- Email: [email protected]
Interleukin-18 protects mice from Enterovirus 71 infection
Cytokine 96 (2017) 132–137
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Interleukin-18 protects mice from Enterovirus 71 infection a,b,1
b,1
b
b
b
b
Zheng Li , Hongbin Wang , Yihui Chen , Junling Niu , Qiuhong Guo , Qibin Leng , ⁎ Zhong Huangb, Zhirui Denga, Guangxun Mengb, a b
MARK
School of Life Sciences, Shanghai University, Shanghai 200444, China CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
A R T I C L E I N F O
A B S T R A C T
Keywords: IL-18 EV71 Inflammasome Cytokine
Previous study has demonstrated that the NLRP3 inflammasome is essential for protecting murine host against Enterovirus 71 (EV71) infection. However, the underlying mechanism remained unknown. Here we discovered that the pleiotropic cytokine interleukin-18 (IL-18), an NLRP3 inflammasome-dependent effector protein, exhibits a protective capability against EV71 challenge. Deficiency of IL-18 in mice exacerbated EV71 infection, which was reflected by increased viral replication, elevated production of interferons (IFN-β, IFN-γ), proinflammatory cytokines (TNF-α, IL-6) and chemokine CCL2, as well as decreased survival of experimental animals. Conversely, administration of recombinant IL-18 considerably restrained EV71 infection in IL-18 deficient mice. Thus, our results revealed a protective role for IL-18 against EV71 challenge, and indicated a novel therapeutic application for IL-18 in EV71 associated hand, foot, and mouth disease (HFMD).
1. Introduction Enterovirus 71 (EV71) is a small RNA virus from genus Enterovirus, family Picornaviridae. The virion is non-enveloped and has an icosahedral capsid, usually 27–30 nm in diameter [1]. Most EV71 infection cases are infants and children younger than 5 years old whose immune systems are not well developed. Since its first identification in 1969, EV71 has caused many epidemics all over the world, especially in the Asia-Pacific region [2]. EV71 infection is usually manifested as hand, foot, and mouth disease (HFMD), occasionally accompanied with severe complications such as encephalitis and neurogenic pulmonary edema [2]. However, currently there is still no effective treatment for EV71-infected severe cases [1]. In a previous study, we found that during EV71 infection, the NLRP3 inflammasome is activated and is protective for experimental mice against lethal EV71 challenge [3]. Another recent paper showed that EV71 3D protein activates the NLRP3 inflammasome through directly binding to NLRP3 and forming a EV71-3D-NLRP3-ASC ring-like structure [4]. The NLRP3 inflammasome is a multi-protein complex that controls the maturation and secretion of the cytokines interleukin18 (IL-18) and IL-1β [5]. Both IL-18 and IL-1β are members of interleukin-1 family [6]. These cytokines are expressed as precursors in the cytoplasm, and inflammasome-dependent activation of caspase-1 leads to the cleavage of the precursors to their mature and active forms. IL-1β is mainly expressed in monocytes and macrophages and the ⁎
1
expression is regulated by proinflammatory stimuli at transcriptional level, whereas IL-18 is expressed in broad cell types such as macrophages, dendritic cells, Kupffer cells, keratinocytes and chondrocytes [6,7]. While IL-1β is a potent proinflammatory cytokine involved in sepsis and some autoinflammatory diseases, IL-18 mainly plays immune regulatory roles in different tissues [7,8]. For example, IL-18 derived from fat tissue has inhibitory effect on obesity and metabolic syndrome [9,10]. Intestinal epithelial cell-derived IL-18 controls colonic microbial homeostasis and regulates mucin production in colon during colitis [11,12]. EV71 mainly infects hosts through stool-oral route and establishes infection in the intestinal tract, so we tested whether the intestine homeostasis regulator IL-18 was involved in EV71 infection in the current study. Of note, our results showed that IL-18 exhibited a protective role against EV71 infection in mice. Furthermore, our study implied that recombinant IL-18 can be a therapeutic candidate for controlling EV71 infection. 2. Materials and methods 2.1. Mice All mice used in this study were on the C57BL/6 genetic background and were bred in specific pathogen-free animal facility at Institut Pasteur of Shanghai. Wild type (WT) mice were obtained from
Corresponding author at: Institut Pasteur of Shanghai, Chinese Academy of Sciences, 320 Yueyang Road, Life Science Research Building, Shanghai 200031, China. E-mail address: [email protected] (G. Meng). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cyto.2017.04.002 Received 26 December 2016; Received in revised form 14 March 2017; Accepted 3 April 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 1. Mortality, morbidity and inflammation in Il18−/− mice are elevated compared to WT controls upon EV71 infection.
Shanghai Laboratory Animal Center. Il18−/− mice were purchased from the Jackson laboratory, USA. All mice care and experimental procedures complied with national guidelines and were approved by the Animal Care and Use Committee at Institut Pasteur of Shanghai.
100 ng, 150 ng, 200 ng and 200 ng of recombinant IL-18 or saline solution with 1% mouse serum on postnatal day 2, 3, 4 and 5, respectively, and EV71 infection was performed on postnatal day 3. 2.4. Histology analysis and disease incidence calculation
2.2. EV71 virus and infection
The mouse leg skeletal muscles were removed intact and fixed instantly with 4% neutral buffered paraformaldehyde on day 9 post infection. For hematoxylin and eosin (H & E) staining, the fixed tissues were embedded in paraffin, cut into 5 μm sections and stained. The sections were examined through microscopic observation, and the inflammatory tissues with distinct tissue damage and leukocyte infiltration were defined under instructions from experienced histopathologists in our pathology core facility. The disease incidence was referring the percentage of inflamed tissues from indicated mice.
