IKKβ-mediated inflammatory myeloid cell activation exacerbates experimental autoimmune encephalomyelitis by potentiating Th1/Th17 cell activation and compromising blood brain barrier
© The Author(s). 2016
Received: 29 October 2015
Accepted: 2 July 2016
Published: 22 July 2016
The inflammatory myeloid cell activation is one of the hallmarks of experimental autoimmune encephalomyelitis (EAE), yet the in vivo role of the inflammatory myeloid cell activation in EAE has not been clearly resolved. It is well-known that IKK/NF-κB is a key signaling pathway that regulates inflammatory myeloid activation.
We investigated the in vivo role of inflammatory myeloid cell activation in myelin oligodendrocyte glycoprotein (MOG) peptides-induced EAE using myeloid cell type-specific ikkβ gene conditional knockout-mice (LysM-Cre/Ikkβ F/F ).
In our study, LysM-Cre/Ikkβ F/F mice had alleviated clinical signs of EAE corresponding to the decreased spinal demyelination, microglial activation, and immune cell infiltration in the spinal cord, compared to the wild-type mice (WT, Ikkβ F/F ). Myeloid ikkβ gene deletion significantly reduced the percentage of CD4+/IFN-γ+ (Th1) and CD4+/IL-17+ (Th17) cells but increased the percentages of CD4+/CD25+/Foxp3+ (Treg) cells in the spinal cord and lymph nodes, corresponding to the altered mRNA expression of IFN-γ, IL-17, IL-23, and Foxp3 in the spinal cords of LysM-Cre/Ikkβ F/F EAE mice. Also, the beneficial effect of myeloid IKKβ deletion in EAE corresponded to the decreased permeability of the blood brain barrier (BBB).
Our findings strongly suggest that IKK/NF-kB-induced myeloid cell activation exacerbates EAE by activating Th1 and Th17 responses and compromising the BBB. The development of NF-κB inhibitory agents with high efficacy through specific targeting of IKKβ in myeloid cells might be of therapeutic potential in MS and other autoimmune disorders.
KeywordsMyeloid cell IKKβ conditional deletion Experimental autoimmune encephalomyelitis T cell Blood brain barrier
Multiple sclerosis (MS) is an autoimmune, demyelinating disease resulting from chronic inflammation in the central nervous system (CNS). The pathological features of MS are characterized by breakdown of the blood-brain barrier (BBB), infiltration/recruitment of peripheral immune cells consisting of lymphocytes and macrophages, demyelination, axonal- and oligodendrocyte loss, and glial scar formation [1, 2, 14, 31, 42]. These pathological features of MS can be recapitulated in the animal model for experimental autoimmune encephalomyelitis (EAE). A series of studies have demonstrated that myeloid cells in the spinal cord, namely microglia and blood-derived macrophages, play pivotal roles in the pathogenesis of MS and EAE. Studies thus far indicate that the activation of these myeloid cells contribute to MS and EAE lesion formation, as they phagocytose myelin, leading to extensive myelin damage and oligodendrocyte dysfunction . In addition, activated microglia and brain-infiltrating macrophages in the demyelinated lesions produce inflammatory mediators including proinflammatory cytokines/chemokines, nitric oxide, and reactive oxygen species, which exert detrimental effects on MS and EAE by directly affecting oligodendrocyte cell death and/or recruiting autoreactive lymphocytes [20, 48, 57]. However, there are also documents indicating that these activated microglia/macrophages can be beneficial in the recovery process after EAE [31, 47, 48]. For instance, alternatively activated microglia and macrophages facilitate recovery from EAE by driving oligodendrocyte differentiation during remyelination . In addition, efficient clearance of myelin debris and apoptotic cells in the lesion site and secretion of neuroprotective mediators from activated microglia/macrophages may promote regeneration of the impaired axons . Thus, the in vivo role of macrophage/microglia activation in EAE is still controversial. Furthermore, recent studies revealed that microglia/macrophages display remarkable plasticity in their activation phenotypes. They can be activated as the “classically activated” M1 phenotype that produces and releases proinflammatory mediators  or the “alternatively activated” M2 phenotype that promotes angiogenesis and matrix remodeling  depending on their microenvironment. Therefore, the specific function of microglia/macrophage activation in the pathogenesis of EAE should be characterized according to the activation phenotype of these cells.
