Modulation of liver regeneration via myeloid PTEN deficiency

Abstract

Molecular mechanisms that modulate liver regeneration are of critical importance for a number of hepatic disorders. Kupffer cells and natural killer (NK) cells are two cell subsets indispensable for liver regeneration. We have focused on these two populations and, in particular, the interplay between them. Importantly, we demonstrate that deletion of the myeloid phosphatase and tensin homolog on chromosome 10 (PTEN) leading to an M2-like polarization of Kupffer cells, which results in decreased activation of NK cells. In addition, PTEN-deficient Kupffer cells secrete additional factors that facilitate the proliferation of hepatocytes. In conclusion, PTEN is critical for inhibiting M2-like polarization of Kupffer cells after partial hepatectomy, resulting in NK cell activation and thus the inhibition of liver regeneration. Furthermore, PTEN reduces growth factor secretion by Kupffer cells. Our results suggest that targeting PTEN on Kupffer cells may be useful in altering liver regeneration in patients undergoing liver resection.

Figure 1

Liver Kupffer cells show upregulated PTEN expression level and an M1-like polarization after 2/3 PHx. (a) The liver weight (LW) to body weight (BW) ratio was analyzed 48 h post-2/3 PHx in PBS-liposome-treated (PBS, n=14) or clodronate-liposome-treated (Clo, n=6) mice. (b) PTEN expression levels in CD45⁺F4/80^hiCD11b^lo Kupffer cells of sham-operated (n=5) or 24 h PHx-treated mice (n=5) were analyzed using flow cytometry. (c) Expression levels of CD206, CD11c, MHC-II and CD80 were analyzed in Kupffer cells from sham (n=5) or 24 h PHx-treated mice (n=5) using flow cytometry

Figure 2

PTEN^mKO mice show a more prominent liver-regenerating capacity after 2/3 PHx. (a) Immunohistochemical staining of PCNA in liver sections from PTEN^f/f (n=32 for 24 h, n=16 for 48 h) and PTEN^mKO (n=30 for 24 h, n=20 for 48 h) mice 24 and 48 h after 2/3 PHx. PCNA-positive hepatocytes were counted in five randomly chosen fields, and the average count of these five fields was set as the PCNA-positive cell number of one mouse. (b) Immunohistochemical staining of Ki-67 in liver sections of PTEN^f/f (n=29 for 24 h, n=15 for 48 h) and PTEN^mKO (n=27 for 24 h, n=20 for 48 h) mice 24 and 48 h after 2/3 PHx. The method for counting Ki-67-positive cells was the same as in a. (c and d) Representative H&E staining (c) and quantification (d) of mitotic hepatocytes (indicated by yellow arrows) of PTEN^f/f (n=8) and PTEN^mKO mice (n=7). The mitotic index was calculated as the average ratio of mitotic hepatocytes (hepatocytes in prometaphase, metaphase and anaphase) to total hepatocytes of five randomly chosen fields. (e) The liver weight (LW) to body weight (BW) ratio was calculated at various time points in sham- or PHx-operated PTEN^f/f (n=3 for sham, n=4 for 12 h, n=5 for 24 h, n=8 for 48 h, n=5 for 10 days) and PTEN^mKO (n=4 for sham, n=6 for 12 h, n=4 for 24 h, n=8 for 48 h, n=5 for 10 days) mice

Figure 3

PTEN-deficient Kupffer cells show an M2-like polarization state. (a) Real-time PCR analysis of M2-related (Ym-1, Cd206, Arginase-1 and Fizz-1) and M1-related (Inos and Il-12p40) markers in collagenase-perfused Kupffer cells from 48 h PHx-operated PTEN^f/f (n=6) and PTEN^mKO (n=6) mice. (b) CD206 and CD11c expression levels in CD45⁺F4/80^hiCD11b^lo Kupffer cells from PTEN^f/f (n=6) and PTEN^mKO (n=4) mice 48 h post PHx were analyzed using flow cytometry

