Interleukin-10-Mediated Lymphopenia Caused by Acute Infection with Foot-and-Mouth Disease Virus in Mice

Figures

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  Figure 1. Lymphopenia coincided with FMDV RNA in the serum during FMDV infection. (A) Representative FACS plots of lymphocytes from mock mice and infected mice at 48 hpi. (B) The number of lymphocytes in the peripheral blood were shown in mock mice and infected mice at 12, 24, 36, 48, and 60 hpi. (C). The number of lymphocytes in the peripheral blood were shown in mock mice, infected mice with severity clinical symptoms (SCS), and infected mice without SCS (including mild clinical symptoms). (D) FMDV RNA copies in the serum from mock mice and infected mice at 12, 24, 36, 48, and 60 hpi were determined by RT-qPCR. The significance was detected using an unpaired t-test. **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns indicates a non-statistically significant difference (p > 0.05).

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  Figure 2. The numbers and frequencies of T cells, B cells, and NK cells in the peripheral blood were decreased in FMDV-infected mice. The numbers of T cell (A), B cells (B), and NK cells (C) in the peripheral blood from mock mice and FMDV-infected mice at 12, 24, 36, 48, and 60 hpi (D). The frequencies of the T cells and B cells were measured from mock mice and FMDV-infected mice at 12, 24, 36, 48, and 60 hpi (E). The frequencies of the CD4+ T cells and CD8+ T cells were measured from mock mice and FMDV-infected-mice at 12, 24, 36, 48, and 60 hpi (F). The frequencies of the T cells, B cells, CD4+ T cells, and CD8+ T cells from mock mice and infected mice with SCS were measured during FMDV infection. The T, B, and NK cell counts were analyzed by an unpaired t-test. The percentages of T, B, CD4+, and CD8+ T cells were analyzed by two-way ANOVA with Tukey’s multiple comparison test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns indicates a non-statistically significant difference (p > 0.05).

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  Figure 3. The apoptosis was detected in T cells, B cells, and NK cells in the peripheral blood from mock mice and FMDV-infected mice. Representative FACS plots of apoptosis in T cells (A), B cells (B), and NK cells (C) from mock mice and FMDV-infected mice. The apoptosis was detected in T cells (D), B cells (E), and NK cells (F) from mock mice and FMDV-infected mice at 12, 24, 36, 48, and 60 hpi by Annexin V/PI detection. Annexin V+/PI− cells were considered early apoptotic cells, and Annexin V+/PI+ cells were considered late apoptotic cells. The apoptosis of the T, B, and NK cells was analyzed by two-way ANOVA with Tukey’s multiple comparison test. Significance was detected using an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01. ns indicates a non-statistically significant difference (p > 0.05).

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  Figure 4. The CFSE-labeled lymphocytes migrate from the blood to tissue. The CFSE-labeled lymphocytes in the heart, liver, kidney, and lung from mock mice and infected mice were observed by fluorescence microscope (10×).

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  Figure 5. The expression of PD-1, CTLA-4, Tim-3, LAG-3, 2B4, TIGIT, and CD160 was analyzed on CD4 and CD8 T cells from mock mice and FMDV-infected mice. The expression of all known seven coinhibitory molecules was analyzed on peripheral blood T lymphocytes, which were harvested from the mock mice and FMDV-infected mice at 48 hpi (n = 10 per group). Significance was detected using an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. ns indicates a non-statistically significant difference (p > 0.05).

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  Figure 6. Potential cytokines/chemokines were detected in the serum from mock mice and FMDV-infected mice. The circulating levels (mean ± SEM) of various cytokines were measured by ELISA in the serum of mock mice and FMDV-infected mice at 12, 24, 36, 48, 60, and 72 hpi. The results are representative of a single experiment with 3 mice per time point. Significance was detected using an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. ns indicates a non-statistically significant difference (p > 0.05).

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  Figure 7. Blocking IL-10/IL-10R signaling with the IL-10R blocker prevented lymphopenia driven by FMDV infection. (A) The survival curve of mice treated with in vivo anti-mouse IL-10R (n = 10) and mice treated with in vivo rat IgG1 isotype control (n = 10) after FMDV infection. (B) The number of lymphocytes in the peripheral blood from mice treated with in vivo rat IgG1 isotype control (n = 5) and mice treated with in vivo anti-mouse IL-10R (n = 5). (C) The apoptosis of T cells and B cells in the peripheral blood from mice treated with in vivo rat IgG1 isotype control (n = 5) and mice treated with in vivo anti-mouse IL-10R (n = 5). (D) The frequencies of CD3, CD19, and NK1.1 cells in the heart from mice treated with in vivo rat IgG1 isotype control (n = 3) and mice treated with in vivo anti-mouse IL-10R (n = 3) are shown. (E) The expression of TIGIT, PD-1, 2B4, CTLA-4, and LAG-3 on CD4 and CD8 in the peripheral blood from mice treated with in vivo rat IgG1 isotype control (n = 5) and mice treated with in vivo anti-mouse IL-10R (n = 5). The survival rates in mice were compared by the log-rank test. The lymphocytes counts and the expression of coinhibitory molecules were analyzed by an…

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  Figure 8. Knocking out IL-10 prevented lymphopenia driven by FMDV infection. (A) The survival curve of IL-10-/- mice (n = 9) and wild-type mice (n = 9) infected with FMDV. (B) The number of lymphocytes in the peripheral blood from IL-10-/- mice (n = 5) and wild-type mice (n = 5) at 48 hpi. (C) The apoptosis of T cells, B cells, and NK cells in the peripheral blood from IL-10-/- mice (n = 5) and wild-type mice (n = 5) at 48 hpi. (D) The frequency of CD3, CD19, and NK1.1 cells in the heart from IL-10-/- mice (n = 5) and wild-type mice (n = 5) at 48 hpi. (E) The expression of TIGIT, PD-1, 2B4, CTLA-4, and LAG-3 on CD4 and CD8 in the peripheral blood of IL-10-/- mice (n = 5) and wild-type mice (n = 5) at 48 hpi. The survival rates in mice were compared by the log-rank test. The lymphocyte counts and the expression of coinhibitory molecules were analyzed by an unpaired t-test. The apoptosis of T, B, and NK cells and the mRNA expression of T, B, and NK cells in the heart were analyzed by two-way ANOVA with Tukey’s multiple comparison test. *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns indicates a non-statistically significant difference (p > 0.05).