Background
Multiple sclerosis (MS) is among one of the most common neurodegenerative diseases affecting an estimated 2.3 million individuals worldwide [
1]. This organ-specific autoimmune disease is characterized by four different types of demyelinating plaques; types I and II which are T cell mediated or T cell and antibody-mediated, while types III and IV are mediated by oligodendrocyte death [
2]. In all four cases, plaques are associated with activated macrophages, microglia, and astrocytes.
Regardless of the type of plaque formation, inflammation plays a central role in MS pathophysiology [
1,
3].
Microglia, the resident immune cells of the central nervous system (CNS), play a major role in maintaining CNS homeostasis. They have been shown to be associated with developing plaques and are thought to contribute to the development of MS [
2,
4], as well as other chronic inflammatory neurodegenerative diseases such as Alzheimer’s [
5]. During MS, activated microglia can play the role of antigen-presenting cells (APCs) and, therefore, skew T cell responses towards a T helper cell 1 (Th1) pro-inflammatory phenotype [
1,
2,
6]. In addition, once activated, microglia upregulate the expression of pro-inflammatory molecules including but not restricted to tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, macrophage inhibitory protein 1 alpha (MIP1α), and inducible nitric oxide synthase (iNOS), all of which have been shown to play a role in demyelination and neuronal damage [
7].
There is a wide variety of immune receptors expressed by microglia that regulate its function. Pathogen-recognition receptors (PRRs) such as nod-like receptors (NLRs) are innate immune receptors and sensors of pathogen-associated molecular patterns (PAMPs) [
8]. NLRs are a group of proteins that share a NACHT and leucine-rich repeat (LRR) domain but differ in their N-terminal effector domain. Upon recognition of their respective ligand, NLRs become activated and it result in the subsequent triggering of multiple pro-inflammatory molecular pathways, such as nuclear factor-kappa B (NF-κB). In addition, they are able to regulate both innate and adaptive immune responses and play a role in pathological processes [
8]. Recently discovered
Nlrp12 is a pyrin-containing intracellular NLR protein. It is largely expressed in the cells of myeloid origin such as monocytes and dendritic cells (DCs). The expression of
Nlrp12 has been shown to play an important role in immune inflammatory responses by negatively regulating the NF-κB pathway and modulatory roles, such as dendritic cell migration [
9,
10]. The NF-κB pathway is one of the major pathways involved in the inflammatory response. Typically, the activation of NF-κB following insults results in the transcription of pro-inflammatory cytokines such as TNFα, IL-1β, and IL-6; chemokines such as CCL5, CCL22, and MIP1α; and proteins, such as iNOS and cyclooxygenase 2 (COX2) [
11,
12].
This study aims to investigate the role of NLRs in neuroinflammation, particularly to uncover the role of Nlrp12 during experimental autoimmune encephalomyelitis (EAE) development. In our study, results show that Nlrp12 acts to downregulate inflammation during the development of EAE. This study may have significant implications in the development of potential novel therapies to treat MS and other neuro-inflammatory degenerative diseases.
Materials and methods
Mice
Nlrp12 knock-out (Nlrp12
−/−
) mice were kindly provided by Dr. Jenny P. Y. Ting (Chapel Hill, NC). All of the protocols and procedures were approved by the University of Sherbrooke at the University of Sherbrooke Animal Facility and Use Committee.
Experimental autoimmune encephalomyelitis
EAE was induced in 8–10-week-old C57BL/6 female mice using a previously established protocol by Miller et al. [
13]
. Briefly, a 1:1 emulsion mixture of myelin oligodendrocyte glycoprotein (MOG
35−55) (Genemed Synthesis Inc., San Antonio, TX) and complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO) supplemented with 100 μg
Mycobacterium tuberculosis H37 RA (Difco Laboratories, Detroit, MI) was prepared using a glass tuberculin syringe. The MOG:CFA emulsion (100 μL) was injected subcutaneously on each side of the midline on the lower back of each mouse for a total of 200 μg MOG
35–55 and 500 μg
Mycobacterium. Pertussis toxin (200 ng) (List Biological Laboratories Inc., Campbell, CA) was injected intraperitoneally on the day of and 48 h following immunization. The mice were monitored every day for the development of disease. Clinical scores were given by two independent observers, using the following scale: 0, no sign of disease; 1, limp tail or weakness in limbs; 2, limp tail and weakness in limb; 3, partial limb paralysis; 4, complete limb paralysis.
