Background
Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disease of the CNS. Pathological studies using autopsy material from patients with MS demonstrate the presence of inflammatory cells and their products in the characteristic brain lesions (plaques) associated with this disease. These and other data generated from animal models of MS have led to the generally accepted hypothesis that the disease is mediated by pathogenic leukocyte responses directed against myelin antigens leading to chronic inflammation and ultimately to a broader neurodegenerative process [
1‐
4]. At the vascular level, migrating inflammatory cells release cytokines thought to induce endothelial activation which further promotes leukocyte migration [
5,
6]. With continued recruitment and infiltration of increased numbers of leukocytes, there is disruption and dissolution of the intact endothelium [
6‐
8]. Loss of endothelial function disrupts the supply of oxygen and glucose to the nearby tissue promoting metabolic stress [
9‐
11]. Bioenergetic imbalance at the vascular level results in a loss of tissue homeostasis. It has been suggested that changes in metabolic homeostasis in MS may share some similarity to that observed in hypoxia/ischemia and has been described as a “virtual hypoxia” [
12,
13]. Thus, therapeutic intervention aimed at the restoration of vascular and metabolic homeostasis should be beneficial in the treatment of MS.
In the brain, maintenance of metabolic homeostasis is the result of a coordinated effort between the cellular constituents of the neurovascular unit (pericytes, endothelial cells, astrocytes, and neurons) [
14‐
16]. These cells make fine-tuned regulatory adjustments that promote survival and maintain the balance between oxygen and glucose availability and neuronal metabolic demand. In response to stress, the brain initiates a number of endogenous adaptive mechanisms that result in a continuous matching of tissue oxygen with capillary density. Adaptive angioplasticity is induced by exercise conditioning [
17‐
19], memory, and learning [
20,
21] and upon exposure to mild 10 % oxygen stress [
14,
22]. We have extensively studied endogenous adaptation to mild oxygen stress [
6,
23,
24]. Exposure to 10 % oxygen is comparable to that encountered at moderate high altitude. Endogenous adaptation to low oxygen is in essence an acclimatization to mild hypoxic stress that promotes tissue survival. We have shown that acclimatization to mild hypoxia ameliorates the signs and symptoms of myelin oligodendrocyte glycoprotein (MOG)-peptide-induced experimental autoimmune encephalomyelitis (EAE) [
25]. Exposure of animals to chronic mild hypoxia on the same day of immunization significantly delayed the onset and severity of clinical symptoms. When animals were subjected to chronic mild hypoxia following the appearance of clinical symptoms, chronic inflammation was suppressed [
25]. Histological evidence of decreased infiltration of leukocytes was observed in both protocols. Hypoxia-inducible factor-1alpha (HIF-1α) was induced in the spinal cord in EAE associated with infiltrating leukocytes and in vascular pericytes in hypoxia-treated animals. However, the exact mechanism responsible for the therapeutic efficacy in EAE is not yet known.
In the present study, we have investigated the potential mechanisms involved in the amelioration of EAE. We have questioned whether exposure to chronic mild hypoxia altered immune mechanisms responsible for induction and/or perpetuation of disease activity in MOG-peptide-induced EAE. Hypoxia significantly delayed the onset of the signs and symptoms of EAE associated with decreased histological evidence of inflammatory activity in the spinal cord. Decreased evidence of leukocyte infiltration was not due to delayed peripheral sensitization of MOG-reactive lymphocytes as splenocytes isolated from MOG-immunized mice exposed to hypoxia exhibited a similar MOG-induced proliferative capacity as splenocytes from EAE animals exposed to normal levels of oxygen. Amelioration of disease did, however, correlate with changes in the cytokine-secreting phenotype of the T-cell subsets. Decreased infiltration of cluster of differentiation (CD)4+ cells along with the increased number of regulatory CD25+FoxP3+ T cells found in the hypoxia-exposed mice suggest a possible mechanism for the therapeutic potential of adaptive chronic mild hypoxia in EAE.
