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The A/VN/1203/04 H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic (DAergic) neurons located in the substantia nigra pars compacta (SNpc), expression of mono- and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines and growth factors. We find that H5N1 induces a transient loss of the DAergic phenotype in SNpc and now report that this loss recovers by 90 days post infection (dpi). A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60 and 90 dpi. We found a significant increase in the number of activated microglia in each of these brain regions that lasted at least 90 days. We also quantified expression of IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, GM-CSF, G-CSF, M-CSF, eotaxin, IP-10, KC, MCP-1, MIP-1α, MIP-1β and VEGF and find that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.
A number of neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis have been shown to have a significant inflammatory component to their pathologies (Appel et al., 2010; Glass et al., 2010; Tansey and Goldberg, 2010). While the proximate cause of inflammation can be varied, it has clearly been demonstrated that viruses, including influenza, can be encephalitic (Hayase and Tobita, 1997; Jang et al., 2008; Sejvar and Uyeki, 2010). In fact, involvement of the CNS during influenza infections can be fatal, particularly in young patients (Smith et al., 1997). Neurological symptoms associated with influenza infection have been reported as far back as 1385, and intermittent outbreaks with similar symptoms have occurred at other times during influenza outbreaks (Menninger, 1926). The most deadly influenza pandemic of the 20th century, the Spanish Flu, occurred in 1918. It is estimated that about 25–30% of the world population was infected and upward of 50 million people died (Taubenberger and Morens, 2006). About the same time, the world was hit by an unusual epidemic of neurological disease. Post-influenza psychosis and post-encephalitic parkinsonism were reported in Europe and in the USA. More than 500,000 patients who survived this influenza infection were thought to have developed some type of nervous system disorder (Ravenholt and Foege, 1982) including encephalitis lethargica aka von Economo’s encephalitis (von Economo, 1917). Additionally, a significant number of persons that recovered from this “sleeping sickness” later developed a form of encephalitic parkinsonism (Dickman, 2001).
Although rare, experimental evidence has shown that some type A influenza viruses are neurotropic, i.e. they can travel into the nervous system following systemic infection (Tanaka et al., 2003; Klopfleisch et al., 2006; Rigoni et al., 2007), a finding confirmed with certain H5N1 influenza viruses (Rimmelzwaan et al., 2006; Jang et al., 2009). Influenza virus, A/VN/1203/04 (H5N1) infection in mice can initiate a parkinsonian pathology that includes bradykinesia, loss of the substantia nigra pars compacta (SNpc) dopaminergic neuron phenotype, increased levels of alpha-synuclein phosphorylation and aggregation, and activation of microglia; each of which persisted at least 60 days after resolution of the infection (Jang et al., 2009). To determine if the pathology worsens with age, we examined: 1) SNpc tyrosine hydroxylase positive DAergic neuron number and striatal dopamine and its metabolites contents through 90 days post infection (dpi), 2) the inflammatory effect of infection by quantitatively measuring the total number of resting and activated microglia in the SNpc and the production of cytokines in regions of the brain infected by H5N1. We found that infection with H5N1 induces a significant, but transient, loss of both DAergic neurons in the SNpc and dopamine (and its metabolites) in the striatum. Examination of other indolamines demonstrated a significant and sustained loss of serotonin in regions of the brain infected with H5N1. We also observed that areas of the brain infected with H5N1 expressed increased levels of pro-inflammatory cytokines, chemokines and growth factors that appeared both dependent and independent of induction of a peripheral cytokine storm (Yuen and Wong, 2005).
All experiments using the highly pathogenic influenza virus (A/VN/1203/04) were conducted in a Biosafety level 3+ laboratory approved for use by the U.S. Department of Agriculture and the Centers for Disease Control. This facility is authorized for the exclusive use of the Division of Virology and other approved scientists at St Jude Children’s Research Hospital.
Stock viruses were prepared by propagating neurotropic A/VN/1203/04 (H5N1) in the allantoic cavity of 10-days-old embryonated chicken eggs for 40 to 48 hours at 37°C. Virus stock was aliquoted, then stored at −70°C until use. Viral infectious titers were determined using the method of Reed and Muench (Reed and Muench, 1938), and expressed in log10 of the 50% egg infectious dose per 1.0 ml of fluid (EID50/mL). 6–8 week old C57BL/6J female mice (Jackson Labs, Bar Harbor ME) were anesthetized by isofluorane inhalation and infected intranasally with 30 µL of allantoic fluid diluted in PBS to the target virus infectious titer (102 EID50). One group of animals received 0.9% saline and was used as age-matched negative control. Infected mice showed a mortality/morbidity rate similar to that described in Jang et al (Jang et al., 2009), so that all measurements taken in animals prior to day 7 were from the complete pool of animals, while only animals that were visibly sick were used for timepoints after 10 dpi.
