Exogenous interleukin-6, interleukin-13, and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice
© Huang et al; licensee BioMed Central Ltd. 2011
Received: 11 August 2011
Accepted: 6 November 2011
Published: 1 December 2011
Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident.
To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection.
After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only.
Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.
Keywordsenterovirus 71 pulmonary edema proinflammatory cytokine mouse model
Enterovirus 71 (EV71), a highly neurotropic, positive-sense single-stranded RNA virus, belongs to the Enterovirus genus of Picornaviridae family. In general, EV71 infections are mild, such as hand, foot, and mouth disease and herpangina in young children. However, central nervous system (CNS) infections with life-threatening pulmonary and cardiac complications have occurred . EV71 has been regarded as the most important neurotropic enterovirus since the effective control of the poliovirus (PV). More than a dozen severe EV71 outbreaks have been reported worldwide since it was first recognized in California in 1969 .
Pulmonary edema (PE) and subsequent rapid onset cardiopulmonary failure are hallmarks of EV71 induced mortality . EV71-induced PE has been considered neurogenic in origin, as it has been observed to be associated with brainstem encephalitis without signs of pneumonia and myocarditis [4, 5]. Most EV71 patients with PE presented symptoms of autonomic nervous system dysregulation and sympathetic excitement, suggesting hemodynamic alterations may underlie the disease mechanism of EV71-induced PE. Elevated levels of plasma catecholamine and epinephrine, coagulative myocytolysis, and myocardial hemorrhage were noted in EV71 patients with brainstem symptoms . Researchers have speculated that systemic and local proinflammatory responses resulting from EV71-related inflammation and brain damage are involved in the development of PE in EV71 patients . However, in one previous study, only 1 out of the 5 PE patients showed systolic hypertension and elevated pulmonary artery pressure .
Our previous studies showed that a mouse-adapted EV71 strain, EV71/MP4, could experimentally infect laboratory mice via oral (p.o.), intramuscular, and intracranial (i.c.) inoculation routes, resulting in CNS infection and death . Clinically, the animals developed neurological disease and pulmonary dysfunction. Viral antigens were concentrated in the cerebellar peduncle of the brainstem beneath the cerebellum and the anterior horn regions of the spinal cord, but not in the heart or lungs . The CNS exhibited obvious pathology. However, while the lungs exhibited emphysema, PE was not observed. Our results suggested that CNS infection alone was not sufficient for the development of PE in EV71-infected mice. In this study, we demonstrated that systemic administration of proinflammatory cytokines IL-6, IL-13, and IFN-γ could exacerbate pulmonary abnormalities and induce mild PE in EV71-infected mice.
Cells and viruses
Rhabdomyosarcoma (RD) cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin and streptomycin. Vero cells (ATCC) were maintained in DMEM containing 5% newborn calf serum, penicillin and streptomycin. Mouse-adapted EV71 strain MP4  was propagated in RD cells. Working stocks contained 4 × 107 PFU/ml. MP4 strains were tested by monoclonal antibodies for EV71 (mAb3324), EV71/coxsackievirus A16 (mAb3323), coxsackievirus A24 (mAb3302), coxsackievirus B3 (mAb3306), echovirus 9 (mAb3313), PV type 1 (mAb3331), PV type 2 (mAb3332), and PV type 3 (mAb3335) (all from Chemicon, Temecula, CA) using indirect immunofluorescence staining of infected RD cell cultures, and RT-PCR using specific primers for EV71. HSV-1/KOS, a clinical isolate strain of human simplex virus-1 (HSV-1) (courtesy of Dr. Shun-Hua Chen, National Cheng Kung University, College of Medicine, Tainan, Taiwan) was grown in Vero cells.