The nuclear factor (NF)-κB signaling pathway plays a central role in the expression of a wide variety of genes controlling immune and inflammatory responses. Upon stimulation, the canonical NF-κB pathway is activated by the IκB kinase (IKK) complex, which is composed of two catalytic subunits, IKKα and IKKβ [28, 41]. In microglia and macrophages, IKK/NF-κB-dependent signaling pathways are critical for M1 phenotype-related proinflammatory gene expression such as inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α [28, 41]. Due to its pivotal role in inflammatory myeloid cell activation, we reasoned that by selectively inhibiting the IKK/NF-κB signaling pathway in macrophages/microglia, we would be able to address the in vivo function of inflammatory myeloid cell activation in EAE. For the targeted analysis of NF-κB signaling, we used IKKβ-conditional knockout mice, in which the ikkβ gene is specifically deleted in myeloid cells, including the majority of microglia and macrophage populations [9, 18], and investigated the in vivo role of the IKK/NF-κB-dependent inflammatory myeloid cell activation during the complex process of demyelination through the development and progression of EAE. Our results showed that IKK/NF-κB-dependent proinflammatory myeloid cell activation exacerbates autoimmmune demyelination, Th17 cell infiltration, and BBB compromise during EAE. These data suggest that pharmacological targeting of the IKK/NF-κB signaling pathway, specifically in myeloid cells, might have therapeutic benefits in autoimmune demyelinating disorders including MS.
Animals, genotyping, and ethic statements
Myeloid cell type-specific IKK-β-deficient (LysM-Cre/Ikkβ F/F ) mice were generated by crossing floxed-ikkβ (Ikkβ F/F ) mice and LysM-Cre knock-in mice expressing Cre under the control of the endogenous lysozyme M promoter, as described previously [9, 10, 37]. The genetic background of both mice was C57BL/6. PCR genotyping was performed using the primers 5′-TGA CCC GGG AAT GAA TAG GA-3′ and 5′-GTC TTC AAC CTC CCA AGC CTT-3′, which amplify both the Ikkβ + (220 bp) and Ikkβ F (310 bp) alleles. LysM-Cre mice were genotyped by PCR using the primer pair NLS-Cre (5′-CCC AAG AAG AAG AGG AAG GTG TCC-3′) and Cre8 (5′-CCC AGA AAT GCC AGA TTA CG-3′) as previously described . Adult (10–11 weeks after birth) female LysM-Cre/Ikkβ F/F and wild-type (WT, Ikkβ F/F ) mice were used.
Mice were housed at 23 ± 3 °C with a 12 h light–dark cycle (light from 08:00 to 20:00) and food and water were provided ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kyung Hee University and Seoul National University. Proper randomization of laboratory animals and handling of data were performed in a blinded manner in accordance with recent recommendations from a NIH Workshop on preclinical models of neurological diseases .
Microglia isolation by CD11b microbeads
Microglial cells were isolated from the lumbar spinal cord of mice using CD11b microbeads (Miltenyil Biotec, Germany). Briefly, spinal cords were dissected after perfusion with PBS and single-cell suspensions were prepared by using the neural tissue dissociation Kit (Miltenyil Biotec) for 30 min at 37 °C. Pure single-cells were acquired by passing the cells through a 40 μm strainer. Myelins were removed by using myelin removal beads (Miltenyil Biotec) for 15 min at 4 °C. Single cells were stained with anti-CD11b antibody (Miltenyi Biotec) for 30 min at 4 °C. The CD11b+ labeled single cells were separated by a magnetic field through MS columns (Miltenyi Biotec). We confirmed that these cells were approximately 95 % pure, as assessed by CD11b+ and CD45+ cytometry analysis. Isolated microglia cells were used in real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis to investigate the level of IKKβ deletion in spinal microglia, as previously described , using the primer summarized in Additional file 1.
Isolation of peritoneal macrophages and lipopolysaccharide-stimulation
Two ml of 2 % thioglycollate (BD Bioscience) was intraperitoneally administered to adult mice (n = 5 per group), to collect peritoneal macrophages. After 3–4 days, the peritoneal macrophages were harvested and isolated. The macrophages were distributed in Eagle’s minimal essential medium (EMEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), seeded onto 24 plates (2.5 × 105 cells/well), and incubated for 3 h at 37 °C in an atmosphere of 5 % CO2. After 4 h, non-adherent cells were removed by washing with Hanks balanced salt solution (HBSS) and the adherent cells (macrophages) were equilibrated with DMEM containing 10 % FBS before incubation with lipopolysaccharide (0111:B4; 100 ng/ml; Sigma-Aldrich) for 6 h. The macrophages were collected to analyze the mRNA levels of cytokines, including TNF-α, IL-1β, IL-6, and iNOS by real time RT-PCR using the primers summarized in Additional file 1.