Histopathology
The immunized mice were anesthetized by intraperitoneal injection of Avertin® (2,2,2-tribromoethanol, approximately 240 mg/kg) (Sigma-Aldrich, St. Louis, MO) diluted in 0.9 % saline solution. The mice were then perfused with ice-cold phosphate-buffered saline (PBS) (Wisent, St. Bruno, QC), and the spinal cords were removed and stored at −80 °C immediately for RNA extraction (thoracic region) and placed in 4 % paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for immunofluorescence analysis (lumbar region). The spinal cord tissues were embedded in paraffin and cut into 5-μm sections.
T cell proliferation assay
T cell proliferation was performed using 3H-thymidine incorporation assay. A single cell suspension was prepared from draining the lymph nodes (more precisely, from the inguinal and axillary lymph nodes) and spleen. CD4+ T cells were then purified using EasySep Mouse CD4+ T Cell Isolation Kit (Stem cell, Vancouver, BC), seeded in a round-bottom 96-well culture plate (1 × 105 cells/well) and activated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) antibodies for 3 days. During the last 18 h of culture, 1 μCi of methyl-[3H]-thymidine (NEN Life Sciences, Boston, MA) was added per well. The cells were harvested onto glass fiber filter mats, and the incorporated radioactivity was measured using Top Count® microplate scintillation counter (PerkinElmer, Wellesley, MA).
Intracellular IL-4 staining for flow cytometry
The purified CD4+ T cells from the wild-type (WT) and Nlrp12
−/− mice were activated by plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) antibodies for 3 days. Then, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL, Sigma Chemical Co., St. Louis, MO) and ionomycin (1 μg/mL, Calbiochem Corp., La Jolla, CA) for 4 h at 37 °C and 5 % CO2 in the presence of Brefeldin A (1 μg/mL, eBioscience, San Diego, CA). After staining the cells with anti-CD4-FITC antibody (eBioscience), the cells were fixed and permeabilized using intracellular fixation and permeabilization buffer (eBioscience) and stained with anti-IL-4-PE antibody, as per the manufacturer’s instructions. Sample analysis was performed with FACSCalibur, and data analysis was done using FlowJo Software (FlowJo, LLC, Ashland, OR).
RNA from the spinal cords and lymph nodes were extracted using TRIzol reagent (Life Technologies Inc., Burlington, ON). The tissues were homogenized with sterile beads (Qiagen, Limburg, Netherlands) at a speed of 20 Hz for 2 min. Chloroform (200 μL) (Fisher Scientific, Ottawa, ON) was added to each tube per 1 mL of TRIzol and incubated at room temperature for 15 min followed by centrifugation at 13,000 rpm for 15 min at 4 °C. Supernatants were collected in new tubes, and 500 μL isopropanol (Fisher Scientific, Ottawa, ON) was added to each tube and incubated for 10 min at −80 °C before spinning down at 13,000 rpm for 10 min at 4 °C. Pellets were washed with 75 % ethanol and re-suspended in 20 μL RNAse-free sterile water (Wisent, St-Bruno, QC). cDNA was synthesized using Oligo(dT) primer (IDT, Coralville, IA), PCR Nucleotide Mix (GE Healthcare, Baie d’Urfe, QC), M-MuLV Reverse Transcriptase, M-MuLV Reverse Transcriptase Buffer (New England BioLabs, Whitby, ON), and RNasin Ribonuclease Inhibitor (Promega, Madison, WI). Reverse transcription PCR (RT-PCR) and quantitative reverse transcription PCR (RT-qPCR) were used to verify the expression of Nlrp12, Mip3α, Cox2, IL-1β, and Ccr5 using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA). Primers (IDT, Coralville, IA) sequences were as follows: Nlrp12
F: 5′-CCT CTT TGA GCC AGA CGA AG-3′, Nlrp12
R: 5′-GCC CAG TCC AAC ATC ACT TT-3′, Mip3α
F: 5′-CTC AGC CTA AGA GTC AAG AAG ATG-3′, Mip3α
R: 5′-AAG TCC ACT GGG ACA CAA ATC-3′, Cox2
F: 5′-CCA GCA CTT CAC CCA TCA GTT-3′,
Cox2
R: 5′-ACC CAG GTC CTC GCT TAT GA-3′,
IL-1β
F: 5′-CAT CCA GCT TCA AAT CTC GCA G-3′, IL-1β
R: 5′CAC ACA CCA GCA GGT TAT CAT C-3′, Ccr5
F: 5′-CGA AAA CAC ATG GTC AAA CG-3′, Ccr5
R: 5′-GTT CTC CTG TGG ATC GGG TA-3′, 18S
F: 5′-CGG CTA CCA CAT CCA AGG AA-3′, and 18S
R: 5′-GCT GGA ATT ACC GCG GCT-3′.