Figure 4

NK cells from PTEN^mKO mice are less activated. (a) Total liver lymphocytes from PTEN^f/f (n=6) and PTEN^mKO (n=4) mice were isolated 48 h post PHx, and treated with a PMA/ionomycin cocktail for 4 h. The fraction of IFN-γ-positive NK cells (CD3^-NK1.1⁺) was analyzed using flow cytometry. (b) The mean fluorescence intensity (MFI) of CD69, ICOS and 2B4 on NK cells from PTEN^f/f (n=5) and PTEN^mKO (n=5) mice 48 h post PHx was analyzed by flow cytometry. (c) FACS-sorted liver Kupffer cells from PTEN^f/f (n=4) or PTEN^mKO (n=4) mice 48 h post PHx were co-cultured with sorted WT liver NK cells at a ratio of Kupffer: NK=1:2, in the presence of 50 IU/ml IL-2. The percentage of IFN-γ-positive NK cells and the mean fluorescence intensity of IFN-γ were analyzed using flow cytometry after 48 h of co-culture. (d) The IFN-γ concentration in the medium of the co-culture described in c was measured by cytometric bead array

Figure 5

Kupffer cells from PTEN^mKO mice express less NK cell-activating factors. (a) Livers of PTEN^f/f (n=6) and PTEN^mKO (n=4) mice 48 h post PHx were perfused by collagenase in situ, and Kupffer cells were analyzed for the expression levels of MHC-II, CD80, CD86 and CD40 by flow cytometry. (b) Livers of PTEN^f/f (n=5) and PTEN^mKO (n=5) mice 3 h post PHx were perfused to isolate Kupffer cells, and the expression levels of Il-15, Il-12p40 and Il-18 were analyzed by real-time PCR. (c) Livers of PTEN^f/f (n=6 for 12 h, n=5 for 24 h, n=5 for 36 h and n=6 for 48 h) and PTEN^mKO (n=6 for 12 h, n=5 for 24 h, n=5 for 36 h and n=6 for 48 h) mice were homogenized in PBS at various time points post PHx, and analyzed by CBA for IL-12 concentration

Figure 6

PTEN-deficient Kupffer cells are more mitogenic for hepatocytes. (a) To isolate Kupffer cells, sham and 48 h PHx-operated livers of PTEN^f/f (n=7 for sham, n=6 for 48 h) and PTEN^mKO (n=5 for sham, n=6 for 48 h) mice were perfused by collagenase in situ, and the expression levels of hepatocyte mitogenic related factors (Pdgf-α, Pdgf-β, Hgf, Osm, Vegf, Il-6 and Tnf-a) were analyzed using real-time PCR. (b) Total liver proteins from PTEN^f/f and PTEN^mKO mice were extracted 48 h after PHx. Levels of p-Stat3, Stat3 and GAPDH were evaluated by western blotting (left), and levels of p-Stat3 (relative to GAPDH and total Stat3) were analyzed with ImageJ (right). (c) FACS-sorted liver Kupffer cells from PTEN^f/f (n=5) or PTEN^mKO (n=5) mice 48 h post PHx were cultured alone for 24 h, and AML-12 cells were stimulated with the Kupffer cell conditioned medium for 48 h. Then, AML-12 cells were dissociated with Trypsin-EDTA and counted

Figure 7

Graphic summary of this article. After PHx in PTEN-sufficient mice, PTEN expression was upregulated in Kupffer cells, which inhibited activation of Akt and thus promoted activation of downstream FoxO1 signaling, resulting in an M1-like polarization state. These M1-like Kupffer cells were more capable of activating NK cells, both through direct cell–cell contact by enhancing CD40, ICOS and CD48 signals and through facilitating secretion of IL-12 and IL-15. Consequently, NK cells in the liver were activated, and more IFN-γ was released, hindering hepatocyte proliferation. Moreover, Kupffer cells with higher PTEN expression levels released less growth factors such as PDGF, HGF and OSM. These effects of PTEN on Kupffer cells combined to hinder liver regeneration (upper panel). On the other hand, after PHx in PTEN-deficient mice, Akt signaling was activated and thus inhibited downstream FoxO1 signaling, resulting in an M2-like polarization state. These M2-like Kupffer cells were less capable of activating NK cells because of less expression levels of direct cell–cell contact molecules and NK cell-activating cytokines mentioned above. Consequently, NK cells in the liver were less activated, and less IFN-γ was released. Moreover, PTEN-deficient Kupffer cells released more growth factors mentioned above. As a result, liver regeneration rate was promoted (lower panel)