Clinical and Immune Responses of Peripheral Chemical Sympathectomy in Enterovirus 71 Infection

The activation of the sympathetic nervous system, release of norepinephrine (NE), and adrenergic receptor signaling participate in and regulate the complicated enterovirus 71 (EV71) brainstem encephalitis (BE). The neurotoxin 6-hydroxydopamine (6-OHDA) selectively ablates sympathetic nerves and markedly depletes NE in innervated organs. Changes in the plasma levels of NE, severity score, cytokine profiles, and percentages of immunophenotype expression in 7-day-old Bltw : CD1 (ICR) mice infected with EV71, with or without 6-OHDA treatment, were compared. The survival rate (76.9%) of EV71-infected and 6-OHDA (30 μg/g)-treated mice was increased significantly. The clinical scores were decreased markedly on days 8-12 in MP4-infected and 6-OHDA-treated mice compared to those without treatment. The results showed that the plasma levels of NE, epinephrine, and dopamine were decreased on days 4–8 after 6-OHDA treatment and at most on day 8. The plasma levels of interleukin (IL)-12p70, tumor necrosis factor, IL-6, and IL-10 did not change significantly after 6-OHDA treatment. Interferon-γ levels decreased evidently on days 4, 6, and 8 after 6-OHDA treatment. The absolute events of CD3+CD4+, CD3+CD8+, and CD3+NK1.1+ cells of peripheral blood mononuclear cells were increased significantly in MP4-infected and 6-OHDA-treated mice compared to those without treatment. In splenocytes, the absolute cells of CD3−NK1.1+, CD3+NK1.1+ and CD11b+Gr-1+ cells of EV71-infected mice were increased significantly after 6-OHDA treatment. These findings suggested that 6-OHDA may be used a probe to explore clinical improvements and immune responses in the complicated EV71 infection. Taken together, peripheral chemical sympathectomy contribute to further understand the immunopathogenesis of EV71 BE with autonomic nervous system dysregulation.


Cell and Viruses
Human rhabdomyosarcoma (RD; no. 60113) cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). The cells were maintained in Dulbecco's modified Eagle's medium (#12800-017, Thermo Fisher Scientific) with heat-inactivated 10% fetal bovine serum (FBS; #04-001-1A, Biological Industries). The Virology Laboratory of the National Cheng Kung University Hospital provided an EV71 strain, Taiwan/4643/98. A mouse-adapted EV71 strain, MP4, was prepared according to a previous study (12). Viruses were propagated in RD cells in DMEM supplemented with 2% heat-inactivated FBS.
Chemical Sympathectomy 6-OHDA hydrochloride (Cat# H4381, Sigma) was dissolved in sterile saline containing 0.02% (w/v) ascorbic acid (vehicle) and was injected intraperitoneally (i.p.). Control mice received injections of an equal volume of the vehicle i.p. To verify the effectiveness of this treatment in depleting catecholamines, plasma was collected and analyzed for the presence of NE, EP, and dopamine (DP).

Mouse Experiments and Experimental Design
Seven-day-old ICR mice were infected with a 50% lethal dose (LD 50 ) of MP4 by i.p. injection. After infection, mock-and MP4infected mice were administered i.p. with 6-OHDA or an equivalent volume of the vehicle. Their body weights, clinical scores, and survival rates were recorded daily for 14 days. Clinical disease was scored as follows: 0, healthy; 1, ruffled fur and hunchback appearance; 2, wasting; 3, limb weakness; 4, limb paralysis; and 5, moribund and death. All animal experiments and protocols of this study were approved according to the rules of the Animal Protection Act of Taiwan by the Institutional Animal Care and Use Committee of National Cheng Kung University (IACUC #107065) and followed the guidelines established by the Ministry of Science and Technology of Taiwan.

