Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis

Abstract

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin⁺- and αSMA⁺ -HSC and CD68⁺- macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Keywords: alkaline phosphatase; hepatic stellate cells; lipid A; lipopolysaccharide; liver fibrosis.

Figure 1

Schematic representation of the chemical structure of lipopolysaccharide (LPS) and the effects of full length LPS, di-phosphoryl lipid A (DPLA), and mono-phosphoryl Lipid A (MPLA) on RAW 264.7 cells in vitro. (A) General structure of wild-type LPS illustrating the lipid A moiety with two phosphate groups, the inner core and the O-antigen. Occasional phosphate groups may also be present in the highly variable O-antigen. The truncated form of LPS, i.e., DPLA, as used in the present study, is also indicated. (B,C) In vitro effects of LPS, DPLA, and MPLA on the expression of MHC class II and the percentage of live cells, as analysed by FACS, in cultures of RAW 264.7 cells. (D–F) In vitro effects of LPS, DPLA, and MPLA on the gene expression of TNFα, IL-1β, and IL-6, respectively, as analysed by qPCR in cultures of RAW264.7 cells. (G) Nitric oxide (NO) production of RAW264.7 cells 24 h after exposure to LPS, DPLA, or MPLA. The results show significant responses of macrophages to LPS, which serves as positive control, and DPLA, but no or limited response to MPLA. n = 5 per group (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 2

Effects of wild-type LPS, di-phosphoryl lipid A (DPLA), and mono-phosphoryl lipid A (MPLA) on gene expression levels in cultures of primary rat hepatic stellate cells (HSCs). (A) Effects of LPS, DPLA, and MPLA on mRNA levels of the fibrogenic parameters Collagen 1a1, αSMA, and TGFβ. (B) Effects of the bacterial toxins on mRNA levels of the LPS receptors TLR-4 and CD14. (C) Effects on mRNA levels of the proinflammatory mediators IL-6 and IL-8. (D) Effects of the bacterial toxins on mRNA levels of MMP-1 and MMP-13. LPS (from E. coli) serves as positive control (* = p < 0.05; ** = p < 0.01; n =5 per group).

Figure 3

LPS-dephosphorylating (A,B) and alkaline phosphatase (AP) activity (C,D) in normal and fibrotic livers of mouse and man. Representative pictures of LPS-dephosphorylating activity in mouse (A) and human liver tissue (B). Brown staining represents sites of enzymatic phosphate release at pH 7.6 LPS from E. coli as substrate (blue is haematoxylin). (C,D) AP activity in mouse and human livers, respectively. Blue staining represents phosphatase activity using the substrate Naphthol-ASMX-phosphate at pH 9.8. (E,F) Quantification of AP activity in liver tissue (E) and in serum (F), at t = 24 h and t = 8 weeks of the CCl₄ administration protocol. Note the LPS-dephosphorylating activity in hepatocytes and hepatic arteries and the increase in fibrotic livers relative to normal, in both mouse and human tissue. Abbreviations: ha = hepatic arterioles, bd = bile duct, pv = portal vein, hep = hepatocytes. * = p < 0.05 compared with normal, ** = p < 0.05 24 h vs. 8 weeks CCl₄, # = p < 0.01 compared with normal. ## p < 0.01 24 h vs. 8 weeks CCl₄.

Figure 4

Effect of exogenously administered iAP on CCl₄-induced liver fibrosis. (A) Scheme depicting the administration schedule of CCl4 (i.p.) to Balb/c mice and the subsequent treatment regimen with iAP (i.v) in the final 2 weeks. (B) Collagen 1a1 gene expression levels in healthy mice and fibrotic animals with and without treatment. (C) Representative pictures of desmin staining in livers of fibrotic mice treated with Phosphase-buffered saline (PBS) or iAP. (D) Quantification of hepatic desmin staining in fibrotic mice treated with PBS or iAP using Cell D image analysing software. (E) α-SMA gene expression levels in healthy mice and fibrotic animals with and without treatment. (F) Representative pictures of CD68 staining in livers of fibrotic mice treated with PBS or iAP. (G) Quantification of intrahepatic CD68 staining in fibrotic mice treated with PBS or iAP using Cell D image analysing software. Magnification of sections: 100×. Bars represent mean ± SEM of 5–6 mice per group (ns = not significant, * = p < 0.05).