Mice were deeply anesthetized with Avertin and transcardially perfused with 0.9% saline followed by 10% neutral buffered formalin at 0, 10, 60 and 90 days post infection. Brains were removed and postfixed for 3 weeks in 10% neutral buffered formalin to ensure that the virus particles present in the tissue have been killed. Brains were cryoprotected with 30% sucrose in PBS, serially sectioned in the coronal plane at 40 micrometers and placed in PBS filled 24 well plates. Free-floating sections were immunolabeled with antibodies directed against tyrosine hydroxylase (TH) (rabbit, P40101-0, PelFreeze, Rogers, AK, 1:500) (to identify dopaminergic neurons) or ionized calcium binding adapter protein-1 (Iba-1) (rabbit, 019-19741, Wako Chemicals, Richmond, VA, 1:500) (to identify microglia) overnight. Primary antibodies were visualized using the ABC method (PK-6101, Vector Laboratories, Burlingame, CA) and visualized using either 3,3-diaminobenzidine (SK-4100 Vector Laboratories, Burlingame, CA) (for DA neurons) or the Vector VIP substrate (SK-4600 Vector Laboratories, Burlingame, CA) (for microglia) as chromogens, respectively.
For identification of neurons undergoing cell death, we used antibodies directed against activated caspase-3 (marking apoptotic neurons, BD Biosciences, Cat # 559565 1:500), TUNEL (TUNEL (TdT-mediated dUTP Nick End Labeling, DeadEnd™ Colorimetric Apoptosis Detection System (Promega, Madison, WI)) or FluoroJadeB (to identify necrotic neurons, (Histo-Chem Inc., Jefferson, AK)). FluoroJadeB staining was carried out as previously described (Faherty et al., 1999; Jensen et al., 2004; Boyd et al., 2007). To examine cell division, we used an antibody directed against Ki-67 (NovoCastra, Newcastle, U.K. 1:2000). Each protocol was performed as previously described (Faherty et al., 1999; McKeller et al., 2002; Jang et al., 2009).
After staining, sections were mounted on Superfrost-Plus (12–550, Fisher, Pittsburgh, PA) slide and counter stained with neutral red to visualize Nissl substance, and then dehydrated, cleared and mounted with Permount (SP15-500, Sigma, St. Louis, MO).
The total number of TH -positive DAergic cell neurons (days 0,10,60 and 90 dpi) and resting and activated microglia (days 0, 60 and 90 dpi) in both hemispheres of the SNpc was estimated using the optical fractionator method (West et al., 1991) in the StereoInvestigator software (version 7.0; MicroBrightField, Colchester, VT, USA) (Baquet et al., 2009). The outline of the SNpc in both hemispheres was delineated at low power (4× magnification) and an unbiased counting frame (60 × 60 µm) was placed at the intersections of a grid (frame size 200 × 200 µm) randomly superimposed on a video image of these contours. Sections were examined under a high power 100× objective lens (n.a. 1.3) on a Olympus BX51 microscope (Olympus, Center Valley, PA) with a MAC5000 motorized XYZ axis computer-controlled stage and a CX9000 CCD video camera (MicroBrightField, Colchester, VT, USA). TH- positive DA neurons and microglial cells were counted at the depth that their nucleus is focused, in each counting area. The reliability of the estimates was measured by calculation of the coefficient of error (West & Gundersen, 1990). Gundersen coefficients of error for m=1 were all less than or equal to 0.10. Statistical significance was calculated using a one-way ANOVA followed by Student-Newman-Keuls post hoc test (Baquet et al., 2009).
Neuron size, determined by the longest length of neuronal cell body was measured using the Neurolucida program (version 7.0; MicroBrightField, Colchester, VT, USA). Raw data was converted to percent of control.