M1/M2 polarization of peritoneal macrophages
To analyze M1/M2 polarization of macrophage cells, macrophages equilibrated with DMEM were treated with lipopolysaccharide (0127:B8; 50 ng/ml; Sigma-Aldrich) for 4 h or IL-4 (0.1 ng/ml; R&D system) for 24 h. These macrophages were incubated with FITC anti-mouse F4/80 (F480, eBioscience), PE anti-mouse CD80 (B7-1; eBioscience), APC anti-mouse CD86 (B7-2, eBioscience), and APC anti-mouse CD206 (MMR, Biolegend) (Additional file 2) for 30 min at 4 °C. After washing twice with 2 % FBS in PBS, macrophages were used for flow cytometry analysis. Data were collected on a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cell Quest Pro software (BD Biosciences). Alteration in protein expression of M1 (iNOS) and M2 marker (arginine-1) was analyzed using protocol as described in 2.11. Western blot analysis.
Preparation of spinal cord for real time RT-PCR and Western blot analyses
At the peak day of neurological impairment (15–18 days post-immunization), the mice used for real-time RT-PCR and Western blot analysis (n = 5 per group) were anesthetized. Each lumbar spinal cord was removed and deep-frozen.
Real-time RT-PCR was performed using SYBR Green PCR Master Mix (ABI, Warrington, UK) as described previously [9, 25, 34]. Reactions were performed in duplicate in a total volume of 10 μl containing 10 pM primer, 4 μl cDNA, and 5 μl SYBR Green PCR Master Mix. The mRNA levels of each target gene were normalized to that of GAPDH mRNA. Fold-induction was calculated using the 2−∆∆CT method, as previously described . All real-time RT-PCR experiments were performed at least thrice and the mean ± SEM values are presented unless otherwise noted. The PCR primer sequences used in this study are listed in Additional file 1.
Histopathological analysis of spinal cord
EAE induction and clinical evaluation
Quantification of demyelination and cell recruitment/infiltration
Methods for quantification of demyelination and cell recruitment/infiltration are presented in Additional file 3.
Western blot analysis
Measurement of weights of spleen and lymph nodes
At the peak day of neurological impairment, five mice in each group were anesthetized. Their spleen and lymph nodes were carefully removed without fat, connective tissue, or fluid. They were weighted using a microbalance (OHAUS, Parsippany, U.S.A.).
For flow cytometry analysis, 5 mice in each group were anesthetized. The lumbar spinal cord and lymph nodes were carefully dissected and dissociated at the time of the peak clinical score as previously described [23, 33, 34]. A single-cell suspension was prepared and fixed with 2 % paraformaldehyde for surface cell analysis. Cells were then washed with 2 % FBS in PBS, incubated with mouse anti-rat CD32 (BD Biosciences, San Jose, CA, USA) for 10 min to block the Fc receptor and washed twice with 2 % FBS in PBS. For surface cell analysis, cells were incubated with APC anti-mouse CD11b (OX-42; Biolegend, San Diego, CA, USA), PE anti-mouse CD45 (OX-1; Biolegend), APC anti-mouse CD4 (OX-35, BD Biosciences), and PE anti-mouse CD8a (OX-8, BD Biosciences) for 30 min at 4 °C. Microglia and macrophages were identified based on their relative CD45 expression levels . Briefly, after acquiring unstained and single color control samples to calculate compensation matrix, we acquired 1 × 104 events within the combined gate based on physical parameters [forward scatter (FSC) and side scatter (SSC)]. CD11b+/CD45+(low) cells and CD11b+/CD45+(high) cells were gated as resident microglia and macrophages, respectively . For intracellular cell analysis, cells were restimulated with phorbol-12-myristate-13-acetate and ionomycin and Golgistop in RPMI media. After 5 h, cells were stained with PerCP-Cy 5.5 anti-mouse CD4 (RM4-5; BD Biosciences), FITC anti-mouse IFN-γ (XMG1.2; BD Biosciences), PE anti-mouse IL-17A (TC11-18H10; BD Biosciences), PE anti-mouse IL-4 (11B11; BD Biosciences), PE anti-mouse CD25 (PC61.5; FJK-16 s; eBioscience), and APC anti-mouse/rat Foxp3 (FJK-16 s; eBioscience) (Additional file 2) for 30 min at 4 °C. The cells were washed twice with 2 % FBS in PBS and used for flow cytometry. To identify CD4+ T cell populations, we first gated on cells (1 × 104) based on FSC and SSC properties. CD4+ T cells were used to analyze populations of Th1, Th2, Th17, and Treg cells on CD4. Three-color staining was performed for one cell for simultaneous analysis. Intracellular cytokine results were indicated as percentages within the CD4+ population. Data were collected on a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cell Quest Pro software (BD Biosciences).