NE, EP, and DP Measurements
The plasma concentrations of NE, EP, and DP were measured using a commercial immunoassay kit (#BA E-5600, Labor Diagnostika Nord GmbH & Co. KG). NE, EP and DP were extracted by using a cis-diol-specific affinity gel in controls, samples and standards, which was then acylated and derivatized enzymatically. Extracted supernatants were quantified using a competitive enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions. To ensure the quality of measurements, the controls were included in the assay.

Cytokine Detection
Cytometric bead array (CBA) kits (BD Pharmingen) were used to detect the presence of IL-12p70, tumor necrosis factor (TNF), monocyte chemoattractant protein-1 (MCP-1), IL-6, IFN-g, and IL-10 in plasma. Briefly, six populations of beads with distinct fluorescence intensities were coated with cytokine-specific capturing antibodies. The cytokine-captured beads were then mixed with 50-mL specimens and phycoerythrin-conjugated detection antibodies to form sandwich complexes. After incubation, washing, and fluorescence data acquisition, the results were generated using the BD FCAP array software (version 1.0.8).

Statistical Analyses
Data are presented as the mean ± standard error of the mean (SEM). The Mann-Whitney test and one-way analysis of variance (ANOVA) with the Kruskal-Wallis test were used to analyze the significance. Kaplan-Meier survival curves were analyzed by the log-rank (Mantel-Cox) test. Differences were considered significant at P < 0.05. All analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.).

RESULTS
Plasma Levels of NE, EP, and DP in MP4-Infected ICR Mice Without or With 6-OHDA Treatment Seven-day-old ICR mice were infected with LD 50 of MP4 by i.p. injection. Plasma levels of NE, EP, and DP were detected by ELISA. NE increased on day 2 and reached a peak on day 5 after infection ( Figure 1A). EP increased on day 2, but decreased slightly on day 4. EP reached the peak on day 5, similar to NE ( Figure 1B). DP increased on day 1 and reached a peak on day 3. The DP levels frequently changed between days 3 and 7. DP dropped on days 4 and 6, but elevated subsequently on days 5 and 7 ( Figure 1C). The NE and EP levels of the mock group had no significant peak-like virus group ( Figures 1A, B). The DP levels of the mock and virus groups had variable changes day by day. Thus, on day 4 after infection, MP4-infected mice were administered i.p. with 30 mg/g/100 mL 6-OHDA or an equivalent volume of the vehicle (0.9% saline, 0.02% ascorbate). After 1 h of 6-OHDA delivery on day 4, cardiac puncture blood collection was performed, and plasma was collected immediately. Plasma NE, EP, and DP levels were detected by ELISA and compared to their levels before the 6-OHDA injection. NE and DP decreased progressively after 6-OHDA treatment on day 4 ( Figure 2A). EP increased after 6-OHDA treatment from days 4 to 6, but decreased from days 6 to 8 ( Figure 2B). The NE, EP, and DA levels in MP4-infected and 6-OHDA-treated mice were lower than those in the MP4-infected and vehicle-treated mice ( Figure 2). Consequently, 6-OHDA reduced sympathetic overactivation on peripheral tissue and systemic compartments in MP4-infected mice.

6-OHDA Treatment in the MP4-Infected Mouse Model
Seven-day-old ICR mice were infected with LD 50 of MP4 by i.p. On day 4 after infection, mock-and MP4-infected mice were administered 6-OHDA (20, 25, or 30 mg/g in 100 mL) or an equivalent volume of vehicle i.p. The survival rate of infected       Figure 5C). The IL-6 levels peaked on day 4, but dropped drastically on day 6 ( Figure 5D). IFN-g peaked on day 4, but decreased progressively from days 6 to 14 ( Figure 5E) Figure 5F).

