Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality of mice with enhanced M1 macrophage polarization and cytokine

Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality of mice with enhanced M1 macrophage polarization and cytokine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality of mice with enhanced M1 macrophage polarization and cytokine Shun-Hua Chen, Cheng-Huei Hung, Yi-Ling Hsiao, Yi-Ping Tsai, Ming-Shi Chang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4131398/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Enterovirus A71 (EV-A71) can cause fatality in patients with increases of cytokines, IL-10, IL-12, and IFN-γ, which are mutually regulated. IFN-γ is induced and protects the host from EV-A71 in a murine infection model. IFN-γ and IL-10 promote the polarization of M1 and M2 macrophages, which produce IL-12 and IL-10, respectively. IL-10 suppresses IL-12, which enhances itself and IFN-γ. The IL-10 family cytokines, IL-19, IL-20, and IL-24, which signal through the two-subunit receptor complex with IL-20RA as one subunit, are therefore designated as IL-20RA cytokines. Previous in vitro T cell studies showed that IL-19 or IL-20 treatment suppresses IFN-γ and that IL-19 treatment enhances IL-10. In the present study of human plasma, IL-19 was detected in healthy controls, and EV-A71 infection increased IL-19 in patients. In the serum of mice, IL-20RA cytokines, but not IL-10, IL-12, and IFN-γ, were detected in mock-infected mice, and EV-A71 infection enhanced IL-19. Compared to wild-type mice, IL-20RA knockout mice were resistant to infection with reduced viral loads in peripheral organs, including the spleen. In infected mice, IL-20RA deficiency sequentially reduced IL-10, but increased IL-12 and IFN-γ, in the serum with T cells expressing IL-10 and macrophages expressing IL-12 and IFN-γ in the spleen. Notably, IL-20RA deficiency increased spleen M1 macrophages. In vitro study showed that treatment with IL-19 or IL-20, but not IL-24, increased IL-10 in CD4 T cells, but reduced IL-12 in macrophages. Our study is novel to show that IL-20RA cytokines affect virus infection, cytokines regulating macrophage polarization, and macrophage polarization. Biological sciences/Immunology/Infectious diseases/Viral infection Biological sciences/Immunology/Cytokines/Interleukins Enterovirus A71 IL-10 IL-12 IL-19 IL-20 IL-24 IFN-γ IL-20RA IL-20RA cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Enterovirus A71 (EV-A71) infects humans by the fecal-oral route and can induce mild illness, such as fever, herpangina, and hand-foot-and-mouth disease [ 1 – 3 ]. This virus can also cause severe symptoms, brainstem encephalitis combined with pulmonary edema complications, which often induce death or long-term neurological sequelae, especially in young children [ 1 ]. Widespread and deadly EV-A71 outbreaks have been frequently reported in the Asia-Pacific region, including Taiwan, for decades [ 2 – 4 ]. Currently, specific and effective antivirals are unavailable for patient treatments. Intravenous immunoglobulin (IVIG) has been used to treat infected patients with fatal symptoms in countries like Taiwan [ 5 ]. Vaccines were developed and used in few places, such as China and Taiwan, but unavailable in other countries [ 6 – 8 ]. Studies of plasma specimens from infected patients show that EV-A71 infection increases IL-10, IL-12, and IFN-γ [ 9 , 10 ] with IL-10 detected before IFN-γ [ 10 ]. Using a murine infection model, we further reveal that EV-A71 induces IFN-γ to protect the host from infection by reducing viral replication [ 11 ]. Despite IFN-γ plays a protective role in infection, the regulation of IFN-γ during EV-A71 infection remains to be elucidated. IFN-γ can be upregulated by following ways. It can enhance its own expression in an autocrine manner [ 12 ]. It 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 ]. IL-12 and IFN-γ can mutually amplify each other [ 15 , 16 ]. However, IFN-γ can be down-regulated by following ways. IL-10 can inhibit IL-12 production [ 17 , 18 ]. IL-10 can promote the polarization of M2 macrophages, which mainly produce IL-10 [ 13 , 14 ]. Additionally, IL-19 or IL-20, but not IL-24, can increase IL-10 and/or decrease IFN-γ in T cells as demonstrated by human and mouse in vitro studies [ 19 – 21 ]. The regulation and interaction of IFN-γ with IL-10, IL-12, IL-19, and IL-20 remain elusive. IL-10, IL-19, and IL-20 belong to the IL-10 family, which contains nine members, IL- 10, IL-20 subfamily members (IL-19, IL-20, IL-22, IL-24, and IL-26), and the distantly related cytokines IL-28A, IL-28B, and IL-29, which are classified as type III IFNs, IFN-λ2, IFN-λ3, and IFN-λ1, respectively [ 22 , 23 ]. The IL-20 subfamily members utilize the heterodimeric receptor complex with two subunits to transduce their signals. IL-19, IL-20, and IL-24 share the receptor composed of IL-20RA (also called IL-20R1 and IL-20Rα) and IL-20RB. IL-20 and IL-24 can also signal through IL-22RA1 paired with IL-20RB. IL-26 transduces signals through IL-20RA and IL-10RB [ 22 ]. Because IL-19, IL-20, IL-24, and IL-26 signal through IL-20RA, they are designated as ‘IL-20RA cytokines’ in the present study. Humans express all four IL-20RA cytokines, and mice express only IL-19, IL-20, and IL-24 [ 22 ]. IL-20RA cytokines are shown to target T cells, macrophages, monocytes, and epithelial cells [ 21 – 24 ]. The interaction of IL-20RA cytokines with virus infection remains unknown, probably because these cytokines are complex in sharing receptors, functions, and activities. Mice with IL-20RA knockout ( IL-20RA −/− mice) were generated [ 25 ], and these mice appear healthy and are available for study. Among IL-20RA cytokines in IL-20RA −/− mice, the signaling of IL-19 is totally blocked, but IL-20 and IL-24 can still signal through the receptor of IL-22RA1 paired with IL-20RB. Using a murine infection model, we found that IL-20RA cytokines (IL-19, IL-20, and IL-24), but not IL-10, IL-12 and IFN-γ, were constitutively expressed in mock-infected mice and that EV-A71 infection enhanced IL-20RA cytokines, especially IL-19, and induced IL-10 in wild-type (WT) mice. By comparing WT and IL-20RA −/− mice, we found that IL-20RA deficiency reduced EV-A71 lethality of mice with a decrease of IL-10 expression in T cells and increases of IL-12 and IFN-γ expression in macrophages and the level of M1 macrophages in manner hardly reported before. MATERIALS AND METHODS Cell, virus, and mice The human muscular (rhabdomyosarcoma, RD) cell line was maintained and propagated according to the instruction of American Type Culture Collection. EV-A71 strain M2, a mouse-adapted virus, was propagated in and titrated on RD cell monolayers by the plaque assay as previously described [ 26 ]. All mouse experiments and care were approved by the Institutional Animal Use and Care Committee of National Cheng Kung University with the approval number of 105283 in accordance with national guidelines and regulations. WT C57BL6/J mice and C57BL6/J-derived IL-20RA −/− mice [ 25 ] were bred and maintained under specific pathogen-free conditions in the Laboratory Animal Center of our university. Infection of mice, tissue collection, and treatments of mice with IFN-γ antibody or liposomes Twelve- to 14-day-old WT and IL-20RA −/− mice were infected with 2 × 10 6 plaque forming units (PFU)/mouse of EV-A71 by intraperitoneal inoculation and monitored for survival and disease scores. The disease score was graded as follows: 0, healthy; 1, ruffled hair; 2, weakness in hind limbs; 3, paralysis in single hind limb; 4, paralysis in both hind limbs, and 5, death. Mice were anaesthetized, and mouse blood was collected. Mice were then perfused by intracardial injection of ice-cold phosphate buffered saline (PBS) containing 0.01 M EDTA and 0.2% BSA, and mouse tissues were harvested. Mouse blood was processed to obtain serum, and the serum was frozen at -80°C and sonicated. Mouse organs and tissues were frozen, thawed, homogenized in 1 mL PBS, frozen, thawed, sonicated, and centrifuged at 13,000 rpm for 15 minutes at 4°C. The resulting sera and organ/tissue supernatants were assayed for viral titers by the plaque assay on RD cell monolayers. Mice were treated with 25 µg of anti-IFN-γ antibody (Clone R4-6A2, Bio X Cell) or normal rat IgG (Sigma) or 50 µL of control liposomes (FormuMax) or clodronate liposomes (Formumax) by intraperitoneal injection one day before infection and on days 1, 3, 5, and 7 post-infection. Treatments of mouse leukocytes with IL-19, IL-20, or IL-24 in vitro Single-cell suspensions were prepared from splenocytes. CD4 T cells were subsequently isolated to 90% purity with positive selection using anti-CD4 magnetic beads (Biolegend) according to the manufacturer instruction. Mouse CD4 T cells were treated without or with IL-19, IL-20, or IL-24 (R&D Systems; 100 ng/mL) and harvested 8 and 24 hours after cytokine treatment. Peritoneal macrophages were harvested from mice, and more than 90% of the cells were positive for F4/80, a marker specific for mouse macrophages, by immunofluorescence staining as previously described [ 12 ]. Macrophages were treated without or with IL-19, IL-20, or IL-24 (100 ng/mL) for 3 days, stimulated without or with poly I:C (Invivogen; 100 ng/mL) for 24 hours, and harvested. The culture supernatants of CD4 T cells and macrophages were harvested to quantify IL-10 and IL-12, respectively by the enzyme-linked immunosorbent assay (ELISA). The remaining cells were processed to extract total RNA by the GENEzol TriRNA Pure Kit (Geneaid). The total RNA of CD4 T cells and macrophages were subjected to RT-PCR to quantify the mRNA encoding IL-10 or