T cell culture and proliferation
Methods for T cell culture and proliferation are presented in Additional file 3.
Co-culture of microglia with T cells
Primary mixed glia was isolated from brains of postnatal 1–3 day WT or LysM-Cre/Ikkβ F/F mice. After removing meninges of brain, single-cells were cultured in DMEM containing 10 mM HEPES, 10 % FBS, 2 mM L-glutamine, and antibiotic/antimycotic in 75 cm2 flasks at 37 °C with 5 % CO2. Culture medium was changed every 2–3 days and glia cultured for 14 days. Detached microglial cells were incubated for 30 min. Non-adherent cells were removed. These cells were approximately 95 % pure based on CD11b+ flow cytometry analysis. At 15 days after EAE induction, 95 % pure CD4+ T cells were harvested from lymph node cells of WT and LysM-Cre/Ikkβ F/F mice by anti-mouse CD4 magnetic beads (Miltenyil Biotec). CD4+ T cells (2 × 106 cells/ml) were re-stimulated with MOG35–55 peptide (25 μg/ml) in the presence IL-2 and IL-12 (20 ng/ml, R&D Systems). After 7 days of culturing, surviving MOG35–55 peptide-specific T cells were co-cultured with microglia in DMEM containing 10 % FBS and MOG35–55 peptide (25 μg/ml). T cells were added to the microglia at an estimated ratio of 1:2 (0.5 × 105 T cells: 1 × 105 microglia). After 24 h, cells were harvested and subjected to T cell differentiation analysis using flow cytometry as described above.
Evaluation of BBB disruption
The level of BBB disruption was detected by quantitative measurement for Evans blue content at the peak day of neurological impairment after immunization, as previously described . Briefly, sterilized 2 % Evans blue solution was intravenously injected at a dose of 4.0 ml/kg per mouse (n = 5 per group). Thirty minutes after injection, the mouse was perfused with saline to remove the Evans blue dye in the vascular system. The brain and lumbar spinal cord were immediately removed, and images were captured. The brain and lumbar lesion of the spinal cord were homogenized with 2.5 ml PBS and mixed with 2.5 ml 60 % trichloroacetic acid to precipitate protein. The samples were centrifuged for 30 min at 1,000 g, and the supernatants were measured at 610 nm for the absorbance of Evans blue by using an enzyme-linked immunosorbent assay reader (Soft Max Pro-5; Molecular devices, CA, USA). The Evans blue content was expressed as micrograms per gram of brain and lumbar spinal cord. To histologically and immunohistochemically investigate BBB permeability, 2 h after Evans blue solution injection to EAE mice, spinal cords were harvested from each group (n = 5 per group) and fixed with 4 % paraformaldehyde solution. Cryo-sections in 30 μm thickness were prepared and used to evaluate extravasation of Evans Blue dye using confocal microscopy. Immunofluorescent staining was performed as described above (Immunohistochemical analysis).
Induction of passive transferred EAE
Induction of transferred EAE was performed with published method [6, 43] with slight modifications. Briefly, T cells were isolated from lymph nodes of WT or LysM-Cre/Ikkβ F/F donor 15–18 days after induction of active EAE and re-stimulated with MOG35–55 peptide (25 μg/ml) in the presence IL-2 and IL-12 (20 ng/ml, R&D Systems, Minneapolis, U.S.A.) in RPMI 1640 medium containing 10 % FBS and 1 % penicillin/streptomycin for 3 days. Purified T cells (1 × 107) were transferred i.v. into sub-lethally irradiated WT or LysM-Cre/Ikkβ F/F recipient mice. Disease development was daily monitored.
Statistical analysis was performed using the SPSS 21.0 package (SPSS Inc, Chicago, USA) for Windows. Neurological scores obtained by EAE induction were analyzed using two-way analysis of variance (ANOVA) with repeated measures with one within-subjects factor (time) and two between-subject factors (WT and LysM-Cre/Ikkβ F/F mice). The data from immunohistochemistry, Western blot, and PCR analysis were analyzed using one-way ANOVA with Tukey post hoc test for comparison of multiple groups. The data were presented as mean ± SEM. P values of less than 0.05 were accepted as statistically significant.
