Livers of fibrotic TLR9^-/- have increased senescence.

<p>SA-β-gal staining of liver sections from CCl₄ and vehicle-treated (control) in WT and TLR9^-/- mice reveals increase in presence of senescent cells in TLR9^-/- fibrotic livers. The data presented were obtained from 2 representative liver samples from different experiments showed in two magnifications (4X and 10X). </p

<i>In</i><i>vivo</i> adoptive transfer model isolates lymphocyte outcome of each strain.

<p>Irradiated WT or TLR9^-/- recipient mice were reconstituted by naive WT or TLR9^-/- donor lymphocytes, along with CCl₄ fibrosis induction, as described in Materials and Methods. Accordingly, there were 4 groups of the WT recipients and another 4 groups of TLR9^-/- recipients. Plain and black bars are reflecting naïve recipients reconstituted with WT or TLR9^-/- donor lymphocytes, respectively. Horizontal and vertical gradient bars reflect fibrotic recipients reconstituted with WT or TLR9^-/- donor lymphocytes, respectively. Recipient livers were assessed for histology collagen area, and serum ALT levels were measured. A) Collagen area significantly increased following CCl₄ induction in both groups. Significant fibrosis exacerbation among WT recipients was observed when donor of lymphocytes was TLR9^-/- compared to WT lymphocytes (<i>P</i><0.01). B) ALT serum levels significantly increased (<i>P</i><0.01) following fibrosis compared to naive recipients. TLR9^-/- fibrotic recipients reconstituted with TLR9^-/- or C) WT lymphocytes achieved a significant fibrosis (<i>P</i><0.01) and D) serum ALT (<i>P</i><0.02) progression when compared to naives. However, both fibrotic groups were similar and showed no hepatic fibrosis or serum ALT changes.</p

Flow cytometry analysis of isolated intra-hepatic lymphocytes.

<p>A) Total percent of CD45 from livers of fibrotic animals showed increased infiltrate in the CpG groups as compared to the vehicle. B) A significant augmentation of liver CD8 content; along with CD4 and NK reductions in vehicle-treated CCl₄ fibrosis as compared to naive animals. The CpG therapy significantly decreased the CD4, and CD8 populations, but markedly increased the NK population up to 3-fold of expression. </p

TLR9^-/- attenuates fibrosis but increases liver injury and senescence.

<p>The CCl₄ fibrosis model was induced for 4 weeks in WT (horizontal gradient bars) and TLR9^-/- (vertical gradient bars) mice as compared to naïve states (plain and black bars, respectively). A) Collagen area following CCl₄ induction in WT and TLR9^-/- animals led to an increased percent of collagen area (<i>P</i><0.001). Hepatic collagen area in the fibrotic TLR9^-/- mice was significantly lower (<i>P</i><0.001) as compared to fibrotic WT rodents. B) Collagen levels from the hepatic hydroxyproline contents showed similar patterns as the collagen area. C) Western blotting of the liver protein extracts revealed a significant reduction in αSMA protein expression as compared to WT (two representative bands for each group are shown. D) Compared to WT, serum ALT levels were significantly higher (<i>P</i>=0.04) in the fibrotic TLR9^-/- mice. The data represent the mean ± SD of 16 animals/group. </p

Direct versus NK-mediated in vitro effects of CpG on HSCs cultures.

<p>Liver NK cells obtained from naïve (plain bars) and fibrotic mice treated with CpG (black bars) <i>versus</i> vehicle (gray bars) were <i>in </i><i>vitro</i> co-cultured with WT isolated HSCs; then HSCs proliferation were determined as described in Materials and Methods. A) Isolated HSCs mono-cultured with 1% FCS-enriched DMEM showed a significant increase of proliferation following direct CpG incubation (vertical gradient bars) as compared to DMEM vehicle-treated cells (horizontal gradient bars). B) Co-culture with the CpG-treated NK cells decreased HSCs proliferation (<i>P</i><0.01) as compared to controls. C) Co-culture of liver NK cells from fibrotic CpG-treated mice with HSCs increased their adherence (<i>P</i><0.01) as compared to the vehicle-treated fibrotic NK cells. D & E) NK increased adherence (<i>P</i><0.01) and cytotoxic marker CD107a (<i>P</i>=0.01) when HSCs were cultured with lymphocytes from fibrotic CpG-treated mice.</p

FIV-α-MSH transduction of HEK293T cells: characterization at the DNA, mRNA and protein levels. A

