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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 myelomonocyte–hepatocyte cellular fusion in vivo.(a,b) Liver tissues of Fah−/− mice transplanted with GKO(a) or Ifngr1−/− (b) BMCs were collected 20 weeks after NTBC withdrawal for immunohistochemical staining of Fah (scale bar, 1000 μm). (c) Fah−/− mice(CD45.2) were transplanted with a 1:1 mixture of WT (CD45.1) and Ifngr1−/− (CD45.2) BMCs. (d,e) The percentage of WT or Ifngr1−/− cells within liver mononuclear cells (LMNC) (d) or CD11b+ hepatocytes(BMDH) (e) are shown 26 weeks after NTBC withdrawal. (f) Hepatocytes were separated from Fah−/− mice 20 weeks after mixed BMT and stained for Fah (red) and CD45.1 (green). Nuclei were counterstained with DAPI (blue) (scale bar, 50 μm). (g) Fah−/− mice transplanted with WT BM were treated with 500ng IFN-γ or PBS control for six weeks, liver tissues were collected 12 weeks after NTBC withdrawal, and immunohistochemical staining of Fah was performed (scale bar, 500 μm).
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f6: IFN-γ–IFN-γR interaction contributes to myelomonocyte–hepatocyte cellular fusion in vivo.(a,b) Liver tissues of Fah−/− mice transplanted with GKO(a) or Ifngr1−/− (b) BMCs were collected 20 weeks after NTBC withdrawal for immunohistochemical staining of Fah (scale bar, 1000 μm). (c) Fah−/− mice(CD45.2) were transplanted with a 1:1 mixture of WT (CD45.1) and Ifngr1−/− (CD45.2) BMCs. (d,e) The percentage of WT or Ifngr1−/− cells within liver mononuclear cells (LMNC) (d) or CD11b+ hepatocytes(BMDH) (e) are shown 26 weeks after NTBC withdrawal. (f) Hepatocytes were separated from Fah−/− mice 20 weeks after mixed BMT and stained for Fah (red) and CD45.1 (green). Nuclei were counterstained with DAPI (blue) (scale bar, 50 μm). (g) Fah−/− mice transplanted with WT BM were treated with 500ng IFN-γ or PBS control for six weeks, liver tissues were collected 12 weeks after NTBC withdrawal, and immunohistochemical staining of Fah was performed (scale bar, 500 μm).

Mentions: In line with the findings that IFN-γ or IFN-γR BMCs failed to rescue the Fah−/− mice, Fah+ BMDH generation was dramatically reduced in those GKO or Ifngr1−/− BM-reconstituted Fah−/− mice compared to WT BM-reconstituted Fah−/− mice (Fig. 6a,b). This reduction of BMDH formation was not likely due to the inhibition of BMDH proliferation, as PCNA staining showed no difference in the proliferation of Fah+ BMDHs between WT BM and GKO BM-reconstituted Fah−/− mice (Supplementary Fig. S5a). The fusion of myelomonocytes with resident hepatocytes was previously reported to be the main mechanism for generating BMDHs78910. We therefore wondered whether the IFN-γ–IFN-γR interaction played a role in this process. First, we determined that IFN-γ did not affect the generation or differentiation of the fusion partners-myelomonocytes from the BM, as no differences in percentage, number, or subsets of hepatic myelomonocytes were observed in GKO or Ifngr1−/− BM-reconstituted Fah−/− mice compared to WT BM-reconstituted Fah−/− mice (Supplementary Fig. S5b–d). We then hypothesized that IFN-γ was required for stimulating the fusion between BM-derived myelomonocytes and resident hepatocytes. To address this hypothesis, we performed a mixed BM–reconstitution experiment in which BM cells containing a 1:1 mixture of CD45.1+ WT and CD45.2+ IFN-γR–deficient BMCs were transplanted into Fah−/− mice (Fig. 6c). The majority of CD11b+ BMDHs in these mixed BM–chimeric Fah−/− mice expressed the CD45.1 marker, indicating that they were derived from WT BM (Fig. 6d,e and Supplementary Fig. S6); importantly, this was likely due to the inability of IFN-γR–deficient myelomonocytes to fuse with the resident hepatocytes rather than an effect of IFN-γ on myelomonocyte survival or migration to the liver, as the similar percentage of CD45.1+ and CD45.2+ cells in the liver mononuclear cells (Fig. 6d) indicated equivalent reconstitution and hepatic migration of these cells after NTBC withdrawal. Immunofluorescence staining of BMDHs also showed that most of the Fah+ hepatocytes expressed CD45.1 (Fig. 6f), further suggesting that BM-intrinsic IFN-γR signaling was required for myelomonocytes to fuse with resident hepatocytes for BMDH generation. Finally, to investigate if IFN-γ could enhance the BMDH production in vivo, Fah−/− mice transplanted with WT BMCs were treated with recombinant IFN-γ or PBS as control, a profound formation of Fah+ BMDH was observed in IFN-γ treated mice, whereas few BMDHs can be seen in controls at that time (Fig. 6g), suggesting that IFN-γ treatment could potentially accelerate the generation of BMDH during BMT-based therapy.