Myeloid-specific ikkβ gene deletion regulates M1/M2 polarization of macrophages
Myeloid-specific ikkβ gene deletion alleviates neurological impairment and spinal demyelination during EAE
Myeloid-specific ikkβ gene deletion ameliorates a subsequent encephalogenic challenge
Mean day of onset (± SEM)
Maximal clinical score (± SEM)
Sun of clinical score (± SEM)
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
7.6 ± 0.7
3.4 ± 0.2#
64.1 ± 1.7#
10.2 ± 0.5
2.6 ± 0.2*
17.3 ± 3.0*
Myeloid-specific ikkβ gene deletion inhibits the recruitment/infiltration of microglia and macrophages in spinal cord lesion during EAE
Myeloid-specific ikkβ gene deletion decreases the size of lymphatic organs and inhibits the differentiation/proliferation of CD4+ T cells during EAE
Myeloid-specific ikkβ gene deletion regulates the percentages of CD4+, CD4+/IFN-γ+, CD4+/IL-17+, and CD4+/CD25+/Foxp3+ T cells
0.52 ± 0.17
0.65 ± 0.21
14.47 ± 1.21##
8.18 ± 0.53**
2.53 ± 1.38
2.89 ± 0.13
3.07 ± 0.74
2.76 ± 0.06
2.97 ± 0.04
2.98 ± 0.95
11.33 ± 0.69##
6.34 ± 0.22**
3.84 ± 0.94
3.01 ± 0.12
14.35 ± 1.21##
5.43 ± 0.63**
0.41 ± 0.09
0.49 ± 0.29
0.87 ± 0.13
0.94 ± 0.27
0.31 ± 0.45
0.37 ± 0.57
4.59 ± 1.97#
13.49 ± 3.21*
0.46 ± 0.06
0.74 ± 0.10
1.55 ± 0.15##
4.05 ± 0.80*
8.43 ± 0.64
8.13 ± 0.84
27.77 ± 2.33##
19.96 ± 2.96*
4.85 ± 0.00
4.39 ± 0.10
4.71 ± 1.08
4.06 ± 0.72
1.01 ± 0.09
1.24 ± 0.01
9.40 ± 1.98##
4.59 ± 0.72*
0.13 ± 0.53
0.10 ± 0.34
14.76 ± 2.64##
4.13 ± 0.66**
4.55 ± 0.60
4.29 ± 0.52
3.49 ± 1.32##
4.01 ± 0.94
16.49 ± 1.02
15.21 ± 0.37
8.01 ± 1.31#
11.31 ± 0.20*
5.00 ± 0.21
4.96 ± 0.57
2.75 ± 0.01##
4.41 ± 0.13**
Myeloid-specific ikkβ gene deletion decreases the percentages of CD4+ T, Th1, and Th17 cells but increases the percentage of Treg cells during EAE
Since naïve CD4 T cells may differentiate into Th1, Th2, Th17, and regulatory T (Treg) cells during T cell receptor (TCR) activation in a particular cytokine milieu involved in autoimmunity, we characterized each subtype of CD4+ T cells in the EAE-induced spinal cord. It is well-known that MS/EAE is mediated by encephalitogenic Th1 and Th17 cells that produce proinflammatory cytokines such as IFN-γ and IL-17, respectively [12, 59]. Thus, we investigated the effects of myeloid IKKβ deletion on the infiltration/activation of CD4+/IFN-γ+ (Th1) and CD4+/IL-17+ T (Th17) cells in the spinal cords and lymph nodes. In the EAE-induced mice, the CD4+/IFN-γ+ Th1 population increased to 11.33 ± 0.69 % and 9.40 ± 1.98 % in the spinal cord (Table 2 and Additional file 4a) and lymph node (Table 2 and Additional file 4b), respectively. Similarly, CD4+/IL-17+ T cell populations in the spinal cord and lymph node increased to 14.35 ± 1.21 % (Table 2 and Additional file 4a) and 14.76 ± 2.64 % (Table 2 and Additional file 4b), respectively. However, in LysM-Cre/Ikkβ F/F mice, Th1 and Th17 cells increased to only 6.34 ± 0.22 % and 5.43 ± 0.63 %, respectively, in the spinal cord (Table 2 and Additional file 4a) and to 4.59 ± 0.72 % and 4.13 ± 0.66 %, respectively, in the lymph nodes (Table 2 and Additional file 4b). Additionally, mRNA induction of IFN-γ, IL-23 (an interleukin that induces differentiation of naïve Th cells to Th17 cells), and IL-17 in the EAE-induced spinal cord was markedly reduced by myeloid IKKβ deletion (Fig. 4d-f). The percentage of CD4+/IL-4+ Th2 cells in the spinal cords (Table 2 and Additional file 4a) or lymph nodes (Table 2 and Additional file 4b) was not significantly altered in WT and LysM-Cre/Ikkβ F/F mice upon EAE induction. Likewise, mRNA expressions of IL-4 (a cytokine that induces the differentiation of naïve Th cells to Th2 cells) and IL-5 (an interleukin produced by Th2 cells) were not significantly affected by immunization or myeloid IKKβ deletion (Fig. 4g and h). Interestingly, the number of CD4+/Foxp3+ and CD4+/CD25+/Foxp3+ Treg cells that maintain tolerance to self-antigens and suppress autoimmune responses increased in the spinal cord of EAE-induced mice compared to normal control mice (0.46 ± 0.06 % vs. 1.55 ± 0.15 %) (Table 2 and Additional file 4a). Notably, this increase was further potentiated by myeloid IKKβ deletion (0.47 ± 0.10 % vs. 4.05 ± 0.80 %), which corresponded with mRNA expression of Foxp3 (a “master gene” for controlling the development and function of Treg cells) in the spinal cord (Fig. 4i). Although the Treg cell populations in the lymph node were reduced in EAE-induced mice compared to the sham-control, there were still more Treg cells in the LysM-Cre/Ikkβ F/F mice compared to the WT mice (Table 2 and Additional file 4b).
