The Gut Microbiota and the Liver: Collaborators in Host Immunity and Metabolism

The gut microbiota consists of over one hundred trillion commensal bacteria required for proper gut immunity development. Commensals also augment the host’s ability to extract energy from the diet. Although restricted to the gut lumen by intestinal barrier epithelia, commensals shed microbial associated molecule patterns (MAMPs) into the circulation where they augment aspects of systemic immunity. Commensals also release fermentation byproducts into the portal blood stream. Since the liver receives 80% of its blood via the portal vein and contains a unique repertoire of immune cells particularly enriched in Kupffer Cells (KC) and Natural Killer T cells, we proposed that gut-derived MAMPs contribute to the development of residential hepatic leukocyte subsets. Because of the contributions of gut bacteria to digestion, we suspected that gut bacteria add an additional level of regulation to host metabolism and would generate a specific hepatic metabolic gene profile. Results showed that a cocktail of MAMPs translocate into the portal circulation of normal conventional (CL) mice stimulating KC expansion. ICAM1 expression, thought to be constitutive on sinusoidal endothelium, was significantly reduced without gut bacteria and was required for KC accumulation. The finding that constitutive ICAM1 expression by LSEC was dependent on gut bacteria lead us to investigate if the frequency of intra-hepatic lymphocytes known to bind ICAM1 were affected by gut bacteria. Results showed that intra-hepatic T lymphocyte populations including NKT (TCRβ+NK1.1+) cells and T helper (CD4+TCRβ+) cells were significantly reduced in GF mice and AVMN mice. In addition to the significant cellular composition changes of the liver related to gut bacteria density, notable changes in murine weight and metabolic gene profiles were observed. The average body mass of CL, GF, and AVMN mice was 37.8g, 33.4g, and 34.1g respectively. Our whole-liver gene array analysis included 217 probe sets mapped to 163 differentially expressed genes between groups, of which forty-eight have roles in lipid metabolism. In conclusion, gut bacteria affect both the hepatic metabolic gene profile and the inflammatory potential of the liver. These finding have implications for many hepatic pathologies including obesity, NAFLD, and autoimmune disease like PBC and AIH mediated by liver leukocytes

Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype

Accessory hepatic lobes in the pediatric population: A report of three cases of torsion and literature review

Congenital liver anomalies are uncommon. Symptomatic accessory hepatic lobes (AHL), either in continuity with the liver or ectopically located, are even less common. AHL have been reported in individuals spanning from neonates to octogenarians and are typically asymptomatic, however when symptomatic often require surgical intervention. We report three new cases of AHL in children (mean = 14.6 years). All three presented with sudden onset of abdominal pain and were diagnosed preoperatively by imaging findings. All three patients had symptom resolution following resection of the torsed accessory liver lobes. We report here the largest series of pediatric AHL torsion at a single institution to date, review the classification schemes, identify diagnostic imaging findings, and summarize associated congenital disorders that should raise suspicion for accessory hepatic lobes

Changes in miR-200c/141 and P300 result in expected EMT/MET responses.

<p>Representative western blot and corresponding real time PCR analysis shows inhibition of miR200c/141 causes partial EMT shifts in vector cells as evidenced by increased Vimentin, increased ZEB1 and phenotypic changes. No change is seen in E-cadherin expression. (84 hr post transfection; cells grown on glass coverslips; n≥2 independent experiments) (<b>A</b>). Representative western blot (48 hr) showing partial MET in SPRR2A cells (increased E-cadherin; phenotypic changes on tissues culture plastic) following transfection with pre-miR-200c and pre-miR-141. Western blot results were verified by real time PCR analysis and immunofluorescence staining (n = 2 independent experiments) (<b>B</b>). Representative western blot (48 hr) and real time PCR showing changes in EMT markers following knock down of P300. Phenotypic changes were not observed, but EP300siRNA significantly reduced vector cell miR200c expression. (n≥2 independent experiments) (<b>C</b>) Real time PCR analysis: comparative 2−ΔΔCT method (U6 or GAPDH internal control); *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; Student's <i>t</i>-test).</p

P300 and PCAF activate the <i>miR-200c/141</i> promoter, while ZEB1 and SPRR2a inhibit this activation.

