Introduction

Sepsis, the leading cause of death in the intensive care unit, is the host’s deleterious systemic inflammatory response to microbial infections. The caecal ligation and puncture (CLP) and treatment with lipopolysaccharide (LPS) are two commonly used sepsis models. In the CLP model, sepsis originates from a polymicrobial infection within the abdominal cavity1. Toll-like receptor 4 (TLR4) has been reported to contribute to bacterial clearance and the host inflammatory response in sepsis2. The bacterial LPS elicits its inflammatory actions through the TLR4, which will lead to the activation of nuclear factor-κB (NF-κB), a transcriptional factor that regulates a battery of inflammatory genes2.

The oestrogen sulfotransferase (EST or SULT1E1) is a cytosolic sulfotransferase best known for its activity in sulfonating and deactivating oestrogen, an anti-inflammatory hormone. This is because the sulfonated estrogens cannot bind to and activate the oestrogen receptor3. Consistent with the role of EST in oestrogen deactivation, EST ablation in mice resulted in structural and functional lesions in the testis4 and placenta5. The basal expression of hepatic EST is low, but its expression is highly inducible in response to ligands for several nuclear receptors6,7,8 and insulin resistance/type 2 diabetes25. Inflammation is also associated with many other diseases, including the sterile inflammation seen in various liver diseases, such as the drug-induced liver injury, non-alcoholic and alcoholic steatohepatitis, and liver ischaemia and reperfusion30. It is interesting to know whether sterile inflammation, such as that caused by the liver ischaemia and reperfusion, can also induce EST and impact the ischaemia and reperfusion responses. Future studies are also necessary to determine whether a chronic inflammation will result in a sustained induction of EST and if so, what will be the pathophysiological relevance and consequence of a sustained EST induction. The reciprocal regulation of inflammation and EST represents a yet-to-be-explored mechanism of endocrine regulation of inflammation, which may be explored to improve the clinical management of sepsis.

Methods

Animals and LPS treatment

The Tet-off EST TG system is composed of two transgenes, TetRE-EST and Lap-tTA. The creation of the TetRE-EST transgenic line was previously reported by us31. The liver-specific Lap-tTA transgenic line was obtained from the Jackson Laboratories (Bar Harbor, ME). Driven by the Lap gene promoter, the Lap-tTA targets the expression of tTA specifically to the hepatocytes32. The TetRE-EST/Lap-tTA bitransgenic mice were generated by crossbreeding. Transgenic mice and their wide-type littermates used in this study were maintained in C57BL/6J background. The EST−/− mice4 and the global TLR4−/− mice19 have been previously described. C3H/HeJ mice and C3H/HeOuJ mice were purchased from the Jackson Laboratories. LPS was dissolved in PBS and given by intraperitoneal (i.p.) injection. When necessary, mice were pretreated with 200 mg kg−1 PDTC by i.p. injection 1 h before challenging with 5 mg per kg LPS. The majority of the experiments were performed on mice of 8 weeks of age except those specified for ovariectomy (5 week old) and measurement of LPS effect on circulating oestrogen levels (4 week old). All mice used were females except those used in Fig. 4g. All mice used were 6–8 weeks old except those used for ovariectomy and measurement of circulating estrogens as described above. The use of mice in this study has complied with all relevant federal guidelines and institutional policies, and was approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Measurement of uterine oestrogen response

Five-week-old virgin females were subjected to ovariectomies. Mice were given a single subcutaneous injection of E2 (20 μg kg−1) 7 days after the surgery. Mice were given a single i.p. injection of bromodeoxyuridine (BrdU, 60 mg kg−1) 18 h after the E2 injection and killed after 2 h. One uterine horn was collected for paraffin section and BrdU immunostaining, and the other was collected for RNA extraction and gene expression analysis by real-time PCR. When necessary, mice were given an i.p. injection of LPS (5 mg kg−1) 12 h before the E2 treatment.

