iNKT Cells Are Responsible for the Apoptotic Reduction of Basophils That Mediate Th2 Immune Responses Elicited by Papain in Mice Following γPGA Stimulation

<div><p>Recent studies have demonstrated that <i>Bacillus subtilis</i>-derived poly-gamma glutamic acid (γPGA) treatment suppresses the development of allergic diseases such as atopic dermatitis (AD). Although basophils, an innate immune cell, are known to play critical roles in allergic immune responses and repeated long-term administration of γPGA results in decreased splenic basophils in an AD murine model, the underlying mechanisms by which γPGA regulates basophil frequency remain unclear. To investigate how γPGA modulates basophils, we employed basophil-mediated Th2 induction <i>in vivo</i> model elicited by the allergen papain protease. Repeated injection of γPGA reduced the abundance of basophils and their production of IL4 in mice, consistent with our previous study using NC/Nga AD model mice. The depletion of basophils by a single injection of γPGA was dependent on the TLR4/DC/IL12 axis. CD1d-dependent Vα14 TCR invariant natural killer T (iNKT) cells are known to regulate a variety of immune responses, such as allergy. Because iNKT cell activation is highly sensitive to IL12 produced by DCs, we evaluated whether the effect of γPGA on basophils is mediated by iNKT cell activation. We found that <i>in vivo</i> γPGA treatment did not induce the reduction of basophils in iNKT cell-deficient CD1d KO mice, suggesting the critical role of iNKT cells in γPGA-mediated basophil depletion at the early time points. Furthermore, increased apoptotic basophil reduction triggered by iNKT cells upon γPGA stimulation was mainly attributed to Th1 cytokines such as IFNγ and TNFα, consequently resulting in inhibition of papain-induced Th2 differentiation via diminishing basophil-derived IL4. Taken together, our results clearly demonstrate that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. Thus, elucidation of the crosstalk between innate immune cells will contribute to the design and development of new therapeutics for Th2-mediated immune diseases such as AD.</p></div

iNKT cells are required for inhibition of papain-induced basophil-specific Th2 differentiation upon γPGA treatment.

<p>(A) Naive CD4⁺CD62L⁺ T cells were cultured with a combination of basophils (6 × 10⁴ cells/well), papain (25 μg/ml), rIL4 (10 ng/ml), or neutralizing anti-IL4 mAbs (5 μg/ml) in the absence or presence of iNKT cells (1.2 × 10⁶ cells/well) purified from Vα14 TCR Tg B6 mice treated i.p. with either PBS or γPGA for 5 days. Intracellular IL4 production was analyzed in CD4⁺ T cells. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001). (B) WT and CD1d KO B6 mice were immunized i.p. with 500 μg papain once a week for 2 weeks. Either PBS or γPGA (2 mg) was i.p. injected into PBS or papain-treated mice a total of 6 times during 2 weeks. Intracellular IL4 production in CD4⁺ T cells (CD3ε⁺CD4⁺) was assessed via flow cytometry on day 14 after the first immunization. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; *P<0.05). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^###P<0.001).</p

Activated iNKT cells by γPGA-stimulated DC stimulation contributes to basophil apoptosis via cytokines but not the Fas/FasL pathway.

