Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition

Background We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. Methods WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. Results In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. Conclusions Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

BackgroundWe have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice.MethodsWT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs.ResultsIn the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2.ConclusionsAltered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation

Antibodies used for Western analysis in this study.

Antibodies used for Western analysis in this study.</p

Administration of recombinant IL-1β and TNFα increases liver injury and rapidly alters transcription of the circadian machinery.

8–10 week old C57BL6 mice were injected with recombinant IL-1β (200ng/mouse/i.p.) or TNFα (200ng/mouse/i.p.) and sacrificed after 4hr. Serum and liver tissue was harvested, and A. Hematoxylin and Eosin staining of liver sections isolated from each condition. B. Liver injury assessed by serum ALT, AST and alkaline phosphatase. C. mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. Values are Mean± SEM. *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001, N = 3–4 per condition.</p

Genetic inhibition of TNFα signaling normalizes expression of hepatic circadian transcription factors following DSS-PN.

8–10 week old TNFRKO mice were subjected to Chow feeding or DSS-PN. A. Liver tissue was harvested, and mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. N = 4 per condition. Values are Mean± SEM. *pB. Western analysis of circadian regulatory proteins. Expression was normalized using Gapdh expression for each blot. Values are Mean± SEM, N = 3/condition, a = significantly different from respective chow control, b = significantly different compared to WT DSS-PN.</p

Deletion of IL-1β signaling ameliorates expression of hepatic circadian transcription factors following DSS-PN.

8–10 week old IL1KO mice were subjected to Chow feeding or DSS-PN. A. Liver tissue was harvested, and mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. Values are Mean± SEM, N = at least 3/condition. *pB. Western analysis of circadian regulatory proteins. Expression was normalized using Gapdh expression for each blot. Values are Mean± SEM, N = 3/condition, a = significantly different from respective chow control, b = significantly different compared to WT DSS-PN.</p

Parenteral nutrition dysregulates circadian regulatory expression in mice.

mRNA expression of circadian transcription factors in hepatic tissue isolated from Chow and DSS-PN mice (Z+2/Z+3). Expression was normalized against HPRT. N = 4 per condition. Values are Mean± SEM. *p<0.05. **p<0.01.</p

Genetic inhibition of IL-1β or TNFα signaling ameliorates DSS-PN induced liver injury.

Wild-type (WT) or (A) IL1KO or (B)TNFRKO mice were treated with chow or DSS-PN and euthanized after 14 days [4, 11]. Liver injury was assessed using serum (Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total serum bile acids). N = 3–9 per condition. Values are Mean± SEM, ***pC. Hematoxylin and Eosin (H&E) staining of liver sections isolated from indicated conditions. D. Immunohistochemical analysis of F4/80+ macrophages in liver sections isolated from indicated conditions, N = 3 per condition, 200X, PT, Portal triad; CV, Central vein. E. Quantification of F4/80 positive staining. Values are Mean± SEM, ****pF. Immunohistochemical analysis of Ki67 positive nuclei in liver sections isolated from indicated conditions, N = 3 per condition, 200X. G. Immunohistochemical analysis of Cytokeratin 7 (CK7) staining liver sections isolated from indicated conditions, N = 2–3 per condition, 200X. H. Quantification of Ki67 positive staining/100X field, values are Mean± SEM. I. Quantification of CK7 staining, values are Mean± SEM.</p