Few reports investigate the influence of virus infection on IL-20RA cytokine expression in vivo, so we studied this issue using a murine model. Mice were infected with EV-A71 (1 × 106 PFU/mouse) by intraperitoneal inoculation to induce systemic infection, and all infected mice succumbed to death 7 days post-infection (d.p.i.). Mouse sera and brains were harvested on 1, 3, and 5 d.p.i. for analyses. Virus was detected in the serum from 1 to 5 d.p.i. and in the brain on 3 and 5 d.p.i. (Fig. 1A, B). We monitored the levels of all mouse IL-20RA cytokines, IL-19, IL-20, and IL-24, by ELISA. In the serum, IL-19, IL-20, and IL-24 were detected in mock-infected mice, showing that these three cytokines are constitutively expressed (Fig. 1C-E). EV-A71 infection enhanced IL-19 levels from 1 to 5 d.p.i. with significant differences found on 5 d.p.i. and slightly increased IL-20 levels on 3 and 5 d.p.i. and the IL-24 level on 1 d.p.i. (Fig. 1C-E). In the brain, IL-19 was detected in mock-infected mice, and EV-A71 infection increased the IL-19 level on 5 d.p.i. (Fig. 1F). However, IL-20 and IL-24 levels were below detection in the brains of mock-infected and infected mice from 1 to 5 d.p.i.
IL-20RA deficiency decreases EV-A71 lethality of mice with reduced viral loads in peripheral organs
IL-20RA cytokines were detected in mice, so we investigated the significance of IL-20RA cytokines on EV-A71 infection by comparing WT and IL-20RA−/− mice. After infection, increases in the death rate and disease scores were detected in WT mice, when compared to IL-20RA−/− mice (Fig. 2A, B). The viral loads in peripheral organs, heart, lung, liver, spleen, and kidney, of WT mice were higher than those of IL-20RA−/− mice on 3 and/or 5 d.p.i. (Fig. 2C). The viral loads in the intestine and central nervous system (CNS), the spinal cord, brain stem, and brain without brain stem, of WT and IL-20RA−/− mice on 3 and 5 d.p.i. were statistically insignificant (Fig. 2C). In our further mouse study, we focused on the peripheral organs, but not in the CNS, for the following two reasons. All IL-20RA cytokines are detected in the serum (periphery), but not in the CNS, of mice. More importantly, the major effect of IL-20RA deficiency on viral loads is found in peripheral organs, but not in the CNS, of mice.
We showed that EV-A71 infection increases serum and brain IL-19 levels of mice. Mice with deletion of the gene encoding IL-19 were generated and available [28], and these mice are viable and appear healthy. Our additional study compared IL-19-deficient mice (kindly provided by Dr. Yasu-Taka Azuma in Osaka Prefecture University) and WT mice infected with EV-A71 (1 × 106 PFU/mouse) by intraperitoneal inoculation. The result showed that all infected IL-19-deficient mice (n = 10) and WT mice (n = 14) succumbed to death in a manner different from that found in infected IL-20RA−/− and WT mice. This result suggests that the effect of IL-20RA cytokines on EV-A71-infection might not be mediated by IL-19 alone. As IL-19, IL-20, and IL-24 share biological activities [22], IL-20 and/or IL-24 may compensate for the loss of IL-19 in mice. These may explain the failure to see the difference in survival rates between infected WT and IL-19-deficient mice and reveal the necessity of using and comparing WT and IL-20RA−/− mice for further study.