The EV71 FY573 strain was cultured in RD cells and concentrated by ultracentrifuge in 120,000 g, 4 °C for 6 h. Then the virus was suspended in STE buffer (100 mM NaCl, 10 mM Tris-HCl (pH 7.4–7.5), 1 mM EDTA (pH 8.0)) and stored at −80 °C. The virus titer was determined through 50% tissue culture infective dose (TCID50). 3day-old Il18−/− and WT mice were infected with EV71 (108 TCID50) through intracranial injection. The mortality and morbidity were checked daily for two weeks and scored as reported [3]. Briefly, the disease scores of the mice were determined as follow: 0, healthy; 10, with paralysis of fore or hind limbs; 20, with paralysis of both fore and hind limbs; 30, death. The survival curve and disease scoring graphs were established by GraphPad Prism. Statistical differences in mouse survival were analyzed by Log-rank (Mantel-Cox) Test. Statistical differences in clinical scores were analyzed by two-way ANOVA. P value < 0.05 was considered significant.
2.5. Quantitative Real Time PCR analysis The leg skeletal muscles of EV71-infected mice were isolated on day 9 post infection and were homogenized in TRIzol reagent (Invitrogen) with a tissue homogenizer. 2 μg of total RNA was reversely transcribed with GoScript Reverse Transcription System (Promega) according to the manufacturer's instructions. The resulting cDNA was amplified using SYBR green Real Time PCR Master Mix (TOYOBO, QPK-201). The Real Time PCR primers were:
2.3. Mouse treatment with recombinant murine IL-18 Recombinant murine IL-18 was purchased from Medical & Biological Laboratories (MBL, B004-5). IL-18 was suspended in 0.9% saline solution with 1% mouse serum and stored at −80 °C. For treatment, Il18−/− mice were intraperitoneally (i.p.) injected with
GAPDH, forward/reverse: 5′-AGGTCGGTGTGAACGGATTTG-3′, 5′TGTAGACCATGTAGTTGAGGTCA-3′. TNF-α, forward/reverse: 5′-GGAACACGTCGTGGGATAATG-3′, 5′-
133
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 2. Proinflammatory cytokine and EV71 RNA levels are increased in Il18−/− mice.
GGCAGACTTTGGATGCTTCTT-3′. IL-6, forward/reverse: 5′-GAGGATACCACTCCCAACAGACC-3′, AAGTGCATCATCGTTGTTCATACA-3′. CCL2, forward/reverse: 5′-TTAAAAACCTGGATCGGAACCAA-3′, GCATTAGCTTCAGATTTACGGGT-3′. EV71, forward/reverse: 5′-TGAATGCGGCTAATCCCAACT-3′, AAGAAACACGGACACCCAAAG-3′. IFN-β, forward/reverse: 5′-AGCTCCAAGAAAGGACGAACA-3, GCCCTGTAGGTGAGGTTGAT-3′. IFN-γ, forward/reverse: 5′-ATGAACGCTACACACTGCATC-3′, CCATCCTTTTGCCAGTTCCTC-3′.
3. Results and discussion 5′3.1. Deficiency of IL-18 leads to more severe disease upon EV71 infection in mice
5′-
Our previous study revealed that IL-18 was secreted from innate immune cells in response to EV71 infection [3]. To assess the effect of IL-18 on EV71 infection, we infected 3-day-old Il18−/− and WT mice with 108 TCID50 of EV71 by intracranial injection and observed the clinical symptoms. The death of mice mainly occurred from day 7 to day 13 post infection. The results showed that Il18−/− mice displayed significantly higher mortality compared with WT mice (Fig. 1A). The disease scores, which were measured based on the paralysis and death, also showed that the Il18−/− mice exhibited more severe disease after EV71 infection (Fig. 1B). Furthermore, histopathological analysis showed that the Il18−/− mouse tissues exhibited severe leukocyte infiltration (indicated by arrows) and potent muscle fiber damage, while the WT mouse tissues had less leukocyte infiltration and relatively complete muscle fibers (Fig. 1C). In order to manifestly present the morbidity levels induced by EV71, we further obtained definite statistical numbers by calculating the percentage of inflamed tissues in Fig. 1C and built the disease incidence diagram. This analysis showed that tissue sections that exhibited obvious inflammation were 10 out of 11 (more than 90%) in Il18−/− mice, but were only 5 out of 13 (less than 40%) in WT mice (Fig. 1D). All these results demonstrated that Il18−/− mice developed more severe disease after EV71 infection,
5′5′5′-
All the quantitative PCR results were analyzed via GraphPad Prism and the statistical differences were analyzed by Student’s t test. P value < 0.05 was considered as significant.
2.6. ELISA analysis The leg skeletal muscles were homogenized in 600 μL ELISA tissue protein extraction buffer (HEPES, 10 mM; KCl, 10 mM; EDTA, 0.1 mM; EGTA, 0.2 mM; DTT, 1 mM; leupeptin, 10 μg/ml; PMSF, 1 mM) with a tissue homogenizer. After centrifugation, the supernatants were examined through TNF-α and IL-6 ELISA kits (eBioscience) according to the manufacturer's instructions. The ELISA data were processed via GraphPad Prism and the statistical differences were analyzed by Student’s t test. P value < 0.05 was considered as significant. 134
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 3. Mortality, morbidity and inflammation were relieved after treatment with recombinant IL-18 in EV71-infected Il18−/− mice.
