Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality in mice by increasing M1 macrophage polarization and cytokine production

Abstract

Introduction: Enterovirus A71 (EV-A71) can cause fatal disease accompanied by increased cytokines, including IL-10, IL-12, and IFN-γ, which are mutually regulated. IFN-γ is induced to protect mice from EV-A71 infection, but its regulation remains unclear. The IL-10 family cytokines, IL-19, IL-20, and IL-24, which signal through a two-subunit receptor complex containing IL-20 receptor subunit A (IL-20RA), are designated as IL-20RA cytokines. IL-20RA cytokines are known to regulate IFN-γ and IL-10 in vitro. We designed this study to investigate the interaction and role of IL-20RA cytokines in viral infection in vivo, which remain unknown.

Methods: Plasma from healthy donors and EV-A71-infected patients was analyzed to detect IL-20RA cytokines. Wild-type (WT) and IL-20RA knockout (IL-20RA ^-/-) mice, as well as isolated T cells and macrophages, were used for functional studies.

Results: In plasma samples, IL-19 was detectable in healthy controls, and EV-A71 infection increased IL-19 levels in infected patients. In sera of WT mice, IL-20RA cytokines, but not IL-10, IL-12, or IFN-γ, were detected in mock-infected animals, and EV-A71 infection significantly increased IL-19 and slightly increased IL-20 levels. Compared with WT mice, IL-20RA ^-/- mice were resistant to EV-A71 infection, with reduced viral loads in peripheral organs, such as the spleen. In sera of infected mice, IL-20RA deficiency sequentially reduced IL-10 levels but increased IL-12 and IFN-γ levels. Abundant T cells expressed IL-10 in splenocytes of infected WT mice, whereas abundant macrophages expressed IL-12 and IFN-γ in splenocytes of infected IL-20RA ^-/- mice. Notably, IL-20RA deficiency reduced M2 macrophages but increased M1 macrophages in splenocytes of infected mice. In vitro, treatment of leukocytes isolated from WT mice with IL-19 or IL-20, but not IL-24, increased IL-10 production in CD4 T cells and reduced IL-12 production in macrophages.

Discussion: EV-A71 infection enhances IL-20RA cytokines, which then increase viral loads and aggravate disease severity in WT mice by elevating the T cell-IL-10-M2 macrophage axis and suppressing the protective M1 macrophage-IL-12-macrophage-IFN-γ axis. This represents a previously unreported mechanism.

Keywords: IFN-γ; IL-10; IL-12; IL-20RA cytokines; enterovirus A71; macrophage polarization.

Figure 1

Levels of viral titers and IL-20RA cytokines in mice. (A) Wild-type mice (WT, n = 10) were infected with EV-A71 (1 × 10⁶ PFU/mouse) and monitored for survival. The sera (B, D-F) and brains (C, G) of mice infected with EV-A71 and collected on 1, 3, or 5 days post-infection (black bars) or of mice mock-infected and collected on the day of infection (M, white bars) were assessed to determine the levels of virus by the plaque assay (B, C) and the indicated cytokines by ELISA (D-G). Data show means + SEM of ≥5 samples per group. *P < 0.05. B.D. stands for below the detection limit.

Figure 2

IL-20RA deficiency reduces viral loads in peripheral organs and EV-A71 lethality of mice. The survival rates (A), disease scores (B), and organ/tissue viral loads (C) of infected WT mice (black circles) and IL-20RA^-/- mice (white circles) are shown. In panels (A–C)n ≥ 10, n ≥ 10, and n = 6 per group or per group or data point, respectively. Data show means ± SEM in panels (B, C) *P < 0.05, **P < 0.01, ***P < 0.001, compared between WT and IL-20RA^-/- groups on the same day (C) or the indicated groups (A, B).

Figure 3

IL-20RA deficiency increases IFN-γ to reduce viral loads in peripheral organs and EV-A71 lethality of mice. (A) The sera of infected WT mice and IL-20RA^-/- mice were harvested to measure IFN-γ by ELISA. ND stands for “not done”, because samples were unavailable due to mouse death. The survival rates on indicated days (B) and organ/tissue viral loads on 7 days post-infection (C) of infected IL-20RA^-/- mice treated with the control or anti-IFN-γ antibody are shown. (A-C)n ≥ 4, n = 8, and n = 8 per data point or group, respectively. Data show means + SEM (A, C) *P < 0.05 and **P < 0.01, compared between WT and IL-20RA^-/- groups on the same day (A, C) or the indicated groups (B).