IL-12, respectively. Quantitative real-time RT-PCR After reverse transcription with reverse primers, the synthesized cDNA was used for quantitative real-time PCR with forward and reverse primers. PCR was performed at 95°C for 10 minutes followed by 40 cycles of denaturation (95°C, 15 seconds) and annealing (60°C, 1 minute) with the kit of Fast SYBR Green Master Mix (Thermo Fisher Scientific). The threshold cycle ( C T ) of each product was determined, normalized to the internal control (β-actin), and shown as Δ C T . All results are shown as the ratio to β-actin calculated as 2 −Δ C T . Primers for the mRNA encoding IL-10 (forward 5’-ATA ACT GCA CCC ACT TCC CA -3’ and reverse 5’-GGG CAT CAC TTC TAC CAG GT-3’), β-actin (forward 5’-AAC CCT AAG GCC AAC CGT GAA AAG ATG ACC-3’ and reverse 5’-CCA GGG AGG AAG AGG ATG CGG C-3’), IL-12 p35 (forward primer 5’-AGG ACT TGA AGA TGT ACC AG-3’ and reverse primer 5’-CTA TCT GTG TGA GGA GGG-3’), or IL-12 p40 (forward 5’-GGA AGC ACG GCA GCA GAA TAA-3’ and reverse 5’-CTT GAG GGA GAA GTA GGA ATG-3’) were used. Cytokine measurement by ELISA Brains harvested from mice were frozen, homogenized in 1 mL PBS containing a protease inhibitor cocktail (Sigma-Aldrich), and centrifuged. Mouse sera and brain supernatants as well as T cell or macrophage culture supernatants were subjected to commercially available ELISA (with detection limits and suppliers shown in parentheses) for assays of cytokines IL-10 (2.7 pg/mL, Biolegend), IL-12 (0.5 pg/mL, Biolegend), IL-19 (62.5 pg/mL, eBioscience), IL-20 (6.4 pg/mL, R&D Systems), IL-24 (18 pg/mL, Elabscience), or IFN-γ (2 pg/mL, R&D Systems) according to the instructions of manufacturers. Flow cytometry Leukocytes were isolated from mouse spleens as previously described [ 27 ] and blocked with the antibody CD16/CD32 (Clone 93; BioLegend) against Fc-receptors to prevent nonspecific binding. The resulting leukocytes were stained with the antibodies against CD3 (clone 17A2, BD Bioscience), CD11b (clone M1/70, BD Bioscience), CD11c (clone HL3, BD Bioscience), CD19 (clone 1D3, BD Bioscience), CD45 (clone 30-F11, Biolegend), CD335 (clone NKp46, BD Biosciences), CD86 (clone GL1, BD Bioscience), F4/80 (clone CI:A3-1, Bio-Rad), MHC-II (clone M5/114.15.2, BD Bioscience) or Ly6G (clone 1A8, BD Biosciences) on the cell surface, fixed, and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) before staining for intracellular IFN-γ (clone XMG1.2, Biolegend), IL-10 (clone JES5, Biolegend), or IL-12 (clone C15.6, Biolegend) and analyzed by the Cytoflex flow cytometer (Beckman Coulter). Statistical analyses Data are expressed as mean ± SE value (error bars). For statistical comparison, levels of IL-mouse 10, IL-12, and IFN-γ, tissue viral titers, and flow cytometry results were analyzed by the Mann-Whitney U test, disease scores were analyzed by two-way ANOVA, and survival rates were analyzed by the log-rank test. In vitro data were analyzed by the Student’s t test. All P values are for two-tailed significance tests. A P value of < 0.05 is considered statistically significant. RESULTS All mouse IL-20RA cytokines, IL-19, IL-20, and IL-24, are detected in the serum of mock-infected mice, and EV-A71 infection increases mouse serum and brain IL-19 levels 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 × 10 6 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. 1 A, 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. 1 C-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. 1 C-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. 1 F). 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. 2 A, 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. 2 C). 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. 2 C). 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 × 10 6 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. 3 A). 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. 3 B, 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. 2 C and 3 C). 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. 4 A). 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. 4 B). 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. 4 B). 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. 4 C). 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. 4 D-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. 5 A). 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. 5 B). 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. 5 C). 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. 5 C). 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. 3 A, 4 B, 5 A, 5 C). 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. 5 E). 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. 6 A). 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. 6 B). 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. 6 C). 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. 6 C). 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. 6 C). 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. 5 C). 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. 8 A). 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. DISCUSSION Very few reports investigate the interaction of moues IL-20RA cytokines with virus infection. Our in vivo study shows that all three IL-20RA cytokines, IL-19, IL-20, and IL-24, are detected in mock-infected mice and that EV-A71 infection enhances IL-20RA cytokines, especially IL-19 in mice. More importantly, IL-20RA cytokines exacerbates EV-A71 infection in mice with an increase of IL-10 expressed by T cells and reduced levels of M1 macrophages as well as IL-12 and IFN-γ expressed by macrophages. Consistently, our mouse in vitro study reveals that both IL-19 and IL-20 enhance IL-10 in T cells, but suppress IL-12 in macrophages. These studies find that all three mouse IL-20RA cytokines are constitutively expressed and suggest that both IL-19 and IL-20 could be the upstream effectors inducing IL-10 to reduce the IL-12/IFN-γ axis as well as M1 macrophages and aggravate EV-A71 infection (Supplementary Fig. 6). Our previous and present mouse studies using IFN-γ receptor-deficient mice and anti-IFN-γ antibody, respectively showed that IFN-γ protects mice from EV-A71 infection [ 11 ]. The present study showed that macrophages produce IFN-γ to fight EV-A71 infection in mice. Overall, our present results showing the interaction of IL-20RA cytokines with EV-A71 infection are unreported and novel. Leukocytes, especially myeloid cells are the primary source of IL-19 and IL-20 [ 22 ], so we performed flow cytometry on the spleen to quantify leukocytes expressing IL-19 or IL-20 in infected mice on 3 and 5 d.p.i., the time points at which EV-A71 infection significantly and slightly increases serum IL-19 and IL-20 levels, respectively, by staining leukocyte markers on the cell surface and the cytokines inside the cells. CD45 + IL-19 + cells (leukocytes expressing IL-19) were detected in both mock-infected and infected mice with a slightly increased level of CD45 + IL-19 + cells found in infected mice on 3 d.p.i. when compared to mock-infected mice (Supplementary Fig. 7A). CD45 + IL-20 + cells (leukocytes expressing IL-20) were detected in infected mice on 3 and 5 d.p.i. and in mock-infected mice (Supplementary Fig. 7B). These results are consistent with the detection of both IL-19 and IL-20 in the serum of mock-infected and infected mice. Macrophages and dendritic cells are reported to be the cellular sources of IL-19 and IL-20, respectively [ 22 ]. We found that a high percentage of dendritic cells, followed by macrophages, T cells, and NK cells expressed IL-19 in infected mice on 3 d.p.i. and in mock-infected mice (Supplementary Fig. 7C). A high percentage of dendritic cells, followed by NK cells, macrophages, and T cells expressed IL-20 in infected mice on 3 and 5 d.p.i. and in mock-infected mice (Supplementary Fig. 7D). Cells other than leukocytes can express IL-19 [ 22 ]. EV-A71 infection is reported to induce the activation of NF-κB signaling [ 42 ], which is shown to upregulate IL-19 expression in the airway epithelia of asthmatic patients [ 43 ]. Future studies are needed to find other cellular sources of IL-19 and IL-20 and address the signaling pathway regarding how EV-A71 infection increases IL-19. Our additional study also tested a 10-fold lower dose of viral inoculum (1 × 10 5 PFU/mouse) and obtained similar results as those of high viral dose (1 × 10 6 PFU/mouse) showing that IL-20RA deficiency ameliorates EV-A71 infection of mice (Supplementary Fig. 8). We used the high viral dose for study, as the differences in IL-10, IL-12, and IFN-γ levels between infected WT and IL-20RA −/− mice are readily detected. Clinical EV-A71 isolates fail to induce death in mice and need to be adapted in mice in order to induce death in mice at the age of 2 weeks old in our model. We also study herpes simplex virus 1 (HSV-1), which can induce death in 6-week-old mice by peripheral (corneal) infection and in 2- or 6-week-old mice by systemic (intraperitoneal) infection. In mice infected with HSV-1 by corneal inoculation, virus mainly spreads by the neuronal route and is detected only in the eye, trigeminal ganglia, and brain, but not in other tissues or organs [ 44 ]. Our additional study found that the survival rate of 2-week-old IL-20RA −/− mice infected with HSV-1 by intraperitoneal injection were higher than that of infected WT mice (Supplementary Fig. 9), in a manner similar to that found in EV-A71-infectied mice. However, the survival rates of 6-week-old IL-20RA −/− and WT mice infected with HSV-1 by corneal or intraperitoneal inoculation were not statistically significant. The EV-A71 and HSV-1 results show that the