<p>(A) Splenocytes were prepared from either PBS- or γPGA-injected mice at 16 hrs after treatment. Expression of FasL was analyzed in NKT cells (NK1.1⁺CD3ε⁺). The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). (B-C) Either PBS or γPGA was i.p. injected into WT, <i>lpr/lpr</i>, and <i>gld/gld</i> B6 mice, and splenocytes were prepared 16 hrs later. (B) Intracellular IFNγ production was analyzed in NKT cells (NK1.1⁺CD3ε⁺). The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors. (C) The absolute number of basophils (FcεRI⁺DX5⁺) among lineage-negative cells (CD3ε^-CD19^-) was determined. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors. (D) Basophils (6 × 10⁴ cells/well) were cultured for 12 hrs with DCs (1.2 × 10⁶ cells/well) purified from WT B6 mice, NK cells (1.2 × 10⁶ cells/well) purified from Jα18 KO B6 mice, or iNKT cells (1.2 × 10⁶ cells/well) purified from Vα14 TCR Tg B6 mice in the presence of either PBS or γPGA (1 or 3 mg/ml). The frequency of apoptotic cells (annexin-V⁺7AAD^-) among basophils was assessed by flow cytometric analysis. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; *P<0.05, **P<0.01). Two-way ANOVA (cells × treatment) showed an interaction between these two factors (^##P<0.01, ^###P<0.001). (E) Basophils (6 × 10⁴ cells/well) were cultured for 12 hrs with or without DCs (1.2 × 10⁶ cells/well) purified from WT B6 mice or iNKT cells (1.2 × 10⁶ cells/well) purified from Vα14 TCR Tg B6 mice in the presence of PBS or γPGA (3 mg/ml). Neutralizing mAbs specific for IFNγ (5 μg/ml), TNFα (5 μg/ml), or IL12 (5 μg/ml) were added during the culture. The frequency of apoptotic cells (annexin-V⁺7AAD^-) among basophils was assessed by flow cytometric analysis. The mean values ± SD are shown (n = 3 per group in the experiment). Two-way ANOVA (neutralizing Ab × treatment) showed an interaction between these two factors (^#P<0.05, ^##P<0.01, ^###P<0.001). (F-G) Total splenocytes purified from WT B6 mice were cultured in the presence of either LPS (1 μg/ml) or γPGA (3 mg/ml). Intracellular IFNγ (F) and TNFα (G) production were analyzed in NK (NK1.1⁺CD3ε^-) or NKT cells (NK1.1⁺CD3ε⁺) at the indicated time points. The mean values ± SD are shown (n = 4 per group in the experiment). (H-I) Total splenocytes purified from WT and CD1d KO B6 mice were cultured in the presence of either LPS (1 μg/ml) or γPGA (3 mg/ml). Intracellular IFNγ (H) or TNFα (I) production was analyzed in NK cell populations (NK1.1⁺CD3ε^-) at the indicated time points. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001).</p

The reduction in basophil number by γPGA stimulation is dependent on CD1d-restricted iNKT cells.

<p>(A) Either PBS or γPGA was i.p. injected into WT B6 mice, and splenocytes were isolated 16 hrs later. The frequencies of NKT cells (NK1.1⁺CD3ε⁺) among total splenocytes were plotted. One representative result is shown (left panel). Intracellular IFNγ and TNFα production (middle panel) and CD69 expression (right panel) were analyzed in NKT cells (NK1.1⁺CD3ε⁺). Representative data of three independent experiments are shown (n = 3 per group in the experiment; Student’s t-test; *P<0.05, **P<0.01). (B) Either PBS or γPGA was i.p. injected into WT B6, C3H/HeN, C3H/HeJ, DT (120 ng/mouse)-treated CD11c-DTR Tg B6, and IL12p35 KO B6 mice, and splenocytes were prepared 16 hrs later. Intracellular IFNγ production was analyzed in NKT cells (NK1.1⁺CD3ε⁺). The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^###P<0.001). (C-D) Either PBS or γPGA was i.p. injected into WT and CD1d KO B6 mice, and splenocytes were prepared 16 hrs later. (C) The frequency of iNKT cells (α-GC/CD1d dimer⁺CD3ε⁺) among total splenocytes was plotted (left panel), and intracellular IFNγ production in iNKT cells was determined via flow cytometry (right panels). The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; *P<0.05). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^##P<0.01). (D) The frequency of basophils (FcεRI⁺DX5⁺) among lineage-negative cells (CD3ε^-CD19^-) among total splenocytes was plotted (left panel). The absolute number of basophils was determined (right panels). The means ± SD are shown (n = 3 per group in the experiment; Student’s t-test; ***P<0.001). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^##P<0.01). (E-F) Either PBS or γPGA was i.p. injected into WT NC/Nga, CD1d KO NC/Nga, and Vα14 TCR Tg NC/Nga mice and 16 hrs later splenocytes were prepared. (E) The absolute cell number of iNKT cells (α-GC/CD1d dimer⁺CD3ε⁺) was determined. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001) (F) The absolute number of basophils (FcεRI⁺DX5⁺) among lineage-negative cells (CD3ε^-CD19^-) was assessed by flow cytometry. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^##P<0.01).</p

DC-derived IL12 is responsible for the reduction of basophils by γPGA stimulation.