Consistent with the ELISA results, TNF-α and IL-6 expression in Il18−/− mice was higher than that in WT mice (Fig. 2C and 2D). In addition, we also found the mRNA level of CCL2, an important leukocyte recruiting chemokine [13], was up-regulated in Il18−/− mice (Fig. 2E). These results demonstrated that the leukocyte infiltration and muscle fiber damage in Il18−/− mice were associated with the increased TNF-α, IL-6 and CCL2. Moreover, quantitative PCR also showed that the viral RNA of EV71 was strongly increased in the muscle tissue from Il18−/− mice (Fig. 2F). Since viral infection usually induce interferon expression [14,15], we also checked interferon β (IFN-β) and interferon γ (IFN-γ). Consistent with EV71 viral replication, the mRNA levels of IFN-β and IFN-γ were also increased in Il18−/− mice (Fig. 2G and 2H). Taken together, these results suggested that increased EV71 replication may be responsible for the increased TNF-α, IL-6, CCL2 and higher mortality in Il18−/− mice, which indicated that IL-18 played an important role in protecting the suckling mice against EV71 infection. 3-day-old WT (n = 13) and Il18−/− (n = 11) mice were infected with EV71 (108 TCID50) through intracranial injection. Muscle tissues from the hind legs on day 9 post infection were examined. (A and B) The levels of proinflammatory cytokines TNF-α and IL-6 were examined via ELISA. (C-H) The RNA levels of cytokines TNF-α, IL-6 (C and D), chemokine CCL2 (E), EV71 virus (F) and the interferons IFN-β and IFNγ (G and H) were examined through Real Time PCR. Every dot represents an individual mouse. The Statistical differences were analyzed with Student’s t test. ∗∗ p < 0.01, ∗ p < 0.05.
indicating that IL-18 played a protective role against EV71 challenge. 3-day-old Il18−/− (n = 28) and WT (n = 37) mice were infected with EV71 (108 TCID50) through intracranial injection. The paralysis and mortality were recorded daily for two weeks. (A) Kaplan–Meier curve of the WT and Il18−/− mice after EV71 infection. Log-rank (Mantel-Cox) Test showed that the P value was < 0.05. (B) Disease scores from indicated experimental mice. The scoring method: +10, with paralysis of fore or hind limbs; +20, with paralysis of both fore and hind limbs; +30, death. A two-way ANOVA analysis of WT versus Il18−/− mice showed the P value was < 0.05. (C) H & E staining of WT (n = 13) and Il18−/− (n = 11) mouse skeletal muscles on day 9 post infection. The representative sections were examined at 100× (upper panel) and 400× (lower panel) magnification. Scale bars: upper, 200 μm; lower, 50 μm. The arrows indicate the leukocytes infiltration. (D) The disease incidence diagram was created by counting the percentage of inflamed tissues carrying obvious tissue damage and leukocyte infiltration from indicated samples in (C). 3.2. Higher EV71 replication and inflammatory cytokines are detected in Il18−/− mice To elucidate the protective mechanism mediated by IL-18 against EV71 infection, we assessed the production of proinflammatory cytokines associated with HFMD. We isolated the leg skeletal muscle tissues on day 9 post infection and first quantified TNF-α and IL-6 by ELISA. The results demonstrated that both TNF-α and IL-6 were increased in Il18−/− compared with WT mice (Fig. 2A and 2B). To further confirm that the transcription of TNF-α and IL-6 were increased in Il18−/− mice after EV71 infection, leg skeletal muscles were subjected to Real Time PCR to examine the mRNA expression levels of these cytokines. 135
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 4. The expression levels of proinflammatory mediators and EV71 were decreased after treatment with recombinant IL-18 in EV71-infected Il18−/− mice.
3.3. Recombinant IL-18 prevents the severe disease in EV71-infected Il18−/ − mice
Il18−/− mice infected with EV71 were treated with recombinant IL18 (R IL-18) (n = 21) or saline (n = 21). (A) Kaplan–Meier curve of mice. Log-rank (Mantel-Cox) test showed that P value was < 0.01. (B) Disease scores showed the morbidity of the mice. The scoring method: +10, with paralysis of fore or hind limbs; +20, with paralysis of both fore and hind limbs; +30, death. A Two-way ANOVA analysis showed that P value was < 0.01. (C) H & E staining of saline treated mice (n = 11) or recombinant IL-18 treated mice (n = 15) on day 9 post infection. The representative sections were examined at 100× (upper panel) and 400× (lower panel) magnification. Scale bars: upper, 200 μm; lower, 50 μm. The arrows indicate the leukocytes infiltration. (D) The disease incidence diagram was built from counting the percentage of inflamed tissues carrying obvious damage and leukocyte infiltration from indicated samples in (C).
To confirm the effect of IL-18 in protecting mice against EV71 infection, we treated Il18−/− suckling mice with recombinant IL-18 or saline on postnatal day 2, 3, 4 and 5 through intraperitoneal injection. On postnatal day 3, these Il18−/− mice were infected with EV71. From this experiment, we found that administration of recombinant IL-18 significantly reduced the mortality induced by EV71 compared with the saline treated control mice (Fig. 3A). Interestingly, the survival of recombinant IL-18 treated Il18−/− mice was even better than the survival of WT mice (Figs. 1A and 3A), indicating a therapeutic potential of recombinant IL-18. As expected, treatment with recombinant IL-18 also relieved the clinical symptoms after EV71 infection (Fig. 3B). H & E staining of skeletal tissues from EV71-infected mice showed that treatment with recombinant IL-18 dramatically relieved inflammatory damage, which was characterized by less leukocyte infiltration (Fig. 3C). The disease incidence showed that tissue sections that exhibited obvious inflammation were 9 out of 11 (more than 80%) in saline treated mice, but were only 7 out of 15 (about 45%) in recombinant IL-18 treated animals (Fig. 3D). Thus, these results suggested that supplement of recombinant IL-18 can compensate for the diminished protective immunity due to IL-18 deficiency in Il18−/− mice.
