Modulation of hepatic cytochrome P450s by Citrobacter rodentium infection in interleukin-6- and interferon-{gamma}-null mice

Abstract

After infection with Citrobacter rodentium, murine hepatic cytochrome P450 (P450) mRNAs are selectively regulated. Several serum proinflammatory cytokines are elevated, the most abundant being interleukin-6 (IL6). To elucidate the role of cytokines in the regulation of P450s during infection, we orally infected wild-type, IL6(-/-), or interferon-γ(-/-) [IFNγ(-/-)] female C57BL/6J mice with C. rodentium and analyzed hepatic P450 expression 7 days later. The majority of P450 mRNAs were equally affected by infection in each genotype, indicating that IL6 and IFNγ are not the primary mediators of P450 down-regulation in this disease model. The down-regulation of CYP3A11 and CYP3A13 and induction of CYP2D9 mRNAs were attenuated in the IL6(-/-) mice, suggesting a role of IL6 in the regulation of only these P450s. Similar evidence implicated IFNγ in the regulation of CYP2D9, CYP2D22, CYP3A11, CYP3A25, and CYP4F18 mRNAs in C. rodentium infection and CYP2B9, CYP2D22, and CYP2E1 in the bacterial lipopolysaccharide model of inflammation. This is the first indication of an in vivo role for IFNγ in hepatic P450 regulation in disease states. The deficiency of IL6 or IFNγ affected serum levels of the other cytokines. Moreover, experiments in cultured hepatocytes demonstrated that tumor necrosis factor α (TNFα) is the most potent and efficacious of the cytokines tested in the regulation of murine P450 expression. It is therefore possible that part of the IFNγ(-/-) and IL6(-/-) phenotypes could be attributed to the reduced levels of TNFα and part of the IFNγ(-/-) phenotype could be caused by reduced levels of IL6.

Fig. 1

Effect of oral C. rodentium infection on mRNA expression of hepatic P450s in WT, IL6(−/−), and IFNγ(−/−) mice. Over 24 h, mice were allowed to drink either sucrose solution (Ctrl) or C. rodentium in sucrose (Inf) then they were sacrificed after 7 days and livers were harvested for measurement of mRNA by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH, with the sucrose-treated (Ctrl) group set to 1. Values represent mean ± S.E.M. (n = 8), and significant differences are in comparison with control groups. *, p < 0.05 by t test.

Fig. 2

Effect of oral C. rodentium infection on expression of hepatic P450 proteins in WT, IL6(−/−), and IFNγ(−/−) mice. Liver microsomes were prepared from the mice described in Fig. 1, and P450 protein levels were measured by Western blotting. For CYP2B and CYP2D the heavy (lower) band is designated (H), light band is designated (L), and the sum of both is designated (T). Top, Western blots of samples from infected and control liver microsomes. Bottom, quantitative analysis of Western blot data. Values represent mean ± S.E.M. (n = 8), and significant differences are in comparison with control groups. *, p < 0.05 by t test.

Fig. 3

Effect of oral C. rodentium infection on cytokine and acute-phase protein mRNA expression in the liver of WT, IL6(−/−), and IFNγ(−/−) mice. Total liver RNA from the mice described in Fig. 1 was analyzed by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH with sucrose-treated (Ctrl) group set to 1. Values represent mean ± S.E.M. (n = 8), and significant differences are in comparison with control groups. *, p < 0.05 by t test.

Fig. 4

Effect of oral C. rodentium infection on serum cytokines of WT, IL6(−/−), and IFNγ(−/−) mice. Mice were treated as described in Fig. 1 then sacrificed after 7 days, at which time blood was collected for measurement of serum IL1β, IL2, IL6, IFNγ, TNFα, KC, IP-10, and MIP1α. Significant IFNγ immunoreactivity was detected in the IFNγ(−/−) mice, which have a targeted mutation in exon 2 of the gene that introduces a termination codon after the first 30 amino acids of the IFNγ protein. The immunoreactivity is presumably caused by expression of the truncated nonfunctional protein; therefore we omitted these measurements from the graphs. Values represent mean ± S.E.M. (n = 8), and significant differences are in comparison with control groups. *, p < 0.05 by t test.

Fig. 5

Effect of LPS injection on hepatic P450 mRNAs in WT and IFNγ(−/−) mice. Animals were injected intraperitoneally with either saline (Ctrl) or 1 mg/kg LPS, and livers were harvested after 24 h for measurement of mRNA expression by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH with the saline-treated group set to 1. Values represent mean ± S.E.M. (n = 6), and significant differences are in comparison with control groups. *, p < 0.05 by t test.

Fig. 6

Effect of cytokine treatment on mRNA expression over time in mouse primary hepatocytes. Three or 6 days after plating, primary cultures were treated with 10 ng/ml each of mouse recombinant IL6, IFNγ, or TNFα, and the cells were harvested after 6, 12, and 24 h. After their isolation, mRNAs were measured by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH with the untreated samples harvested at each time point set to 1. Values represent mean ± S.E.M. (n = 3), and significant differences are in comparison with untreated samples at respective time points. *, p < 0.05 by t test.

Fig. 7

Effect of different concentrations of cytokine treatment on mRNA expression in mouse primary hepatocytes. Three days after plating, primary cultures were treated with 0, 1, 3, 10, and 30 ng/ml each of mouse recombinant IL6, IFNγ, or TNFα, and the cells were harvested after 24 h. After their isolation, measurement of mRNA was done by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH with the untreated samples harvested at each time point set to 1. Values represent mean ± S.E.M. (n = 5 treated; n = 8 control), and significant differences are in comparison with untreated control samples. *, p < 0.05 by Dunnett’s test.