At 10, 60 and 90 days post infection (n=5 at each time point), C57BL/6J mice were deeply anesthetized with Avertin and transcardially perfused with ice-cold 0.9% saline to remove the majority of the blood from the brain vasculature. Brains were rapidly removed and placed in a brain matrix (Model BS-AL-5000C, Braintree Scientific, Braintree. MA) and sliced into 2 mm thick sections and placed on an ice-cooled plate. Tissues were dissected using the following coordinates: SN (Bregma: −2.00- −4.00), striatum (Bregma: +0.00–+2.00mm), brainstem (Bregma: −5.00- −7.00), cortex (Bregma: −1.00- −3.00mm) and the hippocampus (Bregma: −1.00- −3.00mm) (Paxinos and Franklin, 2001). Individual dissected tissues were then homogenized in chilled 0.3M perchloric acid and centrifuged at 10,000g for 15 minutes at 4C. A variety of monoamine transmitters: dopamine (DA), and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA); norepinephrine (NE); 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5 HIAA) were analyzed using reverse-phase ion pairing HPLC combined with electrochemical (EC) detection under isocratic elution conditions. The amount of monoamine neurotransmitters in the tissues were determined by injecting known concentration of monoamine neurotransmitters and extrapolating from a standard curve. Statistical difference was determined using a one-way ANOVA followed by Student-Newman-Keuls post hoc test (Smeyne et al., 2007).
Brains from postnatal days 0–5 (P0-5) C57BL/6J mice were removed from the calvaria, and placed into dissociation media (DM). SN were dissected from the brains as previously described (Smeyne and Smeyne, 2002), and were placed in fresh DM, minced into small pieces and incubated at 37°C in papain following manufacturer’s protocol (Worthington Biochemical Corp., Freehold, N.J.). After digestion in papain, the tissue was rinsed, triturated through a 5 ml serological glass pipette and the cell suspension was layered over plating media (PM) containing Bovine Serum Albumin (BSA, 100 mg/ml) and ovomucoid albumen (100 mg/ml). The cell suspension was centrifuged, resuspended in PM containing 2% rat serum and 8% FBS, and plated at 400,000 cells per well in Lab-Tek (TM) 4 well permanox chamber slides previously coated with laminin (10 µg/ml, Collaborative Biomedical Products) and poly-D-lysine (200 microg/ml, Collaborative Biomedical Products) using .5 ml per well. Cells were placed in a Tri-Gas incubator and maintained at 37°C, 5% CO2, 2% O2. Twenty-four hours post plating, one-third of the media was changed with feeding media complete plus 2% rat serum (RS) with cytosine arabinofuranoside (3 µM final concentration) added to all wells to prevent excessive glial proliferation.
To determine cell number in the cultures, wells were fixed in 3% paraformaldehyde in 1XPBS, followed by 3 rinses with PBS. The individual wells were then processed for TH and Iba-1 staining with the protocol listed above. At the conclusion of antibody staining, wells were removed and coverslips were applied to the permanox slides with Vector aqueous mount containing DAPI. Cells in each well were counted using stereological methods (fractionator) applied to cell culture protocols.
The concentration of interleukin (IL)-1α, IL-1β, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), eotaxin, interferon-inducible protein 10 (IP-10), cytokine-induced neutrophil chemoattractant (KC), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory proteins (MIP)-1α, MIP-1β and vascular endothelial growth factor (VEGF) proteins was simultaneously analyzed from dissected brain regions and lung, as well as sera and cells from SN cell cultures using the Luminex 200 system (Luminex Corp., Austin, TX) and the Milliplex mouse cytokine kit (MPXMCYTO-70K-20, Millipore, Billerica, MA).
For tissue samples, at 3, 10, 21, 60 and 90 days post inoculation, dissected tissues were homogenized in a buffer containing 50 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 0.1% Triton X-100, 150 mM NaCl with a protease and phosphatase inhibitor cocktail (Complete mini, PhosphoStop, Roche, Indianapolis, IN). The tissues lysates were then incubated for 30 minutes on ice and centrifuged at 12,000g for 15 minutes. Supernatants were aliquoted and stored at −70°C until used.
For measurement of cytokine/chemokines in primary SN cultures, one week after plating, dopamine hydrochloride (Sigma, St. Louis, MO) was prepared as a 1M stock in HPLC grade water and diluted to a 500 µm solution in feeding media. Dopamine was diluted to the final concentration added to the cultures at either 500 nM or 5 µm final concentration. Aliquots of media removed at feeding (50–75 µl) were collected in vials on dry ice and stored at −80C after initial media changes and 8, 24 and 48 hours following dopamine addition. At 24 and 48 hours, cells in each of the individual wells corresponding to media aliquots saved at 24h or 48 hours, respectively, were rinsed with 1 time in 1× PBS after media removal, and fixed with 2.5% formalin prepared in PBS for 10 mins at RT.
To quantify cytokine levels, the supernatants were incubated with the microspheres coated with capture antibodies for each analyte for 2 hours. After rinsing, biotinylated detection antibodies were added into each well and incubated for 1 hour. Streptavidin-phycoerythrin conjugates were added and fluorescence intensity was measured by the Luminex 200 reader (Luminex Corp., Austin, TX). The concentration of each cytokine was calculated by generating a standard curve using known concentrations of each cytokine. All results were normalized with total protein concentration measured from each tissue lysate (pg/25 µg of total protein). For cell culture, cytokine levels were measured as described above and corrected based on the total cell number in each well.