Specific-pathogen-free, 7-day-old ICR mice (Laboratory Animal Center, National Cheng Kung University, College of Medicine) were i.c. injected with 10 μl of EV71/MP4 (4 × 105 PUF/mouse) or HSV-1/KOS (2 × 102 PFU/mouse) through the fontanelles using a 26-gauge needle. Control mice were given culture medium. To systemically increase proinflammatory cytokine levels, mice were intraperitoneally (i.p.) injected at three days post-inoculation with recombinant mouse IL-6, IL-13, and IFN-γ (0.05, 0.15, and 0.8 μg/mouse, respectively) (R&D, Minneapolis, MN) 2 or 3 times within 48 h. Control mice were given bovine serum albumin (BSA). Mice were observed twice daily for clinical signs and mortality. Lung function and body weight monitored daily for one week. Lung weight/body weight analysis, histopathology, and immunohistochemistry were performed after these experiments. For i.c. microinjection, EV71/MP4 was bilaterally microinjected into the nucleus tractus solitarii (NTS; AP-7.72 mm, LM+/-0.6 mm, DV-5.5 mm) of 8-week-old ICR mice in a volume of 0.2 μl (3 × 107 PFU/ml) over 10 min through a 27-gauge stainless steel cannula connected to a 10-μl Hamilton syringe (Hamilton, Reno, NV) driven by an injection pump (Legato 200 series, KD Scientific, MA). Mice were observed twice daily for clinical signs and mortality for 5 days. Clinical disease was scored as follows: 0, healthy; 1, ruffled fur and hunchbacked appearance; 2, wasting; 3, limb weakness; 4, limb paralysis; 5, moribund and death. The Institutional Animal Care and Use Committee approved all animal protocols.
Unrestrained whole-body plethysmograph
Pulmonary functions of EV71- and HSV-1-infected mice were measured in unrestrained animals using a whole-body plethysmograph (Buxco, Troy, NY). Readings were collected for 3 min after 3 min of resting in the chamber.
Cytokine and noradrenaline levels
After anesthetization with pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, IL), blood was collected after axilla dissection. The levels of IL-6, IL-10, IL-13, and IFN-γ in serum and noradrenaline in plasma of EV71-infected mice were determined using ELISA kits (ELISA DuoSet, R&D, Minneapolis, MN) and a noradrenaline EIA kit (LDN, Nordhorn, Germany), respectively according to the manufacturer's instructions. The detection limits for IL-6, IL-10, IL-13, IFN-γ, and noradrenaline were 15.6, 31.25, 62.5, 31.25, and 0.027 pg/ml, respectively.
Measurement of wet lung weight
Changes in vascular permeability in lungs were determined by measuring the increment of wet lung weight. Individual lung lobes were removed, blot-dried on gauze, and weighed using a balance with accuracy to 0.0001 gram. Data were expressed as the ratio of total wet lung weight to body weight.
Animals were perfused with isotonic saline containing EDTA. The whole brains and lungs were removed and weighed. Half of the tissues were immersion fixed in 10% buffered formalin for 48 h, bisected, embedded in paraffin, and stained with hematoyxlin and eosin (H & E) or Nissl stain. The remaining tissue samples were frozen in a liquid nitrogen-cold hexane bath in 100% OCT compound (Miles, Elkhart, IN). All samples were stored at -70° C until assayed.
The presence of EV71 virions in cryosections of frozen tissues (8 to 10 μm; Leica CM1800, Wetzlar, Germany) was visualized by a previously described method . In addition, neuronal cells were identified by staining with monoclonal anti-NeuN antibody (Sigma-Aldrich, St. Louis, MO) as previously described . Positively stained cells were enumerated by counting at least 10 fields and expressed as the average number per field.
The clinical scores, lung weight/body weight ratio, and numbers of EV71-positive and NeuN-positive cells were analyzed using either the nonparametric one-way analysis of variance (ANOVA) or Mann-Whitney U tests. The results are expressed as means ± standard errors of the means (S.E.M.). A P value of < 0.05 was considered significant.
EV71 infection provoked proinflammatory cytokine but not noradrenaline production in mice
Cytokine treatment slightly exacerbated the clinical disease and pulmonary dysfunction of EV71-infected mice
Cytokine treatment elevated lung weight and induced mild pulmonary edema in EV71- but not herpes simplex virus-infected mice
EV71 caused neuronal damage in brain regions not associated with the development of pulmonary edema
In the course of the development of a mouse model of human EV71 infection, we demonstrated that EV71 could spread from the gastrointestinal tract or muscle to and accumulate in the CNS, especially in the brainstem of mice after different routes of inoculation . Following neuroinvasion, the animals exhibited paralysis, pulmonary dysfunction, and death, but did not develop PE, the hallmark in human EV71 patients with severe complications . In this study, we demonstrated that EV71-infected mice displayed a cytokine profile that was distinct from that of human patients. The NTS was not the CNS target of EV71 infection as viral titer was extremely low in this area of infected mice, which might explain why PE was absent in the infected animals.
