Interleukin-15 receptor α on hepatic stellate cells regulates hepatic fibrogenesis in mice.

BACKGROUND & AIMS: Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis. METHODS: Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription. RESULTS: IL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-ß) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression. CONCLUSIONS: IL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs. LAY SUMMARY: We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells. TRANSCRIPT PROFILING ACCESSION NUMBER: GSE45612, GSE 68001 and GSE 25097.

Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.

Vitamin D, chronic kidney disease and survival: a pluripotent hormone or just another bone drug?

It is now about 40 years ago that the mechanism of renal 1-α-hydroxylation of vitamin D was discovered and characterized. After this seminal observation, the key role of the active vitamin D derivative 1, 25-(OH)2-vitamin D (calcitriol) in calcium homeostasis and bone mineralization, and its specific role in the course of chronic kidney disease (CKD) and renal osteopathy, was unraveled step by step, while the precursor 25-OH-vitamin D (calcidiol) was gradually ignored. Calcitriol and its synthetic analogue alfa-calcidol became the first-line standard drug to tackle secondary hyperparathyroidism (sHPT) in CKD. Potential side-effects, including hypercalcemia, hyperphosphatemia, and vascular calcification, were partly abrogated by developing less calcemic substances such as paricalcitol or maxacalcitol. Thus, TIME Magazine surprised when nominating vitamin D, with regard to its newly discovered pleiotropic actions, as one of the "top medical breakthroughs" in the December issue of 2007. This vote was driven by novel and spectacular insights into the pivotal regulatory role of vitamin D with regard to autoimmune diseases, immune defense, cancer development and progression, and cardiovascular function and disease. More than 30 cell types express the vitamin D receptor (VDR), and more than ten organs in addition to the kidney are capable of paracrine 1-α-hydroxylation. More than 200 genes are under the control of calcitriol. A MEDLINE search performed in December 2009 focusing on the keywords "vitamin D-and-kidney-and-2009" yielded 523 hits. This review intends to give a subjective and CKD-related update on novel biological and clinical insights with relevance to the steroid hormone vitamin D.

Alcohol Consumption Accumulation of Monocyte Derived Macrophages in Female Mice Liver Is Interferon Alpha Receptor Dependent.

Monocytes develop in the bone marrow from the hematopoietic stem cells and represent heterogeneous phagocyte cells in the circulation. In homeostatic and inflammatory conditions, after recruitment into tissues, monocytes differentiate into macrophages and dendritic cells. Alcohol use causes about 3.3 million worldwide deaths per year, which is about 5.9% of all deaths. In the United States and Europe, alcohol use disorders represent the fifth leading cause of death. Females are more susceptible to alcoholic liver injury in both humans and mice. Strikingly, we still do not know how much of this difference in tissue injury is due to the differential effect of alcohol and its toxic metabolites on a) parenchymal or resident cells and/or b) immune response to alcohol. Therefore, we used a model of chronic alcohol exposure in mice to investigate the dynamics of monocytes, an innate immune cell type showed to be critical in alcoholic liver injury, by using immunophenotypic characterization. Our data reveal a sex-dimorphism of alcohol response of hepatic monocytes in female mice that is interferon receptor alpha dependent. This dimorphism could shed light on potential cellular mechanism(s) to explain the susceptibility of females to alcoholic immunopathogenesis and suggests an additional targetable pathway for alcoholic liver injury in females.

Sex specific effect of alcohol on hepatic plasmacytoid dendritic cells.

Alcoholic liver disease includes a spectrum of clinical and histological entities. They result from the combined direct effect of alcohol and its metabolites on immune cells and resident tissue cells. In humans and mice, females are more susceptible to alcoholic liver injury than males. Despite being involved in sex specific differences of immune mediated tissue injury, plasmacytoid dendritic cells (pDCs) have not been thoroughly assessed as a cellular target of alcohol in humans or mice. Therefore, Meadows-Cook diet was used to study alcohol effect on hepatic dendritic cells. Alcohol consumption for 12 weeks increased hepatic pDCs in female mice. The expression of the C-C chemokine receptor type 2 (CCR2) increased in hepatic pDC of alcohol-fed female mice. Bone marrow transplant chimera showed CCR2 dependent bone marrow egress of pDCs. Chronic alcohol exposure has a sex specific effect on hepatic pDCs population that may explain sex differences to alcoholic liver disease.

Restricted immunological and cellular pathways are shared by murine models of chronic alcohol consumption.