3.4. Treatment with recombinant IL-18 reduced the expression of proinflammatory mediators and the levels of EV71 in Il18−/− mice To further determine the levels of disease associated cytokines and EV71 viral copy, we treated EV71-infected Il18−/− mice with recombinant IL-18 or saline and performed ELISA and Real Time PCR analysis of leg skeletal muscle tissues on day 9 post infection. Consistently, both protein levels and mRNA levels of TNF-α and IL-6 decreased in recombinant IL-18 treated Il18−/− mice compared with the saline 136
Cytokine 96 (2017) 132–137
Z. Li et al.
treated Il18−/− mice (Fig. 4A–4D). Similarly, the mRNA level of CCL2 also decreased in recombinant IL-18 treated Il18−/− mice (Fig. 4E). In addition, the copy of EV71 virus and the transcription of IFN-β and IFNγ were also inhibited after treatment with recombinant IL-18 (Fig. 4F–4H). These results suggested that treatment with recombinant IL-18 in the early stage of EV71 infection blocked the replication of EV71 and reduced the expression of proinflammatory cytokines and chemokine. Il18−/− mice were infected with EV71 (108 TCID50) through intracranial injection and treated with R IL-18 (n = 15) or saline (n = 13). (A and B) The protein levels of TNF-α and IL-6 were examined via ELISA. (C-H) The proinflammatory cytokines (C and D), the chemokine (E), the EV71 viral RNA level (F), and the interferons (G and H) were examined via Real Time PCR. Every dot represents an individual mouse. The statistical differences were analyzed by Student’s t test. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.
Foundation of China (31570895, 91429307, 31370892, 31600146, 81560339), National Key Basic Research Programs (2015CB554302, 2014CB541905), Shanghai Sailing Program (16YF1412400), Shanghai Natural Science Foundation (16ZR1439900), China Postdoctoral Science Foundation (2016M601659), International Partnership Program of the Chinese Academy of Sciences (153831KYSB20160009) as well as the TOTAL foundation for HFMD. We wish to thank the staff of pathology core facility at Institute Pasteur of Shanghai for technical supports. References [1] E.J. Bek, P.C. McMinn, Recent advances in research on human enterovirus 71, Future Virol. 5 (4) (2010) 453–468. [2] M.H. Ooi, et al., Clinical features, diagnosis, and management of enterovirus 71, Lancet Neurol. 9 (11) (2010) 1097–1105. [3] H. Wang, et al., Reciprocal regulation between Enterovirus 71 and the NLRP3 inflammasome, Cell Rep. 12 (1) (2015) 42–48. [4] W. Wang, et al., EV71 3D protein binds with NLRP3 and enhances the assembly of inflammasome complex, PLoS Pathog. 13 (1) (2017) e1006123. [5] M. Chen, et al., Regulation of adaptive immunity by the NLRP3 inflammasome, Int. Immunopharmacol. 11 (5) (2011) 549–554. [6] J.E. Sims, D.E. Smith, The IL-1 family: regulators of immunity, Nat. Rev. Immunol. 10 (2) (2010) 89–102. [7] C.A. Dinarello, et al., Interleukin-18 and IL-18 binding protein, Front. Immunol. 4 (2013) 289. [8] W.P. Arend, G. Palmer, C. Gabay, IL-1, IL-18, and IL-33 families of cytokines, Immunol. Rev. 223 (2008) 20–38. [9] J.N. Fain, D.S. Tichansky, A.K. Madan, Most of the interleukin 1 receptor antagonist, cathepsin S, macrophage migration inhibitory factor, nerve growth factor, and interleukin 18 release by explants of human adipose tissue is by the nonfat cells, not by the adipocytes, Metabolism 55 (8) (2006) 1113–1121. [10] A.J. Murphy, et al., IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome, Cell Metab. 23 (1) (2016) 155–164. [11] E. Elinav, et al., NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis, Cell 145 (5) (2011) 745–757. [12] R. Nowarski, et al., Epithelial IL-18 equilibrium controls barrier function in colitis, Cell 163 (6) (2015) 1444–1456. [13] S.L. Deshmane, et al., Monocyte chemoattractant protein-1 (MCP-1): an overview, J. Interferon Cytokine Res. 29 (6) (2009) 313–326. [14] J. Wu, Z.J. Chen, Innate immune sensing and signaling of cytosolic nucleic acids, Annu. Rev. Immunol. 32 (2014) 461–488. [15] R.E. Randall, S. Goodbourn, Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures, J. Gen. Virol. 89 (Pt 1) (2008) 1–47.