DISCUSSION
EP is an important mediator involved in ANS dysregulation and PE in critical patients with EV71 infection (7,12). At the same time, this naturally occurring neurotransmitter is endogenously overreleased in these patients (12). Stress-induced hormones can alter inflammatory responses to tissue injury; however, the precise mechanism by which EP influences immune and inflammatory responses is not well defined. 6-OHDA selectively destroys peripheral noradrenergic fibers of the SNS. More importantly, 6-OHDA modulates a variety of immune parameters, including cell-mediated responses (19), cytokine production (17), and lymphocyte trafficking and proliferation (20,21). This study demonstrated that catecholamine levels increased in the plasma of EV71-infected mice and then decreased after 6-OHDA administration. Also, mortality and the clinical score were decreased and improved, respectively, in MP4-infected mice after 6-OHDA administration. The roles of NE, EP, and DA in the sympathetic overactivity of severe EV71 infection in light of further correlated evidence of ANS dysfunction complications. Indeed, the levels of proinflammatory cytokines and chemokines, such as IL-6, IFN-g, IP-10, and monokine induced by gamma interferon (MIG), were elevated in the plasma or cerebrospinal fluid of patients with EV71 BE with PE (8). IFN-g plays a cardinal role in the pathogenesis of EV71 BE with PE in both systemic and CNS compartments of patients (9,11). Plasma levels of IFN-g, but not other cytokines, were decreased significantly in EV71-infected mice after 6-OHDA administration. Willemze et al. showed the effect of sympathetic denervation on mucosal innate immune responses using 6-OHDA and found that IFN-g in colon homogenates was normalized for total protein levels (21). ThyagaRajan et al. also reported reductions in sympathetic noradrenaline innervation and NE levels in the spleen of female F344 rats, accompanied by significant reductions in NK cell activity, IL-2 and IFN-g production, and T-and B-cell proliferation (22). Our findings supported the notion that 6-OHDA administration in MP4infected mice possessed anti-inflammatory property and reduced the key cytokine, IFN-g, in the pathogenesis of ANS dysregulation of EV71 infections.
In this study, the absolute cell counts of CD3 + CD4 + , CD3 + CD8 + , and CD3 − NK1.1 + in PBMCs were decreased in EV71-infected mice. However, CD3 + CD4 + , CD3 + CD8 + , and CD3 + NK1.1 + in PBMCs were increased after 6-OHDA administration. In a previous study, patients with severe complications, EV71-associated PE, also had lower circulating CD4 + and CD8 + T and NK cells in the blood (9). Liao et al. demonstrated that NE and EP treatment increased the percentages of EV71-infected cells in THP-1 and Jurkat cells, respectively. In contrast, aand b-blockers decreased the percentages of EV71-infected cells with NE or EP treatment   (25). These findings supported the notion that 6-OHDA administration in MP4-infected mice may block the effects of catecholamine, EP, and NE on PBMCs and splenocytes. The sympathetic innervation of lymphoid tissues provides a pathway for products of the nervous system to modulate immune function. Peripheral chemical sympathectomy in adapted virus infected mouse model to explore the immunopathogenesis of EV71 infection with ANS dysregulation was successfully in this study. Shih and coworkers also testified that immunodeficient neonatal mouse models can be infected with EV71 clinical isolates. Further, crossbreeding between SCARB2 transgenic and stat1 knockout mice may generate a more sensitive and user-friendly hybrid mouse model (26). Recently, a study showed that TLR7 immune modulators in EV71 infections of immunocompetent mouse 129 model, may be another possible alternative (27).
In conclusion, SNS mediators, EP and NE, dysregulate the clinical severity and cytokine profile, constrain the innate immune cell reactivity of host responses, and play critical roles in ANS dysregulation in EV71 infection.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Affidavit of Approval of Animal Use Protocol National Cheng Kung University.

AUTHOR CONTRIBUTIONS
SM-W designed research and revised the manuscript. SM-W obtained funding. YT-L, HP-T, SH-C, and SM-W implemented research. YT-L and SM-W interpreted data, analyzed statistics, and drafted the manuscript. All authors contributed to the article and approved the submitted version.