Murine models of chronic alcohol consumption are frequently used to investigate alcoholic liver injury and define new therapeutic targets. Lieber-DeCarli diet (LD) and Meadows-Cook diet (MC) are the most accepted models of chronic alcohol consumption. It is unclear how similar these models are at the cellular, immunologic, and transcriptome levels. We investigated the common and specific pathways of LD and MC models. Livers from LD and MC mice were subjected to histologic changes, hepatic leukocyte population, hepatic transcripts level related to leukocyte recruitment, and hepatic RNA-seq analysis. Cross-species comparison was performed using the alcoholic liver disease (ALD) transcriptomic public dataset. Despite LD mice have increased liver injury and steatosis by alcohol exposure, the number of CD45+ cells were reduced. Opposite, MC mice have an increased number of monocytes/liver by alcohol. The pattern of chemokine gradient, adhesion molecules, and cytokine transcripts is highly specific for each model, not shared with advanced human alcoholic liver disease. Moreover, hepatic RNA-seq revealed a limited and restricted number of shared genes differentially changed by alcohol exposure in these 2 models. Thus, mechanisms involved in alcohol tissue injury are model-dependent at multiple levels and raise the consideration of significant pathophysiological diversity of human alcoholic liver injury.

Isolation of highly active photosystem II core complexes with a His-tagged Cyt b559 subunit from transplastomic tobacco plants.

Photosystem II (PSII) is a huge multi-protein-complex consisting, in higher plants and green algae, of the PS II core and the adjacent light harvesting proteins. In the study reported here, N-terminal His-tags were added to the plastome-encoded alpha-subunit of cytochrome b559, PsbE, in tobacco plants, thus facilitating rapid, mild purification of higher plant PSII. Biolistic chloroplast transformation was used to replace the wildtype psbE gene by His-tagged counterparts. Transgenic plants did not exhibit an obvious phenotype. However, the oxygen evolution capacity of thylakoids prepared from the mutants compared to the wildtype was reduced by 10-30% depending on the length of the His-tag, although Fv/Fm values differed only slightly. Homoplasmic F1 plants were used to isolate PSII cores complexes. The cores contained no detectable traces of LHC or PsaA/B polypeptides, but the main core subunits of PSII could be identified using immunodetection and mass spectroscopy. In addition, Psb27 and PsbS were detected. The presence of the former was presumably due to the preparation method, since PSII complexes located in the stroma are also isolated. In contrast to previous reports, PsbS was solely found as a monomer on SDS-PAGE in the PSII core complexes of tobacco.

The charge-transfer properties of the S2 state of fucoxanthin in solution and in fucoxanthin chlorophyll-a/c2 protein (FCP) based on stark spectroscopy and molecular-orbital theory.

Fucoxanthin chlorophyll-a/c 2 protein (FCP), the membrane-intrinsic light harvesting complex from the diatom Cyclotella meneghiniana, is characterized by Stark spectroscopy to obtain a quantitative measure of the excited-state dipolar properties of the constituent pigments. The electro-optical properties of the carotenoid fucoxanthin (Fx), the primary light harvester in FCP, were determined from the Stark spectrum measured in a MeTHF glass (77 K) and compared to the results from electronic-structure calculations. On photon absorption by Fx, a 17 D change in the static dipole moment (|Delta mu|exp), and a somewhat larger |Delta mu|exp at the red edge, are measured for the S 0 --> S 2 (1 (1)A g (-)-like -->1 (1)B u *+-like) transition. The large change in dipole moment indicates that Fx undergoes photoinduced charge transfer (CT), and underscores the influence of the S 2 state on the polarity-dependent excited-state dynamics of Fx that has so far been attributed to, and discussed in terms of, the S 0 and the S 1/ICT states. MNDO-PSDCI and SACCI-CISD calculations indicate that the 1 (1)B u (*+)-like state intrinsically possesses a dipole moment much smaller than the 2 (1)A g (*-)-like state, suggesting that solvent fields promote the mixing of these two states and could account for the large dipole moments measured here for the S 0 --> S 2 transition. These CT properties of the 1 (1)B u (*+)-like state of Fx are further enhanced in the protein and underpin its photosynthetic capabilities for light harvesting and energy transfer (ET). In FCP, the CT properties of the Fx's vary according to the energetic position: between 450 and 500 nm there appear to be two sets of Fx's that exhibit |Delta mu| exp values on the order of 5 and 15 D, whereas the red-most Fx's, that are very efficient in ET to chlorophyll-a (Chl-a), exhibit strikingly large |Delta mu| exp values on the order of 40 D. Such magnitudes of |Delta mu| exp suggest a mechanism that enhances Coulombic coupling to promote ET from the S 2 state of Fx to Chl-a. These three sets of Fx's, including a fourth red Fx, are identified by fitting the Stark spectrum of FCP with the Stark spectrum of Fx in MeTHF. In contrast to the Fx's in the protein, the electrostatic properties of the Chl's in FCP are comparatively much smaller. Notably, for the Q y band of Chl-a, a |Delta mu| exp of 0.92 D and a change in polarizability ( Delta alpha exp) of 20 A (3), indicate that the Chl-a's are monomeric in nature and decoupled from each other.

