3.7 PIC Ⅱ inhibited the neutrophil extracellular traps (NETs)
formation and promoted the rupture of aHSCs
Considering the virtual role of intercellular communication between
immune cells and HSCs played in the liver fibrosis, we further
investigated whether PIC Ⅱ influenced neutrophils, another type of
crucial cell existed in the hepatic immune microenvironment. First, we
conducted GSEA plots and revealed that DEGs associated with neutrophil
inhibition was significantly increased in the Mdr2-/-+ PIC Ⅱ group when compared to the Mdr2-/- group
(Fig. 7A ). Based on RNA sequencing results, the expression of
DEGs, enriched in integrins and cytokines, were presented as a heatmap
with 4 clusters by hierarchical cluster analysis. As shown inFig. 7B , PIC Ⅱ markedly decreased the level of neutrophil
chemotaxis markers (Cxcl1 , Cxcl2 ) and notably, increased
the level of neutrophil senescence marker Cxcr4 and decreased the
level of neutrophil makers
(lymphocyte antigen 6
complex, locus G [Ly6g ], Ly6g5b and Ly6g6d ).
These results suggested that PIC Ⅱ might not only recruited NK cellsvia releasing CXCL16 and combining with CXCR6 but also accelerate
the senescence of neutrophils at the same time, which broadens the
possibility of interrelations taken place among these distinct immune
cells in the liver fibrosis process.
The other remarkable thing is that neutrophil-derived fibrous networks,
NETs, often lead a worsen immune and microvascular circumstances
resulting in anabatic hepatic liver fibrosis. Therefore, we performed
immunofluorescence staining of NETs markers CitH3 and MPO and found that
PIC Ⅱ markedly reduced the formation of extracellular NETs in
Mdr2-/- mice (Fig. 7C and Fig.S7 ).
Similarly, the serum level of MPO (Fig. 7D ) and relative mRNA
levels of genes associated with neutrophils, such as the Ly6g,were dramatically increased in the Mdr2-/- group.
Interestingly, although PIC Ⅱ didn’t affect the expression ofLy6g , it markedly decreased MPO level, and
intercellular adhesion
molecule 1 (Icam1) mRNA expression, and increased the senescence
maker of neutrophils (Cxcr4 ) in the fibrotic liver (Fig.
7E ). In consideration of the inflammatory crosstalk between neutrophils
and HSCs in hepatic fibrosis, we further explored the effects of PIC Ⅱ
on primary activated neutrophils and co-cultured system of neutrophils
and HSCs. Consistently, PIC Ⅱ markedly reduced the formation of NETs
induced by IL-2, a classical activator of neutrophils (Fig. 7F )
and dose-dependently promoted the rupture of activated HSCs with the
presence of CM from PIC Ⅱ-treated NK cells (namely CM-Neu) (Fig.
7G ), suggesting the inhibitory effects of PIC Ⅱ on aHSCs might be also
attributed to the restriction of neutrophil activation and NETs
formation.