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Vitamin A deficiency results in an uncontrolled immune response and aggravates liver damage during obstructive cholestasis

ABSTRACT: Background: Vitamin A is an essential nutrient that mammals obtain from dietary carotenoids and retinyl esters. Bile salts aid in the intestinal absorption of vitamin A, after which it is stored in the stellate cells of the liver. Vitamin A-metabolites, such as retinoic acids, are important for cell differentiation, proliferation and immunological processes, and act mostly by activating the nuclear receptors Retinoic Acid Receptor (RAR) and Retinoid Xreceptor (RXR). These receptors also perform key functions in the liver by regulating bile salt, lipid and glucose homeostasisas well is the intrahepatic immune response. Chronic liver diseases are characterized by hepatic vitamin A loss often leading to vitamin A deficiency (VAD). We found that VAD strongly aggravates liver damage in bile duct ligated (BDL) rats and in biliary atresia patients. Here, we performed a transcriptome analysis to characterize the cell biological processes in the rat liver that are disturbed in obstructive cholestasis under VADconditions. Methods: Rats were made VAD by omitting vitamin A from the diet for 16 weeks, followed by ligation of the common bile duct (BDL). 7 days later, the animals were sacrificed. During the 7 days of BDL, half of the animals received daily IP injections with retinyl-palmitate (vitamin A therapy). Bile salt concentrations and liver damage markers (AST/ALT) were determined in serum. Whole genome gene expression in liver tissue was analyzed with the Illumina platform RatRef-12 beadchip. False discovery rate (FDR) was set at a stringent 1%. Data were confirmed by quantitative real time PCR, Western blotting and/or histochemistry. Results: BDL induced dramatic liver damage in VAD rats with serum liver damage markers (AST/ALT)approximately 10-fold increased compared to BDL rats receiving a vitamin A-sufficient (VAS) diet. Only 20 genes were differentially expressed between sham-treated VAS and VAD rats and clustered in the vitamin A metabolism, immune response, carbohydrate and lipid homeostasis. In contrast, 1583 genes were differentially expressed between the BDL-treated VAS and VAD rats. The strongest effects were observed in genes from the vitamin A metabolism, immune response, collagen production and oxidative stress response. Importantly, all the liver damage markers were efficiently reversed by retinyl-palmitate therapy, which was mirrored by an almost complete normalisation of the transcript profile in VAS-BDL rats. Conclusions: We conclude that vitamin A deficiency dramatically aggravates liver damage caused by obstructive cholestasis. The transcriptome analysis suggests that a disturbed immune response may be the predominant causative factor.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE37313 | GEO | 2025/01/01

REPOSITORIES: GEO

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Project description:Extrahepatic cholestasis leads to complex injury and repair processes that result in bile infarct formation, neutrophil infiltration, cholangiocyte and hepatocyte proliferation, extracellular matrix remodeling, and fibrosis. To identify early molecular mechanisms of injury and repair after bile duct obstruction, microarray analysis was performed on liver tissue 24 hours after bile duct ligation (BDL) or sham surgery. The most upregulated gene identified encodes plasminogen activator inhibitor 1 (PAI-1, Serpine 1), a protease inhibitor that blocks urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Because PAI-1, uPA, and tPA influence growth factor and cytokine processing as well as extracellular matrix remodeling, we evaluated the role of PAI-1 in cholestatic liver injury by comparing the injury and repair processes in wild-type (WT) and PAI-1-deficient (PAI-1-/-) mice after BDL. PAI-1-/- mice had fewer and smaller bile infarcts, less neutrophil infiltration, and higher levels of cholangiocyte and hepatocyte proliferation than WT animals after BDL. Furthermore, PAI-1-/- mice had higher levels of tPA activation and mature hepatocyte growth factor (HGF) after BDL than WT mice, suggesting that PAI-1 effects on HGF activation critically influence cholestatic liver injury. This was further supported by elevated levels of c-Met and Akt phosphorylation in PAI-1-/- mice after BDL. In conclusion, PAI-1 deficiency reduces liver injury after BDL in mice. These data suggest that inhibiting PAI-1 might attenuate liver injury in cholestatic liver diseases. Total RNA isolated using TRI Reagent (Sigma, St. Louis, MO) was purified with an RNeasy mini kit (Qiagen, Valencia, CA). Twenty micrograms cRNA was hybridized to a mouse GeneChip (U74Av2, Affymetrix, Santa Clara, CA) at the Siteman Cancer Center GeneChip facility as described by the manufacturer. Analyses used one mouse per chip. Gene expression changes were analyzed using Affymetrix MicroArray Suite 4.0 and GeneChip 3.1 Expression Analysis and Statistical Algorithms (Affymetrix). The complete methodology and full data sets for all 6 analyzed chips are available at http://bioinformatics.wustl.edu.beckerproxy.wustl.edu This study compares the injury and repair processed in wild-type mice after BDL.