Materials and methods
Immunization protocol for MOG-induced EAE
All animal experiments were approved by the Institutional Animal Care and Use Committee, Wayne State University (WSU), and conducted in accordance with the WSU guidelines (protocol # A-09-11-12). Female C57BL/6 mice (6–8 weeks, cat # 000664) purchased from Jackson laboratories (Bar Harbor, MN) were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55, (MEVGWYRSPFSRVVHLYRNGK) (100 μl) (final concentration = 200 μg) emulsified with complete Freund’s adjuvant (CFA) containing 5 mg/ml mycobacterium tuberculosis (Lot # 8183887, Difco Laboratories, Detroit, MI) subcutaneously in two injection sites at the hind flanks of each mouse. Control animals were immunized with an equivalent volume of CFA emulsion that did not contain MOG peptide. All mice received an intraperitoneal (ip) injection with 150 μl of pertussis toxin (300 ng) (List Biological Laboratories, Campbell, CA) immediately after immunization and again 2 days later. Mice were evaluated on a daily basis for changes in body weight, overt signs of illness, and clinical signs of EAE using a five-point scoring system: 0—no symptoms; 1—limp tail; 2—limp tail and hind limb weakness; 3—partial hind limb paralysis; 4—full hind limb paralysis; 5—moribund state or death by EAE. Daily measurements of weight and hematocrit were also made.
Normobaric hypoxia chamber
Hypoxia chambers (Biospherix, Redfield, NY) can be calibrated to create an environment from 0 to 95 % oxygen. There are two mechanisms for maintaining low-oxygen content (8–15 %): (1) selective oxygen vents along the sides can be stopped up or unplugged to allow the free flow of oxygen into the chamber (normoxic controls) and (2) the ProOx controller monitors/feeds nitrogen into the chamber and responds to the current flow of oxygen. This controller calibrates the levels of oxygen and nitrogen using feedback algorithms. Oxygen-sensing equipment was used to verify the concentrations indicated on the controller.
Exposure to normobaric hypoxia
Animals are housed in the Biospherix hypoxia chambers calibrated to administer 10 % oxygen for varying periods of time. In this study, mice were exposed to hypoxia at day 0 (day of immunization). Control normoxic animals were house on the bench top next to the hypoxia chambers under normal oxygen conditions. Following exposure to moderate low oxygen, animals were sacrificed for tissue acquisition at varying times per our approved protocol.
Histology, vascular density, and diaminobenzidine (DAB) staining
Frozen sections (5 μm) were fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100, quenched with 0.3 % H2O2 (in 30 % methanol), and incubated with 5 % normal rabbit serum. The sections were incubated with goat polyclonal anti-glucose transporter protein-1 (Glut-1) Ab (1:100, Santa Cruz Biotechnology, CA) and a biotinylated secondary antibody (Vector Laboratories, CA) for vascular density. Color detection was performed with avidin-biotin horseradish peroxidase solution, ABC kit, and the diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, CA, USA). Sections were prepared at random from a minimum of four samples of the spinal cord. Images were captured using a Pentax K10D camera mounted to an Axiovert 135 light microscope. For vascular density, the number of vessels per field was determined on 20–30 fields per sample from a randomly selected section of the lumbar spinal cord and presented as the mean ± SD.
Immunocytochemistry
Experimental animals were perfused with saline and 4 % paraformaldehyde, and the brain was immersed in the same fixative for 1–2 days. After fixation, the spinal cords were dissected in 1-cm3 blocks, cyroprotected with sucrose, and frozen on dry ice. Sections, 8 μm thick, were prepared with a cryostat (Carl Zeiss, Microm HM505N, Germany). The sections were stored at −20 °C before use. The series consisted of tissue from each group of animals stained with a rabbit anti-mouse HIF-1α IgG2 (Santa Cruz, Santa Cruz, CA) followed by a goat anti-rabbit IgG-FITC-conjugated (Jackson Immunoresearch, West Grove, PA, USA) secondary antibody used at saturation density. When appropriate, DAPI (Molecular Probes, Eugene, OR) was used to visualize the nucleus. Control sections omitting primary antibodies were used to assess nonspecific binding. Some sections were also stained with hematoxylin and eosin (H&E) to visualize infiltrating cells.
Assessment of tissue hypoxia
The assay is based on the chemical reaction of injected pimonidazole which is activated in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids. Hypoxic cells bind pimonidazole (2-nitroimidazoles) to peptide thiols such as glutathione. Although it is difficult to measure Km (O2) for 2-nitroimidazole binding in solid tissue, it has been shown that 10 mmHg is a reasonable threshold value for pimonidazole binding in solid tissue. Sham- or MOG-immunized mice either exposed to 10 % O2 or 21 % normoxia were injected with 50 mg/kg pimonidazole and 5 mg/kg Hoechst dye mixture i.p. and perfused with PBS and 4 % PFA 30 min later. Cryostat sections were stained following the manufacturer’s instructions (Hypoxyprobe, Inc., MA).