The samples were normalized to the internal control 18S rRNA, and relative expression was calculated using the ΔΔC
T method [
14].
Immunofluorescence
Slides were de-paraffinized in xylene (EMD Millipore, Etobicoke, ON) and hydrated in 100, 95, and 70 % ethanol gradient. Antigen unmasking was performed at sub-boiling temperature for 10 min in 10 mM sodium citrate buffer pH 6.0 (Sigma-Aldrich, St. Louis, MO). Immunofluorescence was performed in Sequenza Slide Rack and Coverplate System (Ted Pella, Inc., Redding, CA). The slides were washed with 0.1 % Triton X-100 in PBS solution, blocked in 5 % fetal bovine serum (FBS) plus 0.1 % Triton X-100 in PBS for 1 h and incubated with primary antibody (1:1000) overnight at 4 °C. Secondary antibody (1:2000) incubation was done at room temperature for 2 h. The slides were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL), and photomicrograph pictures were taken with Retiga SRV Mono Cooled numerical camera attached to Zeiss Axioskop 2 Microscope. The pictures were stitched with Adobe Photoshop CS6, and stain density was quantified with Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD).
Antibodies
Rabbit anti-glial fibrillary acidic protein (GFAP) antibody was purchased from Cedarlane (Burlington, ON). Rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) antibody was purchased from Wako (Osaka, Japan). Alexa Fluor 488 AfinniPure Goat Anti-Rabbit IgG (H + L) was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
The percentage of microgliosis and astrogliosis in the spinal cord and gray matter were calculated as follows:
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{gliosis}\ \left(\%\right) = \frac{\mathrm{Density}\ \mathrm{stain}\ }{\mathrm{Total}\ \mathrm{area}} \times \mathsf{100} $$
The percentage of microgliosis and astrogliosis in the white matter were calculated as follows:
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{gliosis}\ \mathrm{in}\ \mathrm{the}\ \mathrm{white}\ \mathrm{matter}\ \left(\%\right) = \left(\frac{\mathrm{Density}\ \mathrm{stain}\ \mathrm{of}\ \mathrm{spinal}\ \mathrm{cord}-\mathrm{Density}\ \mathrm{stain}\ \mathrm{of}\ \mathrm{gray}\ \mathrm{matter}}{\mathrm{Total}\ \mathrm{area}\ \mathrm{of}\ \mathrm{spinal}\ \mathrm{cord}-\mathrm{total}\ \mathrm{area}\ \mathrm{of}\ \mathrm{gray}\ \mathrm{matter}}\right) \times \mathsf{100} $$
Primary cell culture
Cortices from 1-day-old pups were extracted and placed onto a 100-mm petri dish using aseptic techniques. Cortices were sliced with a commercial razor blade, further broken up with a rigorous up-and-down motion in 10 mL of medium, and filtered with a 70-μm filter. The cells were then plated onto a 100-mm petri dish and put in an incubator of 37 °C with 5 % CO2. Cell culture medium DMEM/F12 (Wisent, St. Bruno, QC) was supplemented with 10 % FBS (Invitrogen, Burlington, ON), 1 % penicillin-streptomycin solution (Wisent, St-Bruno, QC), 1 % L-glutamine solution (Wisent, St. Bruno, QC), 0.9 % sodium pyruvate solution (Wisent, St. Bruno, QC), 0.9 % MEM amino acid solution (Wisent, St. Bruno, QC), and 0.9 % amphotericin B solution (Wisent, St. Bruno, QC). The medium of the mixed glial culture was changed every 2 to 3 days. After 3 weeks, primary microglia cells were separated from astrocytes using EasySep CD11b positive selection kit following the manufacturer’s instructions (Stem cell, Vancouver, BC).