<p>Illustration of <i>miR-200c/141</i> promoter, E-box, Z-box, and transcription starting site (TSS) as well as <i>miR-200c/141</i>-luciferase vector and DNA-pull down assay probes (<b>A</b>). Luciferase assay for <i>miR-200c/141</i>-promoter activity in SPRR2a expressing cells: Treatment with TSA and/or AZA shows TSA increased promoter activity (<b>B</b>), as did transfection with a P300 expression vector (0, 0.05, 0.1, 0.2, 0.4 µg) (<b>C</b>). Luciferase assay for <i>miR-200c/141</i>-promoter activity in HuCCT-1 parent cells: transfection with a ZEB1 expression vector (0.1 µg) reduced <i>miR-200c/141</i>-promoter activity, while co-transfection with P300 (0,0.05, 0.1, 0.2, 0.4 µg) (<b>D</b>) or PCAF (0, 0.1, 0.2, 0.4 µg) (<b>E</b>) antagonized this repression. In contrast, transfection with a P300 expression vector (0.4 µg) enhanced <i>miR-200c/141</i>-promoter activity, while co-transfection with SPRR2a (0,0.05, 0.1, 0.2, 0.4 µg) negated this effect (<b>F</b>). (Data represents 2–3 independent experiments; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; (B) Student's <i>t</i>-test; (C–F) one-way ANOVA).</p

A Putative model illustrating the interactions of P300/PCAF/ZEB1 in modulating cell phenotype through control of <i>miR-200c/141</i> transcription.

<p>(AC = acetylation).</p

EMT induced by SPRR2a in HuCCT-1 involves loss of E-cadherin, increased vimentin, and reduction of miR-200 family transcription as compared to vector transfected controls.

<p>Examples of the morphological changes and changes in E-cadherin and vimentin expression in stable SPRR2a clones (NC = negative control) (<b>A</b>). Transcriptional loss of the miR200 family in SPRR2a expressing cells does not involve SH3 domain containing tyrosine kinases. Real-time PCR analysis of miR-200 family after 72 hrs treatment with ABL1 siRNA (<b>B</b>) and PP2 treatment (<b>C</b>) did not alter miR-200 expression. All clones used in this paper stably express SPRR2a (<b>D</b>). Real time PCR analysis: comparative 2-ΔΔCT method (miRNA: U6 internal control; ABL1: GAPDH internal control). (n = 2 independent experiments).</p

P300/PCAF complexes with ZEB1 on the <i>miR-200c/141</i> promoter and requires the CH3 domain of P300 for transcription.

<p>Immunoprecipitation of P300 following transfection with the indicated protein expression vectors verifies P300/ZEB1 and P300/PCAF interactions, which were unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>A</b>). Immunoprecipitation experiments show PCAF acetylates ZEB1 following transfection with ZEB1 ± PCAF expression vectors (24 hrs) (<b>B</b>). DNA pull-down assay using a wild type (wt) or mutational E-box/Z-box sequence for the <i>miR-200c/141</i> promoter after co-transfection of HuCCT-1 shows binding of ZEB1 and PCAF to the wt promoter sequence (<b>C</b>), and p300/PCAF/ZEB1 binding to the wt promoter, which is unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>D</b>). Luciferase assay for <i>miR-200c/141</i> promoter activity following transfection with wild type or CH3 deleted P300 expression vector in HuCCT-1 parent cells shows the CH3 domain is required for miR transcription. (n = 3 independent experiments; ***, <i>P</i><0.001; Student's <i>t</i> -test) (<b>E</b>).</p