Myeloid-specific ikkβ gene deletion inhibits the activity of co-stimulatory factors in macrophages and regulates the differentiation/proliferation of T cells by co-culture with microglia
Myeloid-specific ikkβ gene deletion inhibits BBB permeability with during EAE
Myeloid-specific ikkβ gene deletion maintains BBB integrity in the spinal cord during EAE
Myeloid IKKβ deletion attenuates transferred EAE
Myeloid-specific ikkβ gene deletion ameliorates transferred EAE
Mean day of onset (± SEM)
Maximal clinical score (± SEM)
Sun of clinical score (± SEM)
7.4 ± 1.6
4.2 ± 0.7
43.7 ± 10.6
9.4 ± 0.2
3.6 ± 0.2
33.7 ± 2.1
13.6 ± 1.6*
0.7 ± 0.1**
7.5 ± 2.1**
11.6 ± 1.4
1.4 ± 0.2**
12.9 ± 1.8**
In this study, we investigated the in vivo role of IKK/NF-κB-dependent inflammatory myeloid cell activation in EAE using LysM-Cre/Ikkβ F/F mice. We found that inhibiting inflammatory myeloid cell activation delayed the onset day and alleviated the severity of EAE clinical signs, which was accompanied by reduced demyelination and immune cell infiltration in the spinal cord. The beneficial mechanism of myeloid IKKβ deletion was associated with diminished expansion of Th1 and Th17 cells and increased expression of Treg cells in addition to reduced BBB damage.
In the brain of MS patients or in the EAE animal model, IKK/NF-κB activation is detected in various cell types not only in brain-infiltrating immune and inflammatory cells but also in neurons and oligodendrocytes . The critical role of NF-κB signaling pathways in the development of EAE has long been investigated in in vitro as well as in vivo studies using conventional knockout mice [11, 21]. More recently, the cell-type specific in vivo roles of NF-κB activation in EAE pathology are currently being characterized by different groups using cell type-specific conditional knockout or transgenic mice. For instance, transgenic inactivation of astroglial NF-κB improved the functional and pathological outcomes of EAE by decreasing the astrocyte proinflammatory cytokines and chemokines, indicating the detrimental role of astrocyte NF-κB activation in EAE [3, 4]. Neuronal specific NF-κB ablation through transgenic expression of an IkBα super-repressor (IκBɑ-AA) did not influence neuro-axonal degeneration in EAE, suggesting that neuronal NF-κB signaling is dispensable for EAE .
Although research on the cell type-specific function of NF-κB signaling pathways for autoimmune demyelinating disease in CNS cells (neurons and astrocytes) is relatively well-developed, the exact functions of the NF-κB signaling pathways in myeloid cells for MS/EAE remains elusive. In a study, over-expression of the triggering receptor expressed on myeloid cells 2 (TREM2), a NF-κB target gene, in myeloid cells ameliorated clinical symptoms and reduced demyelination as well as axonal damage in the spinal cord of EAE mice, suggesting a neuroprotective role of myeloid NF-κB signaling . In a more recent study, constitutive activation of NF-κB through myeloid specific IkBα deletion resulted in a more severe clinical course of EAE, indicating a detrimental role of myeloid NF-κB activation in EAE development . Our study using LysM-Cre/Ikkβ F/F mice basically confirmed the previous study by Ellrichmann et al., demonstrating that myeloid NF-κB signalling plays a detrimental role in EAE . Furthermore, in our study, we uncovered the mechanisms underlying these detrimental effects of myeloid NF-κB activation. We found that myeloid NF-κB signaling affects the T cell activation profile during EAE; myeloid IKKβ deletion reduced Th1 and Th17 populations in the spinal cord and lymph nodes, but enhanced the activation of Treg cells in EAE mice. An altered Th cell profile can alleviate demyelination and attenuate the severity of clinical signs of EAE. Therefore, IKKβ/NF-κB-dependent myeloid activation may contribute to EAE pathogenesis by increasing Th1 and Th17 cell responses, while inhibiting Treg cell responses.