Project description:Overall hypothesis: RNAseq can be used to examine and compare the vitamin A (VA) effect, Infection effect caused by Citrobacter rodentium (C. rodentium), and the Interaction effect (VA status × C. rodentium infection) in mouse small intestine (SI) and colon, on the scale of global transcriptome. Methods: Vitamin A deficient (VAD) mice were generated by feeding VAD diet to the pregnant C57/BL6 dams and their post-weaning offspring. Vitamin A sufficient (VAS) mice were fed with purified diet containing normal amount of VA. At weaning, mice were maintained on their respective diets and assigned to one of the four treatment groups (non-infected VAD, infected VAD, non-infected VAS and infected VAS) until the end of the experiments. For the infected groups, age-matched VAD or VAS mice were orally inoculated with C. rodentium. Mice during early (SI study) or peak (colon study) C. rodentium infection were euthanized in two separate studies. The SI study focused on SI and analyses were performed on samples collected 5 days post-infection. The colon study analyzed samples on post-infection day 10, the peak of C. rodentium infection. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Three to six biological replicates were sequenced in each group. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. Results: a) differentially expression genes (DEGs) corresponding to VA effect were present in both the SI and colon. In SI, DEGs altered by VA were mainly involved in the retinoid metabolic pathway and immunity-related pathways. Novel target genes (e.g. Mbl2, Mmp9, Cxcl14, and Nr0b2) and cell types (based on Tuft cell and mast cell markers) under the regulation of VA were suggested by differential expression analysis coupled with the co-expression network analysis. In regard to the colon, VA demonstrated an overall suppressive effect on the cell division pathway. Finally, the comparison of co-expression modules between SI and colon indicated distinct regulatory networks in these two organs. b) In the SI, no Infection effect was detected during early infection, whereas in the colon, during peak of C. rodentium infection: 1) It has been shown that infection upregulated innate and adaptive immune functions, epithelial proliferation, apoptosis, endoplasmic reticulum stress, and reactive oxygen species production, as well as downregulated ion and water absorption. This helped to confirm the findings in previous studies, consolidating the knowledge-base about the host response to C. rodentium, 2) Pathways (e.g. neuron activity, smooth muscle contraction, vascular constriction, and developmental regulation) and additional ion transporters previously not known to be influenced by C. rodentium were suggested as potential mechanisms responsible for the diarrhea pathology and host response. 3) Retinoic acid receptor (RAR) and vitamin D receptor (VDR) signaling pathways appeared to be downregulated by infection, indicating that the malabsorption of micronutrients might be a danger signal for the host to switch from homeostasis to an anti-infection mode. c) In the SI, no Interaction (VA status × C. rodentium infection) effect was detected during early infection. In colon, 1329 genes corresponded to the Interaction effect. During the peak of C. rodentium infection, the sufficient stimulation of cell division, protein catabolism, apoptosis, transcription regulation, MHC class I, interferons, and cytokine signaling might be of key importance for the resistance of VAS mice. In addition, the proper maintenance of the proliferation, differentiation, migration, and activation of T cells and leukocytes during peak infection may also confer the VAS hosts with higher survival and clearance rate. On the other hand, our data suggested that the higher rate of lethal dehydration observed in infected VAD mice, may be attributed to the disrupted of HCO3- metabolism and an improperly high level of Cl- secretion. Conclusions: VA alters the gene expression profile in both SI and colon, whereas the Infection effect and the Interaction effect were only observed in colon during peak of C. rodentium infection, but not in the SI during early infection. Our studies suggest novel target genes and cell types under the regulation of VA in the SI, and indicate that the resistance of the VAS hosts may be attributed to the concordant control over ion/water absorption, immune functions, as well as the balance between epithelial hyperplasia and apoptosis.

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Vitamin A deficiency results in an uncontrolled immune response and aggravates liver damage during obstructive cholestasis

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