Altered inflammatory responses to Citrobacter rodentium infection, but not bacterial lipopolysaccharide, in mice lacking the Cyp4a10 or Cyp4a14 genes

Abstract

Murine hepatic Cyp4a mRNAs are markedly downregulated during inflammation. Here, we investigated the roles of Cyp4a10 and Cyp4a14 in the response to infection with C. rodentium. Absence of either Cyp4a gene attenuated or abrogated the changes in spleen weight, colon crypt length, hepatic cytokine, and acute phase protein mRNAs, and serum acute phase proteins and cytokines caused by infection. Cyp4a10(-/-) mice on a low-salt diet had a similar hepatic acute phase response as those mice on a high-salt diet, suggesting that hypertension associated with this genotype is not the cause of their altered inflammatory response. In contrast, wild-type, Cyp4a10(-/-), and Cyp4a14(-/-) mice showed similar responses to injected LPS. These results implicate Cyp4a10 and Cyp4a14 in the regulation of the host inflammatory response to enteropathogenic bacterial infection but not to acute aseptic inflammation. Understanding the mechanism of this role may lead to novel therapeutic approaches in some inflammatory diseases.

Figure 1

Comparison of the effect of oral C. rodentium infection in wildtype 129X1/SvJ (Jax) and 129X1/SvEvTac (Tac) mice. Over 24 h, female mice were allowed to drink either sucrose solution (Control) or C. rodentium in sucrose (Infected) then sacrificed after 7 days. Livers were harvested for measurement of mRNAs by real-time RT-PCR, and serum was prepared for analysis of serum cytokines A) Hepatic P450 and Fmo3 mRNAs. B) Hepatic cytokine and acute phase protein mRNAs. C) Serum cytokines. mRNA levels are expressed as relative levels of mRNA expression after normalization to GAPDH, with sucrose-treated (Control) group set to 1. Values represent mean ± S.E.M, (n = 6). *, significantly different from control mice of same genotype; #, significantly different from Jax LPS-treated mice, p < 0.05.

Figure 2

Body weight changes in control and infected mice. Over 24 h, male mice were allowed to drink either sucrose solution (Control) or C. rodentium in sucrose (Infected). Body weights were monitored, and expressed as a percentage of the original body weight for each animal. Most groups had n= 5 or 6 animals, but data are missing for some animals on days 2-6. Values represent mean ± S.E.M. *, significantly different from control mice of same genotype; #, significantly different from WT infected mice, p < 0.05.

Figure 3

Liver pathology during C. rodentium infection wildtype (WT), Cyp4a10-deficient (Cyp4a10^−/−) and Cyp4a14-deficient (Cyp4a14^−/−) mice. Liver sections were stained with hematoxylin and eosin and scored for the presence of inflammation using the following scale: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, severe. Points on the graph are livers from individual mice. All livers for uninfected mice had a score of zero, and the data are not shown. *, significantly different from WT group, p<0.05 by Kruskal-Wallace test followed by Dunn's multiple comparison test.

Figure 4

Effect of oral C. rodentium infection on hepatic mRNA expression in wildtype (WT), Cyp4a10-deficient (Cyp4a10^−/−) and Cyp4a14-deficient (Cyp4a14^−/−) mice. Over 24 h, male mice were allowed to drink either sucrose solution (Control) or C. rodentium in sucrose (Infected) then sacrificed after 7 days and livers harvested for measurement of mRNA by real-time RT-PCR. Cytokines and acute phase proteins (A and B); P450s (C). Values are expressed as relative levels of mRNA expression after normalization to GAPDH, with sucrose-treated (Control) group set to 1. Values represent mean ± S.E.M, (n = 5 or 6). *, significantly different from control mice of same genotype; #, significantly different from WT infected mice, p < 0.05.

Figure 5

Effect of oral C. rodentium infection on serum cytokine and acute phase protein concentrations in male wildtype (WT), Cyp4a10-deficient (Cyp4a10^−/−) and Cyp4a14-deficient (Cyp4a14^−/−) mice. Mice were treated as described in Fig. 2 then sacrificed after 7 days, at which time blood was collected for measurement of serum cytokines, chemokines and APPs. IL2, IL3, IL4, IL7, IL9, IL10, IL17, CCL5, CXCL1, MIP1α did not change with infection and are not shown. Values represent mean ± S.E.M, (n = 5 or 6). *, significantly different from control mice of same genotype; #, significantly different magnitude of response from that of WT infected mice, p < 0.05.

Figure 6

Effect of oral C. rodentium infection on serum cytokine and acute phase protein concentrations in female wildtype (WT), Cyp4a10-deficient (Cyp4a10^−/−) and Cyp4a14-deficient (Cyp4a14^−/−) mice. Mice were treated as described in Fig. 2 then sacrificed after 7 days, at which time blood was collected for measurement of serum cytokines, chemokines and APPs. IL2, IL3, IL4, IL7, IL9, IL10, IL17, CCL5, CXCL1, MIP1α did not change with infection and are not shown. Values represent mean ± S.E.M, (n = 5 or 6). *, significantly different from control mice of same genotype; #, significantly different magnitude of response from that of WT infected mice, p < 0.05.

Figure 7

Effect of dietary sodium on hepatic acute phase response to oral C. rodentium infection in wildtype (WT) and Cyp4a10-deficient (Cyp4a10^−/−) mice. Over 24 h, male mice on diets containing either 0.03 or 0.4% sodium were allowed to drink either sucrose solution (Control) or C. rodentium in sucrose (Infected) then sacrificed after 7 days and livers harvested for measurement of mRNA by real-time RT-PCR. Values are expressed as relative levels of mRNA expression after normalization to GAPDH, with sucrose-treated (Control) group set to 1. Values represent mean ± S.E.M, (n = 5 or 6). *, significantly different from control mice of same genotype; #, significantly different from WT infected mice, p < 0.05.

Figure 8

Effect of LPS injection on hepatic P450 and acute phase protein mRNAs in wildtype (WT), Cyp4a10-deficient (Cyp4a10^−/−) and Cyp4a14-deficient (Cyp4a14^−/−) mice. Male mice were injected i.p. with either saline (Control) or 1 mg/kg LPS (LPS), and livers 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 saline-treated group set to 1. Values represent mean ± S.E.M, (n = 5 or 6). *, significantly different from control mice of same genotype; #, significantly different from WT infected mice, p < 0.05.