IL-6 modulates sepsis-induced decreases in transcription of hepatic organic anion and bile acid transporters

Abstract

Sepsis, a lethal inflammatory syndrome, is characterized by organ system dysfunction. In the liver, we have observed decreased expression of genes encoding proteins modulating key processes. These include organic anion and bile acid transport. We hypothesized that the inflammatory mediator IL-6 modulates altered expression of several key hepatic genes in sepsis via induction of the intracellular transcription factor signal transducer and activator of transcription (Stat) 3. Sepsis was induced in IL-6 +/+ and IL-6 -/- mice, and expression of the liver-restricted genes encoding the sodium-taurocholate cotransporter (Ntcp), the multidrug resistant protein (MRP) 2 and the organic anion transporter protein (OATP), was determined. As demonstrated previously, cecal ligation and puncture decreases expression of Ntcp, MRP-2, and OATP in IL-6 +/+ mice. Transcription elongation analysis demonstrated that altered expression resulted from decreased transcription. These changes were not observed in IL-6 -/- animals. Cecal ligation and puncture increased the DNA binding activity of Stat-3 in IL-6 +/+ but not IL-6 -/- mice. Because the promoters of Ntcp, MRP-2, and OATP do not contain Stat-3 binding sites, we postulated that altered Ntcp, MRP-2, and OATP expression resulted from activation of hepatocyte nuclear factor (HNF) 1alpha, which is IL-6 dependent. Cecal ligation and puncture decreased HNF-1alpha expression and DNA binding activity in IL-6 +/+ but not IL-6 -/- mice. Recombinant human IL-6 restored the sepsis-induced decrease in Ntcp, MRP-2, OATP, and HNF-1alpha expression in IL-6 -/- mice. We conclude that sepsis decreases the expression of three key hepatic genes via a transcriptional mechanism that is IL-6, Stat-3, and HNF-1alpha dependent.

Fig. 1. Graphic representation of densitometrically determined steady-state mRNA levels after CLP

Data as mean ± SD. x axis indicates time after CLP; y axis, normalized density; filled squares, IL-6 +/+ mice; open squares, IL-6 +/+ mice administered rhIL-6; filled diamonds, IL-6 -/- mice; open diamonds, IL-6 -/- mice administered rhIL-6. Values were determined using Northern Blotting (see text). Blots were subjected to autoradiography and densitometry. Data for Ntcp, MRP-2, and OATP normalized to values for the constitutively transcribed β2M at the same time point. Values at time 0 were arbitrarily set at 100. For Ntcp, values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than T₀ at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for T₀ at 3, 6, 16, and 24 h. Values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, 16, and 24 h. For MRP-2, values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than T₀ at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for T₀ at 3, 6, and 16 h. Values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, and 16 h. For OATP, values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than T₀ at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for T₀ at 3, 6, and 16 h. Values for IL-6 +/+ and IL-6 +/+ + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, 16, 24, and 48 h. Values for IL-6 -/- + rhIL-6 were significantly different than values for IL-6 -/- at 3, 6, and 16 h. Differences within and between groups were analyzed using ANOVA with Bonferroni correction.

Fig. 2. Graphic representation of densitometrically determined transcription rates after CLP

Data as mean ± SD. x axis indicates time after CLP; y axis, normalized density; filled squares, IL-6 +/+ mice; filled diamonds, IL-6 -/- mice. Values were determined using blots from transcription elongation analysis (see text). Blots were subjected to autoradiography and densitometry. Data for Ntcp, MRP-2, and OATP were normalized to value for the constitutively transcribed β2M at the same time point. Values at time 0 were arbitrarily set at 100. * indicates significantly different than T₀. ^†Significantly different than IL-6 -/- at same time point. Differences within and between groups were analyzed using ANOVA with Bonferroni correction.

Fig. 3. Representative electrophoretic mobility shift assay for Stat-3 DNA binding in IL-6 +/+ and IL-6 -/- mice

Animals were subjected to either CLP or SO. Detection with a radiolabeled DNA sequence homologous to a consensus Stat-3 binding site. T₀ represents unoperated controls. Subscript indicates the time (T) in hours after CLP or SO. A, Five micrograms of liver nuclear extract from IL-6 +/+ mice was used in this assay. No Stat-3 DNA binding was detected after CLP or SO in IL-6 -/- mice (data not shown). B, Five micrograms of liver nuclear extract from IL-6 +/+ and IL-6 -/- mice was used. Some samples (indicated with “+”) were incubated with a monoclonal antibody to the phosphorylated form of Stat-3 (anti-pStat-3) (Santa Cruz Biotechnology) and, thus, “supershifted,” whereas others (indicated by “-”) were not. IL-6 -/- mice given rhIL-6 at the time of surgery are indicated by “+”.

Fig. 4. Representative electrophoretic mobility shift assay of HNF-1α DNA binding in IL-6 +/+ and IL-6 -/- mice

Animals were subjected to either CLP or SO. T₀ represents unoperated controls. Subscript indicates the time (T) in hours after CLP or SO. A, Seven micrograms of liver nuclear extract from IL-6 +/+ mice was used. The first well was loaded with a T₀ sample and “supershifted” via incubation with a polyclonal antibody to HNF-1α (Santa Cruz Biotechnology) as a control. B, Seven micrograms of liver nuclear extract from IL-6 -/- mice was used. Samples indicated with a “+”were incubated with a polyclonal antibody to HNF-1α (Santa Cruz Biotechnology) and, thus, supershifted. Lower bands indicate HNF-1α complex; upper bands, supershifted complex detected after antibody administration. C, Seven micrograms of liver nuclear extract from IL-6 -/- mice was used. “+” indicates that IL-6 -/- mice were injected with rhIL-6 at the time of surgery.

Fig. 5. Representative autoradiograms of Northern blots to determine steady-state mRNA levels of HNF-1α in IL-6 +/+ and IL-6 -/- mice after CLP and SO (top)

Graphic representation of densitometrically determined steady-state mRNA levels of HNF-1α in IL-6 +/+ and IL-6 -/- mice after CLP and SO (bottom). Data as mean ± SD. x axis indicates time in hours after CLP or SO; y axis, normalized density; solid squares, IL-6 +/+ mice subjected to CLP; solid diamonds, IL-6 -/- mice subjected to CLP; open squares, IL-6 +/+ mice subjected to SO; open diamonds, IL-6 -/- mice subjected to SO. Data for HNF-1α normalized to values for the constitutively expressed 18S ribosomal subunit at same time point. Values at time 0 were arbitrarily set at 100. Values for IL-6 +/+ mice subjected to CLP differed significantly from values for IL-6 -/- subjected to CLP and IL-6 +/+ subjected to SO at 6, 16, 24, and 48 h. Values for IL-6 +/+ subjected to CLP differed significantly from values for IL-6 -/- subjected to SO at 3, 6, 16, 24, and 48 h. Differences within and between groups were analyzed using ANOVA with Bonferroni correction.

Fig. 6. Representative immunoprecipitation/immunoblot of HNF-1α using IL-6 +/+ and IL -/- mice

Hepatocyte nuclear factor 1α was immunoprecipitated from 400 μg of liver nuclear extract. Some IL-6 -/- mice received rhIL-6. T₀ represents unoperated controls. Subscript indicates the time in hours after CLP. The negative control is a T₀ sample that was immunoprecipitated with an antibody not related to HNF-1α.