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Natural Killer Cells-Produced IFN-γ Improves Bone Marrow-Derived Hepatocytes Regeneration in Murine Liver Failure Model.

Li L, Zeng Z, Qi Z, Wang X, Gao X, Wei H, Sun R, Tian Z - Sci Rep (2015)

Bottom Line: Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers.Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts.BM-derived myelomonocytes were determined to be the IFN-γ-responding cells.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.

ABSTRACT
Bone-marrow transplantation (BMT) can repopulate the liver through BM-derived hepatocyte (BMDH) generation, although the underlying mechanism remains unclear. Using fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mice as a liver-failure model, we confirmed that BMDHs were generated by fusion of BM-derived CD11b(+)F4/80(+)myelomonocytes with resident Fah(-/-) hepatocytes. Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers. Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts. BM-derived myelomonocytes were determined to be the IFN-γ-responding cells. The IFN-γ-IFN-γR interaction contributed to the myelomonocyte-hepatocyte fusion process, as most of the CD11b(+) BMDHs in mixed BM chimeric Fah(-/-) hosts transplanted with a 1:1 ratio of CD45.1(+) WT and CD45.2(+) Ifngr1(-/-) BM cells were of CD45.1(+) WT origin. Confirming these findings in vitro, IFN-γ dose-dependently promoted the fusion of GFP(+) myelomonocytes with Fah(-/-) hepatocytes due to a direct effect on myelomonocytes; similar results were observed using activated NK cells. In conclusion, BMDH generation requires NK cells to facilitate myelomonocyte-hepatocyte fusion in an IFN-γ-dependent manner, providing new insights for treating severe liver failure.

No MeSH data available.


Related in: MedlinePlus

IFN-γ–IFN-γR interaction contributes to cellular fusion in vitro.(a) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h in the absence (unstimulated [US], top) or presence (bottom) of 1 ng/mL IFN-γ and then stained for Fah (red) and DAPI (blue) (scale bar, 50 μm). (b) Fah-positive hepatocytes were enumerated in the co-culture system from 5 random fields per section. (c) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h with medium alone (US), IFN-γ (1 ng/mL), NK cells pre-stimulated with 200 IU/mL IL-2 for 24 h, or pre-stimulated NK cells plus a neutralizing anti–IFN-γ Ab (20 μg/mL); cells were then stained for Fah (red) and DAPI (blue) (scale bar, 20 μm). Representative pictures and percentage of Fah+ hepatocytes from at least 2 independent experiments are shown. (d) Fah−/− hepatocytes (Hep) or GFP+ splenic myelomonocytes (Mye) were pre-stimulated with (+) or without (−) IFN-γ (1 ng/mL) for 2 h and co-cultured together for additional 14 h. Representative images from at least 2 independent experiments for immunofluorescence staining of Fah (red) and GFP (green) and enumeration of Fah+ hepatocytes are shown(scale bar, 20 μm).
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f7: IFN-γ–IFN-γR interaction contributes to cellular fusion in vitro.(a) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h in the absence (unstimulated [US], top) or presence (bottom) of 1 ng/mL IFN-γ and then stained for Fah (red) and DAPI (blue) (scale bar, 50 μm). (b) Fah-positive hepatocytes were enumerated in the co-culture system from 5 random fields per section. (c) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h with medium alone (US), IFN-γ (1 ng/mL), NK cells pre-stimulated with 200 IU/mL IL-2 for 24 h, or pre-stimulated NK cells plus a neutralizing anti–IFN-γ Ab (20 μg/mL); cells were then stained for Fah (red) and DAPI (blue) (scale bar, 20 μm). Representative pictures and percentage of Fah+ hepatocytes from at least 2 independent experiments are shown. (d) Fah−/− hepatocytes (Hep) or GFP+ splenic myelomonocytes (Mye) were pre-stimulated with (+) or without (−) IFN-γ (1 ng/mL) for 2 h and co-cultured together for additional 14 h. Representative images from at least 2 independent experiments for immunofluorescence staining of Fah (red) and GFP (green) and enumeration of Fah+ hepatocytes are shown(scale bar, 20 μm).

Mentions: To confirm our in vivo findings regarding the essential role of IFN-γ in fusing myelomonocytes and hepatocytes together to generate BMDHs, we determined whether IFN-γ could also promote cell fusion in vitro by co-culturing GFP+ splenic myelomonocytes with Fah−/− hepatocytes in the presence or absence of IFN-γ. Indeed, IFN-γ promoted fusion between myelomonocytes and Fah−/− hepatocytes in a dose-dependent manner as evidenced by the emergence of GFP and Fah expression in Fah−/− hepatocytes (Fig. 7a,b and Supplementary Fig. S7a). The cell fusion was further evidenced by the fact that co-culturing female Fah−/− hepatocytes with male myelomonocytes in the presence of IFN-γ gave rise to Fah and Y chromosome positive cells (Supplementary Fig. S7b). In addition, these fusion derived Fah expressing cells were also positive for cytokeratin (CK)-18, a hepatocyte specific marker (Supplementary Fig. S7c), identifying they were hepatocytes rather than myelomonocytes. Moreover, we found that pre-activated NK cells also promoted cell fusion specifically through IFN-γ secretion (Fig. 7c). Also consistent with the findings from our in vivo experiments, we confirmed that IFN-γR signaling was required for myelomonocyte–hepatocyte cell fusion, as only IFN-γ–stimulated myelomonocytes, but not IFN-γ–stimulated hepatocytes, promoted the fusion of CD11b+ cells with Fah−/− hepatocytes in vitro (Fig. 7d).


