Kupffer cell and interleukin-12-dependent loss of natural killer T cells in hepatosteatosis

Abstract

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL-12-deficient (IL-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines IL-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic IL-12 expression, and Kupffer cell (KC) depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of IL-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells.

Conclusion: Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

Figure 1. Hepatic NKT cell number and function are reduced within the steatotic liver

A. Hepatic mononuclear cells were analyzed by flow cytometry for T cell receptor beta (TCRβ ) and NK1.1 expression or TCRβ and binding to CD1d-tetramer loaded with PBS57 in mice fed choline sufficient diet (CSD) or choline deficient diet (CDD) for 10 weeks. Representative dot plots presented. B. Hepatic IL4 protein expression was measured by ELISA 3 hours following i.v. alpha galactosylceramide (αGal, 200ng/g) administration to 10 week CSD fed or CDD fed mice. n= 6-9 mice per group. *p<0.05 vs. CSD fed vehicle treated mice, ⁺p<0.05 vs. CSD fed, αGal treated mice.

Figure 2. Increased IL12 production does not influence the progression of hepatosteatosis following long-term CDD feeding

A. C57BL/6 mice were fed CDD for 0, 10 or 20 weeks. Hepatic IL-12 expression was determined by quantitative real time PCR of whole liver tissue while serum IL-12 was measured by ELISA. Values are means ± SEM,. B. H&E stained liver histology after 0, 10 and 20 weeks of CDD in C57BL/6 (Wt) and IL-12^−/− mice. Representative photomicrographs are presented at 100x magnification with 400x inserts (bottom right of each image). C. Serum alanine aminotransferase, liver weight to body weight ratio (LW/BW) and hepatic triglycerides were measured following either 0, 10 or 20 weeks of CDD feeding. No significant differences were observed between Wt and IL-12^−/− mice in any of these parameters. Values are means ± SEM, *p<0.05, n=4-8 animals per group.

Figure 3. IL-12 promotes T_h1 cytokine expression within the steatotic liver

Hepatic T_h1 (TNFα and IFNγ) and T_h2 (IL-4 or IL-10) cytokine gene expression was measured by real time PCR in wt and IL12^−/− mice fed CDD for 0, 10 or 20 weeks. Values are means ± SEM, *p<0.05; n = 4-8 animals per group.

Figure 4. IL-12 is involved in steatosis-associated hepatic NKT cell depletion

C57BL/6 (Wt) and Interleukin (IL)-12^−/− mice were fed CDD for 0, 10 or 20 weeks and total liver mononuclear cells were analyzed by flow cytometry. Hepatic mononuclear cells (A) or splenocytes (C) were stained with anti-mouse T cell receptor β (TCR) and natural killer (NK) 1.1 antibodies. T cells, NKT cells and NK cells are outlined in boxes. Values represent percent of total liver mononuclear cells analyzed. B and D. Quantitation of hepatic (B) or splenic (D) NKT cells expressed as percent of total hepatic CD3⁺ cells expressing NK1.1. Figure is representative for 3-6 different experiments. Results are representative of 3-6 animals per group.

Figure 5. Kupffer cells are activated within the steatotic liver

A. Liver sections from wild type mice fed CDD for 0, 10 or 20 weeks were stained with F4/80. Representative 400x photomicrographs presented. B. Quantitation of F4/80 positive cells per 400x field. Ten fields per section from each mouse were analyzed. C. Hepatic CD14 gene expression was measured using real time PCR. D. Hepatic IL12 gene expression 6 hours following LPS injection. E. Serum aspartate aminotransferase levels 6 hours following lipopolysaccharide (LPS, 2.5mg/kg) injection in wt or IL12^−/− mice fed choline sufficient diet (CSD) or CDD diet for 20 weeks. F. IL12 expression 3 hours following treatment with either saline or LPS (10μg/ml) in isolated hepatic macrophage (Mφ) from wt mice fed either a choline sufficient diet or choline deficient diet for 10 weeks. Representative data from two independent experiments shown. Values are mean ± SEM, *p<0.05 versus respective control, ⁺p<0.05 versus LPS-treated choline sufficient diet fed mice; n = 4-8 animals per group.

Figure 6. Clodronate depletes Kupffer cells and ameliorates increased T_h1 associated cytokine expression in steatotic liver tissue

A. Dosing regimen for macrophage depletion studies. Mice with established hepatosteatosis after 10 weeks of CDD were injected every 4-5 days with clodronate for the following 3 weeks, mice after 20 weeks of CDD received clodronate every 4-5 days for the following 5 weeks while continuously fed CDD. B. Liver sections stained with F4/80 antibody from wt mice administered vehicle or clodronate as described in A. Representative photomicrographs at 400x magnification presented. C. Hepatic T_h1 and T_h2 cytokine expression as assessed by real-time PCR in vehicle or clodronate treated animals. Values are mean ± SEM, *p<0.05. n=4-6 animals per group.

Figure 7. Kupffer cells contribute to steatotosis-induced hepatic NKT cell depletion

Hepatic mononuclear cells were isolated from vehicle or clodronate treated wild type mice fed choline deficient diet for indicated time periods and subpopulations of lymphocytes assessed by flow cytometry. Results are representative of 4-6 different experiments, p<0.01.

Figure 8. Hepatosteatosis is associated with decreased numbers of NKT and NK cells in humans

Human liver biopsies were divided into three groups as described in methods. Slides were stained for CD3 and CD57, a marker for human NK cells. CD3 positive cells stained blue, NK positive cells present red. Double positive cells are termed NKT cells. A. Representative microphotographs of stained tissue are presented, NKT cells are pointed out by arrows. B. Quantitation of NKT cells were counted from at least 10 400x pictures of the tissue. Representative results for n = 3-5 per group. Values are means ± SEM.