Oestrogen sulfotransferase activity assay

Liver cytosols were prepared by homogenizing tissues in 5 mmol l−1 KPO4 buffer (pH 6.5) containing 0.25 mol l−1 sucrose. The cytosols were then used for sulfotransferase assay by using [35S]-phosphoadenosine phosphosulfate from Perkin-Elmer (Waltham, MA) as the sulfate donor6. In brief, 20 μg ml−1 total liver cytosolic extract was incubated with 1 μM of oestrone substrate at 37 °C for 30 min. The reaction was terminated by adding ethyl acetate, and the aqueous phase was then counted in the Beckman LS6500 scintillation counter.

Measurement of serum and liver tissue oestrogen levels

The serum concentrations of oestradiol (E2) were measured using the Ultra-Sensitive Estradiol RIA kit (DSL-4800) from Beckman Coulter (Brea, CA). The liver tissue levels of oestrone (E1) and E2 were measured by a Ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS–MS) method with a Waters Acquity UPLC system connected with the Xevo TQ triple quadrupole mass spectrometer as we have previously described33,34. In brief, a Xevo TQ triple quadruple mass spectrometer (Waters, Milford, MA, USA) recorded MS and MS/MS spectra using electrospray ionization in positive-ion and negative-ion modes, capillary voltage of 3.0 kV, extractor cone voltage of 3 V and detector voltage of 650 V. Cone gas flow was set at 50 l h−1, and desolvation gas flow was maintained at 600 l h−1. Source temperature and desolvation temperatures were set at 150 and 350 °C, respectively. Analytical separations were conducted on the UPLC system using an Acquity UPLC HSS T3 1.8 μm 1 × 150 mm analytical column kept at 50 °C and at a flow rate of 0.15 ml min−1.

Kupffer cell isolation and drug treatment

The mouse primary Kupffer cells were isolated as described by others35. Briefly, the mouse liver was digested by collagenase perfusion. After removing the undigested tissue, the cell suspension was processed with gradient centrifugation to isolate Kupffer cells. After 2 h of culture, the non-adherent cells were removed and the remaining adherent cells were further cultured as Kupffer cells. When necessary, Kupffer cells were treated with LPS (1 μg ml−1), Pam3CSK4 (300 ng ml−1) or ODN1826 (10 μg ml−1) for 16 h before RNA collection and real-time PCR analysis. Pam3CSK4, ODN1826 and the control ODN were purchased from InvivoGen (San Diego, CA).

Real-time PCR analysis

Total RNA was extracted from tissues using the TRIzol reagent from Invitrogen. Reverse transcription was performed with iScript cDNA Synthesis Kit from Bio-Rad (Hercules, CA). SYBR Green-based real-time PCR was performed with the ABI 7300 Real-Time PCR System. Data were normalized against the control of cyclophilin signals. The mRNA expression levels in the control groups were arbitrarily set at 1. The sequences of the PCR primers are listed in Supplementary Table 1.

Western blot analysis

Mouse liver tissue was homogenized in radioimmunoprecipitation assay lysis buffer (50 mM Tris (pH 8.0), 150 mM sodium chloride, 1.0%Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). The protein concentrations in the supernatants were quantified, and 50 μg total proteins for each sample was separated on 10% SDS–polyacrylamide gel and transferred to a polyvinylidenedifluoride membrane. The membrane was blocked with 5% milk in 1 × Tris-Buffered Saline and Tween 20 (TBST) buffer followed by overnight incubation at 4 °C with primary polyclonal rabbit antibody against EST/SULT1E1 (Cat # 12522-1-AP; 1:500) from Proteintech (Chicago, IL) and then with a secondary antibody for 1 h at room temperature. After washing the membrane with 1 × TBST, the membrane was incubated with chemiluminescent substrate and exposed against the Kodak X-ray film. Monoclonal mouse antibody against β-actin (1:5,000) was used as a loading control. The original Western blots are shown in Supplementary Figure 5.

