The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice

Abstract

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8⁺ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1^highCD11b^highCD11c^high B220⁺Ly6G^- phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3^-/- mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1⁺CD11b^- and NK1.1⁺Ly6G^- phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1⁺CD11b⁺ cells and partial depletion of CD3⁺NK1.1⁺ and NK1.1⁺CD11b^- cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1^-CD11b⁺ cell population in both liver and blood. Regarding mice with a TLR3^-/- phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b.

Figure 1

Treatment with poly(I:C) induces increased relative numbers of NK cells in the liver but decrease in the blood. Naïve C57BL/6 mice (n = 6 mice/group) were treated with 200 μg poly(I:C) in 200 μl and then sacrificed 4 hr post poly(I:C) injection. Single-cell suspensions were prepared of PBL and liver and stained with antibodies against NK1.1, CD3, and CD11b. Samples were analyzed by 3-color flow cytometry. The frequency of NK cells (NK1.1⁺CD3⁻) and NKT cells (NK1.1⁺CD3⁺) is shown in (a). The frequencies of CD11b⁺NK1.1⁻ and CD11b⁺NK1.1⁺ cells are shown in (b).

Figure 2

Treatment with poly(I:C) induces increased absolute numbers of NK cells in the liver but decrease in the blood. The absolute numbers of NK cells (NK1.1⁺CD3⁻) and NKT cells (NK1.1⁺CD3⁺) as well as CD11b⁺NK1.1⁻ and CD11b⁺NK1.1⁺ cells were calculated by multiplying the relative numbers analyzed in the legend of Figure 1 by the total numbers of the peripheral blood and liver by a haemocytometer. The data represents the mean ± SD of each group. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 3

Kinetics of NK subsets after the treatment with poly(I:C) in the PBL and liver. Naïve C57BL/6 mice (n = 6 mice/group) were treated with 200 μg poly(I:C) in 200 μl and then sacrificed after 1, 4, 8, and 12 hours after injection. Single-cell suspensions were prepared of the PBL and liver and stained with antibodies against NK1.1, CD3, CD11b, and CD11c and analyzed by 3-color flow cytometry. The kinetics of NK1.1⁺CD11b⁺, NK1.1⁺CD11c⁺, NK1.1⁺CD11b⁻, NK1.1⁻CD11b⁺, CD3⁺NK1.1⁺, and CD11c⁺CD11b⁺ are shown in the (a) PBL and (b) liver. The data represents the mean ± SD at each time point of each group. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

The phenotypic analysis of NK cell subsets in the liver after the treatment with poly(I:C). Naïve C57B/6 mice (n = 6 mice/group) were treated with 200 μg poly(I:C) and then sacrificed after 4 hours postinjection. Single-cell suspensions were prepared of liver and stained with antibodies against NK1.1, CD11b, Ly6G, B220, CD25, CD11c, CD80, and CD40 and analyzed by 3-color flow cytometry. The phenotype of NK cell subsets is shown in the liver of control and treated mice with poly(I:C).

Figure 5

Influence of CR3 deficiency on the effect of poly(I:C) on the frequency of NK cells. Naïve C57BL/6 mice and CR3^−/− mice (n = 6 mice/group) were treated with 200 μg poly(I:C) and then sacrificed 4 hours postinjection. Single-cell suspensions were prepared of PBL and liver and stained with antibodies against NK1.1, CD11b, and Ly6G and analyzed by 3-color flow cytometry. (a) The frequency of NK cell (NK1.1⁺CD11b⁺) and NK cell (NK1.1⁺) populations. (b) The frequency of NK1.1⁻ Ly6G⁺ and NK1.1⁺ Ly6G⁻ populations.

Figure 6

Influence of NK cell deficiency on the effect of poly(I:C) on the frequency of NK cells. Naïve C57BL/6 mice and NK-depleted mice (n = 6 mice/group) were treated with 200 μg poly(I:C) and then sacrificed 4 hours post injection. Single-cell suspensions were prepared of PBL and liver and stained with antibodies against NK1.1, CD11b, and CD3 and analyzed by 3-color flow cytometry. (a) The frequencies of NKT cells (CD3⁺NK1.1⁺) cells and CD3⁻NK1.1⁺ cells. (b, c) The frequencies of NK1.1⁻CD11b⁺ and NK1.1⁺CD11b⁺ cells.

Figure 7

Influence of TLR3 deficiency on the effect of poly(I:C) on the frequency of NK cells. Naïve C57BL/6 mice and TLR3^−/− mice (n = 4 mice/group) were treated with 200 μg poly(I:C) and then sacrificed 4 hours postinjection. Single-cell suspensions were prepared of liver and stained with antibodies against NK1.1 and CD11b and then analyzed by 2-color flow cytometry. The frequencies of NK1.1⁻CD11b⁺ and NK1.1⁺CD11b⁺ cells are shown.