4. Conclusion In the present study, we found that the inflammasome-dependent cytokine IL-18 was required for controlling EV71 infection in mice. Il18−/− mice developed much more severe disease, which was characterized by increased levels of EV71, proinflammatory mediators and death. Administration of recombinant IL-18 inhibited EV71 infection, relieved inflammation and increased the survival of experimental animals. Thus, our study suggests that recombinant IL-18 could be a new therapeutic agent for the treatment of EV71 infection. Author contributions G.M. designed the project; Z.L., H.W., Y.C., J.N. and Q.G. performed the experiments; Q.L., Z.H. and Z.D. contributed reagents; Z.L., H.W. and G.M. wrote the manuscript. Competing interests The authors have no financial conflicts of interest. Acknowledgements This work was supported by grants from Natural Science
137
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Interleukin-18 protects mice from Enterovirus 71 infection a,b,1
b,1
b
b
b
b
Zheng Li , Hongbin Wang , Yihui Chen , Junling Niu , Qiuhong Guo , Qibin Leng , ⁎ Zhong Huangb, Zhirui Denga, Guangxun Mengb, a b
MARK
School of Life Sciences, Shanghai University, Shanghai 200444, China CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
A R T I C L E I N F O
A B S T R A C T
Keywords: IL-18 EV71 Inflammasome Cytokine
Previous study has demonstrated that the NLRP3 inflammasome is essential for protecting murine host against Enterovirus 71 (EV71) infection. However, the underlying mechanism remained unknown. Here we discovered that the pleiotropic cytokine interleukin-18 (IL-18), an NLRP3 inflammasome-dependent effector protein, exhibits a protective capability against EV71 challenge. Deficiency of IL-18 in mice exacerbated EV71 infection, which was reflected by increased viral replication, elevated production of interferons (IFN-β, IFN-γ), proinflammatory cytokines (TNF-α, IL-6) and chemokine CCL2, as well as decreased survival of experimental animals. Conversely, administration of recombinant IL-18 considerably restrained EV71 infection in IL-18 deficient mice. Thus, our results revealed a protective role for IL-18 against EV71 challenge, and indicated a novel therapeutic application for IL-18 in EV71 associated hand, foot, and mouth disease (HFMD).
1. Introduction Enterovirus 71 (EV71) is a small RNA virus from genus Enterovirus, family Picornaviridae. The virion is non-enveloped and has an icosahedral capsid, usually 27–30 nm in diameter [1]. Most EV71 infection cases are infants and children younger than 5 years old whose immune systems are not well developed. Since its first identification in 1969, EV71 has caused many epidemics all over the world, especially in the Asia-Pacific region [2]. EV71 infection is usually manifested as hand, foot, and mouth disease (HFMD), occasionally accompanied with severe complications such as encephalitis and neurogenic pulmonary edema [2]. However, currently there is still no effective treatment for EV71-infected severe cases [1]. In a previous study, we found that during EV71 infection, the NLRP3 inflammasome is activated and is protective for experimental mice against lethal EV71 challenge [3]. Another recent paper showed that EV71 3D protein activates the NLRP3 inflammasome through directly binding to NLRP3 and forming a EV71-3D-NLRP3-ASC ring-like structure [4]. The NLRP3 inflammasome is a multi-protein complex that controls the maturation and secretion of the cytokines interleukin18 (IL-18) and IL-1β [5]. Both IL-18 and IL-1β are members of interleukin-1 family [6]. These cytokines are expressed as precursors in the cytoplasm, and inflammasome-dependent activation of caspase-1 leads to the cleavage of the precursors to their mature and active forms. IL-1β is mainly expressed in monocytes and macrophages and the ⁎
1
expression is regulated by proinflammatory stimuli at transcriptional level, whereas IL-18 is expressed in broad cell types such as macrophages, dendritic cells, Kupffer cells, keratinocytes and chondrocytes [6,7]. While IL-1β is a potent proinflammatory cytokine involved in sepsis and some autoinflammatory diseases, IL-18 mainly plays immune regulatory roles in different tissues [7,8]. For example, IL-18 derived from fat tissue has inhibitory effect on obesity and metabolic syndrome [9,10]. Intestinal epithelial cell-derived IL-18 controls colonic microbial homeostasis and regulates mucin production in colon during colitis [11,12]. EV71 mainly infects hosts through stool-oral route and establishes infection in the intestinal tract, so we tested whether the intestine homeostasis regulator IL-18 was involved in EV71 infection in the current study. Of note, our results showed that IL-18 exhibited a protective role against EV71 infection in mice. Furthermore, our study implied that recombinant IL-18 can be a therapeutic candidate for controlling EV71 infection. 2. Materials and methods 2.1. Mice All mice used in this study were on the C57BL/6 genetic background and were bred in specific pathogen-free animal facility at Institut Pasteur of Shanghai. Wild type (WT) mice were obtained from
Corresponding author at: Institut Pasteur of Shanghai, Chinese Academy of Sciences, 320 Yueyang Road, Life Science Research Building, Shanghai 200031, China. E-mail address: [email protected] (G. Meng). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cyto.2017.04.002 Received 26 December 2016; Received in revised form 14 March 2017; Accepted 3 April 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 1. Mortality, morbidity and inflammation in Il18−/− mice are elevated compared to WT controls upon EV71 infection.
Shanghai Laboratory Animal Center. Il18−/− mice were purchased from the Jackson laboratory, USA. All mice care and experimental procedures complied with national guidelines and were approved by the Animal Care and Use Committee at Institut Pasteur of Shanghai.
100 ng, 150 ng, 200 ng and 200 ng of recombinant IL-18 or saline solution with 1% mouse serum on postnatal day 2, 3, 4 and 5, respectively, and EV71 infection was performed on postnatal day 3. 2.4. Histology analysis and disease incidence calculation
2.2. EV71 virus and infection
The mouse leg skeletal muscles were removed intact and fixed instantly with 4% neutral buffered paraformaldehyde on day 9 post infection. For hematoxylin and eosin (H & E) staining, the fixed tissues were embedded in paraffin, cut into 5 μm sections and stained. The sections were examined through microscopic observation, and the inflammatory tissues with distinct tissue damage and leukocyte infiltration were defined under instructions from experienced histopathologists in our pathology core facility. The disease incidence was referring the percentage of inflamed tissues from indicated mice.