Caecal ligation and puncture model

This was performed essentially as described1. In brief, mice were anesthetized with a mixture of ketamine (150 mg kg−1) and xylazine (10 mg kg−1) by i.p. injection. Under aseptic conditions, a 2-cm midline laparotomy was performed to allow the exposure of the caecum with the adjoining intestine. The caecum was 50% ligated with a 4.0 silk suture at its base, below the ileocaecal valve, and was perforated twice with an 18-gauge needle (top and bottom). Twenty-one gauge needles were used in CLP survival experiments when the TG mice were used to avoid excessive injury. The caecum was then gently squeezed to extrude a small amount of faeces from the perforation sites. The caecum was then returned to the peritoneal cavity and the laparotomy was closed with 4.0 silk sutures. The fluid resuscitation was provided after CLP as described1. All mice were then returned to their home cages with free access to food and water, and killed 12 h after the surgery. When necessary, mice were given daily subcutaneous injections of triclosan (10 mg kg−1, Cat #72779 from Sigma) beginning 3 days before CLP. Also when necessary, mice were pretreated with PDTC (200 mg kg−1) by i.p. injection 1 h before CLP, or CLP was performed 7 days after ovariectomy. The bacterial count in the peritoneal fluid was measured as we have previously described2. In brief, the peritoneal cavity was washed with 1 ml PBS, and the peritoneal lavage was collected under sterile conditions. Peritoneal lavage fluid was subjected to serial 10-fold dilutions and cultured overnight in 5% sheep blood agar (Teknova, Hollister, CA). Colony-forming unit was quantified by manual counting.

Kupffer cell depletion

Eight-week-old mice were treated with saline or 20 mg per kg gadolinium chloride (GdCl3, 2 mg ml−1) from Sigma-Aldrich via tail vein injection. Twenty-four hours later, mice were again given the same dose of GdCl3 before being treated with 5 mg kg−1 of LPS by i.p. injection or subjected to CLP. Mice were killed 12 h after LPS or CLP treatment.

Cell culture and transfection and reporter gene assay

HepG2 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. The 2.5- and 1.5-kb 5′-regulatory sequences of the mouse EST gene were amplified by PCR using mouse genomic DNA as the template and subsequently cloned into the pGL3-basic vector from Clontech (Mountain View, CA). The mutant promoter reporter genes were generated by PCR-mediated mutagenesis. HepG2 cells were transiently transfected with the reporter constructs and the p65 expression vector (pCMV-p65) in 48-well plates by using the polyethylenimine polymer transfection agent36. Cells were then collected and measured for luciferase and β-gal activities 24 h after transfection. Transfection efficiency was normalized against β-gal activity derived from the co-transfected pCMX–β-gal plasmid. All EST promoter activities were normalized to EST 2.5-kb vector, which was arbitrarily set at 1.

EMSA and ChIP assay

The p65 protein was synthesized using the T7 Quick Coupled Transcription/Translation System in vitro transcription and translation system from Promega (Madison, WI). EMSA was performed by using 32P-labelled oligonucleotides and p65 protein6. In the ChIP assay, 4-week-old WT female mice received a single i.p. injection of LPS (5 mg kg−1) 12 h before liver collection. The ChIP assay was performed as we have previously described36. In brief, liver tissue lysates were incubated overnight with an anti-p65 antibody (Clone L8F6, Cat # 6956) from Cell Signaling (Danvers, MA) at 4 °C. The precipitated complexes were collected with protein A–agarose/salmon sperm DNA. DNA in the precipitated samples were reverse cross-linked at 65 °C for 4 h and the DNA were recovered by phenol/chloroform extraction and ethanol precipitation before subjecting to real-time PCR analysis. In the ChIP assay, the recruitment of p65 in the vehicle group was arbitrarily set at 1. The original EMSA blots are shown in Supplementary Figure 6.

Statistical analysis

When applicable, results are presented as means±s.d. Statistical analysis was performed using the student’s t-test for comparison between two groups. The Log-rank (Mantel–Cox) test was used to compare the survival profiles. P values of <0.05 were considered statistically significant.

Additional information

How to cite this article: Chai, X. et al. Oestrogen sulfotransferase ablation sensitizes mice to sepsis. Nat. Commun. 6:7979 doi: 10.1038/ncomms8979 (2015).