Figure 4

IL-20RA deficiency increases IFN-γ- expressing macrophages to reduce viral loads in peripheral organs and EV-A71 lethality of mice. The spleens of infected WT and IL-20RA^-/- mice were harvested on 5 days post-infection and processed to quantify splenocytes expressing leukocyte markers (A) CD45 plus (B) macrophages, dendritic cells, B cells, or T cells on the cell surface and IFN-γ in cells. The spleens of mock-infected IL-20RA^-/- mice treated with control or clodronate liposomes for one day were monitored for macrophage levels (C). The survival rates (D) and disease scores (E) on indicated days and organ/tissue viral loads (F) as well as serum IFN-γ protein levels (G) on 5 days post-infection of infected IL-20RA^-/- mice treated with control or clodronate liposomes are shown. Sample sizes per group, n = 5 (C), n = 7 (D, E), and n = 6 for the rest of panels. Except panel (D), data show means + or ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, compared between control and clodronate groups (F) or the indicated groups in the rest of panels.

Figure 5

IL-20RA deficiency increases the levels of serum IL-12 and IL-12-expressing leukocytes in infected mice. (A) The sera of infected WT mice and IL-20RA^-/- mice were harvested to measure IL-12 by ELISA. The spleens of infected WT and IL-20RA^-/- mice were harvested on 3 days post-infection and processed to quantify splenocytes expressing leukocyte markers of (B) CD45 plus (C) macrophages, B cells, neutrophils, or dendritic cells on the cell surface and IL-12 in cells. Data show means ± or + SEM of 6 samples per data point or group. *P < 0.05; **P < 0.01; ***P < 0.001, compared between WT and IL-20RA^-/- groups on the same day (A) or the indicated groups (B, C).

Figure 6

IL-20RA deficiency increases M1 macrophages in the spleen of infected mice. The spleens of WT and IL-20RA^-/- mice infected with EV-A71 for the indicated days or mock-infected (Mock) were harvested and processed to quantify splenocytes expressing markers of macrophages, CD45 and F4/80 (A), M1 macrophages, CD45, F4/80, CD86, and MHC-II (B), or M2 macrophages, CD45, F4/80, CD206, and Arginase-1 (C). Data show means + SEM of 5 samples per group. *P < 0.05 and **P < 0.01.

Figure 7

IL-20RA deficiency decreases levels of serum IL-10 and IL-10-expressing leukocytes in spleens of infected mice. (A) The sera of infected WT mice and IL-20RA^-/- mice were harvested to measure IL-10 by ELISA. The spleens of infected WT and IL-20RA^-/- mice were harvested on 1 day post-infection and processed to quantify splenocytes expressing leukocyte markers of (B) CD45 plus (C) T cells, dendritic cells, macrophages, or NK cells on the cell surface and IL-10 in cells. Data show means ± or + SEM of 6 samples per data point or group. **P < 0.01 and ***P < 0.001, compared between WT and IL-20RA^-/- mice on the same day (A) or the indicated groups (B, C).

Figure 8

Effects of IL-20RA cytokines on mRNA and protein levels of IL-10 in CD4 T cells. (A, B) CD4 T cells harvested from uninfected WT mice were treated without (UN) or with the indicated cytokines for 8 or 24 hours and centrifuged. Total RNA isolated from the cell pellet was subjected to quantitative real-time RT-PCR. The Il10/β-actin mRNA levels are shown, and the levels of control samples without cytokine treatment were set as 1. (C, D) The culture supernatants were collected to measure IL-10 by ELISA. Data show means + SEM of 6 samples per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 9

Effects of IL-20RA cytokines on protein and mRNA levels of IL-12 in macrophages. Peritoneal macrophages were harvested from uninfected WT or IL-20RA^-/- mice, treated without (-) or with the indicated cytokine for 3 days, stimulated without (-) or with poly I:C for 24 hours, and centrifuged. (A) The culture supernatant was collected to measure IL-12 by ELISA. Total RNA isolated from the cell pellet was subjected to quantitative real-time RT-PCR. Levels of Il12a/β-actin(B) and Il12b/β-actin(C) are shown. The levels of control samples without cytokine and poly I:C treatment were set as 1. Data show means + SEM of 6 samples per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 10

Cytokine levels in the plasma of healthy donors and EV-A71 patients. Plasma samples collected from EV-A71-infected children with mild or severe symptoms and healthy donors were assayed for IL-10, IL-12, IL-19, and IFN-γ (A-D) by ELISA. Data show means + SEM of ≥13 samples in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 11

The mechanism of IL-20RA cytokines aggravating EV-A71 infection. EV-A71 infection enhances leukocytes to express IL-20RA cytokines, which increase T cells to produce IL-10 to promote M2 macrophages and suppress M1 macrophage-IL-12-IFN-γ axis to result in increases of viral replication and lethality in WT mice.