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. 2008 Aug 15;29(2):295-305.
doi: 10.1016/j.immuni.2008.06.012.

Posttranscriptional regulation of II10 gene expression allows natural killer cells to express immunoregulatory function

Asher Maroof  1 , Lynette Beattie, Soombul Zubairi, Mattias Svensson, Simona Stager, Paul M Kaye
Affiliations

Posttranscriptional regulation of II10 gene expression allows natural killer cells to express immunoregulatory function

Asher Maroof et al. Immunity. .

Abstract

Natural killer (NK) cells play a well-recognized role in early pathogen containment and in shaping acquired cell-mediated immunity. However, indirect evidence in humans and experimental models has suggested that NK cells also play negative regulatory roles during chronic disease. To formally test this hypothesis, we employed a well-defined experimental model of visceral leishmaniasis. Our data demonstrated that NKp46(+)CD49b(+)CD3(-) NK cells were recruited to the spleen and into hepatic granulomas, where they inhibited host protective immunity in an interleukin-10 (IL-10)-dependent manner. Although IL-10 mRNA could be detected in activated NK cells 24 hr after infection, the inhibitory function of NK cells was only acquired later during infection, coincident with increased IL-10 mRNA stability and an enhanced capacity to secrete IL-10 protein. Our data support a growing body of literature that implicates NK cells as negative regulators of cell-mediated immunity and suggest that NK cells, like CD4(+) T helper 1 cells, may acquire immunoregulatory functions as a consequence of extensive activation.

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Figures

Figure 1
Figure 1. Cellular accumulation of IL-10 mRNA during L. donovani infection
A. Spleen cell were prepared from naïve BALB/c mice and BALB/c mice infected for 14d or 28d with L. donovani. CD4+CD3+ (formula image) and CD8+CD3+ (formula image) T cells, B220+ B cells (formula image), CD49b+ NK cells (formula image), CD11chi DC (formula image) and all residual cells (including macrophages and stromal cells; formula image) were sorted to >99% purity and then examined for IL-10 mRNA accumulation by RT-PCR. Data represents mRNA samples from sorted cells isolated from pools of n=5 spleens and is shown as fold difference over naïve. One of three independent experiments is shown. B. Spleen cell suspensions from naïve BALB/c mice and BALB/c mice infected for 28d with L. donovani were stained for CD49b and NKp46. Plots are gated on CD49b+CD3- cells. C,D. Immunohistology of naïve and d28 infected (D) BALB/c spleen showing distribution of NKp46+ NK cells (green) and F4/80+ red pulp macrophages (red). Sections were counterstained with DAPI (blue). E. Hepatic NK cells co-express CD49b and NKp46. Cells from naïve and 28d-infected BALB/c mice were gated as CD49b+CD3-. For further characterisation, see Supplementary Figure 1. F. NKp46+ NK cells (green) co-localise with F4/80+ macrophages (blue) and CD3+ T cells (red) in hepatic granulomas caused by L. donovani. G,H. Frequency of granulomas identified as containing NKp46+ cells (G) and the frequency of NKp46+ cells located inside granulomas or outside granulomas i.e. in the parenchyma (H), was enumerated from approximately 35-40 granulomas per mouse (in 20 random 8μm sections). Data represents mean ±SD obtained from 3 mice.
Figure 2
Figure 2. Splenic and Hepatic IL-10+ NK cells expand in number during experimental visceral leishmaniasis
Total number of splenic (A) and hepatic (B) CD3-CD49b+ NK cells in naive and 28d infected BALB/c mice (n=4). Spleen (C) and liver (D) cells were prepared from naïve BALB/c mice and BALB/c mice infected for 7d (formula image),14d (formula image)or 28d (formula image)with L. donovani. CD4+CD3+, CD8+CD3+ T cells, NKp46+CD49b+ and all residual cells (including DC, macrophages and stromal cells) were sorted to >99% purity and then examined for IL-10 mRNA accumulation by RT-PCR. Data represents mRNA samples from sorted cells isolated from pools of n=5 spleens and is shown as fold difference over naïve. Data from one of two independent experiments is shown.
Figure 3
Figure 3. Adoptive transfer of NK cells demonstrates their capacity to inhibit host resistance
A. NK cells isolated from naïve BALB/c mice (N-NK) or 21d-infected BALB/c mice (I-NK) were flow sorted and transferred (106 i.v.) into 21d-infected BALB/c recipients. Control mice received no transfer. Splenic (A) and hepatic (B) parasite burdens were determined 7 days post transfer. Symbols represent individual mice and bar represents mean value. Data from one of four independent experiments is shown. C. Immunohistology of d21 infected BALB/c spleen showing distribution of transferred CFSE+ NK cells (white) NK cells. Sections were counterstained with DAPI (blue). D. Frequency of CFSE+ NK cells distributed to the red/white pulp or marginal zone were enumerated 24h post transfer. E. CFSE labelled NK cells purified (2×106 i.v) from naïve and d21 infected BALB/c mice were transferred into day 21 infected recipient mice, then 18h and 7 days post transfer donor NK cells were enumerated in the spleen. F,G. Distribution of CFSE labelled NK cell in liver sections caused by L. donovani using low magnification (F) and high magnification (G) images. (H, I) NK cells naïve mice (N-NK) and from d21-infected BALB/c mice (I-NK) sorted into their CD11c- and CD11clo subsets were transferred into 21d-infected recipients and 7 days later splenic (H) and hepatic (I) parasite burden was determined.
Figure 4
Figure 4. Adoptive transfer of NK cells demonstrates their IL-10-dependent capacity to inhibit host resistance
A. Schematic showing experimental design for experiment using mixed chimeras. B. Infected chimeras have similar frequencies of CD49b+ IL-10-sufficient (CD45.1) and IL-10-deficient (CD45.2) splenic NK cells. Plots are gated on CD49b+CD3- cells. C. Sorted cells from d21 infected chimeric mice were transferred into 21d-infected B6 mice and 7 days later splenic (C) and hepatic (D) parasite burden was determined. Data from one of two independent experiments is shown.
Figure 5
Figure 5. Post-transcriptional regulation of IL-10 gene expression and increased IL-10 secretion by NK cells from L. donovani-infected mice
A. mRNA accumulation was determined in NK cells isolated from naïve mice and mice infected for 24h or 21d with L. donovani. Data is derived from four experiments (mean + SD), with mRNA obtained from NK cells pooled from 3-5 mice per experiment. B,C. NK cells from naïve BALB/c mice (N-NK) and infected BALB/c mice infected for 24h of 21d were transferred into 21d-infected BALB/c recipients and splenic (B) and hepatic (C) parasite burden determined 7 days later. Data represent one of three independent experiments. D-F. CD49b+ NK cells were isolated from naïve BALB/c mice (D) and BALB/c mice infected for 24h (E) or 21d (F) with L. donovani and stimulated in vitro with increasing doses of rIL-12 (□ 0.05ng/ml; ▲ 0.5ng/ml; ○ 5ng/ml) or cultured in medium alone (formula image) . Supernatants were collected each day for 4 days and IL-10 (left panels) and IFNγ (right panels) was determined by ELISA. G. NK cells from mice infected for 24h (○) and 21d (□) were cultured in the presence of actinomycin D. At indicated times, IL-10 mRNA accumulation was determined. Data are expressed as % remaining IL-10 mRNA relative to mRNA levels at time 0 (168 vs 163 molecules of IL-10 mRNA / 1000 molecules HPRT in 24h and d21 samples respectively). Data represent one of two independent experiments.
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