The involvement of NF-κB signaling pathways in encephalitogenic T cell activation has been reported in previous studies. In NF-κB/p50-deficient mice, myelin-specific T-cells are unable to differentiate into either Th1- or Th2-type effector cells in vivo . Mice that received mutated NF-κB essential modifier-binding domain (NBD) peptides did not demonstrate a shift in immune responses from a Th1 to a Th2 profile and exhibited reduced encephalitogenicity of myelin-specific T cells . In addition, highly impaired T cell response is observed in splenocyte cultures from T cell specific IKK2 deficient mice . These Th cell profile changes in previous studies were mainly attributed to NF-κB signaling in T cells. In our study, however, we found for the first time to our knowledge that myeloid NF-κB signaling affects the encephalitogenic T cell activation profile in EAE.
In addition, we found that myeloid NF-κB signalling also affects Th17 cell differentiation. Myeloid IKKβ deletion attenuated the mRNA expression of IL-17 and IL-17α, as well as the population of CD4+/IL-17+ T cells in spinal cords and lymph nodes from EAE mice. NF-κB signalling in T cells has been implicated in Th17 cell activation in previous studies [5, 50]. However, the effect of myeloid NF-κB activation on Th17 cell differentiation has not been reported. Thus far, it is not clear how IKK/NF-κB-mediated myeloid cell activation affect Th1 and Th17 activations. In our study, we observed the Th1/Th17-differentiating effect of myeloid IKK/NF-κB activation both at the spinal cord and the peripheral lymph nodes. Thus, the effects more likely occur during T cell activation in the peripheral lymph node by myeoloid-derived antigen presenting cells (macrophages and myeloid dendritic cells). Indeed, in our study, we found that the expression of various costimulatory molecules and proinflammatory cytokines in the lymph node were down-regulated in the LysM-Cre/Ikkβ F/F mice, which might be responsible for the reduced Th1 and Th17 cell activation. However, it is also possible that altered chemokine expression in the IKK-deleted spinal cord microglia and macrophage affected the recruitment of Th1 or Th17 cells from the circulation, which was not formally tested in our study. In return, we confirmed that T cells co-cultured with microglia derived from LysM-Cre/Ikkβ F/F mice displayed lower percentages of Th1 and Th17 cells but higher percentage of Treg cells than those in the control group. Taken together, our findings suggest that myeloid IKKβ can potentiate Th1/Th17 T cell differentiation in the periphery and activation in the spinal lesion of EAE mice, which can exacerbate the clinical severity of EAE.
It is well-known that Treg cells are critical for controlling disease severity in autoimmunity [7, 12, 53, 62]. Treg cells develop in response to TGF-β and express high levels of CTLA-4 and Foxp3 [7, 53, 62]. The critical role of Treg cells in EAE was demonstrated in a study showing aggravated EAE pathogenesis upon Treg cell depletion . We confirmed that myeloid IKKβ deletion significantly up-regulated the number of CD4+/CD25+/Foxp3+ T cells and the mRNA expression of TGF-β and Foxp3 in the spinal cord and lymph nodes after the peak point of EAE clinical signs. Our findings are in line with recent reports showing that artemisinin analogue SM934 , dexamethasone , and electroacupuncture  ameliorated EAE by enhancing the expansion and functions of Treg cells. Of note, the Treg population in peripheral lymph nodes was decreased upon EAE induction, although it increased in the spinal cord. It is likely that the Th1 and Th17 cell proliferation rate in peripheral lymph nodes outweigh the Treg proliferation rate, thus decreasing the relative Treg percentage upon EAE induction. Nevertheless, our results suggest that the EAE-suppressing effects of myeloid IKKβ deletion may be partly due to the increase in the differentiation/activation of Treg cells in the periphery and recruitment/infiltration into the spinal lesion.