The EV71 FY573 strain was cultured in RD cells and concentrated by ultracentrifuge in 120,000 g, 4 °C for 6 h. Then the virus was suspended in STE buffer (100 mM NaCl, 10 mM Tris-HCl (pH 7.4–7.5), 1 mM EDTA (pH 8.0)) and stored at −80 °C. The virus titer was determined through 50% tissue culture infective dose (TCID50). 3day-old Il18−/− and WT mice were infected with EV71 (108 TCID50) through intracranial injection. The mortality and morbidity were checked daily for two weeks and scored as reported [3]. Briefly, the disease scores of the mice were determined as follow: 0, healthy; 10, with paralysis of fore or hind limbs; 20, with paralysis of both fore and hind limbs; 30, death. The survival curve and disease scoring graphs were established by GraphPad Prism. Statistical differences in mouse survival were analyzed by Log-rank (Mantel-Cox) Test. Statistical differences in clinical scores were analyzed by two-way ANOVA. P value < 0.05 was considered significant.
2.5. Quantitative Real Time PCR analysis The leg skeletal muscles of EV71-infected mice were isolated on day 9 post infection and were homogenized in TRIzol reagent (Invitrogen) with a tissue homogenizer. 2 μg of total RNA was reversely transcribed with GoScript Reverse Transcription System (Promega) according to the manufacturer's instructions. The resulting cDNA was amplified using SYBR green Real Time PCR Master Mix (TOYOBO, QPK-201). The Real Time PCR primers were:
2.3. Mouse treatment with recombinant murine IL-18 Recombinant murine IL-18 was purchased from Medical & Biological Laboratories (MBL, B004-5). IL-18 was suspended in 0.9% saline solution with 1% mouse serum and stored at −80 °C. For treatment, Il18−/− mice were intraperitoneally (i.p.) injected with
GAPDH, forward/reverse: 5′-AGGTCGGTGTGAACGGATTTG-3′, 5′TGTAGACCATGTAGTTGAGGTCA-3′. TNF-α, forward/reverse: 5′-GGAACACGTCGTGGGATAATG-3′, 5′-
133
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 2. Proinflammatory cytokine and EV71 RNA levels are increased in Il18−/− mice.
GGCAGACTTTGGATGCTTCTT-3′. IL-6, forward/reverse: 5′-GAGGATACCACTCCCAACAGACC-3′, AAGTGCATCATCGTTGTTCATACA-3′. CCL2, forward/reverse: 5′-TTAAAAACCTGGATCGGAACCAA-3′, GCATTAGCTTCAGATTTACGGGT-3′. EV71, forward/reverse: 5′-TGAATGCGGCTAATCCCAACT-3′, AAGAAACACGGACACCCAAAG-3′. IFN-β, forward/reverse: 5′-AGCTCCAAGAAAGGACGAACA-3, GCCCTGTAGGTGAGGTTGAT-3′. IFN-γ, forward/reverse: 5′-ATGAACGCTACACACTGCATC-3′, CCATCCTTTTGCCAGTTCCTC-3′.
3. Results and discussion 5′3.1. Deficiency of IL-18 leads to more severe disease upon EV71 infection in mice
5′-
Our previous study revealed that IL-18 was secreted from innate immune cells in response to EV71 infection [3]. To assess the effect of IL-18 on EV71 infection, we infected 3-day-old Il18−/− and WT mice with 108 TCID50 of EV71 by intracranial injection and observed the clinical symptoms. The death of mice mainly occurred from day 7 to day 13 post infection. The results showed that Il18−/− mice displayed significantly higher mortality compared with WT mice (Fig. 1A). The disease scores, which were measured based on the paralysis and death, also showed that the Il18−/− mice exhibited more severe disease after EV71 infection (Fig. 1B). Furthermore, histopathological analysis showed that the Il18−/− mouse tissues exhibited severe leukocyte infiltration (indicated by arrows) and potent muscle fiber damage, while the WT mouse tissues had less leukocyte infiltration and relatively complete muscle fibers (Fig. 1C). In order to manifestly present the morbidity levels induced by EV71, we further obtained definite statistical numbers by calculating the percentage of inflamed tissues in Fig. 1C and built the disease incidence diagram. This analysis showed that tissue sections that exhibited obvious inflammation were 10 out of 11 (more than 90%) in Il18−/− mice, but were only 5 out of 13 (less than 40%) in WT mice (Fig. 1D). All these results demonstrated that Il18−/− mice developed more severe disease after EV71 infection,
5′5′5′-
All the quantitative PCR results were analyzed via GraphPad Prism and the statistical differences were analyzed by Student’s t test. P value < 0.05 was considered as significant.
2.6. ELISA analysis The leg skeletal muscles were homogenized in 600 μL ELISA tissue protein extraction buffer (HEPES, 10 mM; KCl, 10 mM; EDTA, 0.1 mM; EGTA, 0.2 mM; DTT, 1 mM; leupeptin, 10 μg/ml; PMSF, 1 mM) with a tissue homogenizer. After centrifugation, the supernatants were examined through TNF-α and IL-6 ELISA kits (eBioscience) according to the manufacturer's instructions. The ELISA data were processed via GraphPad Prism and the statistical differences were analyzed by Student’s t test. P value < 0.05 was considered as significant. 134
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 3. Mortality, morbidity and inflammation were relieved after treatment with recombinant IL-18 in EV71-infected Il18−/− mice.
