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

doi:10.21203/rs.3.rs-3972516/v1

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

https://doi.org/10.21203/rs.3.rs-3972516/v1

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Objectives

Sepsis is an inflammatory disorder characterized by life-threatening organ dysfunction resulting from a dysregulated host response to infection. In contrast, prostacyclin (PGI₂) 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 PGI₂ 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 PGI₂ 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 PGI₂ 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

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) E₂, PGF_2α, prostacyclin (PGI₂), and thromboxane A₂ (TXA₂), 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/PGD₂ and PGE₂ 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 PGD₂ receptor, improved lethality in a murine sepsis model by decreasing proinflammatory cytokine production [8]. On the other hand, Choudhry et al. reported that PGE₂ has negative roles in sepsis through inhibition of T cells activity by attenuating Ca²⁺ 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.

PGI₂ 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 PGI₂ 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 PGI₂ 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 PGI₂ serving therapeutic targets for sepsis, we here examined the effects of PGIS deficiency on sepsis using a lipopolysaccharide (LPS)-induced mouse sepsis model.

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 PGI₂ 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% CO₂ 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) B₄-d₄, 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).

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. 1A, 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. 1A). Furthermore, LPS-induced changes in mouse movement, such as shivering, were also exacerbated along with decreased body temperature (Fig. 1B). LPS-induced diarrhea was observed at 1 h after LPS injection (Fig. 1C). These LPS-induced symptoms were more severe in PGIS KO mice than in WT mice (Fig. 1B, 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. 1A). In addition, whereas 95% or more of WT survived 72 h after LPS administration, all of the PGIS KO mice died by then (Fig. 1D).

PGI₂ 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 PGI₂, was the most detected prostanoids, followed by TXB₂ (a stable metabolite of TXA₂), PGE₂, PGD₂, and PGF_2a in the peritoneal fluid of nontreated WT mice. At 3 h after LPS administration, PGE₂ production was significantly increased, whereas PGI₂ 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 TXB₂, PGE₂, PGD₂, and PGF_2a was increased compared to WT mice (Fig. 2A). 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. 2B).

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 Ca²⁺-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 PGE₂, TXB₂, PGF_2a, and PGD₂ was increased in PGIS KO MFs compared to WT MFs (Fig. 2C).

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. 3A, 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. 3B). 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 PGI₂ 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. 4B). LPS-induced behavioral changes were attenuated in selexipag-pretreated PGIS KO mice at 3 h, 6 h and 12 h after LPS administration (Fig. 4A). LPS-induced diarrhea was also suppressed (Fig. 4C), and the mortality rate of LPS-administered PGIS KO mice has improved by selexipag administration (Fig. 4E).

In this study, we used genetic and pharmacological approaches to show that PGIS-derived PGI₂-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 PGI₂ 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 PGI₂ 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 PGI₂ without stimulation and that there was a large amount of PGI₂ in the peritoneal cavity of nontreated WT mice (Fig. 2). Resident MFs produced much more PGI₂ in response to proinflammatory stimuli. While an LPS-induced increase in PGI₂ was not observed throughout the entire cavity, it is possible that the production of PGI₂ might be locally increased to suppress inflammatory reactions in response to LPS. Both constitutive and LPS-induced PGI₂ 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 PGI₂ are needed to elucidate the precise mechanism by which PGIS-derived PGI₂-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 PGI₂ has an inhibitory effect on sepsis, similar to PGE₂. 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 PGI₂ 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.

COX

cyclooxygenase

HPLC

high performance liquid chromatography

PGI₂ receptor

i.p.

intraperitoneal

knock out

LC-ESI-MS

liquid chromatography-electrospray ionization-mass spectrometry

LPS

lipopolysaccharide

leukotriene

macrophage

MPO

myeloperoxidase

MRM

multiple-reaction monitoring

NSAIDs

non-steroidal anti-inflammatory drugs

prostaglandin

PGI₂

prostacyclin

PGIS

PGI₂ synthase

qRT-PCR

quantitative RT-PCR

TNF

tumor necrosis factor

thromboxane

wild-type

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.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

Table1.xlsx

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You are reading this latest preprint version

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

Status:

Version 1

Abstract

Objectives

Methodology

Results

Conclusion

Figures

Introduction

Materials and Methods

Mice

LPS-induced sepsis model

Resident peritoneal macrophages (MFs) isolation and culture

Quantitative RT-PCR (qRT-PCR)

Measurement of PGs by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)

Statistics

Results

PGIS deficiency exacerbated sepsis-related symptoms caused by LPS

PGI₂ production in peritoneal cavity of nontreated and LPS-administered mice

PGIS deficiency increased sepsis-related inflammatory responses in the peritoneal cavity

IP agonist treatment improved LPS-induced sepsis-related symptoms and lethality in PGIS KO mice

Discussion

Abbreviations

Declarations

Conflict of Interest

Author Contribution

Acknowledgments

References

Table 1

Additional Declarations

Supplementary Files

Status:

Version 1

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

Status:

Version 1

Abstract

Objectives

Methodology

Results

Conclusion

Figures

Introduction

Materials and Methods

Mice

LPS-induced sepsis model

Resident peritoneal macrophages (MFs) isolation and culture

Quantitative RT-PCR (qRT-PCR)

Measurement of PGs by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)

Statistics

Results

PGIS deficiency exacerbated sepsis-related symptoms caused by LPS

PGI2 production in peritoneal cavity of nontreated and LPS-administered mice

PGIS deficiency increased sepsis-related inflammatory responses in the peritoneal cavity

IP agonist treatment improved LPS-induced sepsis-related symptoms and lethality in PGIS KO mice

Discussion

Abbreviations

Declarations

Conflict of Interest

Author Contribution

Acknowledgments

References

Table 1

Additional Declarations

Supplementary Files

Status:

Version 1

PGI₂ production in peritoneal cavity of nontreated and LPS-administered mice