Clinical findings showed that increases in serum IL-1β, IL-6, IL-10, IL-13, TNF-α, and IFN-γ were associated with PE development in EV71 patients [7, 13, 14]. In this study, the EV71-infected mice exhibited only a subtle and transient elevation of serum IL-6, IL-10, and IFN-γ. Thus, the absence of PE in EV71-infected mice might be attributed to inadequate proinflammatory cytokine levels of IL-6, IL-13, and IFN-γ. Indeed, the introduction of exogenous IL-6, IL-13, and IFN-γ in EV71-infected mice exacerbated pulmonary dysfunction and resulted in increases in lung weight/body weight ratio and intra-alveolar exudation. In addition, treatment with IL-6 and IFN-γ was sufficient for provoking the observed effect. Both IL-6 and IFN-γ are related to PE development via their effects on endothelial cells and inflammation , , and both of these cytokines were important for microvascular angiogenesis and leakage in an atopic dermatitis mouse model . Recent animal study indeed demonstrated the pathological role of IL-6 in the induction of tissue damage and mortality in EV71-infected mice . IL-13 alone exacerbated only pulmonary dysfunction in our EV71-infected mice, which is consistent with the biological function of the cytokine. IL-13 is a potent effector cytokine for asthma, as it can directly cause airway hyperreactivity, mucus over-production, increased pulmonary vessel permeability, and smooth muscle hypertrophy, effects that have been shown to contribute to pulmonary dysfunction and respiratory distress . Taken collectively, our data suggest that elevated serum IL-6 and IFN-γ contribute significantly to PE development during EV71 infection in mice.
Based on the distribution of viral antigens and viral genomic sequences, EV71 is likely propagated throughout the CNS via motor pathways . Our previous study indicated a retrograde axonal transport of the virus in neuronal cells , which serves as strong evidence that EV71 preferentially infects certain tissues or cell types in the CNS. Both histopathological  and magnetic resonance imaging studies ,  of EV71 patients with PE showed that the major CNS lesions were in the posterior medulla oblongata, pons, midbrain, dentate nuclei of the cerebellum, and ventral horns of the cervical spinal cord. In addition, EV71 viral antigens and genomic sequences were detected primarily in neurons and neuronal processes under inflammatory conditions . The fact that exogenous IL-6, IL-13, and IFN-γ worked only in EV71-infected, but not HSV-1-infected mice, suggests a prerequisite for a region-specific CNS infection for PE development.
Surprisingly, PE did not developed in adult mice after a direct inoculation of EV71 to the NTS, an area known to contribute to the development of PE in rats upon injury [24, 25]. In our current study, the neurons in the NTS of adult mice seemed to be more resistant to EV71 than those in the ventral medulla. Thus, it is possible that the inability of EV71 to cause PE was due to the lack of NTS damage and perhaps neurogenic cardiopulmonary complications.
Besides an over-stimulated proinflammatory response, sympathetic excitement has been proposed as another mechanism involved in the development PE in EV71 patients . Imbalances in sympathetic functions have been observed to cause systemic or pulmonary hypertension which may contribute to the development of PE due to hemodynamic alterations and hydrostatic overpressure in mice , other animals [26, 27], and human beings [28, 29]. Moreover, neurogenic stimulation may also induce movement of plasma proteins into the airway lumen in rats . The plasma noradrenaline levels were not altered in EV71-infected mice. Thus, it may worthwhile to test the effects of catecholamine solely or in combination with the cytokines with regard to PE development.
Collectively, we demonstrated that IL-6, IL-13, and IFN-γ over-stimulation exacerbated pulmonary dysfunction and clinical symptoms, and provoked a mild PE in EV71-infected mice. A synergistic proinflammatory cytokine response and damage to specific brain regions, or more precisely specific neuronal cells, may be necessary for the development of EV71-induced PE.
List of abbreviations
bovine serum albumin
herpes simplex virus-1
peak of inspiratory flow
peak of expiratory flow
magnetic resonance imaging
nucleus tractus solitarii
This work was supported by the National Research Program for Genomic Medicine under grants NSC98-3112-B-006-005 and NSC99-3112-B-006-005 from the National Science Council, Republic of China.
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