<p>(A-B) Splenocytes were prepared from either PBS- or γPGA-injected mice at 16 hrs after treatment. (A) The frequency of basophils (FcεRI⁺DX5⁺) among lineage-negative cells (CD3ε^-CD19^-) of total splenocytes was plotted (left panel). The absolute number of basophils was determined (right panels). The means ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). (B) DX5⁺ splenocytes were purified by using anti-biotin MACS after staining total splenocytes with biotin-conjugated anti-DX5 mAb. The frequency of apoptotic cells (annexin-V⁺7AAD^-) among basophils (FcεRI⁺CD3ε^-CD19^-) was assessed by flow cytometry. Left, representative FACS plots; right, summary. The mean values ± SD are shown (n = 4 per group in the experiment; Student’s t-test; ***P<0.001). (C) Expression of Fas on basophils was assessed by flow cytometry. Left, representative FACS plots; right, summary. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). (D) Either PBS or γPGA was i.p. injected into C3H/HeN and C3H/HeJ mice and 16 hrs later splenocytes were prepared. The absolute number of basophils was determined. The mean values ± SD are presented (n = 3 per group in the experiment; Student’s t-test; **P<0.01, ***P<0.001). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^###P<0.001). (E) Splenocytes were prepared from either PBS- or γPGA-injected mice at 16 hrs after treatment. Expression of MHC class II/CD86 and intracellular IL12p40 production were analyzed in DCs (CD11c⁺). The means ± SD are shown (n = 3 per group in the experiment; Student’s t-test; **P<0.01). (F) PBS or DT (120 ng/mouse) was used to treat CD11c-DTR Tg B6 mice, and the frequencies of DCs (CD11c⁺GFP⁺) among total splenocytes was plotted 16 hrs later. One representative result is shown (n = 3 per group in the experiment). (G) Either PBS or γPGA was i.p. injected into WT, DT (120 ng/mouse)-treated CD11c-DTR Tg, and IL12p35 KO B6 mice. The absolute number of basophils was determined. The mean values ± SD are shown (n = 3 per group in the experiment; Student’s t-test; ***P<0.001). Two-way ANOVA (genotype × treatment) showed an interaction between these two factors (^###P<0.001).</p

Differential outcome of EAE pathogenesis depending on the SRG3 over-expression pattern in MBP-specific TCR transgenic B10.PL mice: the development of EAE was suppressed in β-actin-SRG3 Tg mice but was facilitated in CD2-SRG3 Tg mice.

<p>(A-B) Both MBP TCR Tg B10.PL mice and CD2-SRG3/MBP TCR double Tg B10.PL mice (upper panel) or both MBP TCR Tg B10.PL mice and β-acin-SRG3/MBP TCR double Tg B10.PL mice (lower panel) were either non-immunized or s.c. immunized with the MBP-Ac1-11 peptide in CFA. (A) Subsequently, the disease severity was monitored daily according to the classical EAE scoring system after immunization; one representative result out of two independent experiments is shown as the mean ± SD of 5 mice per group (*P<0.05, **P<0.01). (B) Spleens were prepared from the aforementioned four experimental groups, and corresponding photographs were captured (upper panel). Additionally, spleen weight (the middle panel) and total cell number (the lower panel) are shown as the mean values ± SD (n = 5; *P<0.05, **P<0.01).</p

Infiltration of Treg cells into the spinal cord was dramatically increased in β-actin-SRG3 Tg mice compared to CD2-SRG3 Tg mice during EAE pathogenesis.

<p>(A) Splenocytes and (B) MNCs were prepared from the spinal cord of both MBP TCR Tg B10.PL mice and CD2-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. The percentages of CD25⁺FoxP3⁺ Treg cells among the total CD4⁺ T cell population in splenocytes and MNCs were evaluated via flow cytometry (upper panel). Alternatively, splenocytes and MNCs were prepared from the spinal cord of both MBP TCR Tg B10.PL mice and β-actin-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. The percentages of CD25⁺FoxP3⁺ Treg cells among the total CD4⁺ T cell population in splenocytes and MNCs were evaluated via flow cytometry (lower panel). The mean values ± SD are shown (n = 5; *P<0.05).</p

The contrasting effects of differential SRG3 over-expression on the severity of EAE were associated with the alteration of the phenotypes of DCs and macrophages.