Immunoblotting
Proteins were separated in 10 % polyacrylamide gels and transferred onto PVDF (Millipore, Etobicoke, ON) membranes. The membranes were blocked with PBS containing 10 % nonfat milk and 0.05 % Tween-20 (Sigma-Aldrich, St. Louis, MO). The membranes were washed in 1× Tris-buffered saline (TBS) plus 1 % Tween-20 for 15 min and incubated with primary antibody (1:1000) overnight at 4 °C and with secondary antibody (1:2000) for 2 h at room temperature. The membranes were revealed with GE HealthCare Life Sciences Amersham ECL Plus (Baie d’Urfe, QC) and viewed with Molecular Imager VersaDoc from BioRad, and protein bands were quantified using NIH ImageJ software. The antibodies used were as follows: β-actin (rabbit), iNOS (rabbit), and anti-rabbit IgG HRP-linked antibodies were purchased from Cell Signaling Technology (Beverly, MA).
Cytokine measurement
TNFα and IL-6 cytokines in the supernatant of microglia culture were measured using ELISA kits purchased from BioLegend (San Diego, CA). Cerebellum and lymph node samples were homogenized in 0.5 mL of ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitors (Roche Diagnosis, Mannheim, Germany) by rapid agitation for 2 min in the presence of 3-mm stainless beads. The tissue lysate was centrifuged for 10 min at 13,000×g in a cold microfuge, and the supernatant was transferred to a new tube. The concentration of proteins in the lysate was determined by Bradford protein assay. The tissue levels of IL-4 were determined using a high sensitivity IL-4 ELISA Kit (eBioscience, San Diego, CA), and the concentration of IL-4 in serum samples was quantified using Mouse IL-4 DuoSet (R&D Systems), according to the manufacturer’s instruction.
Statistical analysis
All statistical analyses were conducted using GraphPad Prism 6 software. The results were expressed as mean ± SD. Statistical significance was determined using one-way ANOVA Kruskal-Wallis followed by Bonferroni (EAE clinical score), one-way ANOVA followed by Tukey-Kramer (Nlrp12 mRNA expression, iNOS expression in primary microglia, concentration of pro-inflammatory cytokines), two-way ANOVA followed by Tukey’s (percentage of gliosis), or one-way ANOVA followed by Dunet (pro-inflammatory mRNA expression) multiple comparison test. IL-4 results were compared between WT and Nlrp12
−/− mice using Mann-Whitney U test. Statistical significance was accepted at P < 0.05.
Discussion
The process of inflammation is a fundamental response aimed at protecting the body from foreign and detrimental causes. Neuroinflammation can become harmful if it is unregulated and prolonged. A continuous and persistent response will eventually lead to a chronic state of inflammation, a prominent feature of many neurodegenerative diseases, including MS. NLRP12 is of interest to the study of MS notably due to its restricted expression in cells derived from hematopoietic origins such as monocytes, dendritic cells, and granulocytic cells, and most recently, T cells [
16] and its role in attenuating the inflammatory response by interfering in both branches of the NF-κB pathway [
9,
17].
