Infusion of Kupffer Cells Expanded in Vitro Ameliorated Liver Fibrosis in a Murine Model of Liver Injury

Abstract

Transfer of exogenous macrophages represents an alternative technique to treat liver fibrosis. At present, bone marrow-derived monocytes and stem cells are the main sources for exogenous macrophages. Kupffer cells (KCs) are the resident macrophages in the liver and play a critical role in the liver homeostasis and diseases. It is unclear whether infusion of KCs can treat liver fibrosis. In this study, we observed that granulocyte-macrophage colony stimulating factor (GM-CSF) could improve the purity of cultured KCs and significantly up-regulate the expression of Cluster of Differentiation 11b (CD11b). The most important point is that GM-CSF could significantly promote the proliferation of KCs in vitro. KCs expanded in vitro still had the potential of M1/M2 polarization and phagocytosis. Furthermore, infusion of these KCs could ameliorate liver fibrosis induced by carbon tetrachloride (CCl₄) in mice. Together, our results suggest that KCs are likely to be another source for macrophage therapy.

Keywords: GM-CSF; Kupffer cells; cell proliferation; cell therapy; liver fibrosis.

Figure 1.

GM-CSF improved cell purity and up-regulated CD11b expression of Kupffer cells in vitro. KCs were isolated and cultured in the presence of GM-CSF for 7 days. (A) at day 0, day 3, and day 7, cells were collected and analyzed by FACS with PE-labeled anti-mouse F4/80, APC-labeled anti-mouse CD11b or isotype control. (B) Immunofluorescent staining of the cultured KCs with PE-labeled anti-mouse F4/80. The photomicrograph on the right is a larger view of the yellow frame in the middle image. (C) Mouse bone marrow cells were isolated and cultured in the presence of GM-CSF for 7 days to obtain BM-derived macrophages (BMDMs). BMDMs were also analyzed by FACS with PE-labeled anti-mouse F4/80, APC-labeled anti-mouse CD11b or isotype control. FACS: Fluorescence-activated cell sorting.

Figure 2.

GM-CSF promoted the proliferation of Kupffer cells in vitro. KCs were cultured in the presence of GM-CSF. (A) At day 0, day 3, day 7, and day 14, cells were collected and cell numbers were counted. Cells were stained with PE-labeled anti-mouse F4/80, and analyzed by FACS, and then F4/80⁺ cells were calculated (n = 4). (B) BrdU was added into the medium 18 h before the end of the experiment (Day 14). Cells were stained with anti-BrdU and analyzed by FACS. (C) Cell cycle progression of KCs (Day 14) was also determined by FACS analysis. (D) Immunofluorescent staining of KCs (Day 14) with anti-mouse F4/80 and Ki67 antibody. Nuclei were counterstained using Hoechst 33258. The lower row of photomicrographs showed a higher magnification of the yellow frames in the upper row. Data were analyzed using a paired t test. Bars = means ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001. FACS: Fluorescence-activated cell sorting; GM-CSF: granulocyte-macrophage colony stimulating factor.

Figure 3.

Kupffer cells expanded in vitro had the potential of M1/M2 polarization and phagocytosis. (A) KCs were stimulated with LPS (M1) or IL4 (M2), and the expression of key markers were determined by qRT-PCR, iNOS, TNFα, and IL12 as markers of M1, Arg1, YM1/2, and IL10 as markers of M2 Polarization (n = 4). (B) KCs (1 × 10⁵) were cocultured with E. coli BL21 (1 × 10⁶) transformed with an EGFP-expressing plasmid for 2 h. Subsequently, the cells were washed, stained with anti-mouse PE-F4/80 and analyzed by FACS. Data were analyzed using a paired t-test. Bars = means ± SD, *P < 0.05, **P < 0.01. FACS: Fluorescence-activated cell sorting.

Figure 4.

Infusion of Kupffer cells expanded in vitro attenuated liver inflammation in carbon tetrachloride (CCl₄)-induced fibrotic mice. (A) Schematic representation of the experimental procedure. Mice were injected i.p. with CCl₄ twice per week for 8 weeks, and infused with PBS or KCs expanded in vitro (1 × 10⁶ cells/mouse) after the fourth week. Mice were sacrificed 72 h after the last CCl₄ injection. (B) Serum albumin levels of the mice were measured (n = 6). (C) Liver sections were stained by H&E staining and showed less inflammatory cells in the portal region of livers from KCs group mice compared with PBS group. (D) Fibrotic mice infused with KCs showed lower mRNA expression of CCL2, TNF-α, IL1β, IL6, and TGFβ in the liver (n = 4). Unpaired t test for B, and paired t test for D. Bars = means ± SD, *P < 0.05, **P < 0.01.

Figure 5.

Infusion of Kupffer cells expanded in vitro ameliorated liver fibrosis induced by CCl₄ in mice. (A) Liver sections were subjected to immumohistochemical staining for Col1α1. The lower row of photomicrographs was a higher magnification of the blue frames in the upper row. (B) Liver sections were stained with sirius red staining. (C) Liver sections were stained with anti-αSMA, the marker of the activation of hepatic stellate cells (HSCs), and the lower row of photomicrographs showed a higher magnification of the blue frames of the upper row. (D) The Col1α1-positive areas in (A) were quantitatively compared (n = 10). (E) The positive areas of sirius red staining in (B) were quantitatively compared (n = 12). (F) The αSMA-positive areas in (C) were quantitatively compared (n = 6). Data were analyzed using unpaired t test. Bars = means ± SD, **P < 0.01, ****P < 0.0001.