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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

NK-derived IFN-γ increases after NTBC withdrawal.Fah−/− mice were transplanted with WT BMCs, and hepatic NK cell percentage (a) and CD69 expression (b) were evaluated at the indicated time points by flow cytometry. (c)Intracellular cytokine expression in hepatic CD3–NK1.1+ NK cells was analyzed 8 weeks after NTBC withdrawal (NTBC off); mice maintained on NTBC were used as controls (NTBC on). (d) Cytokine mRNA expression in liver tissues of BM-transplanted Fah−/− mice was measured by quantitative PCR 8 weeks after NTBC withdrawal. (e) Fah−/− mice were transplanted with CD4−/−, CD8−/−, Nfil3−/−, or CD1d−/− BMCs, and serum IFN-γ levels were evaluated by ELISA 12 weeks after NTBC withdrawal. (f) Fah−/− mice transplanted with WT BM were treated with anti-NK1.1 mAb or PBS control throughout NTBC withdrawal, and serum IFN-γ levels were detected by ELISA 17 weeks after NTBC withdrawal. U.D., undetectable; Representative data from 2 or 3 independent experiments are shown as the mean ± SEM.
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f3: NK-derived IFN-γ increases after NTBC withdrawal.Fah−/− mice were transplanted with WT BMCs, and hepatic NK cell percentage (a) and CD69 expression (b) were evaluated at the indicated time points by flow cytometry. (c)Intracellular cytokine expression in hepatic CD3–NK1.1+ NK cells was analyzed 8 weeks after NTBC withdrawal (NTBC off); mice maintained on NTBC were used as controls (NTBC on). (d) Cytokine mRNA expression in liver tissues of BM-transplanted Fah−/− mice was measured by quantitative PCR 8 weeks after NTBC withdrawal. (e) Fah−/− mice were transplanted with CD4−/−, CD8−/−, Nfil3−/−, or CD1d−/− BMCs, and serum IFN-γ levels were evaluated by ELISA 12 weeks after NTBC withdrawal. (f) Fah−/− mice transplanted with WT BM were treated with anti-NK1.1 mAb or PBS control throughout NTBC withdrawal, and serum IFN-γ levels were detected by ELISA 17 weeks after NTBC withdrawal. U.D., undetectable; Representative data from 2 or 3 independent experiments are shown as the mean ± SEM.

Mentions: Since we observed that NK cells might be involved in BMDH generation from observing higher NK cell activation at a single time point (12 weeks) in Fig. 2a, we monitored NK cells over time by evaluating hepatic NK cell kinetics during BMDH generation. Although the percentage and total number of NK cells remained similar over time (Fig. 3a and Supplementary Fig. S2b), CD69 expression was greatly induced on NK cells and remained elevated for more than 3 months after NTBC withdrawal (Fig. 3b and Supplementary Fig. S2c). IFN-γ is an important effector molecule produced by NK cells as well as by NKT and conventional CD4+ and CD8+ T cells, among others. Consistent with the presence of more highly activated NK cells, intracellular IFN-γ in NK cells and liver tissue–derived Ifng mRNA were prominently increased after NTBC withdrawal (Fig. 3c,d and Supplementary Fig. S2d), while other measured cytokines did not change. Moreover, serum IFN-γ was detected in WT, CD1d−/−, CD4−/−, and CD8−/− BM-reconstituted liver, but not in Nfil3−/− BM-reconstituted liver (Fig. 3e), suggesting that the IFN-γ produced during this process was mainly derived from NK cells. Supporting this finding, NK1.1+ cell depletion abolished IFN-γ production after WT BM reconstitution (Fig. 3f). Collectively, these results suggest that NK cells are a major producer of IFN-γ in BM-reconstituted livers, prompting us to hypothesize that IFN-γ may play a role during liver regeneration by BMDHs after BMT.


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)

NK-derived IFN-γ increases after NTBC withdrawal.Fah−/− mice were transplanted with WT BMCs, and hepatic NK cell percentage (a) and CD69 expression (b) were evaluated at the indicated time points by flow cytometry. (c)Intracellular cytokine expression in hepatic CD3–NK1.1+ NK cells was analyzed 8 weeks after NTBC withdrawal (NTBC off); mice maintained on NTBC were used as controls (NTBC on). (d) Cytokine mRNA expression in liver tissues of BM-transplanted Fah−/− mice was measured by quantitative PCR 8 weeks after NTBC withdrawal. (e) Fah−/− mice were transplanted with CD4−/−, CD8−/−, Nfil3−/−, or CD1d−/− BMCs, and serum IFN-γ levels were evaluated by ELISA 12 weeks after NTBC withdrawal. (f) Fah−/− mice transplanted with WT BM were treated with anti-NK1.1 mAb or PBS control throughout NTBC withdrawal, and serum IFN-γ levels were detected by ELISA 17 weeks after NTBC withdrawal. U.D., undetectable; Representative data from 2 or 3 independent experiments are shown as the mean ± SEM.
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Related In: Results  -  Collection

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f3: NK-derived IFN-γ increases after NTBC withdrawal.Fah−/− mice were transplanted with WT BMCs, and hepatic NK cell percentage (a) and CD69 expression (b) were evaluated at the indicated time points by flow cytometry. (c)Intracellular cytokine expression in hepatic CD3–NK1.1+ NK cells was analyzed 8 weeks after NTBC withdrawal (NTBC off); mice maintained on NTBC were used as controls (NTBC on). (d) Cytokine mRNA expression in liver tissues of BM-transplanted Fah−/− mice was measured by quantitative PCR 8 weeks after NTBC withdrawal. (e) Fah−/− mice were transplanted with CD4−/−, CD8−/−, Nfil3−/−, or CD1d−/− BMCs, and serum IFN-γ levels were evaluated by ELISA 12 weeks after NTBC withdrawal. (f) Fah−/− mice transplanted with WT BM were treated with anti-NK1.1 mAb or PBS control throughout NTBC withdrawal, and serum IFN-γ levels were detected by ELISA 17 weeks after NTBC withdrawal. U.D., undetectable; Representative data from 2 or 3 independent experiments are shown as the mean ± SEM.
Mentions: Since we observed that NK cells might be involved in BMDH generation from observing higher NK cell activation at a single time point (12 weeks) in Fig. 2a, we monitored NK cells over time by evaluating hepatic NK cell kinetics during BMDH generation. Although the percentage and total number of NK cells remained similar over time (Fig. 3a and Supplementary Fig. S2b), CD69 expression was greatly induced on NK cells and remained elevated for more than 3 months after NTBC withdrawal (Fig. 3b and Supplementary Fig. S2c). IFN-γ is an important effector molecule produced by NK cells as well as by NKT and conventional CD4+ and CD8+ T cells, among others. Consistent with the presence of more highly activated NK cells, intracellular IFN-γ in NK cells and liver tissue–derived Ifng mRNA were prominently increased after NTBC withdrawal (Fig. 3c,d and Supplementary Fig. S2d), while other measured cytokines did not change. Moreover, serum IFN-γ was detected in WT, CD1d−/−, CD4−/−, and CD8−/− BM-reconstituted liver, but not in Nfil3−/− BM-reconstituted liver (Fig. 3e), suggesting that the IFN-γ produced during this process was mainly derived from NK cells. Supporting this finding, NK1.1+ cell depletion abolished IFN-γ production after WT BM reconstitution (Fig. 3f). Collectively, these results suggest that NK cells are a major producer of IFN-γ in BM-reconstituted livers, prompting us to hypothesize that IFN-γ may play a role during liver regeneration by BMDHs after BMT.

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