Isolation and phenotypic characterization of spleen CD11c⁺DCs.

<p>(A) Expression of surface markers in MACS-isolated CD11c⁺DCs. (B) Percentage of CD74 in spleen CD11c⁺DCs. (C) Percentage of CD83 in spleen CD11c⁺DCs. (D) Percentage of CD86 in spleen CD11c⁺DCs. Compare with the NS group, expression of CD74, CD83 and CD86 in the FHF group was lower in the D-GalN group and significantly lower in the FHF group, but there was no significant difference compared with the LPS group (*<i>P</i><0.05 <i>vs</i>. the NS group, #<i>P</i><0.05 <i>vs</i>. the FHF group unpaired t-test; <i>n</i>≥6)</p

Intestinal Dendritic Cells Are Altered in Number, Maturity and Chemotactic Ability in Fulminant Hepatic Failure

<div><p>Fulminant hepatic failure (FHF) is defined as rapid acute liver injury, often complicated with spontaneous bacterial peritonitis (SBP). The precise onset of FHF with SBP is still unknown, but it is thought that SBP closely correlates with a weakened intestinal barrier. Dendritic cells (DCs) play a crucial role in forming the intestinal immune barrier, therefore the number, maturity and chemotactic ability of intestinal DCs were studied in FHF. Mouse intestinal and spleen DCs were isolated by magnetic-activated cell sorting (MACS) and surface markers of DCs, namely CD11c, CD74, CD83 and CD86, were identified using flow cytometry. Immunohistochemistry and Western blotting were performed to detect the distribution and expression of CC-chemokine receptor 7 (CCR7) and CC-chemokine receptor 9 (CCR9), as well as their ligands-CC-chemokine ligand 21 (CCL21) and CC-chemokine ligand 25 (CCL25). Real-time PCR was used to detect CCR7 and CCR9 mRNA, along with their ligands-CCL21 and CCL25 mRNA. Flow cytometry analysis showed that the markers CD74, CD83 and CD86 of CD11c⁺DCs were lower in the D-galactosamine (D-GalN) group and were significantly decreased in the FHF group, while there were no significant changes in the expression of these markers in the lipopolysaccharide (LPS) group. Immunohistochemistry results showed that staining for CCR7 and CCR9, as well as their ligands CCL21 and CCL25, was significantly weaker in the D-GalN and FHF groups compared with the normal saline (NS) group or the LPS group; the FHF group even showed completely unstained parts. Protein expression of CCR7 and CCR9, as well as their ligands- CCL21 and CCL25, was also lower in the D-GalN group and decreased even more significantly in the FHF group. At the gene level, CCR7 and CCR9, along with CCL21 and CCL25 mRNA expression, was lower in the D-GalN group and significantly decreased in the FHF group compared to the NS and LPS groups, consisting with the protein expression. Our study indicated that intestinal DCs were decreased in number, maturity and chemotactic ability in FHF and might contribute to a decreased function of the intestinal immune barrier in FHF.</p></div

Isolation and phenotypic characterization of intestinal CD11c⁺DCs.

<p>(A) Expression of surface markers in MACS-isolated CD11c⁺DCs. (B) Percentage of CD74 in CD11c⁺DCs. (C) Percentage of CD83 in CD11c⁺DCs. (D) Percentage of CD86 in CD11c⁺DCs. Compared with the NS group, expression of CD74, CD83 and CD86 was lower in the D-GalN group and significantly lower in the FHF group, but there was no significant difference compared with the LPS group (*<i>P</i><0.05 <i>vs</i>. the NS group, unpaired <i>t</i>-test; <i>n</i>≥6).</p

Relative mRNA levels of intestinal CCL21, CCR7, CCR9 and CCL25.

<p>(A): CCR7, (B): CCL21, (C): CCR9 and (D): CCL25. Levels of intestinal CCL21, CCR7, CCR9 and CCL25 mRNAs in tissues from the FHF group and D-GalN were significantly decreased compared with the NS group, but there were no significant differences compared with tissues from the LPS group of CCR7, CCR9, CCL21 and CCL25 (*<i>P</i>< 0.05, **<i>P</i>< 0.01 <i>vs</i>. the NS group; #<i>P</i><0.05, ##<i>P</i><0.01 <i>vs</i>. the FHF group, unpaired <i>t</i>-test; <i>n</i>≥8).</p

Immunohistochemical staining of CCR7, CCR9, CCL21 and CCL25.

<p>A, B, C and D: Immunohistochemical staining of CCR7, CCL21, CCR9 and CCL25; E, F, G and H: Integrated optical density of CCR7, CCL21, CCR9 and CCL25. Compared with the NS group, the integrated optical density of CCR7, CCL21, CCR9 and CCL25 was reduced in the D-GalN group and notably reduced in the FHF group, but there was no significant difference compared with the LPS group (**<i>P</i>< 0.01, ***<i>P</i>< 0.001 <i>vs</i>. the NS group; #<i>P</i><0.05, ##<i>P</i><0.01, ###<i>P</i><0.001 <i>vs</i>. the FHF group, unpaired <i>t</i>-test; <i>n</i>≥10).</p

Intestinal CCR7, CCL21, CCR9 and CCL25 protein expression.

<p>A, C, E and G: Electrophoresis banding of intestinal CCR7, CCL21, CCR9 and CCL25. B, D, F and H: Densitometric analysis using the Image-Pro software. Compared with the NS group, absorbance ratios of CCR7, CCL21, CCR9 and CCL25 to GAPDH were lower in the D-GalN group and notably decreased in the FHF group, but there was no significant difference compared with the LPS group (*<i>P</i>< 0.05 <i>vs</i>. the NS group; #<i>P</i><0.05 <i>vs</i>. the FHF group, unpaired <i>t</i>-test; <i>n</i>≥8).</p

Isolation and phenotypic characterization of spleen CD11c⁺DCs.

<p>(A) Expression of surface markers in MACS-isolated CD11c⁺DCs. (B) Percentage of CD74 in spleen CD11c⁺DCs. (C) Percentage of CD83 in spleen CD11c⁺DCs. (D) Percentage of CD86 in spleen CD11c⁺DCs. Compare with the NS group, expression of CD74, CD83 and CD86 in the FHF group was lower in the D-GalN group and significantly lower in the FHF group, but there was no significant difference compared with the LPS group (*<i>P</i><0.05 <i>vs</i>. the NS group, #<i>P</i><0.05 <i>vs</i>. the FHF group unpaired t-test; <i>n</i>≥6)</p