<p>(A) Splenocytes were prepared from both MBP TCR Tg B10.PL mice and CD2-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. The intracellular production of IL6, TNFα, iNOS, and IL10 was assessed in splenic CD11c⁺ DCs via flow cytometry (upper left panel). Additionally, intracellular IL6, TNFα, iNOS, arginase-1, and IL10 production was assessed in splenic macrophages (CD11c^-CD11b⁺F4/80⁺) via flow cytometry (upper right panel). Alternatively, splenocytes were prepared from both MBP TCR Tg B10.PL mice and β-actin-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. Intracellular IL6, TNFα, iNOS, and IL10 production was assessed in splenic DCs (CD11c⁺) (lower left panel) and intracellular IL6, TNFα, iNOS, arginase-1, and IL10 production was assessed in splenic macrophages (CD11c^-CD11b⁺F4/80⁺) via flow cytometry (lower right panel). The mean values ± SD are shown (n = 5; *P<0.05, **P<0.01). (B) Mononuclear cells (MNCs) were isolated from the spinal cord of both MBP TCR Tg B10.PL mice and CD2-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. The intracellular expression of arginase-1 and iNOS was assessed in macrophages/microglia (CD45⁺CD11b⁺F4/80⁺) via flow cytometry (left panel). Alternatively, MNCs were isolated from the spinal cord of both MBP TCR Tg B10.PL mice and β-actin-SRG3/MBP TCR double Tg B10.PL mice immunized with MBP to induce EAE. The intracellular expression of arginase-1 and iNOS was assessed in macrophages/microglia (CD45⁺CD11b⁺F4/80⁺) via flow cytometry (right panel). The mean values ± SD are shown (n = 5; *P<0.05).</p

SRG3 over-expression driven by the β-actin promoter reduced cytokine production in DCs following LPS stimulation.

<p>Flt3L-cultured BMDCs (A) from both WT and CD2-SRG3 Tg B6 mice and (B) from both WT and β-actin-SRG3 Tg B6 mice were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml) for 14 hrs, and subsequently, the percentage of the IL12p40-expressing population among the CD11c⁺ BMDCs was assessed via flow cytometry. Representative data from three independent experiments are shown (upper panel). The graph in lower panel represents mean percentage ± SD for the proportion of IL12p40 (n = 3; **P<0.01). (C) WT, β-actin-SRG3 Tg, and CD2-SRG3 Tg mice were i.p. injected with LPS (2 μg) or vehicle. Sixteen hrs later, intracellular TNFα, IL12p40, iNOS, and IL10 production was assessed in splenic CD11c⁺ DCs via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01).</p

During EAE development, SRG3 over-expression driven by the CD2 promoter enhances Th1 and Th17 differentiation, whereas SRG3 over-expression driven by the β-actin promoter increases Th2 differentiation but decreases Th1 and Th17 differentiation.

<p>(A-C) Both MBP TCR Tg B10.PL mice and CD2-SRG3/MBP TCR double Tg B10.PL mice (upper panel in Fig 5A, 5B, and 5C) or both MBP TCR Tg B10.PL mice and β-actin-SRG3/MBP TCR double Tg B10.PL mice (lower panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132329#pone.0132329.g005" target="_blank">Fig 5A, 5B, and 5C</a>) were either non-immunized or s.c. immunized with the MBP-Ac1-11 peptide in CFA. (A) Purified CD4⁺ splenocytes from the four groups were activated using plate-bound anti-CD3 (10 μg/ml) and anti-CD28 (1 μg/ml) mAbs for 16 hrs and subsequently stimulated with PMA/ionomycin for 2 hrs in the presence of brefeldin A (10 μg/ml). The intracellular expression of IFNγ, IL17, IL4, and IL10 was analyzed via flow cytometry. The mean values ± SD are shown (n = 5; *P<0.05, **P<0.01). (B) The expression of T-bet, RORγt, and GATA-3 in splenic CD4⁺ T cells purified from the four groups was analyzed via flow cytometry. The mean values ± SD are shown (n = 5; *P<0.05). (C) Mononuclear cells (MNCs) were isolated from the spinal cord of the four groups using a Percoll gradient on day 21 or 24 after EAE induction. The isolated MNCs were incubated for 2 hrs in brefeldin A (10 μg/ml), and subsequently, the intracellular expression of the cytokines IFNγ and IL17 was analyzed via flow cytometry. The mean values ± SD are shown (n = 5; *P<0.05, **P<0.01).</p