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.