Infection with H5N1 leads to a wide array of pathologies in humans and mice, including diffuse damage to the lung, hemophagocytic damage to spleen, lymph nodes and blood vessels and alterations in bone marrow (Dybing et al., 2000; Nishimura et al., 2000; Yuen and Wong, 2005; Korteweg and Gu, 2008). Additionally, several strains of H5N1 have been identified that are neurotrophic in mice, inducing inflammation, neuronal death and induction of pathologies seen in Parkinson’s disease, including formation of aggregates of phosphorylated alpha-synuclein (Wang et al., 2008; Jang et al., 2009).
To determine if H5N1 infection can directly induce the parkinsonism pathologies by damaging dopaminergic neurons, we stereologically assessed the number of tyrosine hydroxylase positive (TH+) DAergic neurons in the SNpc (Fig 1A) and empirically determined the total amount of DA, HVA, and DOPAC in the SN, brainstem (Fig 2), cortex and hippocampus (data not shown).
At day 10 dpi, we find an approximately 60% loss of TH-positive neurons in the SNpc, compared to non-infected control mice. By 60 dpi, we saw a recovery in the TH+ DAergic neuron number and by 90 dpi, we saw no difference in the number of SNpc TH+ DA neurons (Fig 1A). From 10 through 60 dpi, the TH+ DAergic neurons appeared shrunken and atrophic. The longest length of TH-positive neuronal cell bodies was reduced by 20%, however the size of these cells recovered and appeared similar to that seen in the non-infected control mice at day 90 dpi (Fig 1B).
To determine if the loss of TH-positive neurons was a result of cell death and the subsequent recovery in number was due to repopulation via neurogenesis we examined expression of activated caspase-3, TUNEL and FluoroJadeB staining as well as expression of Ki-67. In regions of the brain where A/VN/1203/04 virus had been detected, we found a few activated caspase-3-positive apoptotic but not FluoroJadeB necrotic cells (data not shown). The apoptotic cells were only visible through 10dpi, and although not systematically quantitated, did not appear to be numerous enough to account for a 20% reduction in SN TH-positive number. Examination of cells in the SNpc showed no evidence of cell division using immunohistochemical (Ki-67) methods through 90 dpi, and therefore there is no compelling evidence that SNpc DA neurons underwent any form of neurogenesis. Thus, it is most likely that active H5N1 infection induces SNpc DAergic neurons to transiently reduce their metabolic capacity that both compromises TH activity and reduces cell size (Fig 1A,B), each leading to a loss of the DAergic phenotype in neurons.
We used reverse phase HPLC with electrochemical detection to determine if infection with H5N1 affected the levels of dopamine and its metabolites in the striatum, which is the major target of SNpc DAergic neurons, brainstem, substantia nigra and cortex. The amount of striatal DA and its metabolites, DOPAC and HVA, were each significantly decreased by approximately 40% compared to intranasal saline treated control mice at 10 dpi. By 60 dpi, DA levels returned to baseline levels and this change was stable through 90 dpi. This pattern of a transient decrease at 10 dpi followed by recovery at 90 dpi was also seen when examining levels of HVA and DOPAC (Fig 2).
We also compared the turnover ratio of DA in the striatum ((DOPAC + HVA)/DA) to see if infection with H5N1 altered DA metabolism. Despite alterations in levels of DA, its turnover was unchanged due to concurrent fold-changes in DOPAC and HVA.
In brainstem, the pattern of DA levels was reversed. At 10 dpi, we found a transient 300% increase in DA and DOPAC and a 500% increase in HVA (Fig 2) that resolved by 60 dpi. Like, striatum, no changes in DA turnover were detected since the relative ratios of DA to DOPAC and HVA did not change at each timepoint.
No changes in DA, DOPAC or HVA levels were detected in substantia nigra or cortex at any time after influenza infection (data not shown).
We used reverse phase HPLC with electrochemical detection to determine if infection with H5N1 affected levels of NE, 5-HT and its metabolite, 5-HIAA in the SN, striatum, brainstem, cortex and the hippocampus.
In striatum, we observed a slight increase in striatal NE at 10 dpi that resolved to baseline levels by 60 dpi (Fig 3). Unlike NE, 5-HT levels dropped by approximately 60% and remained low through 90 dpi. A similar reduction in 5-HIAA was detected, although there was a slight recovery by 90 dpi to 60 percent of baseline (Fig 4).