important role of IL-20RA cytokines is shown in neonatal mice with systemic virus infections, and further studies are needed to address this issue in future. Previous reports of IL-20RA cytokines mostly focused on T cells to show that IL-19 or IL-20 can increase IL-10 and/or decrease IFN-γ to induce T cell polorazation toward a Th2 profile [ 19 – 21 ]. We previously found that T cell responses, especially the Th2 response, promote the production of antibodies, which protect mice from EV-A71 infection [ 45 – 47 ], and that the Th2 cytokine IL-6 decreases EV-A71 lethality of mice [ 30 ], showing the protective role of Th2 response in EV-A71 infeciton of mice. In the present study, we focus on macrophages, as more abundant macrophages express the protective cytokine, IFN-γ, than T cells in infected IL-20RA −/− mice. Additionally, few studies investigate the effect of IL-20RA cytokines on macrophages until recently [ 24 ], as IL-20RA is detected on macrophages [ 48 ]. Macrophages can differentiate into two distinct subpopulations, classical or inflammatory M1 macrophages and alternative or anti-inflammatory M2 macrophages [ 49 ]. M1 macrophage differentiation can be induced by Th1 cytokines, such as IFN-γ. M1 macrophages produce cytokines, such as IL-12, and express markers, such as CD86 and MHC-II on the cell surface. Although M2 macrophages are more diverse and can be classified into four subtypes depending on the stimuli, the hallmark of all subtypes of M2 macrophages is the secretion of anti-inflammatory cytokine, IL-10 [ 14 ]. Our in vivo results showed that IL-20RA deficiency reduces the level of T cells expressing IL-10 but increases the levels of macrophages expressing IL-12 and IFN-γ and M1 macrophages in mice during infection. Consistently, our in vitro results showed that treatment of IL-19 or IL-20 enhances IL-10 in T cells, but suppresses IL-12 expression in macrophages. IL-20RA cytokines induce STAT3 activation [ 23 ], which is shown to enhance IL-10 [ 50 – 54 ], but inhibits IL-12 [ 55 ]. These findings may explain how IL-20RA cytokines increases IL-10 in T cells, but suppress IL-12 in macrophages. Our previous study used the anti-IL-20RA monoclonal antibody (51D) to ameliorate liver damage (fibrosis) in mice [ 56 ]. Here we show that IL-20RA cytokines aggravates EV-A71 infection in mice. Our additional study tested 51D to reduce EV-A71 infection in mice, but failed. IL-20RA is detected on leukocytes, such as macrophages [ 48 ], 51D treatment may deplete protective leukocytes and result in the failure to reduce EV-A71 infection. Future studies can design small molecules targeting IL-20RA and receptor signaling to test the potential of blocking IL-20RA to reduce virus infections. Declarations DATA AVAILABILITY All data needed to evaluate the conclusions in this study are presented in this manuscript or the Supplementary Information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors. ACKNOWLEDGEMENTS We thank Professors Yee-Shin Lin, Chiou-Feng Lin, and Chih-Peng Chang for helpful suggestions and the technical services provided by the “Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, National Science and Technology Council, Taiwan”. This work was supported by the funding from National Science and Technology Council, Taiwan (NSTC 113-2327-B-006-003) to SHC, SMW, and LCW. AUTHOR CONTRIBUTIONS CHH and YLH contributed equally by performing experiments data analyses, YPT performed experiments, MSC provided mice and suggestions, CCL provided human specimens and suggestions, SMW and LCW provided suggestions, and SHC conducted and obtained funding for the project. COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/. Correspondence and requests for materials should be addressed to Shih-Min Wang, Li-Chiu Wang, or Shun-Hua Chen. Reprints and permission information is available at http://www.nature.com/ reprints References Chang LY, Huang LM, Gau SS, Wu YY, Hsia SH, Fan TY, et al. Neurodevelopment and cognition in children after enterovirus 71 infection. N Engl J Med. 2007;356:1226–34. Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF, et al. An epidemic of enterovirus 71 infection in Taiwan. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4131398","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":282303616,"identity":"0a87cbe1-5e21-4356-a82b-4f8b04a382cd","order_by":0,"name":"Shun-Hua Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYDACCQYGZiAlx8DA2HgAyDCAChLWYgzU0oCsxYCglsQGIEGcFvnZPWaPC9vs0te2H244wLjDztjgAPPB2zwMf8CGYAMGd86YG89sS87ddiYRqOVMspnBAbZkax4GA9xaJHLMpHnbmHO3HQBpaTtgY3CAx0waqCUXlxb5GWAt9elm5x/CtPB/w6uF4QZYy+EEsxsQW4AO42HDq8XgRlqZNM+544bbbgBtSWxLNpY8zGZsOcfAuB63w5K3SfOUVcubnU9/+OBjm51h3/HmhzfeVMgZ43IXKkgAEcxg24nTMApGwSgYBaMAOwAAjlhWHsCmtSsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4585-9279","institution":"National Cheng Kung University","correspondingAuthor":true,"prefix":"","firstName":"Shun-Hua","middleName":"","lastName":"Chen","suffix":""},{"id":282303617,"identity":"2f844b1e-cfa6-4958-9917-bc61a9122b6a","order_by":1,"name":"Cheng-Huei Hung","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Huei","middleName":"","lastName":"Hung","suffix":""},{"id":282303618,"identity":"d1d0f0dc-7087-4b82-8390-cdf9e818ff6f","order_by":2,"name":"Yi-Ling Hsiao","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Ling","middleName":"","lastName":"Hsiao","suffix":""},{"id":282303619,"identity":"ec894b72-c095-44eb-9595-960c8bcfbbe2","order_by":3,"name":"Yi-Ping Tsai","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Ping","middleName":"","lastName":"Tsai","suffix":""},{"id":282303620,"identity":"6e8e9427-0a06-4257-89f8-518f6af8ef58","order_by":4,"name":"Ming-Shi Chang","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Ming-Shi","middleName":"","lastName":"Chang","suffix":""},{"id":282303621,"identity":"6e60ac62-9328-4a01-ac02-8ab008c3dcfb","order_by":5,"name":"Ching-Chuan Liu","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Ching-Chuan","middleName":"","lastName":"Liu","suffix":""},{"id":282303622,"identity":"b1a92be1-1403-4ef5-b5c5-49d22e7b40b6","order_by":6,"name":"Li-Chiu Wang","email":"","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":false,"prefix":"","firstName":"Li-Chiu","middleName":"","lastName":"Wang","suffix":""},{"id":282303623,"identity":"787f77e3-3afb-4041-b30d-0ff4a83fab4f","order_by":7,"name":"Shih-Min Wang","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Shih-Min","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-19 15:07:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4131398/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4131398/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53656373,"identity":"0406c909-9ed5-48d8-bde1-6cdc5ccdb21e","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":584569,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of viral titers and IL-20RA cytokines in mice. The sera (\u003cstrong\u003eA\u003c/strong\u003eand \u003cstrong\u003eC-E\u003c/strong\u003e) and brains (\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003e F\u003c/strong\u003e) of WT mice infected with EV-A71 for the indicated days or mock-infected (M) were collected to determine the levels of virus (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) or the indicated cytokines (\u003cstrong\u003eC-F\u003c/strong\u003e). Data show means + SEM of ≥3 samples per group. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. BD stands for below the detection limit.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/7c8d454cc94ab4a50b5b0a45.png"},{"id":53656372,"identity":"b38e7cc6-f155-44b1-bdfb-2d712ffbcec2","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":626451,"visible":true,"origin":"","legend":"\u003cp\u003eIL-20RA deficiency ameliorates EV-A71 infection in mice with reduced viral loads in peripheral organs. The survival rates (\u003cstrong\u003eA\u003c/strong\u003e), disease scores (\u003cstrong\u003eB\u003c/strong\u003e), and organ/tissue viral loads (\u003cstrong\u003eC\u003c/strong\u003e) of infected WT mice (black circles) and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (white circles) are shown. In panels \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e, \u003cem\u003en\u003c/em\u003e ≥ 10 per group. In panel \u003cstrong\u003eC\u003c/strong\u003e, \u003cem\u003en\u003c/em\u003e = 6 per data point. Data show means ± SEM (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC)\u003c/strong\u003e. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, compared between the indicated groups (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) or WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e groups on the same day (\u003cstrong\u003eC\u003c/strong\u003e).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/db2f0a07f99d4a2d2f4ab526.png"},{"id":53656377,"identity":"121efc34-f22d-4836-867c-aac397c6301a","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335340,"visible":true,"origin":"","legend":"\u003cp\u003eIn infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, the serum IFN-γ level is elevated, and IFN-γ depletion increases viral loads in peripheral organs and EV-A71 lethality.