Consistent with the ELISA results, TNF-α and IL-6 expression in Il18−/− mice was higher than that in WT mice (Fig. 2C and 2D). In addition, we also found the mRNA level of CCL2, an important leukocyte recruiting chemokine [13], was up-regulated in Il18−/− mice (Fig. 2E). These results demonstrated that the leukocyte infiltration and muscle fiber damage in Il18−/− mice were associated with the increased TNF-α, IL-6 and CCL2. Moreover, quantitative PCR also showed that the viral RNA of EV71 was strongly increased in the muscle tissue from Il18−/− mice (Fig. 2F). Since viral infection usually induce interferon expression [14,15], we also checked interferon β (IFN-β) and interferon γ (IFN-γ). Consistent with EV71 viral replication, the mRNA levels of IFN-β and IFN-γ were also increased in Il18−/− mice (Fig. 2G and 2H). Taken together, these results suggested that increased EV71 replication may be responsible for the increased TNF-α, IL-6, CCL2 and higher mortality in Il18−/− mice, which indicated that IL-18 played an important role in protecting the suckling mice against EV71 infection. 3-day-old WT (n = 13) and Il18−/− (n = 11) mice were infected with EV71 (108 TCID50) through intracranial injection. Muscle tissues from the hind legs on day 9 post infection were examined. (A and B) The levels of proinflammatory cytokines TNF-α and IL-6 were examined via ELISA. (C-H) The RNA levels of cytokines TNF-α, IL-6 (C and D), chemokine CCL2 (E), EV71 virus (F) and the interferons IFN-β and IFNγ (G and H) were examined through Real Time PCR. Every dot represents an individual mouse. The Statistical differences were analyzed with Student’s t test. ∗∗ p < 0.01, ∗ p < 0.05.
indicating that IL-18 played a protective role against EV71 challenge. 3-day-old Il18−/− (n = 28) and WT (n = 37) mice were infected with EV71 (108 TCID50) through intracranial injection. The paralysis and mortality were recorded daily for two weeks. (A) Kaplan–Meier curve of the WT and Il18−/− mice after EV71 infection. Log-rank (Mantel-Cox) Test showed that the P value was < 0.05. (B) Disease scores from indicated experimental mice. The scoring method: +10, with paralysis of fore or hind limbs; +20, with paralysis of both fore and hind limbs; +30, death. A two-way ANOVA analysis of WT versus Il18−/− mice showed the P value was < 0.05. (C) H & E staining of WT (n = 13) and Il18−/− (n = 11) mouse skeletal muscles on day 9 post infection. The representative sections were examined at 100× (upper panel) and 400× (lower panel) magnification. Scale bars: upper, 200 μm; lower, 50 μm. The arrows indicate the leukocytes infiltration. (D) The disease incidence diagram was created by counting the percentage of inflamed tissues carrying obvious tissue damage and leukocyte infiltration from indicated samples in (C). 3.2. Higher EV71 replication and inflammatory cytokines are detected in Il18−/− mice To elucidate the protective mechanism mediated by IL-18 against EV71 infection, we assessed the production of proinflammatory cytokines associated with HFMD. We isolated the leg skeletal muscle tissues on day 9 post infection and first quantified TNF-α and IL-6 by ELISA. The results demonstrated that both TNF-α and IL-6 were increased in Il18−/− compared with WT mice (Fig. 2A and 2B). To further confirm that the transcription of TNF-α and IL-6 were increased in Il18−/− mice after EV71 infection, leg skeletal muscles were subjected to Real Time PCR to examine the mRNA expression levels of these cytokines. 135
Cytokine 96 (2017) 132–137
Z. Li et al.
Fig. 4. The expression levels of proinflammatory mediators and EV71 were decreased after treatment with recombinant IL-18 in EV71-infected Il18−/− mice.
3.3. Recombinant IL-18 prevents the severe disease in EV71-infected Il18−/ − mice
Il18−/− mice infected with EV71 were treated with recombinant IL18 (R IL-18) (n = 21) or saline (n = 21). (A) Kaplan–Meier curve of mice. Log-rank (Mantel-Cox) test showed that P value was < 0.01. (B) Disease scores showed the morbidity of the mice. The scoring method: +10, with paralysis of fore or hind limbs; +20, with paralysis of both fore and hind limbs; +30, death. A Two-way ANOVA analysis showed that P value was < 0.01. (C) H & E staining of saline treated mice (n = 11) or recombinant IL-18 treated mice (n = 15) on day 9 post infection. The representative sections were examined at 100× (upper panel) and 400× (lower panel) magnification. Scale bars: upper, 200 μm; lower, 50 μm. The arrows indicate the leukocytes infiltration. (D) The disease incidence diagram was built from counting the percentage of inflamed tissues carrying obvious damage and leukocyte infiltration from indicated samples in (C).
To confirm the effect of IL-18 in protecting mice against EV71 infection, we treated Il18−/− suckling mice with recombinant IL-18 or saline on postnatal day 2, 3, 4 and 5 through intraperitoneal injection. On postnatal day 3, these Il18−/− mice were infected with EV71. From this experiment, we found that administration of recombinant IL-18 significantly reduced the mortality induced by EV71 compared with the saline treated control mice (Fig. 3A). Interestingly, the survival of recombinant IL-18 treated Il18−/− mice was even better than the survival of WT mice (Figs. 1A and 3A), indicating a therapeutic potential of recombinant IL-18. As expected, treatment with recombinant IL-18 also relieved the clinical symptoms after EV71 infection (Fig. 3B). H & E staining of skeletal tissues from EV71-infected mice showed that treatment with recombinant IL-18 dramatically relieved inflammatory damage, which was characterized by less leukocyte infiltration (Fig. 3C). The disease incidence showed that tissue sections that exhibited obvious inflammation were 9 out of 11 (more than 80%) in saline treated mice, but were only 7 out of 15 (about 45%) in recombinant IL-18 treated animals (Fig. 3D). Thus, these results suggested that supplement of recombinant IL-18 can compensate for the diminished protective immunity due to IL-18 deficiency in Il18−/− mice.