In SN, we measured a slight increase in NE at 10 dpi, although this change did not reach statistical significance (Fig 3). In regard to 5-HT, we observed a 50% decrease that stayed significantly below control levels through 90 dpi (Fig 4). A similar reduction was observed in 5-HIAA levels, although by 90 dpi, these levels returned values significantly similar to control animals (Fig 4).
In hippocampus, no changes in NE levels were seen until 60 dpi, where we observed a 75% increase in NE (Fig 3) that returned to baseline levels at 90 dpi. No statistically significant changes were seen in 5-HT or 5-HIAA levels following H5N1 infection compared to control mice (Fig 4).
In cortex, we measured a significant 70% reduction in NE at 10 dpi, that recovered to control levels by 60 dpi (Fig 3). In regard to 5-HT, we observed a 90% decrease that stayed significantly below control levels through 90 dpi. A similar reduction was observed in 5-HIAA levels (Fig 4).
No changes in NE, 5-HT or 5-HIAA were detected in brainstem.
Microglia are the resident immune cells of the CNS, derived from cells in the monocyte lineage (Hickey and Kimura, 1988; Simard and Rivest, 2004). When in their surveillance mode, they are said to be resting (Gehrmann et al., 1993; Raivich, 2005) and have a characteristic histological appearance with long slender tendrils emanating from their cell body (Fig 5A). Once exposed to infection, injury or trauma (Harry and Kraft, 2008) they undergo a transformation in which they retract and thicken their processes and assume a more ameboid morphology (Fig 5B) (Graeber and Streit, 2010). To determine if exposure to H5N1 alters the morphology and number of microglia in the SNpc, we used the optical fractionator to assess the number of resting and activated microglia. Control C57BL/6 mice administered saline intranasally were found to have approximately 7500 total Iba-1-positive microglia in the SNpc. Of these, approximately 10% of these were structurally classified as activated, while 90% were classified as resting. Sixty days after intranasal innoculation of H5N1, we found a 67% increase in total microglial number. Examination of microglial subtype revealed a 300% increase in activated microglia and a 33% increase in resting microglia. The increase in total microglia number, as well as percent increase in activated and resting microglia, were unchanged from day 60 dpi to day 90 dpi (Fig 5C). This suggests that neurotropic influenza exposure in the brain induces a long-term, if not permanent, increase in activated microglia.
Activated microglia have been shown to produce a variety of cytokines, chemokines, and growth factors following exposure to infection as well as other insults to the CNS (Hirsch et al., 2003; Kim and Joh, 2006; Tansey and Goldberg, 2010). This “inflammatory response” has been shown to be varied and specific to the type of insult (Perry et al., 2003). Some cytokines (IL-1α, IL-1β, IL-2, IL-9, IL-12, IFNγ and TNFα) function primarily to induce inflammation (pro-inflammatory), while others (IL-6, IL-10, and IL-13) suppress inflammation (Opal and DePalo, 2000). A class of cytokines function as chemokines, acting as chemoattractants and include eotaxin, KC, IP-10, MCP-1, MIP1α, and MIP1β (Fernandez and Lolis, 2002), while others can act as growth and differentiation factors (GM-CSF, M-CSF and VEGF) (Metcalf, 1985). In this study, we examined cytokine, chemokine and growth factor profiles in regions of the CNS that were infected by H5N1 infection, both during (day 0–21) and after (day 60–90) the acute infectious stage (Jang et al., 2009). To determine if any alterations were specific to the CNS or were in response to, or coincident with, humoral activation of cytokines, we also measured these proteins in lung, which is the primary site of H5N1 influenza infection in mice (Jang et al., 2009) and traditionally, in humans (Yuen and Wong, 2005).
Examination of cytokines expression profiles in tissues generally demonstrated 4 distinct profiles of induction: In the first pattern, proteins were transiently increased during the initial phase of infection through day 10 dpi and then returned to baseline levels. A second pattern of induction showed an initial decrease in expression followed by a continued loss or a return to baseline levels. A third pattern of cytokine/chemokine expression demonstrated an initial transient increase in expression followed by a return to baseline levels and then a re-induction at times after the influenza virus was no longer detectable by immunohistochemical methods (visualization of NP protein). A fourth pattern of cytokine/chemokine expression was observed where there were no changes during the active phase of infection (through day 10 dpi), but at a later time, induction was detected. Examples of these patterns of expression are seen in Fig 6A–D.