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The sera of infected WT mice and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested to measure IFN-γ. ND stands for not done, because samples were unavailable due to the death of all infected WT mice. The survival rates on indicated days (\u003cstrong\u003eB\u003c/strong\u003e) and organ/tissue viral loads on 7 days post-infection (\u003cstrong\u003eC\u003c/strong\u003e) of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice treated with the control or anti-IFN-γ antibody are shown. In panels \u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, and \u003cstrong\u003eC\u003c/strong\u003e, \u003cem\u003en\u003c/em\u003e ≥ 4, \u003cem\u003en\u003c/em\u003e = 8, and \u003cem\u003en\u003c/em\u003e = 8 per data point or group, respectively. Data show means + SEM (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared between WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e groups (\u003cstrong\u003eA\u003c/strong\u003e) or control and anti-IFN-γ antibody groups (\u003cstrong\u003eC\u003c/strong\u003e) on the same day or the indicated groups (\u003cstrong\u003eB\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/3b7ba93b7a363b8af2bdb478.png"},{"id":53656379,"identity":"1e4adc7b-a63c-4069-9081-294496a176e3","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1075194,"visible":true,"origin":"","legend":"\u003cp\u003eIn infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, levels of leukocytes, especially macrophages of the peripheral organ (spleen), expressing IFN-γ are elevated, and macrophage depletion increases viral loads in peripheral organs and EV-A71 lethality and decreases the serum IFN-γ level. The spleens of infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested on 5 days post-infection and processed to quantify cells expressing leukocyte markers (\u003cstrong\u003eA\u003c/strong\u003e) CD45 plus (\u003cstrong\u003eB\u003c/strong\u003e) CD11b and F4/80 for macrophages, CD11c for dendritic cells, CD19 for B cells, or CD3 for T cells on the cell surface and IFN-γ in the cells. (\u003cstrong\u003eC\u003c/strong\u003e) The spleens of mock-infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice treated with control or clodronate liposomes for one day were monitored for macrophage levels. The survival rates (\u003cstrong\u003eD\u003c/strong\u003e) and disease scores (\u003cstrong\u003eE\u003c/strong\u003e) on indicated days and organ/tissue viral loads (\u003cstrong\u003eF\u003c/strong\u003e) as well as serum IFN-γ protein levels (\u003cstrong\u003eG\u003c/strong\u003e) on 5 days post-infection of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice treated with control or clodronate liposomes are shown. Sample sizes per group, \u003cem\u003en\u003c/em\u003e = 5 (\u003cstrong\u003eC\u003c/strong\u003e), \u003cem\u003en\u003c/em\u003e = 7 (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e), and \u003cem\u003en\u003c/em\u003e = 6 for the rest of panels. Data show means + or ± SEM, except panel \u003cstrong\u003eD\u003c/strong\u003e. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, compared between control and clodronate groups (F) or the indicated groups in the rest of panels.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/ade3282e0ae998d36c58f53d.png"},{"id":53656378,"identity":"734e37ee-7c66-4176-85be-036ca3a0c2d4","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":377505,"visible":true,"origin":"","legend":"\u003cp\u003eIL-20RA deficiency increases the levels of serum IL-12 and IL-12-expressing leukocytes (macrophages) as well as M1 macrophages in the peripheral organ (spleen) of infected mice. (\u003cstrong\u003eA\u003c/strong\u003e) The sera of infected WT mice and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested to measure IL-12. The spleens of infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested on 3 days post-infection to quantify cells expressing leukocyte markers of (\u003cstrong\u003eB\u003c/strong\u003e) CD45 plus (\u003cstrong\u003eC\u003c/strong\u003e) macrophages, B cells, neutrophils (Ly-6G), or dendritic cells on the cell surface and IL-12 in the cells or 5 days post-infection to quantify cells expressing markers of macrophages, CD45 and F4/80, (\u003cstrong\u003eD\u003c/strong\u003e) and M1 macrophages, CD45, F4/80, CD86, and MHC-II, (\u003cstrong\u003eE\u003c/strong\u003e) on the cell surface. Data show means ± or + SEM of 5-6 samples per data point or group. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, compared between WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e groups on the same day (\u003cstrong\u003eA\u003c/strong\u003e) or the indicated groups (\u003cstrong\u003eB-E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/510ee2cf8f1df5375dd92f47.png"},{"id":53656381,"identity":"a33f17b3-564c-4fde-869a-da9012b8b7be","added_by":"auto","created_at":"2024-03-28 15:49:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229552,"visible":true,"origin":"","legend":"\u003cp\u003eIL-20RA deficiency decreases the levels of serum IL-10 and IL-10-expressing leukocytes in the peripheral organ of infected mice. (\u003cstrong\u003eA\u003c/strong\u003e) The sera of infected WT mice and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested to measure IL-10. The spleens of infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice were harvested on 1 day post-infection and processed to quantify cells expressing leukocyte markers of (\u003cstrong\u003eB\u003c/strong\u003e) CD45 plus (\u003cstrong\u003eC\u003c/strong\u003e) T cells, dendritic cells, macrophages, or NK cells (CD335) on the cell surface and IL-10 in the cells. Data show means ± or + SEM of 6 samples per data point or group. **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, compared between WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice on the same day (\u003cstrong\u003eA\u003c/strong\u003e) or the indicated groups (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/0dab55531560315268066d50.png"},{"id":53656376,"identity":"17797769-1621-488c-864f-92b9c01aed0f","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":182503,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro assay for the effects of IL-20RA cytokines on the mRNA and protein levels of IL-10 in mouse CD4 T cells. CD4 T cells harvested from uninfected WT mice were treated without (UN) or with the indicated cytokines for 8 and 24 hours and centrifuged. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) Total RNA isolated from the cell pellet was subjected to quantitative real-time RT-PCR. The \u003cem\u003eIl10/β-actin\u003c/em\u003e mRNA levels are shown, and the levels of control samples without cytokine treatment were set as 1. (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) The culture supernatant was collected to measure IL-10 by ELISA. Data show means + SEM of 6 samples per group. *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; and ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/55684a87a8d95d449a237f03.png"},{"id":53656375,"identity":"8f13d35d-4052-4db0-8d84-89da7abe4104","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":284056,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro assay for the effects of IL-20RA cytokines on the protein and mRNA levels of IL-12 in mouse macrophages. Peritoneal macrophages were harvested from uninfected WT or \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice, treated without (-) or with the indicated cytokine for 3 days, stimulated without (-) or with poly I:C for 24 hours, and centrifuged. (\u003cstrong\u003eA\u003c/strong\u003e) The culture supernatant was collected to measure IL-12 by ELISA. (\u003cstrong\u003eB, C\u003c/strong\u003e) Total RNA isolated from the cell pellet was subjected to quantitative real-time RT-PCR. Levels of \u003cem\u003eIl12a/β-actin \u003c/em\u003eand \u003cem\u003eIl12b/β-actin\u003c/em\u003e 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. *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; and ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/c00ce4858186ab6fa18fd103.png"},{"id":54490379,"identity":"de6fdf34-d100-461f-aab5-9c637cad4922","added_by":"auto","created_at":"2024-04-11 10:14:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2088231,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/a1315334-ab16-473c-8c26-4ae1184270ff.pdf"},{"id":53656374,"identity":"f5dba32e-5986-4c5f-afb8-28c9cebfd0fd","added_by":"auto","created_at":"2024-03-28 15:49:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1869570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"319Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-4131398/v1/caeb8ceda6b09b1915b72402.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality of mice with enhanced M1 macrophage polarization and cytokine","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEnterovirus A71 (EV-A71) infects humans by the fecal-oral route and can induce mild illness, such as fever, herpangina, and hand-foot-and-mouth disease [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This virus can also cause severe symptoms, brainstem encephalitis combined with pulmonary edema complications, which often induce death or long-term neurological sequelae, especially in young children [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Widespread and deadly EV-A71 outbreaks have been frequently reported in the Asia-Pacific region, including Taiwan, for decades [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, specific and effective antivirals are unavailable for patient treatments. Intravenous immunoglobulin (IVIG) has been used to treat infected patients with fatal symptoms in countries like Taiwan [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vaccines were developed and used in few places, such as China and Taiwan, but unavailable in other countries [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies of plasma specimens from infected patients show that EV-A71 infection increases IL-10, IL-12, and IFN-γ [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] with IL-10 detected before IFN-γ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Using a murine infection model, we further reveal that EV-A71 induces IFN-γ to protect the host from infection by reducing viral replication [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite IFN-γ plays a protective role in infection, the regulation of IFN-γ during EV-A71 infection remains to be elucidated. IFN-γ can be upregulated by following ways. It can enhance its own expression in an autocrine manner [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It 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 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. IL-12 and IFN-γ can mutually amplify each other [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, IFN-γ can be down-regulated by following ways. IL-10 can inhibit IL-12 production [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. IL-10 can promote the polarization of M2 macrophages, which mainly produce IL-10 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, IL-19 or IL-20, but not IL-24, can increase IL-10 and/or decrease IFN-γ in T cells as demonstrated by human and mouse in vitro studies [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The regulation and interaction of IFN-γ with IL-10, IL-12, IL-19, and IL-20 remain elusive.