3.4. Treatment with recombinant IL-18 reduced the expression of proinflammatory mediators and the levels of EV71 in Il18−/− mice To further determine the levels of disease associated cytokines and EV71 viral copy, we treated EV71-infected Il18−/− mice with recombinant IL-18 or saline and performed ELISA and Real Time PCR analysis of leg skeletal muscle tissues on day 9 post infection. Consistently, both protein levels and mRNA levels of TNF-α and IL-6 decreased in recombinant IL-18 treated Il18−/− mice compared with the saline 136
Cytokine 96 (2017) 132–137
Z. Li et al.
treated Il18−/− mice (Fig. 4A–4D). Similarly, the mRNA level of CCL2 also decreased in recombinant IL-18 treated Il18−/− mice (Fig. 4E). In addition, the copy of EV71 virus and the transcription of IFN-β and IFNγ were also inhibited after treatment with recombinant IL-18 (Fig. 4F–4H). These results suggested that treatment with recombinant IL-18 in the early stage of EV71 infection blocked the replication of EV71 and reduced the expression of proinflammatory cytokines and chemokine. Il18−/− mice were infected with EV71 (108 TCID50) through intracranial injection and treated with R IL-18 (n = 15) or saline (n = 13). (A and B) The protein levels of TNF-α and IL-6 were examined via ELISA. (C-H) The proinflammatory cytokines (C and D), the chemokine (E), the EV71 viral RNA level (F), and the interferons (G and H) were examined via Real Time PCR. Every dot represents an individual mouse. The statistical differences were analyzed by Student’s t test. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.
Foundation of China (31570895, 91429307, 31370892, 31600146, 81560339), National Key Basic Research Programs (2015CB554302, 2014CB541905), Shanghai Sailing Program (16YF1412400), Shanghai Natural Science Foundation (16ZR1439900), China Postdoctoral Science Foundation (2016M601659), International Partnership Program of the Chinese Academy of Sciences (153831KYSB20160009) as well as the TOTAL foundation for HFMD. We wish to thank the staff of pathology core facility at Institute Pasteur of Shanghai for technical supports. References [1] E.J. Bek, P.C. McMinn, Recent advances in research on human enterovirus 71, Future Virol. 5 (4) (2010) 453–468. [2] M.H. Ooi, et al., Clinical features, diagnosis, and management of enterovirus 71, Lancet Neurol. 9 (11) (2010) 1097–1105. [3] H. Wang, et al., Reciprocal regulation between Enterovirus 71 and the NLRP3 inflammasome, Cell Rep. 12 (1) (2015) 42–48. [4] W. Wang, et al., EV71 3D protein binds with NLRP3 and enhances the assembly of inflammasome complex, PLoS Pathog. 13 (1) (2017) e1006123. [5] M. Chen, et al., Regulation of adaptive immunity by the NLRP3 inflammasome, Int. Immunopharmacol. 11 (5) (2011) 549–554. [6] J.E. Sims, D.E. Smith, The IL-1 family: regulators of immunity, Nat. Rev. Immunol. 10 (2) (2010) 89–102. [7] C.A. Dinarello, et al., Interleukin-18 and IL-18 binding protein, Front. Immunol. 4 (2013) 289. [8] W.P. Arend, G. Palmer, C. Gabay, IL-1, IL-18, and IL-33 families of cytokines, Immunol. Rev. 223 (2008) 20–38. [9] J.N. Fain, D.S. Tichansky, A.K. Madan, Most of the interleukin 1 receptor antagonist, cathepsin S, macrophage migration inhibitory factor, nerve growth factor, and interleukin 18 release by explants of human adipose tissue is by the nonfat cells, not by the adipocytes, Metabolism 55 (8) (2006) 1113–1121. [10] A.J. Murphy, et al., IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome, Cell Metab. 23 (1) (2016) 155–164. [11] E. Elinav, et al., NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis, Cell 145 (5) (2011) 745–757. [12] R. Nowarski, et al., Epithelial IL-18 equilibrium controls barrier function in colitis, Cell 163 (6) (2015) 1444–1456. [13] S.L. Deshmane, et al., Monocyte chemoattractant protein-1 (MCP-1): an overview, J. Interferon Cytokine Res. 29 (6) (2009) 313–326. [14] J. Wu, Z.J. Chen, Innate immune sensing and signaling of cytosolic nucleic acids, Annu. Rev. Immunol. 32 (2014) 461–488. [15] R.E. Randall, S. Goodbourn, Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures, J. Gen. Virol. 89 (Pt 1) (2008) 1–47.
4. Conclusion In the present study, we found that the inflammasome-dependent cytokine IL-18 was required for controlling EV71 infection in mice. Il18−/− mice developed much more severe disease, which was characterized by increased levels of EV71, proinflammatory mediators and death. Administration of recombinant IL-18 inhibited EV71 infection, relieved inflammation and increased the survival of experimental animals. Thus, our study suggests that recombinant IL-18 could be a new therapeutic agent for the treatment of EV71 infection. Author contributions G.M. designed the project; Z.L., H.W., Y.C., J.N. and Q.G. performed the experiments; Q.L., Z.H. and Z.D. contributed reagents; Z.L., H.W. and G.M. wrote the manuscript. Competing interests The authors have no financial conflicts of interest. Acknowledgements This work was supported by grants from Natural Science
137