Figure 1.
Steatosis results in increased cellular proliferation in the liver.
The percentage of total Ki67 positive cells and the percent of Ki67/HNF4α double positive hepatocytes are both increased in steatotic livers compared to normal livers of (A, B) murine and (C, D) human samples. Representative images of Ki67 (green), HNF4α (red) and DAPI (blue) immunofluoresence staining in livers of normal and steatotic (B) murine and (D) human liver sections showing increased number of Ki67 positive cells in steatotic livers. Images are taken at 40X and scale bars represent 50 microns.
Figure 2.
Steatosis results in changes in the inflammatory cell populations in the murine liver.
CD45 positive cells were isolated from normal and steatotic digested liver samples by immunomagnetic beads and then stained for the immune markers CD3 and CD11b before being subjected to Flow cytometric analysis. (A) Diagrams are representative scatter plot of flow cytometric analysis for overall CD45 positive cells in the livers. (B) CD45 positive cells were then gated for the percentage of CD3 positive T lymphocytes or CD11b positive myeloid cells. CD3+ and CD11b+ subpopulations were both increased in the steatotic livers versus the control livers.
Figure 3.
Changes in the myeloid cell sub-populations in normal vs steatotic murine livers.
Isolated CD45 positive cells from normal and steatotic livers were stained for myeloid markers, subjected to flow cytometry and gated as a fraction of CD11b positive myeloid cells. (A) CD11b+ cells were analyzed for changes in the total percentage of F4/80 positive macrophages, (B) Gr1hi expressing cells, (C) Ly6C positive Gr1 positive activated monocytes and (D) Ly6C negative Gr1 positive infiltrating neutrophils between normal and steatotic murine livers. Results show a significant decrease in the GR1hi and Ly6CpGr1p subpopulations and a significant increase in the Ly6CnGr1p subpopulation of steatotic versus normal livers, while there was not a significant difference the percentage of F4/80p subpopulation.
Figure 4.
Changes in the lymphoid cell sub-populations in normal vs steatotic murine livers.
Isolated CD45 positive cells from normal and steatotic livers were stained for lymphoid markers, subjected to flow cytometry and gated as a fraction of CD3e positive lymphocytic cells. CD3e+ cells were analyzed for changes in the total percentage of (A) CD4 positive helper T cells, (B) CD8 positive cytotoxic T cells, (C) CD25 positive regulatory T cells, and (D) CD62L negative activated T cells between normal and steatotic murine livers. Results only showed a significant increase in the overall CD25+ subpopulation of CD3e+ cells in the steatotic livers when compared to normal livers.
Figure 5.
Changes in cell sub-populations of B cells, Dendritic cells and Natural Killer cells in steatotic livers.
Isolated CD45 positive cells from normal and steatotic livers were stained for markers for (A) B cells (CD19+), (B) dendritic cells (CD11c+, CD19–) and (C) natural killers cells (NKp46+) before being subjected to flow cytometry and gated as a fraction of CD45+ cells to detect total percentage of subpopulations. Results demonstrate a significant decrease in B cells and a significant increase in total dendritic subpopulations in steatotic murine livers compared to normal livers. Percentage of the overall subpopulation of natural killer cells were not significantly different between groups.
Figure 6.
Quantification of inflammatory cell populations in human liver samples.
Immunohistochemical staining was used to detect and quantify changes in inflammatory cell populations between normal and steatotic human liver samples. Positive immunoreactive staining (dark brown) was calculated as a percentage of total area for (A) CD45 positive inflammatory cells, (B) CD68 positive macrophages (C) CD3 positive T lymphocytes (D) CD8 positive cytotoxic T cells and (E) CD56 positive NK cells in frozen sections of normal and steatotic human livers. Results demonstrate a significant increase in the number of CD45 and CD3 positive cells in the steatotic livers when compared to normal liver samples. CD68 macrophages, CD8 cytotoxic T cells, and CD56 NK cells were not significantly altered between samples. Images are 20x.
Table 1.
Fold change in cytokine levels of high fat diet fed mice.
Figure 7.
Cytokines effect hepatocyte proliferation in vitro.
The effect of different concentrations of Leptin, CXCL1, CXCL2 and CXCL16 (100 ng/ml, 25 ng/ml and 1 ng/ml) on proliferation of (A) HepG2 and (B) HEPT cells determined by MTT assay. 10% FBS was used as a positive control. ** represents P<0.001 and *** P<0.0001 when compared to Serum Free conditions.