In the lung, the expression of proinflammatory cytokines and chemokines, displayed all 3 of these distinct patterns, while 1 showed a unique pattern not seen in any of the brain regions, where there was a sustained decrease in expression. First, we saw that some of these proteins were transiently increased during the initial phase of infection (through day 21 dpi) and then returned or decreased to baseline levels. The pro-inflammatory cytokines/chemokines that expressed this profile were IL-6, IL-12, GM-CSF, G-CSF, IFNγ, IP-10, KC, MCP-1, MIP1β, MIP1α, and TNF IL-10, an aniinflammatorycytokine/chemokine also expressed this profile. A second pattern of induction showed an initial transient decrease in expression followed by a return to baseline levels. The pro-inflammatory cytokines/chemokines that expressed this profile were IL-1β IL-2, eotaxin and VEGF. The cytokine/chemokine pattern 4 expression was also demonstrated in lung where there is an initial transient increase in expression followed by a return to baseline levels, and then a reinduction at times after the influenza virus was no longer detectable by immunohistochemical methods (visualization of NP protein). The pro-inflammatory cytokines/chemokines that expressed this profile were IL-1α and M-CSF. The fourth pattern, where there is induction, return to baseline and then reinduction was seen in the response of IL-1α, eotaxin, and G-CSF. IL-9 had a unique response, where there was a down-regulation without return to baseline, while IL-13 was not detected in lung (Fig 7).
In the brainstem, the expression of cytokines, chemokines and growth factors displayed 2 of the different patterns described above. The pro-inflammatory cytokines, chemokines and growth factors that expressed the first profile were IL-1α, IL-12 (p70), IL-13, eotaxin, G-CSF, GM-CSF, IP-10, KC, M-CSF, MCP-1, MIP-1α, MIP-1β, and TNF-α. The anti-inflammatory cytokines IL-10 also followed this profile. The level of proinflammatory cytokines and growth factors IL-1β, IL-2, and VEGF displayed pattern 4. They were not changed immediately upon detection of the virus, but increased later when NP protein was no longer evident in the region. (Fig 8).
In the substantia nigra, the expression of cytokines, chemokines and growth factors displayed profiles 1, 3, and 4, described above. The pro-inflammatory cytokines, chemokines and growth factors that expressed the first profile were IL-1β, IL-2, IL-6, G-CSF, M-CSF, and MCP-1 where their expression increased at 3 or 10 dpi and then returned to baseline levels. IL-13 exhibited the third pattern listed above, with its levels increasing prior to day 21 followed by a return to baseline and another rise in level at a later point after the active infection (60 dpi) was over. SN levels of the chemokines and growth factors GM-CSF and MIP-1β exhibited pattern 4, where expression did not change immediately upon detection of the virus, but increased later when NP protein was no longer evident in the region. Neither MIP-1α nor TNF-α was detected in the SN following exposure to influenza (Fig 9).
In striatum, the expression of cytokines, chemokines and growth factors displayed profiles 1, 3, and 4, described above. The pro-inflammatory chemokines and growth factors that expressed profile 1 were eotaxin and M-CSF. The cytokine that expressed profile 3 was IL-2, while the anti-inflammatory IL-10 displayed profile 4. IL-1α, IL-6, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFN- γ, MIP-1α, MIP-1 β, and TNF-α were not detected in striatum following exposure to H5N1 influenza (Fig 10).
In cortex, the expression of cytokines, chemokines and growth factors displayed profiles 1, 2 and 4. The pro-inflammatory cytokines that expressed profile 1 were IL-2 and IL-9. The growth factor VEGF expressed profile 2 while an anti-inflammatory cytokine, IL-10 displayed profile 4. IL-1α, IL-6, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFN-γ, MIP-1α, MIP-1 β, or TNF-α were not detected in cortex following exposure to influenza (Fig 11).
The reinduction of cytokine/chemokine expression between 60 and 90 days -long after the H5N1 virus was absent from the CNS- appeared to coincide temporally with the re-expression of tyrosine hydroxylase in the SN and appearance of DA in the striatum. To determine if some of the inflammatory response was induced by this reintroduction of dopamine to the basal ganglia we examined the pattern of cytokine expression using an in vitro postnatal substantia nigra culture system (Smeyne and Smeyne, 2002). We allowed SN cultures to stabilize in a 2% O2, 5% CO2 environment for 1 week and then added either 500 nM or 5 µM DA to each culture. Supernates were taken at 8 hours, 24 hours and 48 hours after the addition of dopamine and 22 cytokines/chemokines were measured. Of the 22 cytokine/chemokines measured, 12 were detected in the supernates (Fig 12). The addition of dopamine increased expression of 4 of the 12 detected cytokine/chemokines including IL-1β (24 hours after addition of 5 µM DA), TNFα (8 hours after 500 nM DA), IL-13 (8 hours after 500 nM DA) and GM-CSF (24 hours after at 5 µm DA).