Natural Killer Cells-Produced IFN-γ Improves Bone Marrow-Derived Hepatocytes Regeneration in Murine Liver Failure Model.

Li L, Zeng Z, Qi Z, Wang X, Gao X, Wei H, Sun R, Tian Z - Sci Rep (2015)

IFN-γ–IFN-γR interaction contributes to cellular fusion in vitro.(a) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h in the absence (unstimulated [US], top) or presence (bottom) of 1 ng/mL IFN-γ and then stained for Fah (red) and DAPI (blue) (scale bar, 50 μm). (b) Fah-positive hepatocytes were enumerated in the co-culture system from 5 random fields per section. (c) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h with medium alone (US), IFN-γ (1 ng/mL), NK cells pre-stimulated with 200 IU/mL IL-2 for 24 h, or pre-stimulated NK cells plus a neutralizing anti–IFN-γ Ab (20 μg/mL); cells were then stained for Fah (red) and DAPI (blue) (scale bar, 20 μm). Representative pictures and percentage of Fah+ hepatocytes from at least 2 independent experiments are shown. (d) Fah−/− hepatocytes (Hep) or GFP+ splenic myelomonocytes (Mye) were pre-stimulated with (+) or without (−) IFN-γ (1 ng/mL) for 2 h and co-cultured together for additional 14 h. Representative images from at least 2 independent experiments for immunofluorescence staining of Fah (red) and GFP (green) and enumeration of Fah+ hepatocytes are shown(scale bar, 20 μm).
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f7: IFN-γ–IFN-γR interaction contributes to cellular fusion in vitro.(a) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h in the absence (unstimulated [US], top) or presence (bottom) of 1 ng/mL IFN-γ and then stained for Fah (red) and DAPI (blue) (scale bar, 50 μm). (b) Fah-positive hepatocytes were enumerated in the co-culture system from 5 random fields per section. (c) Fah−/− hepatocytes were co-cultured with GFP+ splenic myelomonocytes for 16 h with medium alone (US), IFN-γ (1 ng/mL), NK cells pre-stimulated with 200 IU/mL IL-2 for 24 h, or pre-stimulated NK cells plus a neutralizing anti–IFN-γ Ab (20 μg/mL); cells were then stained for Fah (red) and DAPI (blue) (scale bar, 20 μm). Representative pictures and percentage of Fah+ hepatocytes from at least 2 independent experiments are shown. (d) Fah−/− hepatocytes (Hep) or GFP+ splenic myelomonocytes (Mye) were pre-stimulated with (+) or without (−) IFN-γ (1 ng/mL) for 2 h and co-cultured together for additional 14 h. Representative images from at least 2 independent experiments for immunofluorescence staining of Fah (red) and GFP (green) and enumeration of Fah+ hepatocytes are shown(scale bar, 20 μm).
Mentions: To confirm our in vivo findings regarding the essential role of IFN-γ in fusing myelomonocytes and hepatocytes together to generate BMDHs, we determined whether IFN-γ could also promote cell fusion in vitro by co-culturing GFP+ splenic myelomonocytes with Fah−/− hepatocytes in the presence or absence of IFN-γ. Indeed, IFN-γ promoted fusion between myelomonocytes and Fah−/− hepatocytes in a dose-dependent manner as evidenced by the emergence of GFP and Fah expression in Fah−/− hepatocytes (Fig. 7a,b and Supplementary Fig. S7a). The cell fusion was further evidenced by the fact that co-culturing female Fah−/− hepatocytes with male myelomonocytes in the presence of IFN-γ gave rise to Fah and Y chromosome positive cells (Supplementary Fig. S7b). In addition, these fusion derived Fah expressing cells were also positive for cytokeratin (CK)-18, a hepatocyte specific marker (Supplementary Fig. S7c), identifying they were hepatocytes rather than myelomonocytes. Moreover, we found that pre-activated NK cells also promoted cell fusion specifically through IFN-γ secretion (Fig. 7c). Also consistent with the findings from our in vivo experiments, we confirmed that IFN-γR signaling was required for myelomonocyte–hepatocyte cell fusion, as only IFN-γ–stimulated myelomonocytes, but not IFN-γ–stimulated hepatocytes, promoted the fusion of CD11b+ cells with Fah−/− hepatocytes in vitro (Fig. 7d).

Bottom Line: Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers.Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts.BM-derived myelomonocytes were determined to be the IFN-γ-responding cells.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.

ABSTRACT
Bone-marrow transplantation (BMT) can repopulate the liver through BM-derived hepatocyte (BMDH) generation, although the underlying mechanism remains unclear. Using fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mice as a liver-failure model, we confirmed that BMDHs were generated by fusion of BM-derived CD11b(+)F4/80(+)myelomonocytes with resident Fah(-/-) hepatocytes. Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers. Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts. BM-derived myelomonocytes were determined to be the IFN-γ-responding cells. The IFN-γ-IFN-γR interaction contributed to the myelomonocyte-hepatocyte fusion process, as most of the CD11b(+) BMDHs in mixed BM chimeric Fah(-/-) hosts transplanted with a 1:1 ratio of CD45.1(+) WT and CD45.2(+) Ifngr1(-/-) BM cells were of CD45.1(+) WT origin. Confirming these findings in vitro, IFN-γ dose-dependently promoted the fusion of GFP(+) myelomonocytes with Fah(-/-) hepatocytes due to a direct effect on myelomonocytes; similar results were observed using activated NK cells. In conclusion, BMDH generation requires NK cells to facilitate myelomonocyte-hepatocyte fusion in an IFN-γ-dependent manner, providing new insights for treating severe liver failure.

No MeSH data available.


Related in: MedlinePlus