In infected IL-20RA−/− mice, the serum IFN-γ level is elevated, and IFN-γ depletion increases viral loads in peripheral organs and EV-A71 lethality
IL-20RA cytokiens can regulate cytokine expression [21–23, 29]. As type I IFNs (IFN-α and IFN-β), IL-1β, and IL-6 have been shown to protect mice from EV-A71 infection [30–32], we monitored these cytokines and TNF-α using ELISA. The serum and/or brain levels of these cytokines detected in infected WT mice were not significantly lower than those of infected IL-20RA−/− mice from 1 to 5 d.p.i. (Supplementary Fig. 1). Previous in vitro studies showed that treatment with IL-20RA cytokines, IL-19 or IL-20, but not IL-24, decreases IFN-γ levels in primary human T cells in vitro [19, 20]. More importantly, we showed that EV-A71 induces IFN-γ to protect mice from infection by reduceing viral replication [11]. We therefore monitored IFN-γ in mice using ELISA. In mock-infected WT and IL-20RA−/− mice, both serum and brain IFN-γ levels were below detection. EV-A71 infection induced IFN-γ in both WT and IL-20RA−/− mice with reduced serum IFN-γ levels detected in WT mice when compared to IL-20RA−/− mice from 1 to 5 d.p.i. with a significant difference found on 5 d.p.i (Fig. 3A). The brain IFN-γ levels of infected WT mice were slightly lower than those of infected IL-20RA−/− mice on 3 and 5 d.p.i. (Supplementary Fig. 2). To determine the significance of IFN-γ in IL-20RA-deficient mice against EV-A71 infection, we depleted IFN-γ in IL-20RA−/− mice using the monoclonal antibody specific for IFN-γ. IFN-γ depletion reduced the survival rate of infected IL-20RA−/− mice with elevated viral loads in peripheral organs (spleen, lung, and liver) on 7 d.p.i. (Fig. 3B, C), showing that IFN-γ protects IL-20RA−/− mice from EV-A71 infection. We further investigated the regulation of IFN-γ by IL-20RA cytokines during EV-A71 infection, as few studies address this issue.
In infected IL-20RA−/− mice, abundant spleen macrophages express IFN-γ, and macrophage depletion increases viral loads in peripheral organs as well as EV-A71 lethality and decreases the serum IFN-γ level
IFN-γ is mainly expressed by leukocytes, and the spleen is composed by leukocytes. Moreover, abundant EV-A71 was detected in the spleen of infected mice, and both IL-20RA deficiency and IFN-γ depletion affected viral loads in the spleen (Fig. 2C and 3C). As IL-20RA deficiency increases the serum IFN-γ level of infected mice on 5 d.p.i., we therefore monitored the spleen collected on 5 d.p.i. by quantifying leukocytes expressing IFN-γ via staining CD45, a pan leukocyte marker, on the cell surface and IFN-γ inside the cells using flow cytometry (with gating strategy shown in Supplementary Fig. 3). In mock-infected WT and IL-20RA−/− mice, CD45+IFN-γ+ cells, leukocytes expressing IFN-γ were minimal. EV-A71 infection increased the percentages of CD45+IFN-γ+ cells in both WT and IL-20RA−/− mice with a reduced percentage of CD45+IFN-γ+ cells detected in WT mice when compared to IL-20RA−/− mice (Fig. 4A). Macrophages, dendritic cells, B cells, and especially T cells can produce IFN-γ [33]. We further identified and quantified the leukocytes expressing IFN-γ in splenocytes of infected mice on 5 d.p.i. Notably, we found that in infected IL-20RA−/− mice, a high percentage of (CD45+IFN-γ+CD11b+F4/80+) macrophages followed by (CD45+IFN-γ+CD11c+) dendritic cells, (CD45+IFN-γ+CD19+) B cells, and (CD45+IFN-γ+CD3+) T cells expressed IFN-γ in infected IL-20RA−/− mice (Fig. 4B). Moreover, the percentages of all these four types of IFN-γ-expressing leukocytes in infected IL-20RA−/− mice were higher than those of infected WT mice (Fig. 4B).
To assess the importance of macrophages on IL-20RA-deficient mice against EV-A71 infection, IL-20RA−/− mice were treated with the liposomes containing clodronate to deplete macrophages [34]. Clodronate liposome treatment efficiently depleted macrophages by ~ 70%, when compared to control liposomes, in the spleen of mock-infected IL-20RA−/− mice one day after treatment (Fig. 4C). In infected IL-20RA−/− mice, clodronate liposome treatment increased the viral loads in peripheral organs (heart, lung, spleen, intestine, and kidney) with significant differences found on 5 d.p.i., disease scores, and death rate, but decreased the serum IFN-γ level by > 50% on 5 d.p.i., when compared to control liposomes (Fig. 4D-G). These results showed that macrophages produce IFN-γ in infected IL-20RA−/− mice and that macrophages protect IL-20RA−/− mice from EV-A71 infection.