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 myelomonocyte–hepatocyte cellular fusion in vivo.(a,b) Liver tissues of Fah−/− mice transplanted with GKO(a) or Ifngr1−/− (b) BMCs were collected 20 weeks after NTBC withdrawal for immunohistochemical staining of Fah (scale bar, 1000 μm). (c) Fah−/− mice(CD45.2) were transplanted with a 1:1 mixture of WT (CD45.1) and Ifngr1−/− (CD45.2) BMCs. (d,e) The percentage of WT or Ifngr1−/− cells within liver mononuclear cells (LMNC) (d) or CD11b+ hepatocytes(BMDH) (e) are shown 26 weeks after NTBC withdrawal. (f) Hepatocytes were separated from Fah−/− mice 20 weeks after mixed BMT and stained for Fah (red) and CD45.1 (green). Nuclei were counterstained with DAPI (blue) (scale bar, 50 μm). (g) Fah−/− mice transplanted with WT BM were treated with 500ng IFN-γ or PBS control for six weeks, liver tissues were collected 12 weeks after NTBC withdrawal, and immunohistochemical staining of Fah was performed (scale bar, 500 μm).
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Related In: Results  -  Collection

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f6: IFN-γ–IFN-γR interaction contributes to myelomonocyte–hepatocyte cellular fusion in vivo.(a,b) Liver tissues of Fah−/− mice transplanted with GKO(a) or Ifngr1−/− (b) BMCs were collected 20 weeks after NTBC withdrawal for immunohistochemical staining of Fah (scale bar, 1000 μm). (c) Fah−/− mice(CD45.2) were transplanted with a 1:1 mixture of WT (CD45.1) and Ifngr1−/− (CD45.2) BMCs. (d,e) The percentage of WT or Ifngr1−/− cells within liver mononuclear cells (LMNC) (d) or CD11b+ hepatocytes(BMDH) (e) are shown 26 weeks after NTBC withdrawal. (f) Hepatocytes were separated from Fah−/− mice 20 weeks after mixed BMT and stained for Fah (red) and CD45.1 (green). Nuclei were counterstained with DAPI (blue) (scale bar, 50 μm). (g) Fah−/− mice transplanted with WT BM were treated with 500ng IFN-γ or PBS control for six weeks, liver tissues were collected 12 weeks after NTBC withdrawal, and immunohistochemical staining of Fah was performed (scale bar, 500 μm).
Mentions: In line with the findings that IFN-γ or IFN-γR BMCs failed to rescue the Fah−/− mice, Fah+ BMDH generation was dramatically reduced in those GKO or Ifngr1−/− BM-reconstituted Fah−/− mice compared to WT BM-reconstituted Fah−/− mice (Fig. 6a,b). This reduction of BMDH formation was not likely due to the inhibition of BMDH proliferation, as PCNA staining showed no difference in the proliferation of Fah+ BMDHs between WT BM and GKO BM-reconstituted Fah−/− mice (Supplementary Fig. S5a). The fusion of myelomonocytes with resident hepatocytes was previously reported to be the main mechanism for generating BMDHs78910. We therefore wondered whether the IFN-γ–IFN-γR interaction played a role in this process. First, we determined that IFN-γ did not affect the generation or differentiation of the fusion partners-myelomonocytes from the BM, as no differences in percentage, number, or subsets of hepatic myelomonocytes were observed in GKO or Ifngr1−/− BM-reconstituted Fah−/− mice compared to WT BM-reconstituted Fah−/− mice (Supplementary Fig. S5b–d). We then hypothesized that IFN-γ was required for stimulating the fusion between BM-derived myelomonocytes and resident hepatocytes. To address this hypothesis, we performed a mixed BM–reconstitution experiment in which BM cells containing a 1:1 mixture of CD45.1+ WT and CD45.2+ IFN-γR–deficient BMCs were transplanted into Fah−/− mice (Fig. 6c). The majority of CD11b+ BMDHs in these mixed BM–chimeric Fah−/− mice expressed the CD45.1 marker, indicating that they were derived from WT BM (Fig. 6d,e and Supplementary Fig. S6); importantly, this was likely due to the inability of IFN-γR–deficient myelomonocytes to fuse with the resident hepatocytes rather than an effect of IFN-γ on myelomonocyte survival or migration to the liver, as the similar percentage of CD45.1+ and CD45.2+ cells in the liver mononuclear cells (Fig. 6d) indicated equivalent reconstitution and hepatic migration of these cells after NTBC withdrawal. Immunofluorescence staining of BMDHs also showed that most of the Fah+ hepatocytes expressed CD45.1 (Fig. 6f), further suggesting that BM-intrinsic IFN-γR signaling was required for myelomonocytes to fuse with resident hepatocytes for BMDH generation. Finally, to investigate if IFN-γ could enhance the BMDH production in vivo, Fah−/− mice transplanted with WT BMCs were treated with recombinant IFN-γ or PBS as control, a profound formation of Fah+ BMDH was observed in IFN-γ treated mice, whereas few BMDHs can be seen in controls at that time (Fig. 6g), suggesting that IFN-γ treatment could potentially accelerate the generation of BMDH during BMT-based therapy.

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