The A/VN/1203/04 strain of the H5N1 influenza virus induces both short- and long-term effects in the central nervous system of mice. During the acute phase of the infection, which lasts through day 10 post-infection (Jang et al., 2009), the virus induces- in the basal ganglia- a transient loss of the DAergic neurotransmitter phenotype. However, over a period of 80 days from P10 to P90, there is a steady recovery of both TH-positive SN neurons and striatal dopamine. Since we saw no evidence of any significant cell death in the SN, as judged by immunostaining with activated caspase-3 or TUNEL; nor any expression of the mitotic marker Ki-67, we feel that the loss and subsequent recovery was due to injury to the biosynthetic machinery involved in dopamine production rather than a direct influenza-induced cell death and subsequent neuron regeneration. The recovery of dopamine following transient depletion have been well documented in other models of parkinsonism including 6-OHDA (Robinson et al., 1994) and MPTP (Bazzu et al., 2010).
In addition to the transient loss of dopamine in SN and striatum, we also see an acute, but long-lasting, increase in inflammation within the brain, characterized by an increase in the number of resting and activated microglia and differential expression of a number of cytokines and chemokines. Inflammation in the nervous system has been associated with both the initiation and progression of a number of neurological disorders, including Parkinson’s disease (Tansey and Goldberg, 2010). Some of the cardinal pathologies seen in Parkinson’s disease include dopaminergic neuronal death in the substantia nigra pars compacta (and other regions of the brain), aggregation of ubiquinated proteins including alpha-synuclein (Lewy Bodies) throughout the brain and generalized increases in cerebral oxidative stress (Dauer and Przedborski, 2003). Coincident with these processes, there is an activation of the microglia within the brain. Thus, what remains to be determined is whether the inflammatory reaction induced by H5N1 is a primary defect, occurs in response to initiation of these underlying pathologies, or both. There is significant support of the first possibility. For example, increases in microglial number, morphology and production of cytokines have been shown to occur in response to overexpression of alpha-synuclein (Zhang et al., 2005; Su et al., 2008; Reynolds et al., 2009; Sanchez-Guajardo et al., 2010), death of neurons (Minghetti et al., 2005), and increased oxidative stress (Kuhn et al., 2006; Hu et al., 2009; Rojo et al.). Others have shown that activation of the immune system is likely a predisposing factor that contributes to the initiation and progression of these pathologies (Gao et al., 2008; Hu et al., 2008; Liu et al., 2009; Peng et al., 2009).
In this study, we find microglia become activated coincident with the physical presence of the H5N1 virus; and it is only after the microglia become activated that we observe loss of the dopaminergic cellular phenotype (lack of TH production), aggregation of phosphorylated alpha-synuclein (Jang et al., 2009) and induction of cell death. The rapid microglial activation following peripheral inoculation of the influenza virus raises an important question: is the initial microglial activation due to the direct sensing of the virus within the brain parenchyma or does it result from signals initiated outside of the brain, i.e. response to induction of a “cytokine storm” in lung? Support for a direct innate effect comes from studies demonstrating that that both microglia (Wang et al., 2008) and neurons (Yao et al., 2008) are capable of directly interacting with the HA protein on the surface of the influenza virus by binding to sialic acid (SA)-alpha2,3-Galactose receptors, where they can induce inflammatory cytokine production (Yokota et al., 2000; Wang et al., 2008). Other studies, including studies of pandemic 1918 H1N1 influenza, lend support for an indirect adaptive mechanism, which would involve a humoural:microglial interaction (Tumpey et al., 2005; de Jong, 2008). The increased presence of activated T-cells in the brains of Parkinson’s disease patients also supports this possible mode of humoral:innate crosstalk (Appel et al., 2010). In addition to interactions with the physical influenza viral protein, it is also possible that circulating activated T-cells previously exposed to the H5N1 influenza virus may secrete a soluble factor (chemokines/cytokines) that induces microglial activation, through signaling of the B7, CD-40 and CD-23 pathways (Yong and Marks, 2010), of the microglia without the physical presence of viral particles.
