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