The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells - PubMed (original) (raw)
The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells
Luhua H Zhang et al. J Exp Med. 2016.
Abstract
A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3-) cells, defined as CD49a+TRAIL+CXCR6+DX5- cells in the mouse liver, constitutively express AhR. In AhR-/- mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR-/- mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5- liver-resident NK cells and their hapten memory function.
© 2016 Zhang et al.
Figures
Figure 1.
AhR is constitutively expressed in CD49a+TRAIL+DX5− liver-resident NK cells. (A) Representative flow cytometry profile of CD49a and DX5 expression on liver NK (NK1.1+CD3−) cells. (B) Expression of TRAIL (left) and CXCR6 (middle) on CD49a+ liver NK cells. (Right) CXCR6 mRNA expression in FACS-sorted CD49a−DX5+ and CD49a+DX5− liver NK cells, normalized to HPRT and shown relative to CD49a+DX5− liver NK cells. (C) CD49a−DX5+ or CD49a+DX5− NK cells were sorted from the BM, spleen, and liver of WT mice. AhR mRNA expression was assessed by quantitative RT-PCR, normalized to HPRT, and shown relative to BM NK cells (left) or CD49a−DX5+ liver NK cells (right). (D, left) Representative flow cytometry profile of CD49a and AhR expression in liver NK cells. (Right) Mean fluorescent intensity (MFI)–isotype control of AhR staining in CD49a−DX5+ versus CD49a+DX5− liver NK cells. (E, left) Representative flow cytometry profile of TRAIL and AhR expression in liver NK cells. (Right) AhR mRNA expression in FACS-sorted TRAIL− and TRAIL+ liver NK cells, normalized to HPRT and shown relative to relative to TRAIL− liver NK cells. Dot plots are from at least two independent experiments, and n = 3–8 mice per group. All contour plots are representative of at least three independent experiments. Data are shown as mean ± SEM. **, P < 0.01; ***, P < 0.001 (paired Student’s t test).
Figure 2.
AhR−/− mice have a deficiency in CD49a+TRAIL+DX5− liver-resident NK cells. (A) NK (NK1.1+CD3−) cell percentages (left) and expression of CD27 and CD11b on NK cells (right) from the spleen (top) and BM (bottom) of AhR+/+ or AhR−/− mice. (B) Percentage, cell number, and representative plots of CD49a+DX5− (left) and TRAIL+ (right) liver NK cells from AhR+/+ or AhR+/− versus AhR−/− mice. SSC, side scatter. (C) Representative plots of TRAIL+ CD3−NK1.1+ NK cells from the liver of AhR+/+ and AHR−/− mice at 4 wk. (D) CD49a+DX5− NK cells, sorted from the liver of AhR+/− and AhR−/− mice, were assessed for CXCR6 mRNA expression by quantitative RT-PCR, normalized to HPRT, and shown relative to AhR+/− cells. (E) Expression of CD27 and CD11b on CD49a+DX5− liver NK cells from AhR+/+ (circles) and AhR−/− (triangles) mice. (F) Representative histograms of eomes (bottom) and T-bet (top) staining in TRAIL+ (left) and TRAIL− (right) liver NK cells of AhR+/+ (continuous line) and AhR−/− mice (dashed line), as well as an isotype control (shaded). (G) Sublethally irradiated CD45.1+ recipient host mice were reconstituted i.v. with an equal mixture of CD45.1+ WT and CD45.2+ AhR+/− or AhR−/− fetal liver cells. (Top) At 2 mo, mice received three i.v. injections of 3 µg FICZ over the course of 1 wk before analysis of liver NK cells. The ratio of CD45.2+ to CD45.1+ cells in TRAIL+ liver NK cells from recipients of AhR+/− versus AhR−/− fetal liver cells is shown. (Bottom) Representative plots of CD45.2 and CD45.1 staining on TRAIL+ liver NK cells. Scatter plots are from at least two independent experiments, and n = 4–11 mice per group. All contour plots are representative of at least two independent experiments. Data are shown as mean ± SEM. **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test).
Figure 3.
AhR regulates the homeostasis of NK cells. (A) NK (NK1.1+CD3−) cells in the BM, spleen, and liver of WT mice were assessed for proliferation by BrdU incorporation. (B) Enriched splenic NK cells from AhR+/+ and AhR−/− mice were labeled with CFSE and cultured in the presence of 1,000 U/ml IL-2. CFSE dilution staining in NK cells is shown at day 3. (C) Representative growth curves for liver mononuclear cells (left) or enriched splenic NK cells (middle) isolated and cultured in 1,000 U/ml IL-2 and 200 nM FICZ or vehicle control. (Right) Representative growth curves of enriched splenic NK cells cultured in 100 ng/ml IL-15 and 200 nM FICZ or vehicle control. (D) Expression of KLRG1 on NK cells in IL-2 or IL-15 assessed by flow cytometry on day 7. (E) Expression of Tim-3 on IL-2–cultured liver (left) and splenic (right) NK cells at day 7. (F) Expression of annexin V on IL-2–cultured splenic NK cells at day 7. Circles, AhR+/+ or AhR+/−; triangles, AhR−/−; closed, vehicle control; open, FICZ. Scatter plots are from at least two independent experiments, and n = 3–8 mice per group. All representative data are from at least three independent experiments. Data are shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test for AhR+/+ vs. AhR−/−; paired Student’s t test for vehicle vs. FICZ).
Figure 4.
AhR-deficient mice have impaired NK cell–mediated delayed CHS. (A) Schematic of CHS assay after T cell depletion. Mice were injected with 50 µg of anti-CD3–depleting antibody 1 d before sensitization with OXA. At 1 mo, mice were again injected with 50 µg anti-CD3 1 d before challenging with OXA by painting the ears. Ears were measured by micrometer, and ears were harvested 4 d after rechallenge. (B) Ear swelling of AhR+/+ and AhR−/− mice relative to control ears. Data are representative of two independent experimental cohorts, and n = 3–4 mice per group. (C) Representative H&E stains of OXA-challenged ears from AhR+/+ (top) and AhR−/− (bottom) mice at day 4 after rechallenge. (D) Schematic of adoptive transfer CHS assay. AhR+/+ and AhR−/− mice were sensitized with OXA, and 40,000 CD49a+DX5− liver NK cells were adoptively transferred into B10;B6 Rag2−/−γc−/− mice. At 1 mo, mice were challenged with OXA by painting the ears. Ears were measured by micrometer, and ears were harvested 4 d after rechallenge. (E) Ear swelling of mice receiving 40,000 AhR+/+ or AhR−/− CD49a+DX5− liver NK cells. n = 4–5 mice per group. (F) Representative H&E stains of OXA-challenged ears from AhR+/+ and AhR−/− recipient mice at day 4 after rechallenge. (C and F) Bars, 50 µm. Data are shown as mean ± SEM (two-way ANOVA).
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