Based on our findings of virus in the brain and probable interactions with circulating T-cells, we suggest that a combination of mechanisms occur. We find that cytokine expression, a measure of immune activation/response, in most cases closely follows a pattern initiated in lung including IL-1α, IL-1β, IL-6, eotaxin G-CSF, IFNγ, IP-10, KC, MCP-1 MIP1α, MIP1β, TNFα and VEGF (acutely) and GM-CSF (with a delayed response,) and thus are likely to be, at least in part, a response in brain from a lung-initiated cytokine storm. However, there are clear examples, including IL-9, IL-12, and IL-13, where we find cytokine expression in the brain, that have no apparent relationship to the response observed in lung. In substantia nigra, the region of the brain whose cells that is highly susceptible to oxidative stress in mice (Jenner, 1998; Yokoyama et al., 2008) and is most affected by Parkinson’s disease in humans (Parent and Parent, 2010), the pattern of activation of IL-13 and GM-CSF were particularly interesting. IL-13 showed a rapid induction followed by return to baseline levels and then a re-induction long after H5N1 virus, marked by anti-NP immunohistochemistry, is absent from the brain. GM-CSF was never induced until 60 days after H5N1 infection. The stimulus for this cytokine re-induction is unknown. However, we find it interesting that the timing of cytokine re-induction closely approximates the timing of TH re-expression in both the SN and striatum. A comparison of our in vivo and in vitro data shows that IL-13 and GM-CSF are induced by addition of DA in SN culture supernates. We also found a rapid increase in TNFα in our cultures, although we were not able to detect this in the tissue samples. Several studies have examined the role of dopamine in microglial activation and found that dopamine can act as an inducer of oxidative stress via mechanisms that include DA quinone formation (Smith et al., 1994; Kuhn et al., 2006; Mastroeni et al., 2009; Goncalves et al., 2010) and microglia cell activation (Goncalves et al., 2010). Thus, at least for a subpopulation of the measured cytokines, our results support the hypothesis that dopamine, itself, may be an inducer of an inflammatory reaction and perhaps even a factor in Parkinson’s disease progression
Based on our previous study (Jang et al., 2009), we found that the earliest and most severely affected regions of the CNS affected by H5N1 influenza infection were the brainstem (BS) and midbrain. Each of these subdivisions contains structures that degenerate in Parkinson’s disease, including locus coeruleus and substantia nigra (SN), respectively. Based on our cytokine analysis, these regions also show the largest cytokine inductions. In these regions, 16 of the 22 cytokine/chemokines analyzed show -at least- a 2-fold increase in protein levels at the peak of expression, while 9 of these had at change of at least 500%. The largest increases were in IL-6 (500% BS, 400% SN), IL-13 (500% BS, 800% SN), eotaxin (500% BS, 200% SN), G-CSF (1000% BS, 8000% SN), IFNγ (6000%, BS, SN), IP-10 (100,000% BS, 500,000% SN), KC (5000% BS, 10,000% SN), MCP-1 (60,000% BS, SN), and MIP1α (6000% BS, SN). Additionally, the brainstem was the only place in the CNS we were able to still see an increase in TNFα (100%, BS) when examined 10 days after the infection; despite the fact that this inflammatory protein is usually downregulated soon after the initial insult (Ghoshal et al., 2007).
The source of the secreted cytokines/chemokines/growth factors in brain is critical to understanding the process of inflammation following influenza infection. We clearly see an increase in the number of activated microglia, and these have been shown to secrete a number of cytokines when challenged by bacterial or viral infection (Hayashi et al., 1995; Persidsky et al., 1999; Wang et al., 2008). Additionally, these cells secrete other factors (MCP-1, MIP1α) that are involved in macrophage homing (Carr et al., 1994; Menten et al., 2002) and IFNγ (Dufour et al., 2002) that has been shown to inhibit viral replication, which may be part of the brain’s innate neuroprotective mechanism.
Our findings therefore suggest that any neurotropic influenza virus that activates the immune system in the brain, whether directly or indirectly, could contribute to CNS disorders of protein aggregation; and more generally that viruses may be an important etiological agent in the developmental sequalae of neurodegenerative diseases including Parkinson’s disease. Our studies and the aforementioned papers from other labs suggest that activation of an immune response may be a precipitating factor for developing later parkinsonism. Our work using the A/VN/1203/04 H5N1 influenza virus, shows that this strain can get into the CNS and activate the innate immune system. This is sufficient to induce transient damage to neurons as well as mediate long-term changes (deposition of phosphorylated alpha synuclein and permanent activation of microglia); but is insufficient alone to cause Parkinson’s disease. Further work on the role of influenza as well as immune system modulation by viruses will be necessary to determine if modulation of these changes can alter the pathogenesis of this disease.
This project was funded in part by the Michael J. Fox Foundation for Parkinson’s Research (RJS), National Institute of Neurological Disease and Stroke, NIH, NS058310 (RJS), National Institute of Allergy and Infectious Diseases, NIH, Department of Health and Human Services, under contract No. HHSN266200700005C (RW) and by the American Lebanese Syrian Associated Charities (ALSAC).
The authors declare that they have no conflicts of interest.