Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced mouse model through tumor necrosis factor-α production

Abstract

Background: The mechanisms triggering nonalcoholic steatohepatitis (NASH) remain poorly defined.

Results: Kupffer cells are the first responding cells to hepatocyte injuries, leading to TNFα production, chemokine induction, and monocyte recruitment. The silencing of TNFα in myeloid cells reduces NASH progression.

Conclusion: Increase of TNFα-producing Kupffer cells is crucial for triggering NASH via monocyte recruitment.

Significance: Myeloid cells-targeted silencing of TNFα might be a tenable therapeutic approach. Nonalcoholic steatohepatitis (NASH), characterized by lipid deposits within hepatocytes (steatosis), is associated with hepatic injury and inflammation and leads to the development of fibrosis, cirrhosis, and hepatocarcinoma. However, the pathogenic mechanism of NASH is not well understood. To determine the role of distinct innate myeloid subsets in the development of NASH, we examined the contribution of liver resident macrophages (i.e. Kupffer cells) and blood-derived monocytes in triggering liver inflammation and hepatic damage. Employing a murine model of NASH, we discovered a previously unappreciated role for TNFα and Kupffer cells in the initiation and progression of NASH. Sequential depletion of Kupffer cells reduced the incidence of liver injury, steatosis, and proinflammatory monocyte infiltration. Furthermore, our data show a differential contribution of Kupffer cells and blood monocytes during the development of NASH; Kupffer cells increased their production of TNFα, followed by infiltration of CD11b(int)Ly6C(hi) monocytes, 2 and 10 days, respectively, after starting the methionine/choline-deficient (MCD) diet. Importantly, targeted knockdown of TNFα expression in myeloid cells decreased the incidence of NASH development by decreasing steatosis, liver damage, monocyte infiltration, and the production of inflammatory chemokines. Our findings suggest that the increase of TNFα-producing Kupffer cells in the liver is crucial for the early phase of NASH development by promoting blood monocyte infiltration through the production of IP-10 and MCP-1.

FIGURE 1.

Blood monocytes represent the major source of infiltrated cells after 10 days of NASH-inducing diet. A, the FACS plots and cell numbers of neutrophils (CD11b^hiF4/80⁻), monocytes (CD11b^intF4/80^low), and Kupffer cells (CD11b⁺F4/80^hi) are shown in CD45⁺NK1.1⁻-gated whole liver cells isolated from mice fed CT and MCD diets. Representative FACS plots and histograms show an increase in frequency and cell numbers of monocytes. Data are representative of at least four independent experiments with a total of n = 8 mice per condition. B, hepatic Ccr2 and Tnfa transcript expression was determined by qPCR from whole liver samples of mice fed CT and MCD diets. The RNA level is expressed as fold-induction compared with control diet. Data represent mean ± S.D. C, cell number of monocytes within CD45⁺NK1.1⁻-gated mononuclear liver cell fraction isolated from mice fed CT and MCD diets after 0, 2, 6, 8, and 10 days, with a total of n = 3 mice per condition. *, p < 0.05 and ***, p < 0.0005 as determined by two-tailed t test.

FIGURE 2.

The initiation of NASH disease depends on resident liver macrophages. A, C57BL/6 mice fed CT or MCD diets were treated either with two injections of control liposomes (CT Lipo.) or clodronate-containing liposomes (clod. Lipo.). H&E-stained (a, d, g, and j) and Oil-Red O-stained (b, e, h, and k) tissue sections were examined under a bright field microscope at ×200 magnification. Liver sections were also stained with anti-F4/80 (green), anti-CD31 (red), and DAPI (blue), and examined under a apotome fluorescent microscope at ×200 magnification (c, f, i, and l). Arrows and arrowheads indicate ballooning hepatocytes and lipid inclusion, respectively. B, serum ALT was measured to assess liver injury. C, Oil-Red O staining revealed lipid accumulation in hepatocytes. Area of Oil-Red O-stained vesicles (μm²) was quantified using Zeiss axiovision software. Counting was done on 2–3 different views on each mouse/treatment group. D, macrophage depletion was assessed by the loss of AF488 anti-F4/80 fluorescence. The ratio of green pixel (AF488 anti-F4/80) was normalized to DAPI pixel (cell nucleus) on each tissue section and was measured by NIH ImageJ 64. *, p < 0.05 and ***, p < 0.0005 as determined by two-tailed t test.

FIGURE 3.

Kupffer cells induce lipid accumulation and monocyte infiltration into the liver. A, C57BL/6 mice fed CT and MCD diets for 10 days were injected twice with control liposomes (CT Lipo.) or clodronate-containing liposomes (clod. Lipo.) at days −2 and +5 of feeding (see schematic). The representative FACS plots (left panels) and cell numbers of monocytes (CD11b^intF4/80^low) and Kupffer cells (CD11b⁺F4/80^hi) (right panel) are shown in CD45⁺NK1.1⁻-gated liver mononuclear cells. The neutrophils (CD11b^hiF4/80⁻) are gated out of the analysis. Representative FACS plots and compiling data of cell numbers show an increase in frequency and cell numbers of monocytes. Data are from 3 independent experiments with a total of n = 6 mice per condition. B, hepatic Tnfa transcript expression was determined by qPCR from whole liver samples of mice fed CT and MCD diets. The RNA level is expressed as fold-induction compared with control diet. Data represent mean ± S.D. *, p < 0.05 and **, p < 0.005 as determined by two-tailed t test.