Western analysis
The expression of HIF-1α in the spinal cords of mice was evaluated by Western blot analysis. Protein extracts were prepared by lysing tissue homogenates in 500 μl of RIPA buffer [1 % NP-40, 0.1 % sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS), pH 7.4] supplemented with a CompleteTM protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). Lysates were allowed to incubate on ice for 30 min followed by centrifugation at 13,000 rpm for 15 min at 4 °C to pellet debris. Calculation of total protein in microglial extracts was determined by using a standard protein assay [bicinchoninic acid (BCA) protein assay reagent; Pierce, Rockford, IL, USA]. Protein extracts (40 μg of the total protein) were electrophoresed on 4–10 % Tris–HCl Ready Gels (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Bedford, MA, USA) using a wet transfer apparatus (Bio-Rad). Blots were probed using a rabbit anti-mouse HIF-1α (Santa Cruz) followed by a donkey anti-rabbit IgG–HRP conjugate (Jackson Immunoresearch, West Grove, PA, USA). Blots were stripped and re-probed with a mouse anti-actin monoclonal antibody (Santa Cruz) to verify uniformity in gel loading. Blots were developed using the ChemiGlow West substrate (Alpha Innotech, San Leandro, CA, USA) and visualized by exposure to X-ray film (Kodak Biomax, Rochester, NY, USA).
Isolation of spinal cord leukocytes and flow cytometry
To determine whether exposing immunized mice to hypoxia influenced the influx of immune cells, T cells, polymorphonuclear leukocytes (PMNs), macrophages, and microglia were quantitated by fluorescence-activated cell sorting (FACS) analysis, as previously described [
26]. Briefly, mice were perfused to eliminate leukocytes from the vasculature, whereupon the brain and spinal cord were collected to recover infiltrating cells. Following vascular perfusion, tissues were minced in HBSS (HyClone, S. Logan, UT) and digested filtered through a 70-μm nylon mesh cell strainer using a rubber policeman. The resulting slurry was digested for 30 min at 37 °C in HBSS supplemented with 0.2 mg/ml collagenase type I (Sigma-Aldrich, St Luis, MO), 5000 U/ml DNase I (Invitrogen, NY), 1 mg/ml dispase (Invitrogen), and 0.025 % trypsin to obtain a single-cell suspension. Following enzyme neutralization, cells were layered onto a discontinuous OptiPrep (Fisher) gradient and centrifuged at 2000 rpm for 15 min at room temperature in a swinging bucket rotor. After centrifugation, myelin debris was carefully aspirated and the cell interface was collected. Following extensive washes and incubation in Fc block (BD Biosciences, CA) to minimize nonspecific Ab binding to FcRs, cells were stained with directly conjugated Abs for the four-color FACS to detect PMNs and macrophages (CD11b+, CD45
high) and microglia (CD11b+, CD45
low-intermediate). To enumerate EAE-associated T-cell infiltrates, cells were analyzed using antibodies to CD3, CD4, and CD25. For the FoxP3, staining cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % saponin before incubating with the antibody. All Abs were purchased from BD Biosciences. Cells were analyzed using a BD FACSAria with the compensation set based on the staining of each individual fluorochrome alone and correction for autofluorescence with unstained cells.
Isolation of splenocytes
Spleens of both hypoxia-exposed and normoxic EAE mice were removed and minced in PBS having 2 % FBS. Then they passed through a 70-μm cell strainer, washed and then centrifuged. The pellet is resuspended and red blood cells lysed with ACK lysing buffer. After the red cell lysis cells are washed and suspended in Dulbecco’s modified Eagle’s medium (DMEM), cell suspensions are kept in complete DMEM medium on ice.
T-cell stimulation
Single-cell suspensions of splenocytes were prepared from animals exposed to normoxia (immunized only) or to hypoxia (immunized plus low oxygen) as well as from naïve mice. The cells were plated into 96-well plates at a concentration of 1 × 105 cells/well and incubated with MOG peptide (50 μg/ml) and nonspecific mitogen concanavalin A (ConA). Cultures were maintained at 37 °C in humidified 5 % CO2/air. Cells were pulsed with 1 mCi [3H]-thymidine after 72 h and then harvested 18 h later on a Filtermat for scintillation counting. The results were determined as means from triplicate cultures and are shown with SEM.
IFN-γ and IL-17A ELISPOT assay
The MOG-specific response of T cells isolated from immunized mice either exposed to hypoxia or normoxia was analyzed for cytokine-secreting phenotype by ELISPOT assay. Briefly, splenocytes (2 × 105 cells/well) were plated into IL-17A or IFN-γ-coated 96-well filtration plates (Millipore) and stimulated with MOG peptide overnight at 37 °C in humidified 5 % CO2/air incubators. After 24 h, the plates were incubated with the appropriate biotinylated antibodies (all antibodies from ebiosciences), and the cytokine-producing cells were visualized with streptavidin-alkaline phosphatase system (Vector Laboratories), and the plates were analyzed using the CTL ImmunoSpot Analyzer (Cellular Technology Limited, Shaker Heights, OH) with ImmunoSpot software. The frequency of cytokine-producing cells was expressed as the difference between the mean number of spots and the mean background for each experiment. The data were presented as the mean ± SEM of each group, n = 3 mice, and performed in triplicate.