To investigate the implication of Nlrp12 in MS, EAE was induced in the WT and in
Nlrp12
−/−
mice. Our results demonstrated that in mice lacking the
Nlrp12 gene, EAE developed earlier compared to the WT mice, and the
Nlrp12
−/−
mice showed increased severity throughout the course of the disease. Interestingly, after EAE induction,
Nlrp12 mRNA expression was significantly increased in the WT mice compared to the healthy WTs. These results suggest that Nlrp12 plays an important role in maintaining the level of inflammation and ensuring that a hyper-inflammatory state does not occur. In fact, the expression profile of
Nlrp12 over the course of the disease is suggestive of this regulatory role. Indeed, previous studies have shown that in response to live bacteria such as
M. tuberculosis, TNFα, and IFNγ, a reduction in
Nlrp12’s expression is in accordance with an increase in the inflammatory response [
18,
19]. Moreover,
Nlrp12’s over-expression has been previously shown to attenuate the inflammatory response by negatively regulating the NF-κB pathways [
17]. A study conducted by Shami et al. demonstrated that the expression of
Nlrp12 is increased in response to nitric oxide [
20]. NO is a reactive molecule that is produced in iNOS at sites of inflammation in MS, and it is involved in lesion development [
21]. As T cell responses play a crucial role in the development of EAE [
12], we evaluated purified CD4 T cell proliferative response after CD3/CD28 activation and we saw significantly elevated proliferation of T cells from the
Nlrp12
−/− mice. We then evaluated T cells proliferation using recall response 10 days after EAE induction by stimulating purified CD4 T cells with MOG peptide in the presence of splenocytes. We observed the tendency of T cells from the
Nlrp12
−/− mice for a higher proliferation rate; however, these differences never reached a statistical significance. These results are similar to those published by Lukens et al. [
16], where authors observed that pure anti-CD3/CD 28 activation resulted in significantly higher proliferation in T cells from
Nlrp12
−/−
mice, while in the presence of splenocytes, differences between T cell proliferation of different genotypes were greatly reduced. In this work, the authors propose that the cell autonomous effect of
Nlrp12 in T cells shifts T cell differentiations to a Th2-IL-4 producing phenotype [
16].
We measured the concentration of IL-4 in the serum, lymph nodes, and brain samples from the WT and from
Nlrp12
−/−
mice at 3 weeks after EAE and did not find any differences in the expression of IL-4. These results are consistent with our observation that there were no differences in percentage of IL-4 producing cells and that results from experiments in complex cellular interaction at the tissue level in vivo can be different from results of clean anti-CD3/CD28 activation in vitro. Till today, no exact mechanism has been described that explains
Nlrp12 activity in different cell types.
Nlrp12 has been shown to inhibit classical and alternative pathways of NF-κB in different cell types and different stimulations; for extensive review, please read Tuncer et al. [
9]. In light of these controversies, the different KO strategies to remove
Nlrp12 may have produced an uncontrolled variable that resulted in different phenotypes [
22,
23]. Future studies should address these differences.
In our studies we observed that
Nlrp12
−/−
mice demonstrated more severe course of EAE according to classical evaluation of clinical scores, while in the work by Lukens and co-workers, the authors noted appearances of the atypical EAE. These results are intriguing, as overall effect of
Nlrp12 on the EAE pathology was similar to our observations. Furthermore, EAE is a well-characterized and the most widely used mouse model to study MS [
13]. It exhibits the main features of MS pathology such as inflammation, destruction of myelin, and reactive gliosis. Moreover, many of the current therapies for MS, such as Tysabri were developed following EAE studies [
24]. However, it is important to note that the evaluations of clinical scores are subjective. In our studies, we did not measure the degree of atypical EAE as there is no quantifiable scale to evaluate this pathology. Observing video clips published by Lukens et al. (supplemental materials), we can tell that
Nlrp12 mouse was severely compromised and had impaired righting reflex, which suggests severe weakness/paralysis of the hind limbs as well as paralysis of the trunk muscles.