IL-20RA deficiency increases the levels of serum IL-12, spleen macrophages expressing IL-12, and spleen M1 macrophages in infected mice
IL-12 can enhance macrophages to express IFN-γ [16], which can further amplify the production of both IL-12 and IFN-γ [15], so we monitored IL-12. In mock-infected WT and IL-20RA−/− mice, the serum IL-12 levels were below detection. EV-A71 infection induced IL-12 in both WT and IL-20RA−/− mice with reduced serum IL-12 levels detected in WT mice when compared to IL-20RA−/− mice from 1 to 5 d.p.i. with significant differences found on 3 and 5 d.p.i (Fig. 5A). The biggest difference in serum IL-12 levels of infected WT and IL-20RA−/− mice was detected on 3 d.p.i. We therefore performed flow cytometry to quantify the leukocytes expressing IL-12 in splenocytes on 3 d.p.i. by staining leukocyte markers on the cell surface and IL-12 inside the cells. In mock-infected WT and IL-20RA−/− mice, CD45+IL-12+ cells (leukocytes expressing IL-12) in splenocytes were minimal. EV-A71 infection increased the percentages of CD45+IL-12+ cells in both WT and IL-20RA−/− mice with a reduced percentage of CD45+IL-12+ cells detected in WT mice when compared to IL-20RA−/− mice (Fig. 5B). Dendritic cells, neutrophils, and especially macrophages as well as B cells can produce IL-12 [17]. We further identified and quantified the leukocytes expressing IL-12 in splenocytes of infected mice on 3 d.p.i. and found that in infected IL-20RA−/− mice, high percentages of (CD45+IL-12+CD11b+F4/80+) macrophages and (CD45+IL-12+CD19+) B cells followed by (CD45+IL-12+CD11c+) dendritic cells and (CD45+IL-12+Ly6G+) neutrophils expressed IL-12 in infected IL-20RA−/− mice (Fig. 5C). Moreover, the percentages of macrophages, B cells, and neutrophils expressing IL-12 in infected IL-20RA−/− mice were significantly higher than those of infected WT mice (Fig. 5C).
IL-20RA deficiency increases the levels of serum IFN-γ and IL-12 as well as spleen macrophages expressing IFN-γ and IL-12 in infected mice (Fig. 3A, 4B, 5A, 5C). IFN-γ can promote the polarization of M1 macrophages, which express markers, such as CD86 and MHC-II, on the cell surface and produce cytokines, such as IL-12 [13, 14]. We quantified M1 macrophages in infected mice and found that the percentage of (CD45+F4/80+CD86+MHC-II+) M1 macrophages in the spleen of infected IL-20RA−/− mice was higher than that of infected WT mice (Fig. 5E).
IL-20RA deficiency decreases the levels of serum IL-10 and spleen T cells expressing IL-10 in infected mice
As IL-10 is shown to inhibit IL-12 production in macrophages [18, 35–37], we therefore monitored IL-10. In mock-infected WT and IL-20RA−/− mice, the serum IL-10 levels were below detection. EV-A71 infection induced IL-10 in both WT and IL-20RA−/− mice with an elevated serum IL-10 level detected in WT mice when compared to IL-20RA−/− mice on 1 d.p.i. (Fig. 6A). We performed flow cytometry to quantify the leukocytes expressing IL-10 in splenocytes on 1 d.p.i. by staining leukocyte markers on the cell surface and IL-10 inside the cells. In mock-infected WT and IL-20RA−/− mice, the percentages of CD45+IL-10+ cells (leukocytes expressing IL-10) in splenocytes were minimal. After infection, the percentage of CD45+IL-10+ cells in splenocytes of WT mice was higher than that of IL-20RA−/− mice (Fig. 6B). Dendritic cells, macrophages, NK cells, and especially T cells can produce IL-10 [38]. We further identified and quantified the leukocytes expressing IL-10 in splenocytes of infected mice on 1 d.p.i. and found that in infected WT mice, a high percentage of (CD45+IL-10+CD3+) T cells followed by (CD45+IL-10+CD11c+) dendritic cells, (CD45+IL-10+CD11b+F4/80+) macrophages, and (CD45+IL-10+CD19+) NK cells expressed IL-10 (Fig. 6C). Moreover, the percentages of all these four types of IL-10-expressing leukocytes in infected WT mice were higher than those of infected IL-20RA−/− mice (Fig. 6C).