Statistics
All histological and immunocytochemical analyses are conducted using four to six sections per animal with three to six areas of analysis per section and four to six animals per group (see the “
Materials and methods” section). Data is expressed as an average of each area of analysis. Between-group analysis was accomplished using one-way analysis of variance (ANOVA) with least significant difference post hoc testing. Significance is set at
p value <0.05. Based on the variability in these data from our previous studies [
25], we can distinguish a difference in individual proteins and in capillary density with 95 % power.
Discussion
In the brain, metabolic homeostasis is finely tuned and maintained by the cellular constituents of the neurovascular unit (pericytes, endothelial cells, astrocytes, and neurons) [
14‐
16] and is essential for the survival of neurons and the other resident cells of CNS. To achieve balance, endogenous mechanisms have been developed to continuously match oxygen utilization and demand to capillary density. As such, structural adaptations that result in vascular remodeling have been shown to underlie endogenous adaptation to oxygen stress and are thought to promote tissue survival and plasticity [
10,
43]. We questioned whether an endogenous adaptive mechanism induced in response to mild stress would alter inflammatory mechanisms in EAE and neurodegenerative disease. In this study, adaptive vascular remodeling was assessed by the up-regulation of hypoxia-responsive elements and evidence of increased vascular density in exposed animals. Both sham-immunized and MOG-immunized animals respond to mild hypoxia associated with an increase in expression of HIF-1α and the HIF-1-dependent genes such as vascular endothelial growth factor (VEGF). VEGF promotes survival of endothelial cells in in vivo and in vitro [
44] and induces angioplasticity [
44] increasing vascular density. HIF-1α was induced in both MOG-immunized mice, MOG-immunized mice plus hypoxia, and sham-immunized mice plus hypoxia. However, the specific cellular localization was significantly different in the experimental groups. HIF-1 cellular localization in healthy mice exposed to mild hypoxia is associated with pericytes, glial cells, and ultimately neurons. In MOG-immunized animals housed under normoxic conditions, HIF-1α expression was confined to the infiltrating leukocyte populations in plaque areas confirming reports by others [
25,
45]. Expression of HIF-1α in immunized animals exposed to low oxygen was associated with a population of cells evenly distributed within the spinal cord. Preliminary evidence suggests that at least a proportion of these cells may be vascular pericytes. Upon exposure to mild hypoxia, pericytes migrate from their microvascular location and proliferate before renewing coverage of newly formed vessels. Pericyte responses to chronic mild hypoxia are discussed in more detail in a review by Dore-Duffy and LaManna [
14]. In response to chronic mild hypoxic stress, HIF-1α is stabilized and can in turn bind to hypoxia-responsive elements (HREs) that regulate the transcription of target genes, including those encoding erythropoietin, glucose transporters, glycolytic enzymes, antimicrobial factors, and the angiogenic factor VEGF. Stabilization of HIF-1α is an early event in the adaptive response to low oxygen that leads to a cascade of HIF-1-dependent and HIF-1-independent events that culminates with increased vascular density by 2–3 weeks. The transient expression of signaling molecules in this cascade and that hypoxia did not alter T-cell peripheral sensitization may account for the fact that animals eventually developed disease. HIF-1 levels can also be regulated by a number of signaling pathways important in immune responses as well, notably by those of NFκB and STAT3 [
46].
Inflammatory sites can also be characterized as hypoxic milieu due to a focal increase in oxygen demand due to accumulation of activated inflammatory cells [
47,
48]. Vascular cuff formation is associated with evidence of BBB damage and undoubtedly compromised vascular homeostasis. Recently, Davies et al. [
49] has shown pimonidazole staining in the spinal cords of EAE-induced rats. In our study, we found that pimonidazole staining was restricted to splenocytes and leukocytes infiltrating the spinal cord in MOG-immunized EAE mice. We further show that endogenous adaptation to chronic mild hypoxia reduced pimonidazole staining in EAE mice and produced a clinical benefit in EAE. In the Davis study, induction of hyperoxia did not significantly alter clinical disease in EAE in rats. They interpreted their studies to indicate that hypoxia was detrimental and necessary for the development of disease and suggested that hyperoxia may be beneficial as a therapeutic modality in MS. However, they did not distinguish between endogenous and inflammatory-mediated hypoxia. Hyperoxygenation of hypoxic tissue has been shown to increase cellular oxidative stress and further induce tissue damage [
50]. Following exposure to chronic mild hypoxia, adaptation is associated with increased vascularity and restoration of metabolic homeostasis. Therefore, adaptation is protective. These results might explain the lack of clinical benefit from hyperoxia as reported by Davies et al. [
50].