\u003c/p\u003e \u003cp\u003eIL-10, IL-19, and IL-20 belong to the IL-10 family, which contains nine members, IL- 10, IL-20 subfamily members (IL-19, IL-20, IL-22, IL-24, and IL-26), and the distantly related cytokines IL-28A, IL-28B, and IL-29, which are classified as type III IFNs, IFN-λ2, IFN-λ3, and IFN-λ1, respectively [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The IL-20 subfamily members utilize the heterodimeric receptor complex with two subunits to transduce their signals. IL-19, IL-20, and IL-24 share the receptor composed of IL-20RA (also called IL-20R1 and IL-20Rα) and IL-20RB. IL-20 and IL-24 can also signal through IL-22RA1 paired with IL-20RB. IL-26 transduces signals through IL-20RA and IL-10RB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Because IL-19, IL-20, IL-24, and IL-26 signal through IL-20RA, they are designated as \u0026lsquo;IL-20RA cytokines\u0026rsquo; in the present study. Humans express all four IL-20RA cytokines, and mice express only IL-19, IL-20, and IL-24 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. IL-20RA cytokines are shown to target T cells, macrophages, monocytes, and epithelial cells [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe interaction of IL-20RA cytokines with virus infection remains unknown, probably because these cytokines are complex in sharing receptors, functions, and activities. Mice with \u003cem\u003eIL-20RA\u003c/em\u003e knockout (\u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) were generated [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and these mice appear healthy and are available for study. Among IL-20RA cytokines in \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the signaling of IL-19 is totally blocked, but IL-20 and IL-24 can still signal through the receptor of IL-22RA1 paired with IL-20RB. Using a murine infection model, we found that IL-20RA cytokines (IL-19, IL-20, and IL-24), but not IL-10, IL-12 and IFN-γ, were constitutively expressed in mock-infected mice and that EV-A71 infection enhanced IL-20RA cytokines, especially IL-19, and induced IL-10 in wild-type (WT) mice. By comparing WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, we found that IL-20RA deficiency reduced EV-A71 lethality of mice with a decrease of IL-10 expression in T cells and increases of IL-12 and IFN-γ expression in macrophages and the level of M1 macrophages in manner hardly reported before.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell, virus, and mice\u003c/h2\u003e \u003cp\u003eThe human muscular (rhabdomyosarcoma, RD) cell line was maintained and propagated according to the instruction of American Type Culture Collection. EV-A71 strain M2, a mouse-adapted virus, was propagated in and titrated on RD cell monolayers by the plaque assay as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. All mouse experiments and care were approved by the Institutional Animal Use and Care Committee of National Cheng Kung University with the approval number of 105283 in accordance with national guidelines and regulations. WT C57BL6/J mice and C57BL6/J-derived \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] were bred and maintained under specific pathogen-free conditions in the Laboratory Animal Center of our university.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInfection of mice, tissue collection, and treatments of mice with IFN-γ antibody or liposomes\u003c/h2\u003e \u003cp\u003eTwelve- to 14-day-old WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were infected with 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e plaque forming units (PFU)/mouse of EV-A71 by intraperitoneal inoculation and monitored for survival and disease scores. The disease score was graded as follows: 0, healthy; 1, ruffled hair; 2, weakness in hind limbs; 3, paralysis in single hind limb; 4, paralysis in both hind limbs, and 5, death. Mice were anaesthetized, and mouse blood was collected. Mice were then perfused by intracardial injection of ice-cold phosphate buffered saline (PBS) containing 0.01 M EDTA and 0.2% BSA, and mouse tissues were harvested. Mouse blood was processed to obtain serum, and the serum was frozen at -80\u0026deg;C and sonicated. Mouse organs and tissues were frozen, thawed, homogenized in 1 mL PBS, frozen, thawed, sonicated, and centrifuged at 13,000 rpm for 15 minutes at 4\u0026deg;C. The resulting sera and organ/tissue supernatants were assayed for viral titers by the plaque assay on RD cell monolayers. Mice were treated with 25 \u0026micro;g of anti-IFN-γ antibody (Clone R4-6A2, Bio X Cell) or normal rat IgG (Sigma) or 50 \u0026micro;L of control liposomes (FormuMax) or clodronate liposomes (Formumax) by intraperitoneal injection one day before infection and on days 1, 3, 5, and 7 post-infection.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eTreatments of mouse leukocytes with IL-19, IL-20, or IL-24 in vitro\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions were prepared from splenocytes. CD4 T cells were subsequently isolated to 90% purity with positive selection using anti-CD4 magnetic beads (Biolegend) according to the manufacturer instruction. Mouse CD4 T cells were treated without or with IL-19, IL-20, or IL-24 (R\u0026amp;D Systems; 100 ng/mL) and harvested 8 and 24 hours after cytokine treatment. Peritoneal macrophages were harvested from mice, and more than 90% of the cells were positive for F4/80, a marker specific for mouse macrophages, by immunofluorescence staining as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Macrophages were treated without or with IL-19, IL-20, or IL-24 (100 ng/mL) for 3 days, stimulated without or with poly I:C (Invivogen; 100 ng/mL) for 24 hours, and harvested. The culture supernatants of CD4 T cells and macrophages were harvested to quantify IL-10 and IL-12, respectively by the enzyme-linked immunosorbent assay (ELISA). The remaining cells were processed to extract total RNA by the GENEzol TriRNA Pure Kit (Geneaid). The total RNA of CD4 T cells and macrophages were subjected to RT-PCR to quantify the mRNA encoding IL-10 or IL-12, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time RT-PCR\u003c/h2\u003e \u003cp\u003eAfter reverse transcription with reverse primers, the synthesized cDNA was used for quantitative real-time PCR with forward and reverse primers. PCR was performed at 95\u0026deg;C for 10 minutes followed by 40 cycles of denaturation (95\u0026deg;C, 15 seconds) and annealing (60\u0026deg;C, 1 minute) with the kit of Fast SYBR Green Master Mix (Thermo Fisher Scientific). The threshold cycle (\u003cem\u003eC\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) of each product was determined, normalized to the internal control (β-actin), and shown as Δ\u003cem\u003eC\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e. All results are shown as the ratio to β-actin calculated as 2\u003csup\u003e\u0026minus;Δ\u003cem\u003eC\u003c/em\u003eT\u003c/sup\u003e. Primers for the mRNA encoding IL-10 (forward 5\u0026rsquo;-ATA ACT GCA CCC ACT TCC CA -3\u0026rsquo; and reverse 5\u0026rsquo;-GGG CAT CAC TTC TAC CAG GT-3\u0026rsquo;), β-actin (forward 5\u0026rsquo;-AAC CCT AAG GCC AAC CGT GAA AAG ATG ACC-3\u0026rsquo; and reverse 5\u0026rsquo;-CCA GGG AGG AAG AGG ATG CGG C-3\u0026rsquo;), IL-12 p35 (forward primer 5\u0026rsquo;-AGG ACT TGA AGA TGT ACC AG-3\u0026rsquo; and reverse primer 5\u0026rsquo;-CTA TCT GTG TGA GGA GGG-3\u0026rsquo;), or IL-12 p40 (forward 5\u0026rsquo;-GGA AGC ACG GCA GCA GAA TAA-3\u0026rsquo; and reverse 5\u0026rsquo;-CTT GAG GGA GAA GTA GGA ATG-3\u0026rsquo;) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCytokine measurement by ELISA\u003c/h2\u003e \u003cp\u003eBrains harvested from mice were frozen, homogenized in 1 mL PBS containing a protease inhibitor cocktail (Sigma-Aldrich), and centrifuged. Mouse sera and brain supernatants as well as T cell or macrophage culture supernatants were subjected to commercially available ELISA (with detection limits and suppliers shown in parentheses) for assays of cytokines IL-10 (2.7 pg/mL, Biolegend), IL-12 (0.5 pg/mL, Biolegend), IL-19 (62.5 pg/mL, eBioscience), IL-20 (6.4 pg/mL, R\u0026amp;D Systems), IL-24 (18 pg/mL, Elabscience), or IFN-γ (2 pg/mL, R\u0026amp;D Systems) according to the instructions of manufacturers.