<p>. Genomic DNA extracted either before (293T) or 24 and 72 h after transduction of HEK293T cells with FIV-α-MSH (293T-α-MSH) was analyzed by PCR using the recombinant α-MSH-specific primers. The plasmid pLionII- α -MSH was used to demonstrate the expected size fragment. <b>B</b>. RNA samples, prepared from similarly treated cell cultures 20 days after transduction, were analyzed by RT-PCR using the same primers. <b>C</b>. The integrity of the mRNA in the control and transduced culture cells was demonstrated using the GAPDH primers. <b>D</b>. Production and secretion of α-MSH peptide was demonstrated by RIA. α-MSH peptide was detected in cell lysate (L) and culture medium (CM) of HEK293T cells, 10 days after transduction with FIV-α-MSH. No signal was detected in cell lysate or culture medium of control HEK293T cells, transduced with FIV-YFP (293T-YFP).</p

Viability of transduced cells in liver of chicks, as estimated using TUNEL and PCNA.

<p>Liver paraffin sections of FIV-treated chicks (<b>A</b>) and controls (<b>B</b>), immunostained with anti-GFP antibody (green) and DAPI (blue), were analyzed for apoptosis using TUNEL assay (A′, B′, red). Very low incidence of cell death was observed in both groups of chicks. The areas of the TUNEL positive cells were enlarged in the white boxes (inserts). Scale = 100 µm. Liver paraffin sections were immunostained with anti-GFP and DAPI (C, green and blue, respectively) and anti-PCNA (C′, red). Co-labeling of a group of hepatocytes is demonstrated (C′′), indicating actively proliferating FIV-transduced cells. Scale = 50 µm.</p

Immunofluorescence analyses of YFP expression in the liver and spleen of post-hatch chicks following <i>in ovo</i> FIV administration.

<p>Whole-mount immunostaining with anti-GFP antibody (green) and anti-SMA antibody (red) was performed on pieces of livers from: 2-day-old chicks treated with either PBS (<b>A</b>) or FIV-YFP (<b>B</b>), and from 40-day-old FIV-YFP-treated chicks (<b>C</b>). Images were obtained using epi-fluorescent stereomicroscope. Scale bar = 200 µm for A and C, and 0.5 mm for B. Immunostaining of paraffin sections with anti-GFP antibody was performed on liver (<b>D & E</b>) and spleen (<b>F & G</b>) tissues from 2-day-old chicks treated with FIV-YFP. For each of these sections, DAPI staining (blue) is shown in the corresponding images (<b>D′–G′</b>) to indicate cell nuclei. Arrowheads mark nuclei of YFP-expressing cells with splenocyte morphology (F′). Merged YFP and DAPI staining is shown in the corresponding <b>D′′–G′′</b>. Images were obtained by confocal microscopy except for <b>E</b>, <b>E′</b> and <b>E</b>′′, which were obtained by epi-fluorescenct microscope. The arrow in <b>E′′</b> indicates a YFP-expressing cell with endothelial morphology located next to transduced cells with hepatocyte morphology. <b>H</b>. Confocal images of whole-mount immunostained liver sample, using both anti-GFP (<b>H</b>) and anti-SMA (<b>H′</b>) antibodies, confirmed the association of some of the YFP-expressing cells with blood vessels, which appear in yellow in the merged image (<b>H′′</b>). Scale bar = 20 µm (D-G) and 50 µm (H).</p

YFP expression following transduction of primary cultures of chick embryonic cells with FIV-YFP vectors.

<p>Primary cell cultures from E11 chick embryos were transduced with FIV-YFP (2×10⁴ TU/well, in a 24-well plate) and YFP-expressing cells (green) were detected by fluorescence microscopy, 48 h after transduction (<b>A–F</b>). The corresponding bright-field micrographs of the same cultures (<b>A</b>′<b>–F</b>′) demonstrate the tissue-specific morphology of the cells. Scale bar = 20 µm. Rate of transduction in each culture, determined by FACS analysis, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036531#pone.0036531.s002" target="_blank">Figure S2</a>.</p

Estimation of transduction efficiency in livers of <i>in ovo</i> FIV-YFP transduced chicks by flow cytometry.

<p>Liver samples from 2-day-old control chicks treated with PBS (<b>A</b>) or FIV-YFP (<b>B</b>) were analyzed by flow cytometry. One representative image from the control PBS and FIV-YFP treated chick groups is shown. Cells in each sample were selected (window P1 in A) and YFP fluorescence was analyzed (B). While no obvious signals were obtained in the control samples, the rate of transduced liver cells in the samples from FIV-YFP treated chicks was 0.46±0.19% (n = 3). Blue and red mark the YFP-positive and negative cells, respectively. The sample with the highest transduction rate (0.8%) is presented.</p