CD4 T cells, especially Th2 cells, were the major producer of IL-10 [39], and our additional results showed that the percentages of CD45+IL-10+CD4+cells, CD4 T cells expressing IL-10 in the splenocytes of infected WT mice were indeed higher than that of infected IL-20RA−/− mice (Supplementary Fig. 4A). We further identified the CD4 T cell subsets, which produce IL-10. Our additional results showed that a high percentage of (CD45+IL-10+CD4+IL-4+) Th2 cells, followed by (CD45+IL-10+CD4+CD25+) Treg cells and (CD45+IL-10+CD4+CXCR3+IFN-γ+) Th1 cells expressed IL-10 in the splenocytes of infected WT mice (Supplementary Fig. 4B-D). Moreover, the percentages of all these three subsets of CD4 T cells expressing IL-10 in infected WT mice were higher than those of infected IL-20RA−/− mice (Supplementary Fig. 4B-D).
Treatment with IL-19 or IL-20, but not IL-24, increases IL-10 in mouse CD4 T cells, but reduces IL-12 in mouse macrophages, in vitro
Our in vivo results showed that the percentage of T cells expressing IL-10 was elevated in infected WT mice when compared to infected IL-20RA−/− mice (Fig. 6C). A mouse in vitro study showed that IL-19 treatment increases IL-10 production in CD4 T cells [21]. We therefore performed in vitro studies to investigate the effect of IL-20RA cytokines on mouse CD4 T cells to express IL-10. Mouse CD4 T cells were harvested from uninfected WT mice and treated with IL-19, IL-20, or IL-24. Our results showed that treatment of IL-19 or IL-20, but not IL-24, for 8 and 24 hours significantly enhanced the mRNA and protein levels of IL-10 in CD4 T cells (Fig. 7).
Our in vivo results showed that abundant macrophages expressed IL-12 in infected IL-20RA−/− mice and that the percentage of macrophages expressing IL-12 was reduced in infected WT mice when compared to infected IL-20RA−/− mice (Fig. 5C). We further assessed whether 20RA cytokines can suppress IL-12 in macrophages using in vitro study, as few studies investigate this issue. IL-12 is a 70-kDa heterodimeric cytokine composed of two subunits, p35 and p40 [40], encoded by the mouse genes of Il12a and Il12b, respectively. Macrophages harvested from uninfected WT mice were used for studies. Our ELISA results showed that a very low level of IL-12 p70 protein was detected in the culture supernatant of unstimulated macrophages obtained from WT mice (Fig. 8A). In order to boost the IL-12 level in macrophages, we tested EV-A71, but the virus failed to do so. We then assessed the RNA virus mimic, poly I:C, which is reported to enhance IL-12 in mouse bone marrow-derived macrophages [41]. We measured Il12a and Il12b mRNA by the quantitative real-time RT-PCR assay and p70 protein by the ELISA. Stimulation with poly I:C for 24 hours enhanced the levels of Il12a and Il12b mRNA and p70 protein of IL-12 in macrophages obtained from WT or IL-20RA−/− mice (Fig. 8). IL-10 is reported to suppress IL-12 [17, 18] and was therefore used as a positive control for the assay. Macrophages were treated with cytokines for 3 days, stimulated with poly I:C for 24 hours, and harvested for assays. IL-10 treatment reduced the Il12a and Il12b mRNA as well as protein levels of IL-12 in macrophages obtained from WT or IL-20RA−/− mice (Fig. 8). Treatment with IL-19 or IL-20, but not IL-24, decreased the Il12a and Il12b mRNA as well as protein levels of IL-12 in macrophages obtained from WT mice, but not from IL-20RA−/− mice (Fig. 8).
IL-19 is detected in the plasma of healthy controls, and EV-A71 infection increases plasma IL-19 levels in patients.
As both IL-19 and IL-20 enhance IL-10 in mouse CD4 T cells, but suppress IL-12 in mouse macrophages, in vitro, we measured these four cytokines and IFN-γ in the plasma specimens collected from patients tested positive for EV-A71 and from healthy controls. EV-A71-infected patients were divided into two groups, one with severe symptoms, brainstem encephalitis with or without pulmonary edema complication, and the other one with mild symptoms, fever, herpangina, or hand-foot-and-mouth disease. IL-19 was detected in healthy controls (Supplementary Fig. 5A). The IL-19 levels of infected patients with severe or mild symptoms were higher than that of healthy controls. The IL-19 level of EV-A71-infected patients with severe symptoms were slightly higher than those of mild symptoms. The results of IL-10, IL-12, and IFN-γ were similar to that of IL-19 (Supplementary Fig. 5). The IL-20 levels of both infected patients and healthy controls were below detection.