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eLeukocytes were isolated from mouse spleens as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and blocked with the antibody CD16/CD32 (Clone 93; BioLegend) against Fc-receptors to prevent nonspecific binding. The resulting leukocytes were stained with the antibodies against CD3 (clone 17A2, BD Bioscience), CD11b (clone M1/70, BD Bioscience), CD11c (clone HL3, BD Bioscience), CD19 (clone 1D3, BD Bioscience), CD45 (clone 30-F11, Biolegend), CD335 (clone NKp46, BD Biosciences), CD86 (clone GL1, BD Bioscience), F4/80 (clone CI:A3-1, Bio-Rad), MHC-II (clone M5/114.15.2, BD Bioscience) or Ly6G (clone 1A8, BD Biosciences) on the cell surface, fixed, and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) before staining for intracellular IFN-γ (clone XMG1.2, Biolegend), IL-10 (clone JES5, Biolegend), or IL-12 (clone C15.6, Biolegend) and analyzed by the Cytoflex flow cytometer (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE value (error bars). For statistical comparison, levels of IL-mouse 10, IL-12, and IFN-γ, tissue viral titers, and flow cytometry results were analyzed by the Mann-Whitney U test, disease scores were analyzed by two-way ANOVA, and survival rates were analyzed by the log-rank test. In vitro data were analyzed by the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. All \u003cem\u003eP\u003c/em\u003e values are for two-tailed significance tests. A \u003cem\u003eP\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eAll mouse IL-20RA cytokines, IL-19, IL-20, and IL-24, are detected in the serum of mock-infected mice, and EV-A71 infection increases mouse serum and brain IL-19 levels\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFew 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 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIL-20RA deficiency decreases EV-A71 lethality of mice with reduced viral loads in peripheral organs\u003c/h2\u003e \u003cp\u003eIL-20RA cytokines were detected in mice, so we investigated the significance of IL-20RA cytokines on EV-A71 infection by comparing WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. After infection, increases in the death rate and disease scores were detected in WT mice, when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The viral loads in peripheral organs, heart, lung, liver, spleen, and kidney, of WT mice were higher than those of \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on 3 and/or 5 d.p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The viral loads in the intestine and central nervous system (CNS), the spinal cord, brain stem, and brain without brain stem, of WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on 3 and 5 d.p.i. were statistically insignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe 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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], 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 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PFU/mouse) by intraperitoneal inoculation. The result showed that all infected IL-19-deficient mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) and WT mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14) succumbed to death in a manner different from that found in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e 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 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice for further study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn infected\u003c/b\u003e \u003cb\u003eIL-20RA\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, the serum IFN-γ level is elevated, and IFN-γ depletion increases viral loads in peripheral organs and EV-A71 lethality\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIL-20RA cytokiens can regulate cytokine expression [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As type I IFNs (IFN-α and IFN-β), IL-1β, and IL-6 have been shown to protect mice from EV-A71 infection [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice from 1 to 5 d.p.i. (Supplementary Fig.\u0026nbsp;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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. More importantly, we showed that EV-A71 induces IFN-γ to protect mice from infection by reduceing viral replication [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We therefore monitored IFN-γ in mice using ELISA. In mock-infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, both serum and brain IFN-γ levels were below detection. EV-A71 infection induced IFN-γ in both WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with reduced serum IFN-γ levels detected in WT mice when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice from 1 to 5 d.p.i. with a significant difference found on 5 d.p.i (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The brain IFN-γ levels of infected WT mice were slightly lower than those of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on 3 and 5 d.p.i. (Supplementary Fig.\u0026nbsp;2). To determine the significance of IFN-γ in IL-20RA-deficient mice against EV-A71 infection, we depleted IFN-γ in \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice using the monoclonal antibody specific for IFN-γ. IFN-γ depletion reduced the survival rate of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with elevated viral loads in peripheral organs (spleen, lung, and liver) on 7 d.p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), showing that IFN-γ protects \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn infected\u003c/b\u003e \u003cb\u003eIL-20RA\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice, 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\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIFN-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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 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.\u0026nbsp;3). In mock-infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e cells, leukocytes expressing IFN-γ were minimal. EV-A71 infection increased the percentages of CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e cells in both WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with a reduced percentage of CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e cells detected in WT mice when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Macrophages, dendritic cells, B cells, and especially T cells can produce IFN-γ [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, a high percentage of (CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e) macrophages followed by (CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) dendritic cells, (CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e) B cells, and (CD45\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e) T cells expressed IFN-γ in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, the percentages of all these four types of IFN-γ-expressing leukocytes in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were higher than those of infected WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the importance of macrophages on IL-20RA-deficient mice against EV-A71 infection, \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were treated with the liposomes containing clodronate to deplete macrophages [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Clodronate liposome treatment efficiently depleted macrophages by ~\u0026thinsp;70%, when compared to control liposomes, in the spleen of mock-infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice one day after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e 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 \u0026gt;\u0026thinsp;50% on 5 d.p.i., when compared to control liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G). These results showed that macrophages produce IFN-γ in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and that macrophages protect \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice from EV-A71 infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIL-20RA deficiency increases the levels of serum IL-12, spleen macrophages expressing IL-12, and spleen M1 macrophages in infected mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIL-12 can enhance macrophages to express IFN-γ [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which can further amplify the production of both IL-12 and IFN-γ [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], so we monitored IL-12. In mock-infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the serum IL-12 levels were below detection. EV-A71 infection induced IL-12 in both WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with reduced serum IL-12 levels detected in WT mice when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice from 1 to 5 d.p.i. with significant differences found on 3 and 5 d.p.i (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The biggest difference in serum IL-12 levels of infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003e cells (leukocytes expressing IL-12) in splenocytes were minimal. EV-A71 infection increased the percentages of CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003e cells in both WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with a reduced percentage of CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003e cells detected in WT mice when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Dendritic cells, neutrophils, and especially macrophages as well as B cells can produce IL-12 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, high percentages of (CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e) macrophages and (CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e) B cells followed by (CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) dendritic cells and (CD45\u003csup\u003e+\u003c/sup\u003eIL-12\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) neutrophils expressed IL-12 in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, the percentages of macrophages, B cells, and neutrophils expressing IL-12 in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were significantly higher than those of infected WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIL-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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 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 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We quantified M1 macrophages in infected mice and found that the percentage of (CD45\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003eMHC-II\u003csup\u003e+\u003c/sup\u003e) M1 macrophages in the spleen of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was higher than that of infected WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIL-20RA deficiency decreases the levels of serum IL-10 and spleen T cells expressing IL-10 in infected mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs IL-10 is shown to inhibit IL-12 production in macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], we therefore monitored IL-10. In mock-infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the serum IL-10 levels were below detection. EV-A71 infection induced IL-10 in both WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with an elevated serum IL-10 level detected in WT mice when compared to \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on 1 d.p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). 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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the percentages of CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e cells (leukocytes expressing IL-10) in splenocytes were minimal. After infection, the percentage of CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e cells in splenocytes of WT mice was higher than that of \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Dendritic cells, macrophages, NK cells, and especially T cells can produce IL-10 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e) T cells followed by (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) dendritic cells, (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e) macrophages, and (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e) NK cells expressed IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, the percentages of all these four types of IL-10-expressing leukocytes in infected WT mice were higher than those of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCD4 T cells, especially Th2 cells, were the major producer of IL-10 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and our additional results showed that the percentages of CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003ecells, CD4 T cells expressing IL-10 in the splenocytes of infected WT mice were indeed higher than that of infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;4A). We further identified the CD4 T cell subsets, which produce IL-10. Our additional results showed that a high percentage of (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eIL-4\u003csup\u003e+\u003c/sup\u003e) Th2 cells, followed by (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e) Treg cells and (CD45\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR3\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e) Th1 cells expressed IL-10 in the splenocytes of infected WT mice (Supplementary Fig.\u0026nbsp;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 \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;4B-D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment 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\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur in vivo results showed that the percentage of T cells expressing IL-10 was elevated in infected WT mice when compared to infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). A mouse in vitro study showed that IL-19 treatment increases IL-10 production in CD4 T cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur in vivo results showed that abundant macrophages expressed IL-12 in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and that the percentage of macrophages expressing IL-12 was reduced in infected WT mice when compared to infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], encoded by the mouse genes of \u003cem\u003eIl12a\u003c/em\u003e and \u003cem\u003eIl12b\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). 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 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We measured \u003cem\u003eIl12a\u003c/em\u003e and \u003cem\u003eIl12b\u003c/em\u003e 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 \u003cem\u003eIl12a\u003c/em\u003e and \u003cem\u003eIl12b\u003c/em\u003e mRNA and p70 protein of IL-12 in macrophages obtained from WT or \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). IL-10 is reported to suppress IL-12 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] 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 \u003cem\u003eIl12a\u003c/em\u003e and \u003cem\u003eIl12b\u003c/em\u003e mRNA as well as protein levels of IL-12 in macrophages obtained from WT or \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Treatment with IL-19 or IL-20, but not IL-24, decreased the \u003cem\u003eIl12a\u003c/em\u003e and \u003cem\u003eIl12b\u003c/em\u003e mRNA as well as protein levels of IL-12 in macrophages obtained from WT mice, but not from \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIL-19 is detected in the plasma of healthy controls, and EV-A71 infection increases plasma IL-19 levels in patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs 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.\u0026nbsp;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.\u0026nbsp;5). The IL-20 levels of both infected patients and healthy controls were below detection.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eVery few reports investigate the interaction of moues IL-20RA cytokines with virus infection. Our in vivo study shows that all three IL-20RA cytokines, IL-19, IL-20, and IL-24, are detected in mock-infected mice and that EV-A71 infection enhances IL-20RA cytokines, especially IL-19 in mice. More importantly, IL-20RA cytokines exacerbates EV-A71 infection in mice with an increase of IL-10 expressed by T cells and reduced levels of M1 macrophages as well as IL-12 and IFN-γ expressed by macrophages. Consistently, our mouse in vitro study reveals that both IL-19 and IL-20 enhance IL-10 in T cells, but suppress IL-12 in macrophages. These studies find that all three mouse IL-20RA cytokines are constitutively expressed and suggest that both IL-19 and IL-20 could be the upstream effectors inducing IL-10 to reduce the IL-12/IFN-γ axis as well as M1 macrophages and aggravate EV-A71 infection (Supplementary Fig.\u0026nbsp;6). Our previous and present mouse studies using IFN-γ receptor-deficient mice and anti-IFN-γ antibody, respectively showed that IFN-γ protects mice from EV-A71 infection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The present study showed that macrophages produce IFN-γ to fight EV-A71 infection in mice. Overall, our present results showing the interaction of IL-20RA cytokines with EV-A71 infection are unreported and novel.\u003c/p\u003e \u003cp\u003eLeukocytes, especially myeloid cells are the primary source of IL-19 and IL-20 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], so we performed flow cytometry on the spleen to quantify leukocytes expressing IL-19 or IL-20 in infected mice on 3 and 5 d.p.i., the time points at which EV-A71 infection significantly and slightly increases serum IL-19 and IL-20 levels, respectively, by staining leukocyte markers on the cell surface and the cytokines inside the cells. CD45\u003csup\u003e+\u003c/sup\u003eIL-19\u003csup\u003e+\u003c/sup\u003e cells (leukocytes expressing IL-19) were detected in both mock-infected and infected mice with a slightly increased level of CD45\u003csup\u003e+\u003c/sup\u003eIL-19\u003csup\u003e+\u003c/sup\u003e cells found in infected mice on 3 d.p.i. when compared to mock-infected mice (Supplementary Fig.\u0026nbsp;7A). CD45\u003csup\u003e+\u003c/sup\u003eIL-20\u003csup\u003e+\u003c/sup\u003e cells (leukocytes expressing IL-20) were detected in infected mice on 3 and 5 d.p.i. and in mock-infected mice (Supplementary Fig.\u0026nbsp;7B). These results are consistent with the detection of both IL-19 and IL-20 in the serum of mock-infected and infected mice. Macrophages and dendritic cells are reported to be the cellular sources of IL-19 and IL-20, respectively [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We found that a high percentage of dendritic cells, followed by macrophages, T cells, and NK cells expressed IL-19 in infected mice on 3 d.p.i. and in mock-infected mice (Supplementary Fig.\u0026nbsp;7C). A high percentage of dendritic cells, followed by NK cells, macrophages, and T cells expressed IL-20 in infected mice on 3 and 5 d.p.i. and in mock-infected mice (Supplementary Fig.\u0026nbsp;7D). Cells other than leukocytes can express IL-19 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. EV-A71 infection is reported to induce the activation of NF-κB signaling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], which is shown to upregulate IL-19 expression in the airway epithelia of asthmatic patients [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Future studies are needed to find other cellular sources of IL-19 and IL-20 and address the signaling pathway regarding how EV-A71 infection increases IL-19.