). To quantify PGs, LC-ESI-MS-based analysis was performed on a Prominence high performance liquid chromatography (HPLC) system (Shimadzu (Kyoto, Japan)) coupled with a QTRAP 5500 hybrid triple-quadrupole linear ion-trap mass spectrometer (AB SCIEX (Framingham, MA, USA)). PGs were subsequently analyzed via multiple-reaction monitoring (MRM) in negative-ion mode, as described previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Comparisons among all experimental groups were performed using one-way analysis of variance (ANOVA) followed by the Tukey‒Kramer post hoc test (dependent on the results of Levene\u0026rsquo;s test to determine the equality of variances) or a Kruskal\u0026ndash;Wallis test with a Steel‒Dwass multiple comparisons test (if the data were not normally distributed). Survival curves were compared by the Kaplan‒Meier method, and statistical analyses in the survival experiments were performed by a log-rank test. All statistical analyses were performed using JMP Pro version 16 (SAS Institute Inc. (Cary, NC, USA)). The level of significance is indicated in each figure (i.e., **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePGIS deficiency exacerbated sepsis-related symptoms caused by LPS\u003c/h2\u003e \u003cp\u003eSince it has been reported that administration of LPS causes sepsis-related symptoms such as hypothermia [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we first intraperitoneally injected LPS into WT and PGIS KO mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, in WT mice, rectal temperature began to decrease at 1 h, peaked at 24 h and gradually recovered by day 7 after LPS administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, LPS-induced changes in mouse movement, such as shivering, were also exacerbated along with decreased body temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). LPS-induced diarrhea was observed at 1 h after LPS injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These LPS-induced symptoms were more severe in PGIS KO mice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). PGIS deficiency also exacerbated the reduction in rectal temperature of PGIS KO mice. The peak of hypothermia occurred earlier in PGIS KO mice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, whereas 95% or more of WT survived 72 h after LPS administration, all of the PGIS KO mice died by then (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePGI\u003csub\u003e2\u003c/sub\u003e production in peritoneal cavity of nontreated and LPS-administered mice\u003c/h2\u003e \u003cp\u003eWe next analyzed prostanoid production in the peritoneal cavity of nontreated and LPS-administered mice using LC-MS/MS. 6-keto-PGF\u003csub\u003e1a\u003c/sub\u003e, a stable metabolite of PGI\u003csub\u003e2\u003c/sub\u003e, was the most detected prostanoids, followed by TXB\u003csub\u003e2\u003c/sub\u003e (a stable metabolite of TXA\u003csub\u003e2\u003c/sub\u003e), PGE\u003csub\u003e2\u003c/sub\u003e, PGD\u003csub\u003e2\u003c/sub\u003e, and PGF\u003csub\u003e2a\u003c/sub\u003e in the peritoneal fluid of nontreated WT mice. At 3 h after LPS administration, PGE\u003csub\u003e2\u003c/sub\u003e production was significantly increased, whereas PGI\u003csub\u003e2\u003c/sub\u003e production tended to be decreased in WT mice. In PGIS KO mice, 6-keto-PGF\u003csub\u003e1a\u003c/sub\u003e was not detected, prostanoid production was shunted, and the production of TXB\u003csub\u003e2\u003c/sub\u003e, PGE\u003csub\u003e2\u003c/sub\u003e, PGD\u003csub\u003e2\u003c/sub\u003e, and PGF\u003csub\u003e2a\u003c/sub\u003e was increased compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We further investigated the expression of genes involved in prostanoid biosynthesis in the peritoneal cells by qRT-PCR analysis. Expression of the \u003cem\u003ePtgis\u003c/em\u003e (PGIS) gene was downregulated in WT mice following LPS administration. PGIS-expressing resident cells may decrease in response to LPS stimulation. \u003cem\u003ePtgs1\u003c/em\u003e (COX-1) gene expression was also downregulated, but \u003cem\u003ePtgs2\u003c/em\u003e (COX-2) gene expression was up-regulated after LPS administration. The upregulation of COX was increased in PGIS KO mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next investigated the prostanoid production from peritoneal resident MFs \u003cem\u003ein vitro\u003c/em\u003e. Among the prostanoids detected, 6-keto-PGF\u003csub\u003e1a\u003c/sub\u003e was the most prominently observed in MFs of WT mice, and its production was increased upon stimulation by both Ca\u003csup\u003e2+\u003c/sup\u003e-ionophore A23187 and LPS. In contrast, 6-keto-PGF\u003csub\u003e1a\u003c/sub\u003e was not detected in culture medium from MFs of PGIS KO mice with or without A23187 or LPS. In response to LPS stimulus, prostanoid production was shunted and the productions of PGE\u003csub\u003e2\u003c/sub\u003e, TXB\u003csub\u003e2\u003c/sub\u003e, PGF\u003csub\u003e2a\u003c/sub\u003e, and PGD\u003csub\u003e2\u003c/sub\u003e was increased in PGIS KO MFs compared to WT MFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePGIS deficiency increased sepsis-related inflammatory responses in the peritoneal cavity\u003c/h2\u003e \u003cp\u003eWe further investigated the effects of PGIS deficiency on LPS-induced inflammation-related gene expression in the peritoneal cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the gene expressions levels of proinflammatory cytokines, including \u003cem\u003eTnf\u003c/em\u003e (TNF-α) and \u003cem\u003eIl6\u003c/em\u003e (IL-6), were elevated at 12 h after LPS administration in peritoneal cells from WT mice, and the increases in these gene expressions were upregulated by PGIS deficiency. LPS-induced induction of myeloperoxidase (MPO), which is specifically expressed in neutrophils, and CXCL1, a chemokine related to neutrophil migration, was also enhanced in PGIS KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In PGIS KO mice, LPS-induced neutrophil migration into the peritoneal cavity might be facilitated, leading to the exacerbation of LPS-induced sepsis-related symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIP agonist treatment improved LPS-induced sepsis-related symptoms and lethality in PGIS KO mice\u003c/h2\u003e \u003cp\u003eWe next pretreated WT or PGIS KO mice with selexipag, a selective agonist for the PGI\u003csub\u003e2\u003c/sub\u003e receptor (IP), to reveal the effects of IP stimulation on LPS-induced sepsis-related symptoms. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, selexipag treatment significantly palliated LPS-induced sepsis-related symptoms in WT mice. Selexipag significantly improved hypothermia at 12 h after LPS administration in WT mice. LPS-induced behavioral change such as shivering was also suppressed by selexipag at 12 h after LPS administration in WT mice. The effects of selexipag on LPS-induced sepsis-related symptoms in PGIS KO mice was more striking than those in WT mice. In PGIS KO, selexipag treatment significantly relieved hypothermia at 12 h and 24 h, followed by a gradual recovery until the endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). LPS-induced behavioral changes were attenuated in selexipag-pretreated PGIS KO mice at 3 h, 6 h and 12 h after LPS administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). LPS-induced diarrhea was also suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and the mortality rate of LPS-administered PGIS KO mice has improved by selexipag administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used genetic and pharmacological approaches to show that PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e-IP signaling suppresses LPS-induced sepsis-related symptoms. PGIS deficiency exacerbated the sepsis-related symptoms, which the IP agonist conversely alleviated. It has been indicated that PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e is an ambivalent regulator of inflammatory reactions. We previously reported that PGIS deficiency suppressed hemorrhagic cystitis, contact dermatitis, and acetic acid-induced writhing reaction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. On the other hand, IP deficiency augmented acute lung injury and allergic lung inflammation [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the anti-inflammatory functions of PGIS have not been fully elucidated. This is the first report to show the anti-inflammatory roles of PGIS \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIpseiz \u003cem\u003eet al\u003c/em\u003e. demonstrated that the transcription factor Gata6 controlled PGIS expression, and PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e suppressed the LPS-induced proinflammatory IL-1b production via IL-10 induction in resident peritoneal MFs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We here found that mouse peritoneal resident MFs produced PGI\u003csub\u003e2\u003c/sub\u003e without stimulation and that there was a large amount of PGI\u003csub\u003e2\u003c/sub\u003e in the peritoneal cavity of nontreated WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Resident MFs produced much more PGI\u003csub\u003e2\u003c/sub\u003e in response to proinflammatory stimuli. While an LPS-induced increase in PGI\u003csub\u003e2\u003c/sub\u003e was not observed throughout the entire cavity, it is possible that the production of PGI\u003csub\u003e2\u003c/sub\u003e might be locally increased to suppress inflammatory reactions in response to LPS. Both constitutive and LPS-induced PGI\u003csub\u003e2\u003c/sub\u003e productions were absent in PGIS KO mice. As a results, PGIS KO mice may exhibit excessive inflammatory responses to LPS and then die.\u003c/p\u003e \u003cp\u003eIt was noteworthy that PGIS deficiency increased LPS-induced gene expression levels of proinflammatory cytokines such as TNF-a and IL-6, as well as CXCL1 chemokine, in peritoneal cells while also increasing neutrophil infiltration into the peritoneal cavity. Oppositely, in murine hemorrhagic cystitis, PGIS gene deletion suppressed the expressions levels of proinflammatory cytokines and chemokines and neutrophil infiltration. These differences might arise from differences in the types of cells expressing the IP receptor involved in each inflammatory response. Additional studies aimed at identifying the sources and targets of PGI\u003csub\u003e2\u003c/sub\u003e are needed to elucidate the precise mechanism by which PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e-IP signaling suppresses sepsis-related symptoms.\u003c/p\u003e \u003cp\u003eAs described above, several reports using experimental animal models demonstrated that each prostanoids has a different role in the formation of sepsis symptoms. Our study indicated that PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e has an inhibitory effect on sepsis, similar to PGE\u003csub\u003e2\u003c/sub\u003e. Furthermore, it demonstrated that selexipag, an IP agonist, effectively suppresses sepsis-related symptoms. Selexipag, known as a novel IP receptor stimulator, has a milder impact on other PG receptors compared to PGI\u003csub\u003e2\u003c/sub\u003e analogs like beraprost and ilopstost [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Selexipag is extensively utilized for the treatment of pulmonary hypertension and has been shown to inhibit LPS-induced lung damage [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our study indicated that selexipag might be safely used for the treatment of sepsis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclooxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePGI\u003csub\u003e2\u003c/sub\u003e receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.p.