Sphingosine-1-phosphate prevents egress of hematopoietic stem cells from liver to reduce fibrosis

BACKGROUND & AIMS: There is growing interest in the use of bone marrow cells to treat liver fibrosis, however little is known about their anti-fibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSC) to liver fibrosis in mice.

METHODS: Purified (c-kit+/sca1+/lin-) hematopoietic stem cells (HSC) were repeatedly infused into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on methionine/choline deficient (MCD) diets. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC cell lines was quantified in trans-well assays. Levels of S1P in liver, bone marrow and lymph fluid were measured using an ELISA. Liver tissues were collected and analyzed by immunohistochemical, quantitative PCR and sphingosine kinase activity assays. We performed quantitative PCR analyses of expression of sphingosine kinase 1 and 2 (SphK), sphingosine-1-phosphate lyase 1 (SGPL1), and sphingosine-1-phosphate phosphatase 1 (SGPP1) in normal human liver and cirrhotic liver from patients with alcohol related liver disease (n=6).

RESULTS: Infusions of HSC into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSC vs control mice; P<.001), and hepatic hydroxyproline content (328 mg/g in mice given HSC vs 428 mg/g in control mice; P<.01). HSC infusion also reduced hepatic expression of alpha smooth muscle actin (0.19±0.007-fold compared with controls; P<.0001) and collagen type I alpha 1 chain (0.29±0.17-fold compared with controls; P<.0001). These anti-fibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSC to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients hepatic S1P levels increased due to elevated hepatic sphingosine kinase-1 expression, that contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSC had increased hepatic retention of HSC (1697±247 cells in mice given FTY720 vs 982±110 cells in controls; P<.05) and further reductions in fibrosis.

CONCLUSIONS: In studies of mice with chronic liver injury, we demonstrated the anti-fibrotic effects of repeated infusions of purified HSC. We found that HSC promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSC in the liver could increase their anti-fibrotic activities and developed for treatment of patients with liver fibrosis.