Besides the effects on peripheral T cell activation, our data suggest that attenuated BBB compromise may be another mechanism for the EAE-suppressing effects of myeloid IKK/NF-κB signaling. In our study, the EAE-induced BBB leakage measured by Evans blue staining was clearly reduced by myeloid IKKβ deletion, demonstrating that myeloid IKK/NF-κB activation potentiates BBB damage. The effects of myeloid IKKβ on BBB compromise was also confirmed by immunostaining of PECAM-1 and fibronectin proteins, other markers of BBB damage, which were increased in the spinal cords of WT EAE mice in correlation with astrocytic activation. However, the increased immunofluorescence was diminished in LysM-Cre/Ikkβ F/F mice in agreement with the reduction of astrocytic activation. These data suggest that the suppression of astrocytic activation by myeloid IKKβ deletion may be associated with reduced permeability of the BBB. These findings were similar but distinct from the previous studies in which PECAM-1 and fibronectin were increased in proportion to the level of inflammation in EAE and were observed around capillaries in the CNS of EAE mice [15, 29, 45, 61]. In our study, we found that PECAM-1 and fibronectin are upregulated in activated astrocytes. Although we have not elucidated how myeloid NF-κB activation might have induced astrocyte activation and PECAM-1/fibronectin upregulation, our result suggests putative reciprocal interactions among myeloid cells (specifically, M1/M2 phenotype cells), astrocyte, proinflammatory cytokines/chemokines, cell adhesion molecule, and endothelial cells in the spinal cord. Further studies regarding the molecular interaction of myeloid NF-κB activation and BBB are merited. Since astrocytes are critical in maintaining BBB integrity [51, 60], IKK/NF-κB-mediated myeloid activation might compromise the normal integrity of the BBB by inducing astrocyte activation. Additionally, although pertussis toxin is very effective in permeabilizing an intact BBB  and thus allowing myelin-specific T cells to enter the CNS, other mechanisms by which pertussis toxin promotes neuroinflammation in EAE have been suggested . Our results suggest that the enhancement effect of pertussis toxin and inhibitory effect of myeloid IKKβ deletion for permeability of the BBB might be in a mixture in the present study. The BBB of myeloid IKKβ deleted mice might be more resistant to pertussis toxin than WT mice, although the physiologic mechanism remains unclear.
We found that myeloid cell-specific IKKβ expression plays a pivotal role for demyelination in the EAE model. The inhibition of NF-κB activation by conditional deletion of IKKβ in myeloid cells resulted in alleviated clinical signs of EAE compared with WT mice, corresponding with decreased spinal demyelination and glial activation, decreased infiltration of peripheral immune cells, and reduced expression of proinflammatory mediators. Interestingly, these beneficial effects were associated with the suppression of Th1 and Th17 T cells, the up-regulation of Treg cells, and reduced BBB damage. Our findings imply that therapies targeting IKKβ function in myeloid cells may be effective for the treatment of MS and other demyelinating pathologies of the CNS.
BBB, blood–brain barrier; CFA, complete Freund’s adjuvant; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; EB, Evans blue; EMEM, Eagle’s minimal essential medium; Iba-1, ionized calcium binding adaptor molecule-1; IKK, IkB kinase; IL, interleukin; iNOS, inducible nitric oxide synthase; LFB, luxol fast blue; LPS, lipopolysaccharide; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NF-κB, nuclear factor kappa B; PECAM-1, platelet endothelial cell adhesion molecule-1; PTX, pertussis toxin; TCR, T cell receptor; TGF-β, transforming growth factor-beta; Th, T-helper; TNF-α, tumor necrosis factor-alpha; Tregs, regulatory T cells; TREM2, triggering receptor expressed on myeloid cells 2; WT, wild-type; ZO-1, zona occluden-1
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2010–0010858 and 2014R1A2A1A11051240).
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All supporting data help readers understand manuscript and main data.
MJL performed the behavioral experiment, immunohistochemistry, PCR analysis, flow cytometry analysis, Western blots analysis, and prepared figures. SJB and YJ performed the thymidine incorporation. GL assisted with flow cytometry analysis. JC, MJ, and HL assisted with behavioral experiments, genotyping, mouse maintenance, or cell culture. BSC performed the morphological analysis using semithin sections. SJL contributed to the interpretation of data and supervised the project. IHC conceived all experiments, analyzed the results, and wrote the manuscript. All authors have read and approved the final manuscript.
All the authors of this manuscript have no competing interest in this subject.
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Ethical approval and consent to participate
All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kyung Hee University. Proper randomization of laboratory animals and handling of data were performed in a blinded manner in accordance with recent recommendations from a NIH Workshop on preclinical models of neurological diseases .
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