To further elucidate how
Nlrp12 is playing a protective role in the disease, the spinal cords of both the WT and
Nlrp12
−/−
mice were analyzed for the expression of genes implicated in EAE as well as in MS. Our results demonstrated a significant increase in the mRNA expression of
Cox-2,
IL-1β, and
Ccr5 genes in the
Nlrp12
−/−
mice compared to the WT mice, suggesting a protective role played by
Nlrp12 in EAE at the level of pro-inflammatory gene expression. The increase in expression of pro-inflammatory molecules in
Nlrp12-deficient phenotype has been demonstrated by multiple studies [
22,
25].
Next, we demonstrated that
Nlrp12 inhibits inflammation during EAE at the level of microglia. We showed that
Nlrp12 deficiency augments pro-inflammatory microglial phenotypes by using purified primary microglia cells from the WT and
Nlrp12
−/−
mice. Consistent with our in vivo observation, stimulation of microglia with LPS resulted in a significant increase of iNOS expression, NO, TNFα, and IL-6 secretion from the
Nlrp12
−/−
microglia cells compared to the WT microglia. These results are consistent with the suppressive role of
Nlrp12 in cells of myeloid origin [
26]. A report by Lukens et al. also found increased inflammatory response in the CNS tissue of
Nlrp12
−/−
mice compared to WT controls, although, microglia responses per se were not verified. Furthermore, the notion of inhibitory NLRs is not new. Similar to our results, stimulation of primary
Nlrx1
−/−
microglia cells revealed a significant increase in the pro-inflammatory response, thus, showing a suppressive role for Nlrx1 in microglial activation [
27].
The roles of microglia and astrocytes are well defined in the pathology of MS. Previous studies on Nlrp3 have demonstrated that the absence of this receptor results in better disease outcome and reduced gliosis following EAE [
28]. The spinal cords of the
Nlrp12
−/−
mice and WT mice were stained with Iba1 and GFAP in order to assess the extent of microgliosis and astrogliosis, respectively. Surprisingly, no differences in the percentage of gliosis were observed between the two genotypes at the third week; however, after 9 weeks, the
Nlrp12
−/−
mice demonstrated significantly increased gliosis compared to the WT mice. Additionally, in both genotypes, the majority of gliosis occurs within the white matter area of the spinal cord. Although a quantitative difference was not observed at the third week, in vitro study suggests qualitative changes in microglia activation. Indeed upon LPS stimulation, microglia from the
Nlrp12
−/−
mice released significantly more pro-inflammatory mediators. Furthermore, the remarkable increase in
Ccr5 mRNA expression observed in the
Nlrp12
−/−
mice suggests that Nlrp12 may be playing a crucial role in the influx of inflammatory infiltrates. CCR5 is a chemokine receptor that is expressed primarily by monocytes, macrophages, effector T cells, immature dendritic cells, and NK cells [
29]. Moreover, previous studies in both animal and in MS patients have demonstrated the upregulation of CCR5 in inflammatory lesions [
30‐
32]. Also, a chronic over-expression of IL-1β has been shown to result in the disruption of the blood-brain barrier (BBB) and in the infiltration of leukocytes such as macrophages, DCs, and neutrophils [
33,
34]. Thus, the increase of
Ccr5 and
IL-1β mRNA in the spinal cords of the
Nlrp12
−/−
compared to the WT mice during EAE supports the notion of an increased influx of inflammatory cells in these mice. In fact, the entry of pro-inflammatory leukocytes into the CNS is an early phenomenon capable of initiating events that result in BBB disruption and neuroinflammation [
35]. Interestingly, previous studies have demonstrated a reduction in inflammatory infiltrates within the CNS in EAE-induced
Nlrp3
−/−
mice, where Nlrp3 was shown to play an inflammatory role by inducing immune cell migration whereas, our results suggest that
Nlrp12 plays a protective role by maintaining the level of inflammatory influx [
36,
37]. Thus, future studies should focus on evaluating in details the presence of inflammatory infiltrates in order to clarify the driving force responsible for the differences observed between WT and
Nlrp12
−/−
mice.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MG, TM, and DG designed the experiments and wrote the manuscript. MG, TM, and EI performed the experiments. PG made the silica analysis. EZ, BD, SI, and AA helped for the complementary experiments in T cells. All authors read and approved the manuscript.