NPA Tumor inductions increase the severity of CCl4 related hepatic injury.

<p>CCl4-hepatic injury was evaluated by hematoxylin and eosin (H&E) staining of necro-inflammatory liver lesions and ALT serum levels. Immunohistochemical staining with H&E (5X magnification) for the four major animal groups showed necro-inflammatory lesions and cell infiltrations that were increased in the fibrotic mice receiving the NPA-tumor cells (D) as compared to fibrotic alone (C). Arrows indicate the area with lymphocyte infiltrations. No inflammatory infiltrates were seen in H&E staining of (A) naïve WT and (B) naïve mice receiving the NPA-tumor cells. (E) Serum ALT levels were in line with histological findings and showed increase from (60±25/L) in fibrotic animals without tumor to (85.5 ± 20.5 U/L) in animals with tumor and hepatic fibrosis; p-value = 0.021.</p

VEGF serum levels.

<p>Quantikine Mouse VEGF immunoassay serum levels was significantly increased in the fibrotic group bearing tumor as compared to tumor alone (p = 0.01) or fibrosis alone groups (p = 0.04). No statistical significant differences were found in VEGF serum levels between the fibrotic and non-fibrotic group with tumor induction.</p

In vitro co-culture of lymphocytes with NPA cell line.

<p>Adhered-NPA cells post co-culture with NK cells from different animal groups were analyzed for proliferation by CFSE using flow cytometry. (A) Direct co-culture of NPA cells with spleen NKs from fibrotic mice with tumor significantly decreased NPA tumor cell proliferation compared to the fibrotic mice without tumor, indicating highly stimulated NK cells effects; p-value = 0.001. (B) A representative histogram of the NPA cells following incubations with NK cells of fibrosis and tumor mice. The histogram shows CSFE-proliferations changes in day 3 and day 5 as compared to day 0 of CSFE staining. Proliferations fold changes were calculated by divided day 0 to day 5.</p

Hepatic fibrosis increase NPA tumor weight and size.

<p>In vivo S.C injection of NPA cells model was performed as described in M&M. In this model; S.C NPA-cells tumor was induced in fibrotic and naïve (no fibrosis) animals. S.C tumors were explanted at the end of 6 weeks post S.C injection, and evaluated for tumor weight and volume. (A) and (B) show the external appearance of the tumor in the animal’s back. Tumor weight (C) and volume (D) was increased significantly from (0.13±0.06gr) and (0.28±0.18ml) in the fibrotic group to (0.05±0.025 gr) and (0.09±0.01ml) in the non-fibrotic group; p-value = 0.02 and 0.04, respectively.</p

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