Adaptation to chronic mild hypoxia may also induce an anti-inflammatory environment. It has been shown that under inflammatory conditions, enhanced T-cell activity can lead to lower oxygen levels and HIF-1 induction [
51], which in turn can lead to induction of several genes involved in the glycolytic pathway. Glut-1 (a glucose transporter), hexokinase 2, glucose-6-phosphate isomerase, enolase 1, pyruvate kinase muscle, and lactate dehydrogenase [
52,
53] are inducted in T cells. On the other hand, numerous nonhypoxic stimuli including T-cell receptor (TCR) activation [
54], nitric oxide, lipopolysaccharides, and infectious microbes can result in HIF-1α up-regulation even under normoxic conditions [
55] suggesting that HIF-1 may play multiple roles in leukocytes [
51]. It will be important to study the effect of hypoxia as well as EAE using animals with targeted HIF-1 deficiency. Studies by others suggest that HIF-1 modulates T-cell differentiation towards Th17 cytokine-secreting phenotype. Decreased HIF-1α resulted in diminished Th17 development but enhanced T-regulatory cell differentiation protecting mice from autoimmune neuroinflammation [
52]. Clambey et al. [
53] also showed that HIF-1α induced FoxP3+ Tregs during inflammation. Similarly, we found in our EAE model CD4+ cells were significantly decreased, while the CD4+CD25+FoxP3 Tregs subset was higher in the spinal cords of EAE mice exposed to chronic mild hypoxia when compared to normoxic EAE mice. MOG-specific Th17 responses were found delayed. Thus, exposure to low oxygen appeared to alter key inflammatory pathways important in EAE pathogenesis.
For many years, Th1 cells were deemed responsible for the initiation of autoimmune demyelination. IL-17-secreting cells are also considered a critical effector population in autoimmune demyelinating disease. IL-23 polarizes IL-17-secreting myelin-reactive CD4+ T cells. These cells are highly encephalitogenic upon transfer into naïve syngeneic recipients [
1,
56,
57]. Recently, more focus was on Tregs, since it has been shown that there was a resistance to autoimmune demyelination in mice reconstituted with CD4+CD25+FoxP3+ regulatory T cells [
28,
58,
59], and Tregs were correlated with the reduced inflammation in EAE [
60]. These studies suggested that development of EAE is due to the balance between encephalithogenic and suppressor cells, and adaptation to mild hypoxia favors the induction of suppressor cells. In addition to T cells, chronic exposure to mild hypoxia modulated inflammatory mediators in favor of anti-inflammation. In our studies, we found anti-inflammatory cytokine IL-10 was induced and the inflammatory chemokine CXCL13 was attenuated in sham-immunized animals exposed to hypoxia and during the early phases of hypoxic EAE. CXCL13 has been shown to be detrimental both in EAE and MS [
38,
61]. Its levels increase at the onset of MS in a clinically isolated syndrome (CIS) [
36,
62], increasing further with exacerbations in RRMS [
61] regarded as a biomarker of inflammation in MS [
61,
63].
Results in animal models may be useful in human disease. It has been known for some time that a high altitude can promote wound repair [
64,
65]. Moderate or intermittent stimulation of deep tissue healing by reduced tissue oxygenation has been shown to promote plasticity. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, HIF-1α was identified as a central regulator [
66]. The investigators showed that when HIF-1α was inhibited by small molecule inhibitors (YC-1 and 2-methyoxyestradiol) there was delayed lymphatic repair, decreased vascular endothelial growth factor-C (VEGF-C) expression, reduced numbers of VEGF
+ cells, and an overall reduction in inflammation. A recent study in human spinal cord injury has shown that daily exposure to intermittent hypoxia enhanced motor function in injured patients [
67].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NE, the first author, has done the EAE-induction injections, majority of immune-based analysis of leukocyte regulatory subsets, WB and ELISA assays, and drafted the manuscript. VK and SK have performed many EAE hypoxia experiments leading to the development of baseline observations. ZS performed immunocytochemistry and flow cytometry. D-D is the PI of the project and designed and oversaw the experiments conducted and reported in the manuscript. All authors have read and approved the final version of the manuscript.