FIGURE 4.

Kupffer cells trigger NASH development by promoting the recruitment of blood monocytes. C57BL/6 mice fed CT and MCD diets were treated once either with control liposomes (CT Lipo.) or clodronate-containing liposomes (clod. Lipo.) at 2 days before (A–C) or 5 days after (D–F) starting feeding (see schematic). A and D, liver damage is measured by the level of serum ALT. B and E, hepatic lipid inclusion is determined by Oil-Red O staining on frozen liver sections. C and F, depletion of infiltrating monocytes (CD11b^intF4/80^low) and Kupffer cells (CD11b⁺F4/80^hi) are assessed by the flow cytometry. Data are from 2 independent experiments with a total of n = 4 mice per condition. *, p < 0.05 and **, p < 0.005 as determined by two-tailed t test.

FIGURE 5.

CD11b^lowF4/80^hi Kupffer cells produce TNFα and IL-1α at the early phase of NASH development. A, representative FACS plots of CD11b-stained cells versus F4/80 (left), or intracellular TNFα (right) expression within CD45⁺NK1.1⁻-gated mononuclear liver cells from mice fed with CT or MCD diets for 2 days. The neutrophils (CD11b^hiF4/80⁻) are gated out of the analysis. Monocytes are defined as CD11b^intF4/80^low cells and Kupffer cells as CD11b^lowF4/80^hi. B, frequency of CD11b⁺ cells versus (C) cell number of monocytes (CD11b^intF4/80^low) and Kupffer cells (CD11b^lowF4/80^hi) after 2 days of diet. D, measurement of secreted IL-1α from ex vivo culture of sorted Kupffer cells and monocytes. E, hepatic expression of Tnfa, Ccr2, and Timp-1 were determined by qPCR from whole liver samples from mice fed CT and MCD diets for 2 days. The RNA level is expressed as fold-induction compared with control diet. These results are compiled data from n = 3–4 mice per condition, gated on CD45⁺NK1.1⁻ liver cells from mice fed CT or MCD diets for 2 days. Data represent mean ± S.D. *, p < 0.05 and ***, p < 0.0005 as determined by two-tailed t test.

FIGURE 6.

TNFα-producing CD11b^intLy6C^hi monocytes are involved in later phase of NASH disease. A, representative FACS plots show CD11b versus F4/80 (left), or CD11b versus intracellular TNFα (right) expression in CD45⁺NK1.1⁻-gated mononuclear liver cells from mice fed CT and MCD diets for 10 days. B, histogram of MFI of TNFα from gated monocytes (CD11b^intF4/80^low) and Kupffer cells (CD11b^lowF4/80^hi). Representative FACS data of two independent experiments with a total of n = 4 mice per condition. C, representative FACS plots of TNFα in monocytes (CD11b^intF4/80^lowLy6C^hi) and Kupffer cells (CD11b^lowF4/80^hiLy6C^low) within CD11b⁺F4/80⁺CD45⁺NK1.1⁻ liver cells. D, frequency and E, MFI histograms are compiled data from three independent experiments with a total of n = 4–6 mice per condition. *, p < 0.05 and ***, p < 0.0005, as determined by two-tailed t test.

FIGURE 7.

Silencing of TNFα in targeted myeloid cells reduces NASH development. C57BL/6 mice fed CT and MCD diets were intravenously injected either with control luciferase (Luc) or TNFα siRNA liposomes on days −3, 0, 4 and 7. A, H&E staining (×100 and 200 magnification) (a–h), and Oil-Red O-stained tissue sections (i–l) were examined under bright field microscope. Representative images from three independent experiments with a total of n = 6 mice per condition. Arrows and arrowheads indicate ballooning hepatocytes and lipid inclusion, respectively. B, area of Oil-Red O-stained vesicles (μm²) was quantified using zeiss axiovision software. Counting was done on 2–3 different views on each mouse per treatment group. C, serum ALT was measured. D, silencing of TNFα in targeted myeloid cells abrogated the increased expression of hepatic Tnfa transcript. Tnfa transcript was determined by qPCR from whole liver samples. *, p < 0.05; **, p < 0.005; and ***, p < 0.0005, as determined by two-tailed t test.

FIGURE 8.

Silencing of TNFα in targeted myeloid cells reduces monocyte infiltration. A, representative FACS plots of CD11b versus F4/80 expression within CD45⁺NK1.1⁻-gated mononuclear liver cells isolated from mice fed CT and MCD diets treated with control luciferase (Luc) or TNFα siRNA liposomes. The neutrophils (CD11b^hiF4/80⁻) are gated out of our analysis. Representative FACS plots are from a total of n = 6 mice per condition. B, frequency and cell number of monocytes from three independent experiments with a total of n = 6 per condition. C, knockdown of TNFα in myeloid cells (Kupffer cells and monocytes). D, fold-induction of hepatic Timp-1 transcript expression determined by qPCR in whole livers. E, level of chemokine/cytokine from whole liver cell lysates with a total of n = 4–6 mice per condition. Data represent mean ± S.D. *, p < 0.05; **, p < 0.005; and ***, p < 0.0005, as determined by two-tailed t test.

FIGURE 9.

Schematic presentation of the proposed model for the early initiation of NASH development. See text for details.