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eknock out\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-ESI-MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliquid chromatography-electrospray ionization-mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eleukotriene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emacrophage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emyeloperoxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultiple-reaction monitoring\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSAIDs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-steroidal anti-inflammatory drugs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprostaglandin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGI\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprostacyclin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePGI\u003csub\u003e2\u003c/sub\u003e synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative RT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethromboxane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors state explicitly that there are no conflicts of interest in connection with this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTsubasa Ochiai, Hiroshi Kuwata, and Shuntaro Hara designed the experiments for this study. Tsubasa Ochiai, Toshiya Honsawa and Keishi Yamaguchi performed the experiments, analyzed the results, and made the figures. Tsubasa Ochiai and Hiroshi Kuwata adjusted LC-MS/MS. Chieko Yokohama generated and provided the PGIS KO mice. Yuka Sasaki participated in discussions and provided technical support. Tsubasa Ochiai and Shuntaro Hara wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by JSPS KAKENHI Grants-in-Aid for Scientific Research (B) (No. 19H03375 to S.H.), and for Early Career Scientists (No. 22K15283 to T.O.) from the Japan Society for the Promotion of Science, and by Grants-in-Aid for Scientific Research on Innovative Areas (Nos. 23116515 and 25116720 to S.H.) from the Ministry of Education, Sports, Science, Culture, and Technology of Japan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003evan der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG. The immunopathology of sepsis and potential therapeutic targets. 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Br J Pharmacol. 2002;137:315\u0026thinsp;\u0026ndash;\u0026thinsp;22; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.bjp.0704872\u003c/span\u003e\u003cspan address=\"10.1038/sj.bjp.0704872\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuwano K, Hashino A, Asaki T, et al. 2-[4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy]-N-(methylsulfonyl)acetamide (NS-304), an orally available and long-acting prostacyclin receptor agonist prodrug. J Pharmacol Exp Ther. 2007;322:1181\u0026ndash;8; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1124/jpet.107.124248\u003c/span\u003e\u003cspan address=\"10.1124/jpet.107.124248\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbramovitz M, Adam M, Boie Y, et al. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta. 2000;1483:285\u0026thinsp;\u0026ndash;\u0026thinsp;93; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s1388-1981(99)00164-x\u003c/span\u003e\u003cspan address=\"10.1016/s1388-1981(99)00164-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSitbon O, Channick R, Chin KM, et al. Selexipag for the treatment of pulmonary arterial hypertension. N Engl J Med. 2015;373(26):2522\u0026ndash;33; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMoa1503184\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa1503184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"prostacyclin, prostaglandin terminal synthase, lipopolysaccharide, sepsis, selexipag","lastPublishedDoi":"10.21203/rs.3.rs-3972516/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972516/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eSepsis is an inflammatory disorder characterized by life-threatening organ dysfunction resulting from a dysregulated host response to infection. In contrast, prostacyclin (PGI\u003csub\u003e2\u003c/sub\u003e) is a bioactive lipid produced by PGI synthase (PGIS) and is known to play important roles in inflammatory reactions as well as cardiovascular regulation. However, little is known about the roles of PGIS and PGI\u003csub\u003e2\u003c/sub\u003e in sepsis.\u003c/p\u003e\u003ch2\u003eMethodology\u003c/h2\u003e \u003cp\u003eSepsis was induced by intraperitoneal injection of 5 mg/kg lipopolysaccharide (LPS) in wild type (WT) or PGIS knockout (KO) mice. Selexipag, a selective PGI\u003csub\u003e2\u003c/sub\u003e receptor (IP) agonist, was administered 2 h before LPS injection and again given every 12 h for 3 days.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIntraperitoneal injection of LPS induced diarrhea, shivering and hypothermia. These symptoms were more severe in PGIS KO mice than in WT mice. The expression of \u003cem\u003eTnf\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e genes was notably increased in PGIS KO mice. In contrast, over 95% of WT mice survived 72 h after the administration of LPS, whereas all of the PGIS KO mice had succumbed by that time. The mortality rate of LPS-administrated PGIS KO mice was improved by selexipag administration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study suggests that PGIS-derived PGI\u003csub\u003e2\u003c/sub\u003e negatively regulates LPS-induced symptoms via the IP receptor. Selexipag shows promise for the clinical treatment of human sepsis.\u003c/p\u003e","manuscriptTitle":"Prostacyclin synthase deficiency exacerbates inflammatory reactions in lipopolysaccharide-induced sepsis in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 04:38:45","doi":"10.21203/rs.3.rs-3972516/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-05T13:40:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-23T09:09:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4404784c-b686-455c-a651-77662e93900b","date":"2024-03-19T16:11:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-18T17:25:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-26T13:22:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-26T13:22:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation Research","date":"2024-02-20T10:32:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3af39a63-e092-47da-881b-29f6ddecd8b5","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-30T08:18:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 04:38:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3972516","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972516","identity":"rs-3972516","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}