Decrease of CXCL1 message and neutrophil counts in TLR4 deficient liver and after antibiotic treatment.

<p><b>(A)</b> CXCL1 expression in the total liver as analyzed by microarrays. Mean values were obtained from three Genechips for three WT and three TLR4 deficient mice. Statistically significant differences between WT and TLR4 deficient mice are indicated by an asterisk, *<i>P</i><.05, Student <i>t</i> test. (<b>B)</b> CXCL1 expression measured by quantitative RT-PCR. The relative quantity of CXCL1 mRNA in the total liver of WT and TLR4 deficient mice is indicated (*<i>P</i><.01). <b>(C)</b> Relative expression of CXCL1 in the liver from untreated or antibiotic-treated (ABT) WT mice and TLR4 deficient mice; *<i>P</i><.01 <b>(D)</b> Neutrophils counts in the total liver. CD11+ Gr1^high TCR- cells among total live leukocytes isolated from WT and TLR4 deficient liver. In Fig 1B, 1C and 1D, data are representative of five separate experiments with six WT mice (treated or not with antibiotics) and five TLR4 mice; ^<b>#</b><i>P</i><.05; unpaired Mann -Whitney test.</p

Neutrophils migrate in response to CXCL1 secretion following TLR4 activation in hepatic stellate cells.

<p><b>(A)</b> Schematic representation of the neutrophil chemotaxis assay. <b>(B)</b> Quantification of neutrophil migration in response to secretory WT or TLR4 deficient stellate cells. WT stellate cells were treated (WT HSC + anti CXCL1) or not with anti-CXCL1 antibody. As for internal positive control, the migration of neutrophils towards TLR4 deficient stellate cells supplemented with CXCL1 protein (TLR4 HSC + CXCL1) and with CXCL1 protein only (CXCL1) was quantified in only one experiment. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between WT HSCs and TLR4 deficient HSCs, as well as between WT HSCs treated or not with anti-CXCL1, are indicated.</p

Hepatic stellate cells are the major source of CXCL1, as shown by both quantification of secretion and <i>in situ</i> localization.

<p><b>(A)</b> Quantification of CXCL1 secretion in enriched fractions of hepatocytes, KCs, LSECs and HSCs, freshly isolated and stimulated <i>in vitro</i> with LPS (1 ng/mL LPS, black squares) during 24 hours. Data are representative of three separate experiments with six mice in each group; ^<b>#</b><i>P</i><.05. <b>(B)</b> <i>In-situ</i> localization of CXCL1 in the liver. Immunofluorescent detection for CXCL1 (red) and liver cells nuclei (blue) for nuclei first shows CXCL1 expression in the sinusoids throughout liver parenchyma. <b>(C)</b> Higher resolution shows that CXCL1 (red) is expressed by sub-endothelial cells, which also store retinol droplets in separate compartments, as shown by CRBP1 staining (green). The Cellular Retinol Binding Protein-1 (CRBP-1) is the best marker to detect simultaneously both resting (Glial Fibrillary Acidic Protein, GFAP+) and activated (α-Smooth Muscle Actin, αSMA+) stellate cells <i>in situ</i>. Alexa Fluor-546-CXCL1 (red) staining does not colocalize either with Tie2-GFP in LSECs (green, <i>upper panel</i>), or F4/80 in KCs (blue, <i>middle panel</i>), but with AlexaFluor-488-CRBP1 (green, <i>lower panel</i>), staining both resting and activated HSCs. TOPRO3 was used for nuclei vizualisation.</p

Cytokine secretion by hepatocytes, KCs, LSECs and HSCs after isolation from the same liver and in response to low levels of LPS.

<p>Liver cells were freshly isolated on density gradient followed by cell sorting and stimulated with LPS (1ng/mL LPS, black bars or 100ng/mL LPS, hatched bars). Cytokine secretion was measured in the same supernatant with a multiplex assay, run in triplicates. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between basal LPS stimulation (1ng/mL) and higher LPS stimulation (100ng/mL) are indicated. Lower panel: bright field images of cells right after isolation (Hepatocytes, LSECs, KCs). Images of HSCs at higher resolution show the retinol droplets at Day 0 and the typical shape of the activated stellate cells after 4 days in culture.</p

Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.

<p>Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.</p