Osteopontin deletion drives hematopoietic stem cell mobilization to the liver and increases hepatic iron contributing to alcoholic liver disease.

The aim of this study was to investigate the role of osteopontin (OPN) in hematopoietic stem cell (HPSC) mobilization to the liver and its contribution to alcoholic liver disease (ALD). We analyzed young (14-16 weeks) and old (>1.5 years) wild-type (WT) littermates and global Opn knockout (Opn-/- ) mice for HPSC mobilization to the liver. In addition, WT and Opn-/- mice were chronically fed the Lieber-DeCarli diet for 7 weeks. Bone marrow (BM), blood, spleen, and liver were analyzed by flow cytometry for HPSC progenitors and polymorphonuclear neutrophils (PMNs). Chemokines, growth factors, and cytokines were measured in serum and liver. Prussian blue staining for iron deposits and naphthol AS-D chloroacetate esterase staining for PMNs were performed on liver sections. Hematopoietic progenitors were lower in liver and BM of young compared to old Opn-/- mice. Granulocyte colony-stimulating factor and macrophage colony-stimulating factor were increased in Opn-/- mice, suggesting potential migration of HPSCs from the BM to the liver. Furthermore, ethanol-fed Opn-/- mice showed significant hepatic PMN infiltration and hemosiderin compared to WT mice. As a result, ethanol feeding caused greater liver injury in Opn-/- compared to WT mice. Conclusion: Opn deletion promotes HPSC mobilization, PMN infiltration, and iron deposits in the liver and thereby enhances the severity of ALD. The age-associated contribution of OPN to HPSC mobilization to the liver, the prevalence of PMNs, and accumulation of hepatic iron, which potentiates oxidant stress, reveal novel signaling mechanisms that could be targeted for therapeutic benefit in patients with ALD. (Hepatology Communications 2018;2:84-98).

New multi protein patterns differentiate liver fibrosis stages and hepatocellular carcinoma in chronic hepatitis C serum samples.

AIM: To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for detection and differentiation of liver fibrosis (F1-F2), liver cirrhosis (F4) and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). METHODS: Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. RESULTS: A 4 peptide/protein multimarker panel (7486, 12,843, 44,293 and 53,598 Da) correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples (AUROC 0.943). Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern (2873, 6646, 7775, 10,525 and 67,867 Da) with a specificity of 100% and a sensitivity of 85% in training samples (AUROC 0.976) and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APRI score and alfa-fetopotein (AFP) improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. CONCLUSION: SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Most probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.

Spectroscopic characterization of the excitation energy transfer in the fucoxanthin-chlorophyll protein of diatoms.

We characterized the energy transfer pathways in the fucoxanthin-chlorophyll protein (FCP) complex of the diatom Cyclotella meneghiniana by conducting ultrafast transient absorption measurements. This light harvesting antenna has a distinct pigment composition and binds chlorophyll a (Chl-a), fucoxanthin and chlorophyll c (Chl-c) molecules in a 4:4:1 ratio. We find that upon excitation of fucoxanthin to its S2 state, a significant amount of excitation energy is transferred rapidly to Chl-a. The ensuing dynamics illustrate the presence of a complex energy transfer network that also involves energy transfer from the unrelaxed or 'hot' intermediates. Chl-c to Chl-a energy transfer occurs on a timescale of a 100 fs. We observe no significant spectral evolution in the Chl-a region of the spectrum. We have applied global and target analysis to model the measured excited state dynamics and estimate the spectra of the states involved; the energy transfer network is discussed in relation to the pigment organization of the FCP complex.

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex.

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.