\u003c/p\u003e \u003cp\u003eOur additional study also tested a 10-fold lower dose of viral inoculum (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e PFU/mouse) and obtained similar results as those of high viral dose (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PFU/mouse) showing that IL-20RA deficiency ameliorates EV-A71 infection of mice (Supplementary Fig.\u0026nbsp;8). We used the high viral dose for study, as the differences in IL-10, IL-12, and IFN-γ levels between infected WT and \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice are readily detected. Clinical EV-A71 isolates fail to induce death in mice and need to be adapted in mice in order to induce death in mice at the age of 2 weeks old in our model. We also study herpes simplex virus 1 (HSV-1), which can induce death in 6-week-old mice by peripheral (corneal) infection and in 2- or 6-week-old mice by systemic (intraperitoneal) infection. In mice infected with HSV-1 by corneal inoculation, virus mainly spreads by the neuronal route and is detected only in the eye, trigeminal ganglia, and brain, but not in other tissues or organs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our additional study found that the survival rate of 2-week-old \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice infected with HSV-1 by intraperitoneal injection were higher than that of infected WT mice (Supplementary Fig.\u0026nbsp;9), in a manner similar to that found in EV-A71-infectied mice. However, the survival rates of 6-week-old \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and WT mice infected with HSV-1 by corneal or intraperitoneal inoculation were not statistically significant. The EV-A71 and HSV-1 results show that the important role of IL-20RA cytokines is shown in neonatal mice with systemic virus infections, and further studies are needed to address this issue in future.\u003c/p\u003e \u003cp\u003ePrevious reports of IL-20RA cytokines mostly focused on T cells to show that IL-19 or IL-20 can increase IL-10 and/or decrease IFN-γ to induce T cell polorazation toward a Th2 profile [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We previously found that T cell responses, especially the Th2 response, promote the production of antibodies, which protect mice from EV-A71 infection [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and that the Th2 cytokine IL-6 decreases EV-A71 lethality of mice [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], showing the protective role of Th2 response in EV-A71 infeciton of mice. In the present study, we focus on macrophages, as more abundant macrophages express the protective cytokine, IFN-γ, than T cells in infected \u003cem\u003eIL-20RA\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Additionally, few studies investigate the effect of IL-20RA cytokines on macrophages until recently [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], as IL-20RA is detected on macrophages [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Macrophages can differentiate into two distinct subpopulations, classical or inflammatory M1 macrophages and alternative or anti-inflammatory M2 macrophages [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. M1 macrophage differentiation can be induced by Th1 cytokines, such as IFN-γ. M1 macrophages produce cytokines, such as IL-12, and express markers, such as CD86 and MHC-II on the cell surface. Although M2 macrophages are more diverse and can be classified into four subtypes depending on the stimuli, the hallmark of all subtypes of M2 macrophages is the secretion of anti-inflammatory cytokine, IL-10 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our in vivo results showed that IL-20RA deficiency reduces the level of T cells expressing IL-10 but increases the levels of macrophages expressing IL-12 and IFN-γ and M1 macrophages in mice during infection. Consistently, our in vitro results showed that treatment of IL-19 or IL-20 enhances IL-10 in T cells, but suppresses IL-12 expression in macrophages. IL-20RA cytokines induce STAT3 activation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which is shown to enhance IL-10 [\u003cspan additionalcitationids=\"CR51 CR52 CR53\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], but inhibits IL-12 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These findings may explain how IL-20RA cytokines increases IL-10 in T cells, but suppress IL-12 in macrophages. Our previous study used the anti-IL-20RA monoclonal antibody (51D) to ameliorate liver damage (fibrosis) in mice [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Here we show that IL-20RA cytokines aggravates EV-A71 infection in mice. Our additional study tested 51D to reduce EV-A71 infection in mice, but failed. IL-20RA is detected on leukocytes, such as macrophages [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], 51D treatment may deplete protective leukocytes and result in the failure to reduce EV-A71 infection. Future studies can design small molecules targeting IL-20RA and receptor signaling to test the potential of blocking IL-20RA to reduce virus infections.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in this study are presented in this manuscript or the Supplementary Information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professors Yee-Shin Lin, Chiou-Feng Lin, and Chih-Peng Chang for helpful suggestions and the technical services provided by the \u0026ldquo;Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, National Science and Technology Council, Taiwan\u0026rdquo;. This work was supported by the funding from National Science and Technology Council, Taiwan (NSTC 113-2327-B-006-003) to SHC, SMW, and LCW. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCHH and YLH contributed equally by performing experiments data analyses, YPT performed experiments, MSC provided mice and suggestions, CCL provided human specimens and suggestions, SMW and LCW provided suggestions, and SHC conducted and obtained funding for the project. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information The online version contains supplementary material available at https://doi.org/.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Shih-Min Wang, Li-Chiu Wang, or Shun-Hua Chen. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permission information\u003c/strong\u003e is available at http://www.nature.com/\u003c/p\u003e\n\u003cp\u003ereprints\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChang LY, Huang LM, Gau SS, Wu YY, Hsia SH, Fan TY, et al. 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Hepatology. 2014;60:1003\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Enterovirus A71, IL-10, IL-12, IL-19, IL-20, IL-24, IFN-γ, IL-20RA, IL-20RA cytokines","lastPublishedDoi":"10.21203/rs.3.rs-4131398/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4131398/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnterovirus A71 (EV-A71) can cause fatality in patients with increases of cytokines, IL-10, IL-12, and IFN-γ, which are mutually regulated. IFN-γ is induced and protects the host from EV-A71 in a murine infection model. IFN-γ and IL-10 promote the polarization of M1 and M2 macrophages, which produce IL-12 and IL-10, respectively. IL-10 suppresses IL-12, which enhances itself and IFN-γ. The IL-10 family cytokines, IL-19, IL-20, and IL-24, which signal through the two-subunit receptor complex with IL-20RA as one subunit, are therefore designated as IL-20RA cytokines. Previous in vitro T cell studies showed that IL-19 or IL-20 treatment suppresses IFN-γ and that IL-19 treatment enhances IL-10. In the present study of human plasma, IL-19 was detected in healthy controls, and EV-A71 infection increased IL-19 in patients. In the serum of mice, IL-20RA cytokines, but not IL-10, IL-12, and IFN-γ, were detected in mock-infected mice, and EV-A71 infection enhanced IL-19. Compared to wild-type mice, \u003cem\u003eIL-20RA\u003c/em\u003e knockout mice were resistant to infection with reduced viral loads in peripheral organs, including the spleen. In infected mice, IL-20RA deficiency sequentially reduced IL-10, but increased IL-12 and IFN-γ, in the serum with T cells expressing IL-10 and macrophages expressing IL-12 and IFN-γ in the spleen. Notably, IL-20RA deficiency increased spleen M1 macrophages. In vitro study showed that treatment with IL-19 or IL-20, but not IL-24, increased IL-10 in CD4 T cells, but reduced IL-12 in macrophages. Our study is novel to show that IL-20RA cytokines affect virus infection, cytokines regulating macrophage polarization, and macrophage polarization.\u003c/p\u003e","manuscriptTitle":"Deficiency of IL-20 receptor subunit A decreases enterovirus A71 lethality of mice with enhanced M1 macrophage polarization and cytokine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 15:49:14","doi":"10.21203/rs.3.rs-4131398/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f742854-229f-4395-b665-89be64857cda","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29725714,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"},{"id":29725715,"name":"Biological sciences/Immunology/Cytokines/Interleukins"}],"tags":[],"updatedAt":"2024-04-11T10:06:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-28 15:49:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4131398","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4131398","identity":"rs-4131398","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}