Probiotic antigens stimulate hepatic natural killer T cells - PubMed (original) (raw)

Probiotic antigens stimulate hepatic natural killer T cells

Shuwen Liang et al. Immunology. 2014 Feb.

Abstract

Increasing evidence suggests that gut flora play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high-fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed-strain probiotics (VSL#3); single-strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT cells. Toll-like receptor 4 (TLR4) knockout mice were also evaluated for their responses to HF diet. High-dose VSL#3 was more effective than low-dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids from B. infantis decreased α-GalCer-mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF-diet-induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain-specific and dose-dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells.

Keywords: interleukin-2; natural killer T cells; non-alcoholic fatty liver disease; probiotics; steatosis.

© 2013 John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1

The dose- and strain-dependent effects of probiotics on high-fat (HF) diet-induced obesity, insulin resistance and hepatic steatosis. Wild-type (WT) C57BL/6 mice were fed either normal diet (ND) or HF diet and some of HF-diet-fed mice were also treated with high-dose VSL#3 (HF+hi VSL#3), or low-dose VSL#3 (HF+lo VSL#3), or antibiotics (HF+anti) or high dose of _Bifidobacterium infantis (_HF+hi B infantis), or continuous ND or HF diet. (a) Animal weight; (b) glucose tolerance tests; (c) serum insulin level; (d) insulin resistance measured by HOMA-IR; (e) hepatic triglyceride content; (f) representative haematoxylin & eosin of liver histology. *P < 0·05 versus HF-diet group.

Figure 2

Figure 2

The dose- and strain-dependent effect of probiotics on hepatic natural killer T (NKT) cells. Animals were treated as described in Fig. 1. Hepatic mononuclear cells (HMNC) were isolated and labelled with various surface markers. (a) A representative dot plot of NKT cell staining (gated on total HMNC); (b) means ± SD of three independent experiments (n = 5 per group). *P < 0·05 versus high-fat (HF) -diet group.

Figure 3

Figure 3

Probiotic antigens stimulate natural killer T (NKT) cells in vitro and in vivo. (a) Bacterial glycolipids extracted were co-cultured with NKT hybridoma. Interleukin-2 (IL-2) released by NKT hybridoma indicates their activation. 1 = medium only; 2 = artificial antigen-presenting cells (aAPCs) only; 3 = aAPCs loaded with low-dose VSL#3 extract; 4 = aAPCs loaded with high-dose VSL#3 extract; 5 = aAPCs loaded with high-dose VSL#3 extract plus α-GalCer; 6 = aAPCs loaded with low-dose Bifidobacterium infantis extract; 7 = aAPCs loaded with high-dose B. infantis extract; 8 = aAPCs loaded with high-dose B. infantis extract plus α-GalCer; and 9 = aAPCs loaded with α-GalCer. (b) Lipid extracts from VSL#3 or B. infantis, or α-GalCer (2 μg per mouse) were injected into C57BL/6 wild-type mice fed a normal diet. After 24 hr, the animals were killed and their hepatic NKT cells were evaluated as described in Fig 2. Means ± SD of the percentages of hepatic NKT cells (gated on CD3+ and CD1d Tetramer+) among hepatic mononuclear cells (HMNC) are shown (n = 5 per group). (c) A representative histogram of NKT cell proliferation assay. HMNC were labelled with CFSE and stimulated with aAPCs loaded with VSL#3 lipid extract or unloaded empty beads. *P < 0·025, #P < 0·002 versus control.

Figure 4

Figure 4

Disrupting Toll-like receptor 4 (TLR4) signalling did not protect mice from high-fat (HF) -diet-induced non-alcoholic fatty liver disease (NAFLD). Wild-type and TLR4 knockout C57BL/6 mice were fed normal diet (ND) or HF diet for 12 weeks. (a) Hepatic natural killer T (NKT) cell content; (b) animal weight; (c) glucose tolerance tests; (d) insulin resistance as measured by HOMA-IR; (e) representative haematoxylin & eosin staining of liver histology; (f) hepatic triglyceride content. Results were means ± SD (n = 5 per group)

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